# **Special Issue in Honor of Dr. Michael Weber's 70th Birthday**

**Photodynamic Therapy: Rising Star in Pharmaceutical Applications**

> Edited by Eduard Preis, Matthias Wojcik, Gerhard Litscher and Udo Bakowsky

Printed Edition of the Special Issue Published

## **Special Issue in Honor of Dr. Michael Weber's 70th Birthday: Photodynamic Therapy: Rising Star in Pharmaceutical Applications**

## **Special Issue in Honor of Dr. Michael Weber's 70th Birthday: Photodynamic Therapy: Rising Star in Pharmaceutical Applications**

Editors

**Eduard Preis Matthias Wojcik Gerhard Litscher Udo Bakowsky**

MDPI ' Basel ' Beijing ' Wuhan ' Barcelona ' Belgrade ' Manchester ' Tokyo ' Cluj ' Tianjin

*Editors* Eduard Preis Department of Pharmaceutics and Biopharmaceutics University of Marburg Marburg Germany

Matthias Wojcik Department of Pharmaceutics and Biopharmaceutics University of Marburg Marburg Germany

Gerhard Litscher Research Unit of Biomedical Engineering in Anesthesia and Intensive Care Medicine Medical University of Graz Graz Austria

Udo Bakowsky Department of Pharmaceutics and Biopharmaceutics University of Marburg Marburg Germany

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Pharmaceutics* (ISSN 1999-4923) (available at: www.mdpi.com/journal/pharmaceutics/special issues/Honor Michael).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-5960-5 (Hbk) ISBN 978-3-0365-5959-9 (PDF)**

© 2022 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.

## **Contents**


In Vivo Quantification of the Effectiveness of Topical Low-Dose Photodynamic Therapy in Wound Healing Using Two-Photon Microscopy

Reprinted from: *Pharmaceutics* **2022**, *14*, 287, doi:10.3390/pharmaceutics14020287 . . . . . . . . . **127**


## **Daphne N. Dorst, Marti Boss, Mark Rijpkema, Birgitte Walgreen, Monique M. A. Helsen and Desir ´ee L. Bos et al.**

Photodynamic Therapy Targeting Macrophages Using IRDye700DX-Liposomes Decreases Experimental Arthritis Development

Reprinted from: *Pharmaceutics* **2021**, *13*, 1868, doi:10.3390/pharmaceutics13111868 . . . . . . . . **289**


Strategies Reprinted from: *Pharmaceutics* **2021**, *13*, 1995, doi:10.3390/pharmaceutics13121995 . . . . . . . . **463**

## *Editorial* **Editorial on the "Special Issue in Honor of Dr. Michael Weber's 70th Birthday: Photodynamic Therapy: Rising Star in Pharmaceutical Applications"**

**Eduard Preis 1,\* , Matthias Wojcik 1,\*, Gerhard Litscher 2,\* and Udo Bakowsky 1,\***


Thousands of years ago, phototherapy or heliotherapy was performed by ancient Egyptians, Greeks, and Romans. However, from the mid-19th century onward, names such as Arnold Rikli, Niels Ryberg Finsen, Downes and Blunt, Oscar Raab, and Hermann von Tappeiner started to appear and paved the way for current experts in the field of photodynamic therapy (PDT), such as Wainwright, Maisch, and Hamblin. Still, only a tiny fraction of PDT's potential has been realized in the clinical practice guidelines [1].

This Special Issue of *Pharmaceutics* commemorates the tremendous influence Michael Weber's work had on the use and practical applications of PDT. Looking back after his 70th birthday, in addition to his inventions, patents, and publications, he always found room for hands-on experience as a practicing doctor. He pioneered the clinical use of lasers and photodynamic therapy for nearly 25 years in Germany and several other countries.

Multiple contributions from all over the world emphasize the importance of PDT and signal that there is much more to expect. The mechanism of PDT generally relies on three main components, i.e., light, a photosensitizer (PS), and molecular oxygen; however, it can be subdivided into different applications, which sometimes leads to confusing abbreviations and definitions. When applied against bacteria and fungi, it is often referred to as antimicrobial photodynamic therapy (aPDT) or photodynamic antimicrobial chemotherapy (PACT); against viruses, it is called antiviral photodynamic therapy (aPDT); against cancer cells, most researchers use the unmodified term photodynamic therapy (PDT). To simplify this matter, we divide photodynamic therapy into two parts, i.e., cancer treatment (PDT) or antimicrobial and antiviral therapy (aPDT).

The most extensive section in this Special Issue is aPDT, which comprises five articles and one communication covering bacteria treatment, three articles focusing on wound healing, and one article each looking at antifungal and antiviral therapy. González et al. and Núñez et al. used transition metal complexes, i.e., a homo-bimetallic Re(I) complex [2] and a polypyridine Ir(III) complex [3]. Both proved that these complexes could be effectively used in aPDT. Whereas the first showed an enhanced effect by combining the PS with cefotaxime, the latter successfully used imipenem. Garcia et al. applied Fotoenticine®, a new PS derived from chlorin e-6, on a microcosm biofilm [4]. In this ex vivo model, which is closer to the complex in vivo conditions, a qualitative and quantitative reduction in bacterial viability was shown. Regarding biofilms, Battisti et al. highlighted the new fluorescence lifetime imaging, which might offer more insights into the biofilm dynamics and facilitate treatment optimization [5].

**Citation:** Preis, E.; Wojcik, M.; Litscher, G.; Bakowsky, U. Editorial on the "Special Issue in Honor of Dr. Michael Weber's 70th Birthday: Photodynamic Therapy: Rising Star in Pharmaceutical Applications". *Pharmaceutics* **2022**, *14*, 1786. https://doi.org/10.3390/ pharmaceutics14091786

Received: 23 August 2022 Accepted: 24 August 2022 Published: 26 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Cuadrado et al. and Ayoub et al. used a more technological approach and processed the PS. They incorporated their PS (i.e., zinc menthol-phthalocyanine or parietin) into magnetic nanocomposites [6] or cyclodextrin-inclusion complexes [7].

Additionally, three articles took bacteria treatment one step further and focused more on wound healing. They bridge the gap between in vitro and in vivo conditions [8] and show new in vivo quantification methods [9,10].

Pérez-Laguna et al. assessed the effect of combination therapy against different strains of *Candida* spp. using methylene blue as PS and chlorhexidine [11]. They achieved a reduction in methylene blue concentration while maintaining the same photodynamic efficacy. Another study by Sadraeian et al. compared the effects of UV-C light and aPDT with photodithazine against SARS-CoV-2 pseudovirus [12].

The PDT section consists of five articles covering different aspects. Chai et al. synthesized a new PS that showed beneficial properties (i.e., tracking and ablation) against HepG2 human hepatocellular carcinoma cells [13]. Dobre et al. investigated the gene expression pattern of HT29 cells treated with a new porphyrin derivate that they had previously synthesized and analyzed [14]. Nanosized drug delivery systems are vital when applying the most PS in PDT. Thus, Lehmann et al. and Yeh et al. incorporated their PS in liposomes and lipid-calcium phosphate nanoparticles, respectively [15,16]. While the first group reported the feasibility of liposome nebulization and pulmonary drug delivery, the second successfully treated SCC4 and SAS cells in vitro and in a xenograft model with a combination therapy using EGFR siRNA and PDT. Bartosi´nska et al. compared three different forms of 5-aminolevulinic acid in treating actinic keratosis and showed that 5-aminolevulinic acid phosphate was superior to the other forms at present due to its higher tolerability and lesser pain [17].

Two articles covering immunomodulatory therapy extended the scope of the two defined sections, PDT and aPDT. Dorst et al. assessed the efficacy of IRDye700DX-loaded liposomes in the treatment of arthritis and provided insights into the difficulties of these treatment regimens [18]. Christensen et al. presented the first-in-human study of 5-aminolevulinic acid against chronic graft-versus-host disease [19]. They used extracorporeal photopheresis combined with photoactivation of the generated protoporphyrin IX and proved the tolerability and safety of the procedure. Apart from these captivating research articles, seven profound review articles deal with different fields in PDT and aPDT. A broad overview of PDT, from history to future perspectives, was given by Correia et al. [20]. They summarized the essential parameters, discussed advantages and limitations, and emphasized that PDT is a promising therapeutic option. Lange et al. and Ailioaie et al. focused on the use of cyanine-derived dyes and curcumin in PDT, respectively [21,22]. Whereas the first group thoroughly described all used dyes, the second included all technological advances and presented the use of curcumin against different cancer types in detail. Fahmy et al. focused their article on liposomal formulations in PDT, highlighted the versatility of liposomes, and listed an astonishing number of the most recent state-of-the-art studies [23]. The comprehensive review by Piaserico et al. deals with the possible applications of PDT against actinic keratoses. They provided important information on the benefits of post- and pre-treatment strategies that improve the therapeutic efficiency of PDT [24].

A systematic review by Dalvi et al. on using aPDT against periodontitis demanded more robust and well-designed studies due to the substantial flaws limiting their reproducibility [25]. Starting from single PS, along with their different classes and drug-delivery systems, Youf et al. continued to thoroughly describe all possible combinations with aPDT [26].

Overall, photodynamic therapy is a lively topic that can be used in various fields. In 2017, despite some breakthroughs, the world did not seem prepared for PDT and aPDT. We look forward to the changes in clinical practice in the upcoming years.

We thank *Pharmaceutics* (MDPI) and their excellent team for permanently staying by our side and helping us to produce this Special Issue.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Article* **Effective Treatment against ESBL-Producing** *Klebsiella pneumoniae* **through Synergism of the Photodynamic Activity of Re (I) Compounds with Beta-Lactams**

**Iván A. González <sup>1</sup> , Annegrett Palavecino <sup>2</sup> , Constanza Núñez <sup>2</sup> , Paulina Dreyse <sup>3</sup> , Felipe Melo-González <sup>4</sup> , Susan M. Bueno <sup>4</sup> and Christian Erick Palavecino 2,\***


**Abstract:** Background: Extended-spectrum beta-lactamase (ESBL) and carbapenemase (KPC<sup>+</sup> ) producing *Klebsiella pneumoniae* are multidrug-resistant bacteria (MDR) with the highest risk to human health. The significant reduction of new antibiotics development can be overcome by complementing with alternative therapies, such as antimicrobial photodynamic therapy (aPDI). Through photosensitizer (PS) compounds, aPDI produces local oxidative stress-activated by light (photooxidative stress), nonspecifically killing bacteria. Methodology: Bimetallic Re(I)-based compounds, PSRe-µL1 and PSRe-µL2, were tested in aPDI and compared with a Ru(II)-based PS positive control. The ability of PSRe-µL1 and PSRe-µL2 to inhibit *K. pneumoniae* was evaluated under a photon flux of 17 µW/cm<sup>2</sup> . In addition, an improved aPDI effect with imipenem on KPC<sup>+</sup> bacteria and a synergistic effect with cefotaxime on ESBL producers of a collection of 118 clinical isolates of *K. pneumoniae* was determined. Furthermore, trypan blue exclusion assays determined the PS cytotoxicity on mammalian cells. Results: At a minimum dose of 4 µg/mL, both the PSRe-µL1 and PSRe-µL2 significantly inhibited in 3log<sup>10</sup> (>99.9%) the bacterial growth and showed a lethality of 60 and 30 min of light exposure, respectively. Furthermore, they were active on clinical isolates of *K. pneumoniae* at 3–6 log10. Additionally, a remarkably increased effectiveness of aPDI was observed over KPC<sup>+</sup> bacteria when mixed with imipenem, and a synergistic effect from 3 to 6log<sup>10</sup> over ESBL producers of *K. pneumoniae* clinic isolates when mixed with cefotaxime was determined for both PSs. Furthermore, the compounds show no dark toxicity and low light-dependent toxicity in vitro to mammalian HEp-2 and HEK293 cells. Conclusion: Compounds PSRe-µL1 and PSRe-µL2 produce an effective and synergistic aPDI effect on KPC<sup>+</sup> , ESBL, and clinical isolates of *K. pneumoniae* and have low cytotoxicity in mammalian cells.

**Keywords:** photodynamic therapy; multi-drug resistance; antibiotic synergy; *Klebsiella pneumoniae*

#### **1. Introduction**

Due to the emergence of multi-drug resistance (MDR) pathogenic bacteria, the deficit of new antibiotics is one of the most pressing threats to human health in the 21st century [1]. The world health organization has presented a ranking of the most relevant MDR bacteria that require the urgent development of new antimicrobial therapies. Strains of

**Citation:** González, I.A.; Palavecino, A.; Núñez, C.; Dreyse, P.; Melo-González, F.; Bueno, S.M.; Palavecino, C.E. Effective Treatment against ESBL-Producing *Klebsiella pneumoniae* through Synergism of the Photodynamic Activity of Re (I) Compounds with Beta-Lactams. *Pharmaceutics* **2021**, *13*, 1889. https://doi.org/10.3390/ pharmaceutics13111889

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 14 September 2021 Accepted: 1 November 2021 Published: 8 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*Klebsiella pneumoniae* producing extended-spectrum β-lactamase (ESBL) and carbapenemase (KPC) are among the most relevant [2,3]. *K. pneumoniae* is a Gram-negative bacillus associated with pneumonia and urinary tract infections (UTI) [4,5]. Additionally, *K. pneumoniae*is one of the most relevant agents of healthcare-associated infections (HAIs) [6]. The HAIs produced by *K. pneumoniae* can be severe, producing mortalities as high as 30 to 70% [7,8]. The use of polymyxins (colistin) or tigecycline antibiotics are the only therapeutic options to treat severe KPC<sup>+</sup> infections [9]. The global increase in pan-resistant Enterobacteriaceae has resulted in increased use of colistin, which has accelerated the onset of resistance to polymyxins; the emergence of the mcr-1 gene is a good example [1,10]. Therefore, MDR-*K. pneumoniae* strains have a great potential to become a "superbug"; therefore, they are an excellent model for discovering new antimicrobial treatments [8].

In this scenario, non-antibiotic therapeutic options with antimicrobial properties should be explored. An alternative is the antimicrobial photodynamic inactivation (aPDI) based on light-activated photosensitizer compounds (PS) [11–13]. The PSs are chemical compounds that absorb and accumulate the quantized energy of a specific wavelength accessing a triplet excited state by intersystem crossing processes [14]. The accumulated energy is transferred to the molecular oxygen commonly present in biological solutions through two mechanisms of action to produce reactive oxygen species (ROS): The Type I effect transfers energetic electrons that produce superoxide (O<sup>2</sup> •−); the O<sup>2</sup> •− produces other ROS, such as hydrogen peroxide (H2O2) and hydroxyl radical (HO• ) [15,16]. The Type II effect transfers the energy (with no electrons) to generate singlet oxygen (1O2) [17,18]. ROS such as <sup>1</sup>O<sup>2</sup> produces photooxidative stress by concerted addition reactions of alkene groups in closer organic macromolecules such as protein alkylation, lipid carboxylation, and DNA degradation [12,19,20]. Hence, photooxidative stress results in non-specific bacterial cell death produced by damage over bacterial structures such as plasma membranes or DNA [17,21]. Many initiatives have developed PS compounds with aPDI properties for bacteria such as *K. pneumoniae* [13,20,22–24]. PS with a longer-lived excited state lifetime improve the probability of interacting with triplet oxygen and must produce more <sup>1</sup>O<sup>2</sup> [25–28]. Moreover, it has been identified that cationic PSs produce more significant inhibition in bacterial growth [29], probably due to a more intimate interaction with the negatively charged bacterial envelope [30]. Therefore, cationic PSs may show a better photodynamic effect on *K. pneumoniae* than anionic PSs [29–31]. Our laboratory has tested various cationic Ir(III) organometallic PSs with antimicrobial properties against *K. pneumoniae* [23,29,32]. Other authors have also developed PS for *K. pneumoniae* [31,33–35], where some of these are organic molecules able to inhibit bacterial growth in vitro [33]. The PS molecule must show low levels of cytotoxicity to reduce the probability of adverse pharmacological effects. In this regard, the PSs based on organic molecules should be less toxic [19]. Other coordination compounds based on transition metals into tetrapyrrole structures or 5-aminolevulinic acids (ALA) have been successfully used for photodynamic treatment of cancer [36,37]. The Ru(II) complex containing three phenanthroline ligands has shown highlighted antimicrobial activity [38]. Additionally, the Re(I) complexes have been used in vitro against a broad spectrum of bacteria [39]. In this sense, the complexes with transition metals such as Re(I) can be considered good options according to their photophysical properties to produce reactive oxygen species useful for antimicrobial treatment [40].

Here we verify that PS compounds that absorb in a wide range of the visible spectrum, such as bimetallic Re (I) bimetallic complexes with polypyridine bridging ligand, may be helpful in aPDI over *K. pneumoniae*. These complexes have (Re(CO)3Cl)2µ-NˆN general formula and here were evaluated with the following NˆN ligands: 2,3-Dicarboxypyrazino [2,3-f] [4,7] phenanthrolinedicarboxylic (L1) and 2,3-Diethoxycarbonylypyrazino [2,3-f] [4,7] phenanthroline (L2) to obtain the corresponding PSRe-µL1 and PSRe-µL2 compounds [41]. The photodynamic effect of the PSRe-µL1 and PSRe-µL2 compounds was tested in vitro in two laboratory strains of *K. pneumoniae*: the carbapenem susceptible (KPC−) KPPR-1 and the carbapenem-resistant (KPC<sup>+</sup> ) ST258 strain, and also in a previously characterized population of 118 clinical isolates of *K. pneumoniae*, including 66 ESBL-producers [42]. In

addition, the PSs capacity to inhibit bacterial growth was verified, as well as the pharmacological properties, such as minimum effective dose (MEC), lethality time, and synergy with imipenem (Imp) and cefotaxime (Cfx) antibiotics. Finally, the low cytotoxicity in mammalian cells determined in vitro makes these PSs a promising alternative to complement the treatment of complicated infections.

#### **2. Materials and Methods**

#### *2.1. Synthesis of the Photosensitizer Compounds*

Our group had previously synthesized and characterized the structure, photophysics, and purity of the PSRe-µL1 and PSRe-µL2 compounds, published in González et al., 2020 [41]. The characterizations included nuclear magnetic resonance (NMR), Fourier transforms infrared spectroscopy (FT-IR), elemental analysis TD-DFT calculations, and cyclic voltammetry. Additionally, the absorption spectra measured in acetonitrile solution were performed in a Shimadzu UV–Vis-NIR 3101-PC spectrophotometer. Finally, the molar extinction coefficients of the characteristic absorption bands and the area under the curve between 500–700 nm were determined. The complexes can be described using the following general formula: (Re(CO)3Cl)2µ-NˆN, where NˆN is 2,3-Dicarboxypyrazino [2,3-f] [4,7] phenanthrolinedicarboxylic (L1) or 2,3-Diethoxycarbonylypyrazino [2,3-f] [4,7] phenanthroline (L2). The Re(I) PSs obtained with both L1 and L2 are then designated as PSRe-µL1 and PSRe-µL2, respectively.

#### *2.2. Antimicrobial Activity of Photosensitizers Compounds*

Stock solutions of 2 mg/mL of each photosensitizer compound were prepared in dimethyl sulfoxide DMSO (Sigma-Aldrich, Saint louis, MO, USA), from which suitable work concentrations were obtained by dilution in an aqueous medium. The imipenem susceptible (KPC-) strain KPPR1 and the imipenem resistant strain ST258 (KPC<sup>+</sup> ) of *K. pneumoniae* were used for the antimicrobial assay. Additionally, 118 clinical isolates of *K. pneumoniae* were used. Those clinical isolates were previously characterized for sensitivity and pathogenicity patterns [42]. For photodynamic experiments, all bacteria were grown as axenic culture in Luria Bertani medium and suspended at 1 <sup>×</sup> <sup>10</sup><sup>7</sup> colony forming units (CFU)/mL cation-adjusted Muller Hinton broth (ca-MH). Bacteria were mixed with each PS in 24-well plates, in a final volume of 500 µL and light-irradiated immediately after adding the PS in a chamber with a white LED lamp at a photon flux of 17 µW/cm<sup>2</sup> . Controls plates with bacteria but no PS and with PS and no light were also included. All plates, including controls, were incubated for 1 h or the indicated time, and broth-micro dilution and sub-cultured on ca-MH agar plates were used to determine the CFUs of the viable bacteria. The agar plates were incubated at 37 ◦C, and colony count was recorded using a stereoscopic microscope after 16–20 h of incubation in the dark, following the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2017) [43]. Control wells with bacteria, with or without photosensitizer but not exposed to light, were also included.

#### *2.3. Determination of the Synergy between PSs and Cfx*

To determine the fractional inhibitory concentration index (FIC) value, the following formula was used [44,45]:

$$\text{FIC Index} = \frac{\text{MICac}}{\text{MIC}\_{\text{a}}} + \frac{\text{MICbc}}{\text{MIC}\_{\text{b}}}$$

MICac is the MIC of compound A, combined with compound B, and MICbc is the MIC of compound B combined with compound A. The MICa and MICb are the MIC of the A and B compounds alone, respectively. Values in the FIC index ≤ 0.5 are considered synergistic, and values > 4 are considered antagonistic [44]. To determine the MIC- Cfx in combination with each PS, 1 <sup>×</sup> <sup>10</sup><sup>7</sup> UFC/mL of ESBL-producing bacteria were aPDI treated for 30 min with 4 µg/mL of each PSRe mixed with serial dilution (32–0.125 µg/mL) of Cfx in ca-MH broth, as stated above. To determine the modification in the PS-MEC when combined with Cfx, 1 <sup>×</sup> <sup>10</sup><sup>7</sup> UFC/mL of ESBL-producing bacteria were added to 4 <sup>µ</sup>g/mL of Cfx and mixed with a serial dilution of each PS (32–0.125 µg/mL) in ca-MH broth, as mentioned previously.

#### *2.4. Cell Culture*

The human cell lines from the American-type culture collection (ATCC), HEp-2 (CCL-23), and HEK293T (CRL-3216) were grown in DMEM with a mix of 1% streptomycin/penicillin antibiotics. The medium was supplemented with 10% fetal bovine serum (FBS), and the cultures were incubated in a 5% CO<sup>2</sup> atmosphere. Initial cultures of <sup>5</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> cells per well were incubated for 24 to 48 h to a 70–90% confluence in triplicate in 24-well plates.

#### *2.5. Cytotoxicity Tests in Eukaryotic Cells*

The cytotoxic effect of PS compounds on HEp-2 and HEK293T cells was determined by evaluating cell death by the exclusion of trypan blue. The 24-well plates with cells at 70–90% confluence were mixed with 4 µg/mL of PSRe-µL1 and PSRe-µL2 compounds or control with D-PBS and incubated for 1 h in the dark or light-activated in a white LED light chamber with 17 µW/ cm<sup>2</sup> of photon flux. Subsequently, the PSs were removed by washing twice with D-PBS, and the cells were incubated in the dark for an additional 24 h in DMEM supplemented with antibiotics + 10% FBS in a 5% CO<sup>2</sup> atmosphere. After incubation, the cells were trypsinized, and the cell death was determined by trypan blue exclusion in a hemocytometer chamber.

#### *2.6. Statistical Analysis*

The Prism version 9.0 Software (GraphPad Software, LLC, San Diego, CA, USA) was used to perform the statistical analysis. Statistical significance was assessed using a two-tail *T*-test for parametric pairing groups or one-way ANOVA followed by the Tukey post-test for the lethality curves.

#### **3. Results**

#### *3.1. Absorption Properties of the PSRe-µL1 and PSRe-µL2 Compounds*

We previously showed that cationic Ir(III) are effective photosensitizers against *K. pneumoniae* [29]. However, these compounds present absorption peaks below 400 nm, limiting their excitation with wavelengths that better penetrate tissues [11]. Two bimetallic Re (I) compounds have been tested to enhance the PS activation in deeper tissue infections. Those bimetallic Re(I) complexes present polypyridine ligands (Figure 1A,B), they are fully structurally characterized, and they are thermodynamically stable in acetonitrile solution [41,46]. As shown in Table 1 and Figure 1D, the PSRe-µL1 and PSRe-µL2 present similar absorption processes at 358 and 360 nm, respectively. However, they show dark coloration attributed to a wide range of absorption bands in the visible range, with the maximums at 587 and 588 nm (Figure 1C,D). As shown in Figure 1D, both PSRe-µL1 and PSRe-µL2 compounds have an intense dark color, which shows their characteristic of absorbing light with a significant molar extinction coefficient in a wide visible range. The broad range absorption (500–700 nm) shows a significant area under the curve of 16.28 and 14.39 for PSRe-µL1 and PSRe-µL2, respectively. The compounds also present significantly high molar absorption of 3384 and 5600 M−<sup>1</sup> cm−<sup>1</sup> for the PSRe-µL1 and PSRe-µL2, respectively (Table 1). The absorptions are attributable to the singlet metal-to-ligand (1MLCT) and singlet ligand-to-ligand (1LLCT) charge transfer transitions from the metal and chloride ligand to ligand bridge (L1 or L2), following the behavior of analogous compounds and the trends of cyclic voltammetry experiments and TD-DFT calculations [41]. In addition, the cyclic voltammetry showed a first oxidation process at 1.51 and 1.70 for PSRe-µL1 and PSRe-µL2, respectively. A second oxidation process was obtained only for PSRe-µL1

at 1.65 V (Table 1). The HOMO-LUMO energy gaps calculated for the PSRe-µL1 and PSRe-µL2 complexes were of similar magnitude, 2.73 and 2.84 eV, respectively (Table 1). µL1 and PSRe-µL2 complexes were of similar magnitude, 2.73 and 2.84 eV, respectively (Table 1).

and chloride ligand to ligand bridge (L1 or L2), following the behavior of analogous compounds and the trends of cyclic voltammetry experiments and TD-DFT calculations [41]. In addition, the cyclic voltammetry showed a first oxidation process at 1.51 and 1.70 for PSRe-µL1 and PSRe-µL2, respectively. A second oxidation process was obtained only for PSRe-µL1 at 1.65 V (Table 1). The HOMO-LUMO energy gaps calculated for the PSRe-

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 5 of 16

**Figure 1.** Chemical structure of the Re(I) bimetallic photosensitizer compounds and their L ligands and absorption spectra. The chemical structure of Re(I) complexes show the L1 and L2 ligands (**A**) and the (Re(CO)3Cl)2µ-N^N, whose replacement of R<sup>1</sup> results in the PSRe-µL1 and PSRe-µL2 compounds (**B**). Macroscopic appearance of PSRe-µL1 and PSRe-µL2 compounds concentrated in solution at 2 mg/mL (**C**). The absorption spectra for the PSRe-µL1 and PSRe-µL2 compounds in acetonitrile solution (**D**). **Figure 1.** Chemical structure of the Re(I) bimetallic photosensitizer compounds and their L ligands and absorption spectra. The chemical structure of Re(I) complexes show the L1 and L2 ligands (**A**) and the (Re(CO)3Cl)2µ-NˆN, whose replacement of R<sup>1</sup> results in the PSRe-µL1 and PSRe-µL2 compounds (**B**). Macroscopic appearance of PSRe-µL1 and PSRe-µL2 compounds concentrated in solution at 2 mg/mL (**C**). The absorption spectra for the PSRe-µL1 and PSRe-µL2 compounds in acetonitrile solution (**D**).

**Table 1.** Photophysical and electrochemical evaluation of photosensitizers. **Table 1.** Photophysical and electrochemical evaluation of photosensitizers.


\* Extracted from Campagna et al., 2007 [47]. λabs, wavelength. Area, the area under the curve. Eox, oxidation state. , molar absorption. \* Extracted from Campagna et al., 2007 [47]. λabs, wavelength. Area, the area under the curve. Eox, oxidation state. ε, molar absorption.

The PS-Ru [Ru(bpy)3](PF6)<sup>2</sup> (bpy = 2,2′-bipyridine) compound was used as a control to compare the aPDI activity of the PSRe-µL1 and PSRe-µL2 compounds [38]. According to the literature, in acetonitrile, the PS-Ru shows a charge-transfer absorption process at 450 nm [48] and a significantly low molar extinction coefficient at 550 nm of ~600 M−1cm−1 [47]. In addition, we determine its area under the curve of 0.83 between 500–700 nm wavelength, which is significantly smaller than the PS-Re compounds (Table 1). The PS-Ru [Ru(bpy)3](PF6)<sup>2</sup> (bpy = 2,2<sup>0</sup> -bipyridine) compound was used as a control to compare the aPDI activity of the PSRe-µL1 and PSRe-µL2 compounds [38]. According to the literature, in acetonitrile, the PS-Ru shows a charge-transfer absorption process at 450 nm [48] and a significantly low molar extinction coefficient at 550 nm of ~600 M−<sup>1</sup> cm−<sup>1</sup> [47]. In addition, we determine its area under the curve of 0.83 between 500–700 nm wavelength, which is significantly smaller than the PS-Re compounds (Table 1).

#### *3.2. Antimicrobial Photodynamic Inactivation Activity of Compounds PSRe-µL1 and PSRe-µL2 3.2. Antimicrobial Photodynamic Inactivation Activity of Compounds PSRe-µL1 and PSRe-µL2*

The photodynamic antimicrobial capacity of the two new Re (I) compounds was determined by inhibiting the bacterial growth of *K. pneumoniae*. PSRe-µL1 and PSRe-µL2 at 16 µg/mL were then mixed with each *K. pneumoniae* strain, KPPR1 (KPC−) and ST258 The photodynamic antimicrobial capacity of the two new Re (I) compounds was determined by inhibiting the bacterial growth of *K. pneumoniae*. PSRe-µL1 and PSReµL2 at 16 µg/mL were then mixed with each *K. pneumoniae* strain, KPPR1 (KPC−) and ST258 (KPC<sup>+</sup> ) at 1 <sup>×</sup> <sup>10</sup><sup>7</sup> CFU/ml. The aPDI activity of the PSRe-µL1 and PSRe-µL2 compounds was compared with the reference PS-Ru antimicrobial activity as a positive control [34,38,49]. Initial tests were performed with 16 µg/mL of each compound in an aqueous solution (ca-MH). Figure 2 shows the photodynamic treatment with PSRe-µL1 and PSRe-µL2 (Green bars), which inhibits in 3 log<sup>10</sup> (<99.9%) the growth of both strains of *K. pneumoniae* (\* *p* < 0.05) compared to untreated control bacteria (Blue bars). The results

(KPC<sup>+</sup>

show that the bactericidal effect produced by both PSs is dependent on light (Red bars) (ns *p* > 0.05, compared to untreated control) because growth inhibition is observed after light activation. Therefore, the PSRe-µL1 and the PSRe-µL2 compounds must be activated by light to exhibit their bactericidal effect. Comparable results were obtained with the 16 µg/mL PS-Ru control, as the bacterial growth inhibition was observed only after light activation (*p* < 0.05). that the bactericidal effect produced by both PSs is dependent on light (Red bars) (ns *p* > 0.05, compared to untreated control) because growth inhibition is observed after light activation. Therefore, the PSRe-µL1 and the PSRe-µL2 compounds must be activated by light to exhibit their bactericidal effect. Comparable results were obtained with the 16 µg/mL PS-Ru control, as the bacterial growth inhibition was observed only after light activation (*p* < 0.05).

) at 1 × 10<sup>7</sup> CFU/ml. The aPDI activity of the PSRe-µL1 and PSRe-µL2 compounds was compared with the reference PS-Ru antimicrobial activity as a positive control [34,38,49]. Initial tests were performed with 16 µg/mL of each compound in an aqueous solution (ca-MH). Figure 2 shows the photodynamic treatment with PSRe-µL1 and PSReµL2 (Green bars), which inhibits in 3 log<sup>10</sup> (<99.9%) the growth of both strains of *K. pneumoniae* (\* *p* < 0.05) compared to untreated control bacteria (Blue bars). The results show

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**Figure 2.** The Photodynamic antimicrobial inhibition of PSRe-µL1 and PSRe-µL2 compounds. The imipenem-sensitive strain (KPPR1) and the imipenem resistant strain (ST258) of *K. pneumoniae* were used at a 1 × 10<sup>7</sup> CFU/mL and mixed in triplicate with 16 µg/mL of PSRe-µL1, PSRe-µL2, or PS-Ru compounds. The mix was incubated for 1 h at 17 µW/cm<sup>2</sup> with light for aPDI (green bars) or in the dark for controls (red bars). The results were compared to control of bacteria not combined with the PSs (blue bars). Colony count enumerated viable bacteria on ca-MH agar after serial micro-dilution. The CFU/mL values are presented as means ± SD on a log<sup>10</sup> scale. The + and − signs indicate the presence or absence of a compound or condition. Not significant (ns) *p* > 0.05 by Student's *t*-test among bacteria treated with PS without light compared to untreated control bacteria; \* *p* < 0.05 by Student's *t*-test among bacteria treated with PS exposed to light compared to untreated control bacteria. **Figure 2.** The Photodynamic antimicrobial inhibition of PSRe-µL1 and PSRe-µL2 compounds. The imipenem-sensitive strain (KPPR1) and the imipenem resistant strain (ST258) of *K. pneumoniae* were used at a 1 <sup>×</sup> <sup>10</sup><sup>7</sup> CFU/mL and mixed in triplicate with 16 <sup>µ</sup>g/mL of PSRe-µL1, PSRe-µL2, or PS-Ru compounds. The mix was incubated for 1 h at 17 µW/cm<sup>2</sup> with light for aPDI (green bars) or in the dark for controls (red bars). The results were compared to control of bacteria not combined with the PSs (blue bars). Colony count enumerated viable bacteria on ca-MH agar after serial micro-dilution. The CFU/mL values are presented as means ± SD on a log<sup>10</sup> scale. The + and − signs indicate the presence or absence of a compound or condition. Not significant (ns) *p* > 0.05 by Student's *t*-test among bacteria treated with PS without light compared to untreated control bacteria; \* *p* < 0.05 by Student's *t*-test among bacteria treated with PS exposed to light compared to untreated control bacteria.

#### *3.3. Determination of the Minimum Effective Concentration and Lethality for the Compounds PSRe-µL1 and PSRe-µL2 3.3. Determination of the Minimum Effective Concentration and Lethality for the Compounds PSRe-µL1 and PSRe-µL2*

Because the PSRe-µL1 and PSRe-µL2 compounds showed a significant aPDI effect on the two *K. pneumoniae* strains, a further two pharmacologic parameters were determined: the minimum effective concentration (MEC) and the minimum light exposition time (lethality) on the KPPR1 and the ST258 *K. pneumoniae* strains. The PS-MEC was determined by exposition of 1 × 10<sup>7</sup> CFU/mL of each *K. pneumoniae* strain to serial dilutions Because the PSRe-µL1 and PSRe-µL2 compounds showed a significant aPDI effect on the two *K. pneumoniae* strains, a further two pharmacologic parameters were determined: the minimum effective concentration (MEC) and the minimum light exposition time (lethality) on the KPPR1 and the ST258 *K. pneumoniae* strains. The PS-MEC was determined by exposition of 1 <sup>×</sup> <sup>10</sup><sup>7</sup> CFU/mL of each *K. pneumoniae* strain to serial dilutions (ranging from 0 to 32 µg/mL) of each PS in ca-MH broth. The aPDI treatment was performed for 1 h at a fluence rate of 17 µW/cm<sup>2</sup> . After treatment, viable bacteria were enumerated as above by serial micro-dilution. We established the MEC as the concentration where the bacterial viability decreased in 3 log<sup>10</sup> (99.9%). As shown in Figure 3, the MEC was determined at 4 µg/mL for both PSRe-µL1 (Figure 3A) and PSRe-µL2 (Figure 3B) (\*\* *p* < 0.01, \*\*\* *p* < 0.001, Student's *t*-test comparing to control with 0 µg/mL). The MEC of 4 µg/mL of PSRe-µL1 or PSRe-µL2 were used to determine the lethality time on 1 <sup>×</sup> <sup>10</sup><sup>7</sup> CFU/mL of each bacterial

strain in ca-MH broth. The mix was exposed to 17 µW/cm<sup>2</sup> of a white LED light for 5, 15, 30, 60, and 120 min. Control wells with bacteria without PS were also included. For each time, viable bacteria were enumerated by serial micro-dilution and colony counted in ca-MH agar. We established the lethality when the bacterial viability decreased in 3 log<sup>10</sup> (99.9%). As seen in Figure 3C, although PSRe-µL1 produced a significant reduction after 30 min of incubation (*p* = 0.013, compared to time 0), a 3log<sup>10</sup> reduction was observed after 60 min of light exposure (*p* < 0.01, compared to time 0). In comparison, the PSRe-µL2 significantly (*p* < 0.01) reduced in 3log<sup>10</sup> the bacterial viability after 30 min (Figure 3D). also included. For each time, viable bacteria were enumerated by serial micro-dilution and colony counted in ca-MH agar. We established the lethality when the bacterial viability decreased in 3 log<sup>10</sup> (99.9%). As seen in Figure 3C, although PSRe-µL1 produced a significant reduction after 30 min of incubation (*p* = 0.013, compared to time 0), a 3log<sup>10</sup> reduction was observed after 60 min of light exposure (*p* < 0.01, compared to time 0). In comparison, the PSRe-µL2 significantly (*p* < 0.01) reduced in 3log10 the bacterial viability after 30 min (Figure 3D).

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formed for 1 h at a fluence rate of 17 µW/cm<sup>2</sup>

(ranging from 0 to 32 µg/mL) of each PS in ca-MH broth. The aPDI treatment was per-

merated as above by serial micro-dilution. We established the MEC as the concentration where the bacterial viability decreased in 3 log<sup>10</sup> (99.9%). As shown in Figures 3, the MEC was determined at 4 µg/mL for both PSRe-µL1 (Figure 3A) and PSRe-µL2 (Figure 3B) (\*\* *p* < 0.01, \*\*\* *p* < 0.001, Student's *t*-test comparing to control with 0 µg/mL). The MEC of 4 µg/mL of PSRe-µL1 or PSRe-µL2 were used to determine the lethality time on 1 × 10<sup>7</sup> CFU/mL of each bacterial strain in ca-MH broth. The mix was exposed to 17 µW/cm<sup>2</sup> of a white LED light for 5, 15, 30, 60, and 120 min. Control wells with bacteria without PS were

. After treatment, viable bacteria were enu-

**Figure 3.** Determination of minimum effective concentration and time lethality. The minimum effective concentration (MEC) and lethality of the PSRe-µL1 and PSRe-µL2 were determined using the imipenem-sensitive (KPPR1) and the imipenem resistant (ST258) strains of *K. pneumoniae*. For MEC determination, bacteria at 1 × 10<sup>7</sup> CFU/mL were incubated with increasing concentrations (0–32 µg/mL) of the compounds PSRe-µL1 (**A**) or PSRe-µL2 (**B**) and exposed for 1 h to 17 µW/cm<sup>2</sup> of white LED light. The time lethality was determined, mixing the bacteria with 4 µg/mL of PSRe-µL1 (**C**) or PSRe-µL2 (**D**), and exposure for increasing times (5, 15, 30, 60, and 120 min) to 17 µW/cm<sup>2</sup> of white LED light. Colony count enumerated viable bacteria on ca-MH agar after serial-microdilution. The CFU/mL values are presented as means ± SD on a log<sup>10</sup> scale (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 by Student's *t*-test among bacteria treated with PS exposed to light compared to untreated control bacteria). *3.4. Increased Photodynamic Effect of PSRe-µL1 and PSRe-µL2 in Combination with Imipenem* **Figure 3.** Determination of minimum effective concentration and time lethality. The minimum effective concentration (MEC) and lethality of the PSRe-µL1 and PSRe-µL2 were determined using the imipenem-sensitive (KPPR1) and the imipenem resistant (ST258) strains of *K. pneumoniae*. For MEC determination, bacteria at 1 <sup>×</sup> <sup>10</sup><sup>7</sup> CFU/mL were incubated with increasing concentrations (0–32 µg/mL) of the compounds PSRe-µL1 (**A**) or PSRe-µL2 (**B**) and exposed for 1 h to 17 µW/cm<sup>2</sup> of white LED light. The time lethality was determined, mixing the bacteria with 4 µg/mL of PSRe-µL1 (**C**) or PSRe-µL2 (**D**), and exposure for increasing times (5, 15, 30, 60, and 120 min) to 17 µW/cm<sup>2</sup> of white LED light. Colony count enumerated viable bacteria on ca-MH agar after serial-microdilution. The CFU/mL values are presented as means ± SD on a log<sup>10</sup> scale (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 by Student's *t*-test among bacteria treated with PS exposed to light compared to untreated control bacteria).

#### The desired quality for photosensitizer compounds is to be used as adjunctive therapy with antibiotics. The improvement in combined therapy of the compounds PSRe-µL1 *3.4. Increased Photodynamic Effect of PSRe-µL1 and PSRe-µL2 in Combination with Imipenem*

The desired quality for photosensitizer compounds is to be used as adjunctive therapy with antibiotics. The improvement in combined therapy of the compounds PSReµL1 and PSRe-µL2 with imipenem was used to verify their usefulness in eradicating carbapenemase-producing *K. pneumoniae*. The strains of *K. pneumoniae* susceptible to carbapenem KPC<sup>−</sup> (KPPR1), and the resistant strain KPC<sup>+</sup> (ST258), were exposed to the preparation of 4 µg/mL of imipenem mixed in aqueous solution with 4 µg/mL of each PSRe-µL1, PSRe-µL2 (corresponding to their MECs), or the control compound PS-Ru [29]. Additionally, control bacteria exposure to light but without imipenem were included. As expected, when mixed with imipenem, the PSRe-µL1 and PSRe-µL2 compounds showed a significantly (\*\*\* *p* < 0.001) increased effect over bacterial viability, increasing from 3 to

6log<sup>10</sup> the bactericidal effect for the KPC**<sup>+</sup>** strain (Figure 4). As seen previously, this behavior was not observed when combining the imipenem with the PS-Ru control compound, keeping the 3log<sup>10</sup> inhibitory effect (*p* < 0.05) [29]. 6log<sup>10</sup> the bactericidal effect for the KPC**<sup>+</sup>** strain (Figure 4). As seen previously, this behavior was not observed when combining the imipenem with the PS-Ru control compound, keeping the 3log<sup>10</sup> inhibitory effect (*p* < 0.05) [29].

and PSRe-µL2 with imipenem was used to verify their usefulness in eradicating carbapenemase-producing *K. pneumoniae*. The strains of *K. pneumoniae* susceptible to car-

aration of 4 µg/mL of imipenem mixed in aqueous solution with 4 µg/mL of each PSReµL1, PSRe-µL2 (corresponding to their MECs), or the control compound PS-Ru [29]. Additionally, control bacteria exposure to light but without imipenem were included. As expected, when mixed with imipenem, the PSRe-µL1 and PSRe-µL2 compounds showed a significantly (\*\*\* *p* < 0.001) increased effect over bacterial viability, increasing from 3 to

(ST258), were exposed to the prep-

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(KPPR1), and the resistant strain KPC<sup>+</sup>

bapenem KPC<sup>−</sup>

**Figure 4.** Increased photodynamic effect with imipenem. The effect of carbapenem on the PSRe-µL1 and PSRe-µL2 activity was determined in the strains of *K. pneumoniae* sensitive (KPPR1) or resistant (ST258) to imipenem. The bacteria at 1 × 10<sup>7</sup> UFC/mL were exposed to a mixture of 4 µg/mL of imipenem and with 4 µg/mL of each PS or the control PS-Ru. For aPDI treatment, the mixes were incubated for 1 h at 17 µW/cm<sup>2</sup> of light (green bars) or in the dark (red bars). Control also includes bacteria not treated (blue bars). Colony count enumerated viable bacteria on ca-MH agar after serial microdilution. The CFU/mL values are presented as means ± SD on a log<sup>10</sup> scale. The + and - signs indicate the presence or absence of a compound or condition. Not significant (ns) *p* > 0.05 by Student's *t*-test among bacteria treated with PS + imipenem without light compared to untreated control bacteria; \* *p* < 0.05, \*\* *p* < 0.01 \*\*\* *p* < 0.001 by Student's *t*-test among bacteria treated with PS + imipenem exposed to light compared to untreated control bacteria. **Figure 4.** Increased photodynamic effect with imipenem. The effect of carbapenem on the PSRe-µL1 and PSRe-µL2 activity was determined in the strains of *K. pneumoniae* sensitive (KPPR1) or resistant (ST258) to imipenem. The bacteria at <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>7</sup> UFC/mL were exposed to a mixture of 4 <sup>µ</sup>g/mL of imipenem and with 4 µg/mL of each PS or the control PS-Ru. For aPDI treatment, the mixes were incubated for 1 h at 17 µW/cm<sup>2</sup> of light (green bars) or in the dark (red bars). Control also includes bacteria not treated (blue bars). Colony count enumerated viable bacteria on ca-MH agar after serial microdilution. The CFU/mL values are presented as means ± SD on a log<sup>10</sup> scale. The + and signs indicate the presence or absence of a compound or condition. Not significant (ns) *p* > 0.05 by Student's *t*-test among bacteria treated with PS + imipenem without light compared to untreated control bacteria; \* *p* < 0.05, \*\* *p* < 0.01 \*\*\* *p* < 0.001 by Student's *t*-test among bacteria treated with PS + imipenem exposed to light compared to untreated control bacteria.

#### *3.5. Antimicrobial Photodynamic Inhibition of the PSRe-µL1 and PSRe-µL2 Compounds over Clinical Isolates* We have in our laboratory a collection of 118 clinical isolates of *K. pneumoniae* from *3.5. Antimicrobial Photodynamic Inhibition of the PSRe-µL1 and PSRe-µL2 Compounds over Clinical Isolates*

patients who had an active infection [42]. We used these isolates to verify the photodynamic activity of the PSRe-µL1 and PSRe-µL2 compounds on community bacteria and We have in our laboratory a collection of 118 clinical isolates of *K. pneumoniae* from patients who had an active infection [42]. We used these isolates to verify the photodynamic activity of the PSRe-µL1 and PSRe-µL2 compounds on community bacteria and compared them with PS-Ru positive control [49]. As seen in Figure 5, photodynamic treatment with 4 µg/mL PSRe-µL1 or PSRe-µL2 significantly (\*\*\* *p* < 0.001; compared to untreated control) inhibits bacterial growth > 3log<sup>10</sup> (>99.9%) of clinical isolates of *K. pneumoniae*. The results show that the bactericidal effect produced by PSRe-µL1 and PSRe-µL2 is light-dependent (ns = *p* > 0.05; compared to the untreated control). Those results are comparable with the positive control PS-Ru (4 µg/mL) (\*\*\* *p* < 0.001). Because this collection of strains has been characterized by its resistance profile and presents 66 ESBL-producing isolates, we analyzed whether a synergic effect occurred with cefotaxime. It was first determined whether the combined treatment with Cfx increases the inhibition of bacterial growth of aPDI with

PSRe-µL1 or PSRe-µL2. The clinical isolates were exposed to the preparation of 4 µg/mL of cefotaxime with 4 µg/mL of PSRe-µL1 or PSRe-µL2. As expected, the PSs compounds mixed with cefotaxime significantly (\*\*\* *p* < 0.001) increased the bactericidal effect on the clinical isolate population from 3 to 6log<sup>10</sup> reduction (Figure 5). No significantly increased inhibitory effect was observed for the PS-Ru control combined with cefotaxime (ns *p* > 0.05). the preparation of 4 µg/mL of cefotaxime with 4 µg/mL of PSRe-µL1 or PSRe-µL2. As expected, the PSs compounds mixed with cefotaxime significantly (\*\*\* *p* < 0.001) increased the bactericidal effect on the clinical isolate population from 3 to 6log<sup>10</sup> reduction (Figure 5). No significantly increased inhibitory effect was observed for the PS-Ru control combined with cefotaxime (ns *p* > 0.05).

compared them with PS-Ru positive control [49]. As seen in Figure 5, photodynamic treatment with 4 µg/mL PSRe-µL1 or PSRe-µL2 significantly (\*\*\* *p* < 0.001; compared to untreated control) inhibits bacterial growth > 3log<sup>10</sup> (>99.9%) of clinical isolates of *K. pneumoniae*. The results show that the bactericidal effect produced by PSRe-µL1 and PSRe-µL2 is light-dependent (ns = *p* > 0.05; compared to the untreated control). Those results are comparable with the positive control PS-Ru (4 µg/mL) (\*\*\* *p* < 0.001). Because this collection of strains has been characterized by its resistance profile and presents 66 ESBL-producing isolates, we analyzed whether a synergic effect occurred with cefotaxime. It was first determined whether the combined treatment with Cfx increases the inhibition of bacterial growth of aPDI with PSRe-µL1 or PSRe-µL2. The clinical isolates were exposed to

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**Figure 5.** Antimicrobial photodynamic inactivation of clinical isolates of *K. pneumoniae*. (**A**) Growth inhibition of 118 clinical isolates of *K. pneumoniae* subjected to antimicrobial photodynamic inactivation (aPDI) with PSRe-µL1 and PSRe-µL2 compounds compared to control, PS-Ru. The bacteria were utilized at 1 × 10<sup>7</sup> CFU/mL and mixed in triplicate with 4 µg/mL of PSRe-µL1 and PSRe-µL2 compounds. For the aPDI, the mixture of bacteria with the PSs was exposed for 1 h at 17 µW/cm<sup>2</sup> of white light. As a control, bacteria combined with the PSs not exposed to light and bacteria not combined with the control PS-Ru were included. Colony count enumerated of viable bacteria on ca-MH agar after serial micro-dilution. The CFUs/mL values are presented as means ± SD on a log<sup>10</sup> scale. Not significant (ns) *p* > 0.05 by Student's *t*-test among treated bacteria compared to control; \*\* *p* < 0.01, \*\*\* *p* < 0.001 by Student's *t*-test among treated bacteria compared to control. **Figure 5.** Antimicrobial photodynamic inactivation of clinical isolates of *K. pneumoniae*. (**A**) Growth inhibition of 118 clinical isolates of *K. pneumoniae* subjected to antimicrobial photodynamic inactivation (aPDI) with PSRe-µL1 and PSRe-µL2 compounds compared to control, PS-Ru. The bacteria were utilized at 1 <sup>×</sup> <sup>10</sup><sup>7</sup> CFU/mL and mixed in triplicate with 4 <sup>µ</sup>g/mL of PSRe-µL1 and PSRe-µL2 compounds. For the aPDI, the mixture of bacteria with the PSs was exposed for 1 h at 17 µW/cm<sup>2</sup> of white light. As a control, bacteria combined with the PSs not exposed to light and bacteria not combined with the control PS-Ru were included. Colony count enumerated of viable bacteria on ca-MH agar after serial micro-dilution. The CFUs/mL values are presented as means ± SD on a log<sup>10</sup> scale. Not significant (ns) *p* > 0.05 by Student's *t*-test among treated bacteria compared to control; \*\* *p* < 0.01, \*\*\* *p* < 0.001 by Student's *t*-test among treated bacteria compared to control.

#### *3.6. Synergism between aPDI with Cefotaxime and FIC Index Determination 3.6. Synergism between aPDI with Cefotaxime and FIC Index Determination*

The fractional inhibitory concentration (FIC) index was determined to verify when the PS and Cfx combination increases the bactericidal activity synergistically or additively. We used the MEC determination for the photosensitizer compounds, and the results of the mixture with antibiotics were tabulated as the MIC for simplicity. The set of 66 ESBL-producing clinical isolates of *K. pneumoniae* [42] were mixed with 4 µg/mL PSReµL1, PSRe-µL2, or PS-Ru and added to a serial log2 dilutions of Cfx (from 0 to 32 µg/mL). The mixes were incubated for 1 h for aPDI or in the dark, and the Cfx-MIC was determined 16–20 h after. As seen in Figure 6A, compared to the aPDI untreated group, a significant reduction (\*\*\* *p* < 0.001) from 8 µg/mL (8–8) to 0.17 µg/mL (0.09–0.34) with PSRe-µL1 and 0.23 µg/mL (0.15–0.41) with PSRe-µL2 on Cfx-MIC was observed (Table 2). In addition, The fractional inhibitory concentration (FIC) index was determined to verify when the PS and Cfx combination increases the bactericidal activity synergistically or additively. We used the MEC determination for the photosensitizer compounds, and the results of the mixture with antibiotics were tabulated as the MIC for simplicity. The set of 66 ESBLproducing clinical isolates of *K. pneumoniae* [42] were mixed with 4 µg/mL PSRe-µL1, PSRe-µL2, or PS-Ru and added to a serial log2 dilutions of Cfx (from 0 to 32 µg/mL). The mixes were incubated for 1 h for aPDI or in the dark, and the Cfx-MIC was determined 16–20 h after. As seen in Figure 6A, compared to the aPDI untreated group, a significant reduction (\*\*\* *p* < 0.001) from 8 µg/mL (8–8) to 0.17 µg/mL (0.09–0.34) with PSRe-µL1 and 0.23 µg/mL (0.15–0.41) with PSRe-µL2 on Cfx-MIC was observed (Table 2). In addition, the combined treatment also reduced the PSRe-µL1-MEC from 4 to 0.5 µg/mL and the PSRe-µL2 MEC from 4 to 0.5 µg/mL (Figure 6B, Table 2). Both PSRe-µL1 and PSRe-µL2 compounds produced a significant change in Cfx-susceptibility with an FIC index of 0.15 for (Table 2). Figure 6B and Table 2 shows that the control compound, PS-Ru, did not significantly change Cfx-susceptibility and shows an FIC index of 1.58. Given that synergy is defined with an FIC index ≤ 0.5 [44], the increase in the inhibitory effect shown by the combination of PSRe-µL1 or PSRe-µL2 with Cfx is synergistic and not additive.

**Figure 6.** Determination of FIC index using combined photodynamic inactivation and cefotaxime antibiotic. A population of 66 ESBL-producing clinical isolates of *K. pneumoniae* was used to determine the modification on Cfx-MIC by the PSs and the modification in PSs-MECs by cefotaxime. To determine the Cfx-MIC modification, 1 × 10<sup>7</sup> CFU/mL of bacteria were mixed in triplicate with 4 µg/mL of PSRe-µL1, PSRe-µL2, or PS-Ru. The mix was added to serial dilutions of Cfx and incubated for 1 h with 17 µW/cm<sup>2</sup> of white light or in the dark. The Cfx-MIC was then determined after 16–20 h at 37 °C in the dark (**A**). The MEC for PSRe-µL1 and PSRe-µL2 were determined in combination with 4 µg/mL of Cfx, performed in triplicate in ca-MH agar for 16–20 h (**B**). The MIC values are presented as median ± SD of µg/mL on a log<sup>2</sup> scale. Not significant (ns) *p* > 0.05 by Student's *t*-test among treated bacteria compared to control; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 by Student's *t*-test among treated bacteria compared to control. **Figure 6.** Determination of FIC index using combined photodynamic inactivation and cefotaxime antibiotic. A population of 66 ESBL-producing clinical isolates of *K. pneumoniae* was used to determine the modification on Cfx-MIC by the PSs and the modification in PSs-MECs by cefotaxime. To determine the Cfx-MIC modification, 1 <sup>×</sup> <sup>10</sup><sup>7</sup> CFU/mL of bacteria were mixed in triplicate with 4 µg/mL of PSRe-µL1, PSRe-µL2, or PS-Ru. The mix was added to serial dilutions of Cfx and incubated for 1 h with 17 µW/cm<sup>2</sup> of white light or in the dark. The Cfx-MIC was then determined after 16–20 h at 37 ◦C in the dark (**A**). The MEC for PSRe-µL1 and PSRe-µL2 were determined in combination with 4 µg/mL of Cfx, performed in triplicate in ca-MH agar for 16–20 h (**B**). The MIC values are presented as median ± SD of µg/mL on a log<sup>2</sup> scale. Not significant (ns) *p* > 0.05 by Student's *t*-test among treated bacteria compared to control; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 by Student's *t*-test among treated bacteria compared to control.

**Table 2.** FIC index calculation. **Table 2.** FIC index calculation.


the combined treatment also reduced the PSRe-µL1-MEC from 4 to 0.5 µg/mL and the PSRe-µL2 MEC from 4 to 0.5 µg/mL (Figure 6B, Table 2). Both PSRe-µL1 and PSRe-µL2 compounds produced a significant change in Cfx-susceptibility with an FIC index of 0.15 for (Table 2). Figure 6B and Table 2 shows that the control compound, PS-Ru, did not significantly change Cfx-susceptibility and shows an FIC index of 1.58. Given that synergy is defined with an FIC index ≤ 0.5 [44], the increase in the inhibitory effect shown by the

combination of PSRe-µL1 or PSRe-µL2 with Cfx is synergistic and not additive.

<sup>a</sup>MIC values are the median for the ESBL-producing *K. pneumoniae*, *n* = 66. \* The PS-MEC values modified by Cfx. <sup>a</sup> MIC values are the median for the ESBL-producing *K. pneumoniae*, *n* = 66. \* The PS-MEC values modified by Cfx.

#### *3.7. The Cytotoxic Effect of PSRe-µL1 and PSRe-µL2 on Mammalian Cells 3.7. The Cytotoxic Effect of PSRe-µL1 and PSRe-µL2 on Mammalian Cells*

The PSRe-µL1 and PSRe-µL2 compounds were tested in vitro and found to be secure for mammalian cells. This work tested the intrinsic cytotoxicity (dark cytotoxicity) and light-dependent cytotoxicity of the PSs compounds in the human HEp-2 and HEK293 cell lines. The trypan blue exclusion technique allowed us to determine cell death of 500,000 cells in the presence of 4 µg/mL PSRe-µL1 or PSRe-µL2. The cells were incubated with PSs for 1 h in the dark or activated with 17 µW/cm<sup>2</sup> of white LED light when the PSs were removed. Fresh medium DMEM with 10% FBS without PS was replaced, and cells were incubated 24 h more in the dark at 37 °C in a 5% CO<sup>2</sup> atmosphere. As shown in Figure 7, when HEp-2 and HEK293T cells were exposed to 4 µg/mL of PSRe-µL1 or PSRe-µL2 in the dark, no significant reduction in the cell survival was observed (ns = *p* > 0.05, Student's The PSRe-µL1 and PSRe-µL2 compounds were tested in vitro and found to be secure for mammalian cells. This work tested the intrinsic cytotoxicity (dark cytotoxicity) and lightdependent cytotoxicity of the PSs compounds in the human HEp-2 and HEK293 cell lines. The trypan blue exclusion technique allowed us to determine cell death of 500,000 cells in the presence of 4 µg/mL PSRe-µL1 or PSRe-µL2. The cells were incubated with PSs for 1 h in the dark or activated with 17 µW/cm<sup>2</sup> of white LED light when the PSs were removed. Fresh medium DMEM with 10% FBS without PS was replaced, and cells were incubated 24 h more in the dark at 37 ◦C in a 5% CO<sup>2</sup> atmosphere. As shown in Figure 7, when HEp-2 and HEK293T cells were exposed to 4 µg/mL of PSRe-µL1 or PSRe-µL2 in the dark, no significant reduction in the cell survival was observed (ns = *p* > 0.05, Student's *t*-test, comparing treated cells with the control). Similarly, the light exposition induced no significant cell death for HEp-2 cells, although a slight (12.5 ± 5%) but significant (*p* > 0.05) reduction in cell viability of PSRe-µL1 over HEK293T cells were shown.

*t*-test, comparing treated cells with the control). Similarly, the light exposition induced no significant cell death for HEp-2 cells, although a slight (12.5 ± 5%) but significant (*p* > 0.05)

reduction in cell viability of PSRe-µL1 over HEK293T cells were shown.

**Figure 7.** Cytotoxicity of the PSRe-µL1 and PSRe-µL2 compounds. Survival of 5 × 10<sup>5</sup> cells of HEp-2 and HEK293 human cell lines exposed to 4µg/mL of each PSRe-µL1 and PSRe-µL2 compounds were normalized to control untreated cells and expressed as a percentage of dead cells. Dark cytotoxicity was evaluated with cells exposed to the compound but not activated with light. Light-dependent cytotoxicity was evaluated, exposing the cells for 1 h at 17 µW/cm<sup>2</sup> of white LED light. Not significant (ns) *p* > 0.05, by Student's *t*-test between cells exposed to each PS compared to control cells; \* *p* < 0.05 by Student's *t*-test cells exposed to each PS compared to control cells. **Figure 7.** Cytotoxicity of the PSRe-µL1 and PSRe-µL2 compounds. Survival of 5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells of HEp-2 and HEK293 human cell lines exposed to 4µg/mL of each PSRe-µL1 and PSRe-µL2 compounds were normalized to control untreated cells and expressed as a percentage of dead cells. Dark cytotoxicity was evaluated with cells exposed to the compound but not activated with light. Light-dependent cytotoxicity was evaluated, exposing the cells for 1 h at 17 µW/cm<sup>2</sup> of white LED light. Not significant (ns) *p* > 0.05, by Student's *t*-test between cells exposed to each PS compared to control cells; \* *p* < 0.05 by Student's *t*-test cells exposed to each PS compared to control cells.

#### **4. Discussion 4. Discussion**

Previously, we reported photosensitizing compounds based on Ir (III), which exhibited absorption processes close to 400 nm wavelength [29]. Therefore, although those compounds are microbicide in vitro, they may be more challenging to access exciting light into the tissues [11]. In this sense, this work tested Re(I) PS compounds that exhibit absorption processes at longer wavelengths, improving the possibility to access excited states into the tissues. Considering the sustained decrease in antibiotic options, these SPs could greatly complement the treatment of infections with MDR microorganisms such as *K. pneumoniae* [1,3]. In this context, using aPDI as complementary therapy becomes viable due to their usefulness as a rescue therapy for infections with MDR bacteria and reverse resistance to antibiotics of choice, reducing the spread of MDR strains [50]. Previously, we reported photosensitizing compounds based on Ir (III), which exhibited absorption processes close to 400 nm wavelength [29]. Therefore, although those compounds are microbicide in vitro, they may be more challenging to access exciting light into the tissues [11]. In this sense, this work tested Re(I) PS compounds that exhibit absorption processes at longer wavelengths, improving the possibility to access excited states into the tissues. Considering the sustained decrease in antibiotic options, these SPs could greatly complement the treatment of infections with MDR microorganisms such as *K. pneumoniae* [1,3]. In this context, using aPDI as complementary therapy becomes viable due to their usefulness as a rescue therapy for infections with MDR bacteria and reverse resistance to antibiotics of choice, reducing the spread of MDR strains [50].

The effective microbial activity of the PSRe-µL1 and PSRe-µL2 photosensitizers rests on the photophysical properties provided by their chemical structure. Although these compounds are neutral (do not have positive charges in the coordination sphere), they can easily polarize by absorption of light, as described in a previous report [41]. This characteristic may improve their molecular proximity to the *K. pneumoniae* cell envelope. Additionally, by exciting these high-rate light-absorbing dark compounds, they can produce high-energy triplet states, which promote energy transfer to molecular oxygen [51]. At first glance, the antimicrobial effect observed with PSRe-µL1 and PSRe-µL2 can be explained by access to a triplet state, as reported [41]. It will then depend on the nature of the Re (I) complex; the excited state responsible for the generation of the ROS producing photooxidative stress could be states 3MLCT, 3LC, or a mixture of both states [52]. Although aPDI performance is similar for both PSs, the PSRe-µL2 requires less time exposition than PSRe-µL1 to get the same effect. This difference could be associated with the higher coefficients of molar extinction presented for the PSRe-µL2 compound. The difference in the chemical structure of the L2 polypyridine ligand involving more complex hydrocarbon chains may be related [52]. A more extensive photophysical characterization The effective microbial activity of the PSRe-µL1 and PSRe-µL2 photosensitizers rests on the photophysical properties provided by their chemical structure. Although these compounds are neutral (do not have positive charges in the coordination sphere), they can easily polarize by absorption of light, as described in a previous report [41]. This characteristic may improve their molecular proximity to the *K. pneumoniae* cell envelope. Additionally, by exciting these high-rate light-absorbing dark compounds, they can produce high-energy triplet states, which promote energy transfer to molecular oxygen [51]. At first glance, the antimicrobial effect observed with PSRe-µL1 and PSRe-µL2 can be explained by access to a triplet state, as reported [41]. It will then depend on the nature of the Re (I) complex; the excited state responsible for the generation of the ROS producing photooxidative stress could be states <sup>3</sup>MLCT, <sup>3</sup>LC, or a mixture of both states [52]. Although aPDI performance is similar for both PSs, the PSRe-µL2 requires less time exposition than PSRe-µL1 to get the same effect. This difference could be associated with the higher coefficients of molar extinction presented for the PSRe-µL2 compound. The difference in the chemical structure of the L2 polypyridine ligand involving more complex hydrocarbon chains may be related [52]. A more extensive photophysical characterization should be carried out to corroborate these hypotheses, such as determining the bacterial cell envelope damage by TEM or the production of reactive oxygen species.

At concentrations as low as 4 µg/mL, both the PSRe-µL1 and the PSRe-µL2 compounds showed an effective aPDI activity, inhibiting the growth of *K. pneumoniae* (>3log10). These results are comparable to using cyclometalated Ir (III) complexes as aPDI, as previously reported [23,29]. Similar values have been reported for other PS compounds against Gram-negative bacteria [53–56]. When combined with imipenem, the PSReµL1 and PSRe-µL2 compounds produced a similar effect, shown previously by Ir(III) based cyclometalated compounds [29] and other photosensitizers such as rose bengal for *Acinetobacter baumannii* [57]. Similarly, alternative compounds such as anti-biofilm peptides mixed with conventional antibiotics reported good antimicrobial activity in an in vivo model [58]. This behavior could be related, as was mentioned before, to the external chemical structures of the polypyridine ligands. In the Re(I)-PSRs, polypyridine ligands have heteroatoms, allowing dipole–dipole interactions that may weaken the bacterial cell envelope facilitating the β-lactam action. The synergism shown by these compounds with β-lactams suggests that they damage the bacterial cell wall structure. Oxidative stress is known to modify the membrane permeability of *K. pneumoniae* [59]. However, at the moment, we have not carried out experiments, such as transmission microscopy, to confirm this idea. Our results also show the capacity of the Re(I)-PSRs compounds to inhibit the bacterial growth of a collection of 118 clinical isolates of *K. pneumoniae*. We used this collection of strains because its antibiotic resistance is characterized and presents a well-established sub-population of ESBL-producing bacteria [42]. The aPDI was not only effective; the FIC also demonstrates the synergic combination with Cfx over the total population and a significant reduction in Cfx-MIC in an ESBL-producers subpopulation. The synergistic effect exhibited by these compounds is one of their most remarkable qualities. The use of photodynamic therapy may resolve the lost susceptibility of MDR bacteria. Therefore, photodynamic therapy combined with more conventional antibiotics avoids the use of rescue therapy [50,57]. Similar to Cfx-MIC being reduced in combination, PSs-MEC was also reduced, indicating that lower concentration antibiotic/PS regimens could be used effectively.

The antimicrobial activity of the PSRe-µL1 and PSRe-µL2 is photodynamic and, therefore, its activation is dependent on light. The dependence on light suggests that the PS compounds themselves and in the dark are not toxic, as the results with mammalian cells show us. The slight but significant light-dependent cytotoxicity exhibited by PSRe-µL1 on HEK293T cells reinforces the low intrinsic toxicity. However, it may imply potential damage to the host tissues that must be considered and prevented with a better characterization of the exposure times and concentration of the compound. Furthermore, the cytotoxicity shown by the PSRe-µL1 and PSRe-µL2 compounds is of low significance compared to that shown by the antitumor photosensitizer compounds [60]. However, these compounds must be tested in vivo to establish whether a significant cytotoxic effect may arise, such as an anaphylactic reaction [61]. A murine model may then probe if these compounds could treat infectious diseases in vivo.

For now, it is difficult to accurately calculate the dose of light necessary to activate these PSRe compounds fully; however, we observed in vitro that with photon flux as low as 17µW/cm<sup>2</sup> of white light, they are bactericidal. Compounds with optimum absorbance at higher wavelengths ranging from 450–750 nm would improve the exposure of PS to light into the tissues, but being less energetic, the PSs must have a triplet excited state of easy access to promote energy transfer [62]. The dark color of PSRe-µL1 and PSRe-µL2 may imply that light of different wavelengths can also be absorbed by this Re-PS, increasing the possibility for it to be excited into the tissues [51]. Therefore, we need to characterize its antimicrobial activity better when activated with defined wavelengths before starting in vivo studies in infection models [63].

#### **5. Conclusions**

The present study shows that the increased MICs of resistant bacteria could be reverted by aPDI, turning resistant strains into susceptible ones. Thus, aPDI would effectively treat MDR bacteria, greatly complementing antibiotic therapy. Furthermore, therapeutic regimens with lower doses of antibiotics and PS can be used effectively due to the synergis-

tic effect. The requirement for lower doses of antibiotics will help reduce the generation of resistance. In this work, two bimetallic Re(I) compounds were tested as PSs to be used in the aPDI. According to their UV-vis absorption characterization, it was identified that the absorptions at lower energies occur at wavelengths higher than 450 nm, improving its use in infections compared to the PSs based on Ir(III), previously studied. The dark quality in both powder solids and liquid solutions of the Re(I) compounds may imply that light of a wide wavelength range can be absorbed [51], increasing the possibility of it being excited into the tissues. Although its low excitation at more penetrating wavelengths, during infections of internal organs such as UTI, optical fibers can be used through a catheter to deliver the required light dose [64,65]. Furthermore, the bactericidal activity of these PSs compounds occurred at similar concentrations to those used in antibiotic therapies (CLSI 2017). At these concentrations, the PSs showed no dark cytotoxicity or low light-dependent toxicity on mammalian cells.

**Author Contributions:** Conceptualization, C.E.P. and I.A.G.; methodology, C.E.P. and I.A.G.; software, C.E.P. and I.A.G.; validation, C.E.P., P.D., S.M.B. and I.A.G.; formal analysis, C.E.P., P.D., S.M.B. and I.A.G.; investigation, C.N., A.P., and F.M.-G.; resources, C.E.P., P.D., S.M.B. and I.A.G.; data curation, C.E.P., S.M.B. and I.A.G.; writing—original draft preparation, C.E.P., P.D. and I.A.G.; writing—review and editing, C.E.P., P.D. and I.A.G.; visualization, C.E.P. and I.A.G.; supervision, C.E.P., S.M.B. and I.A.G.; project administration, C.E.P.; funding acquisition, C.E.P., S.M.B., P.D. and I.A.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** Authors are supported by grants: UCEN grants CIP2019007 (awarded to C.E.P.), ANID-Fondecyt grant N◦ 11180185 (awarded to I.A.G.), 11200764 (awarded to F.M.-G.), 1201173 (awarded to P.D.), PI\_M\_2020\_31 USM project (awarded to P.D.), Millennium Institute of Immunology and Immunotherapy P09/016-F, ICN09\_016, (awarded to S.B.).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Faculty of Health Sciences of the Universidad Central de Chile N◦ : 01/2017 approved on 01-04-2017.

**Informed Consent Statement:** The informed consent form was approved by the ethics committee of the Faculty of Health Sciences of the Central University of Chile and by the Bioethics Committee of the Central Metropolitan Health Service of Chile (MHSC) act number: N◦ 124/07.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available because they are confidential data of patients protected by the informed consent protocol.

**Acknowledgments:** We would like to thank Myriam Pizarro, head of the "Hospital El Carmen de Maipú" laboratory, for her collaboration and provision of the clinical isolates of Klebsiella pneumoniae. We also would like to thank Susan Bueno from Pontificia Universidad Católica de Chile for providing the control strains of *K. pneumoniae*, KPPR1, and the typo strain ST258.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Ethical Approval:** The study protocol was approved by the ethics committee of the Faculty of Health Sciences of the Central University of Chile and by the Bioethics Committee of the Central Metropolitan Health Service of Chile (MHSC), Act number: N◦ 124/07.

#### **References**


## *Article* **Effective Photodynamic Therapy with Ir(III) for Virulent Clinical Isolates of Extended-Spectrum Beta-Lactamase** *Klebsiella pneumoniae*

**Constanza Núñez <sup>1</sup> , Annegrett Palavecino <sup>1</sup> , Iván A. González <sup>2</sup> , Paulina Dreyse <sup>3</sup> and Christian Erick Palavecino 1,\***


**Abstract:** Background: The extended-spectrum beta-lactamase (ESBL) *Klebsiella pneumoniae* is one of the leading causes of health-associated infections (HAIs), whose antibiotic treatments have been severely reduced. Moreover, HAI bacteria may harbor pathogenic factors such as siderophores, enzymes, or capsules, which increase the virulence of these strains. Thus, new therapies, such as antimicrobial photodynamic inactivation (aPDI), are needed. Method: A collection of 118 clinical isolates of *K. pneumoniae* was characterized by susceptibility and virulence through the determination of the minimum inhibitory concentration (MIC) of amikacin (Amk), cefotaxime (Cfx), ceftazidime (Cfz), imipenem (Imp), meropenem (Mer), and piperacillin–tazobactam (Pip–Taz); and, by PCR, the frequency of the virulence genes K2, magA, rmpA, entB, ybtS, and allS. Susceptibility to innate immunity, such as human serum, macrophages, and polymorphonuclear cells, was tested. All the strains were tested for sensitivity to the photosensitizer PSIR-3 (4 µg/mL) in a 17 µW/cm<sup>2</sup> for 30 min aPDI. Results: A significantly higher frequency of virulence genes in ESBL than non-ESBL bacteria was observed. The isolates of the genotype K2+, ybtS+, and allS+ display enhanced virulence, since they showed higher resistance to human serum, as well as to phagocytosis. All strains are susceptible to the aPDI with PSIR-3 decreasing viability in 3log10. The combined treatment with Cfx improved the aPDI to 6log10 for the ESBL strains. The combined treatment is synergistic, as it showed a fractional inhibitory concentration (FIC) index value of 0.15. Conclusions: The aPDI effectively inhibits clinical isolates of *K. pneumoniae*, including the riskier strains of ESBL-producing bacteria and the K2+, ybtS+, and allS+ genotype. The aPDI with PSIR-3 is synergistic with Cfx.

**Keywords:** antibiotic resistance; virulence factors; *Klebsiella pneumoniae*; photodynamic therapy

## **1. Introduction**

*Klebsiella pneumoniae* is one of the major health-associated infection (HAIs) producers worldwide, including pneumonia, urinary tract, and bloodstream infections [1,2]. Moreover, HAI-producing *K. pneumoniae* strains progressively accumulate more multiple-drugs resistance (MDR), including extended-spectrum β-lactamases (ESBL) and carbapenemases, such as KPC [3–5]. Treatment options for infections caused by MDR strains of *K. pneumoniae* are severely reduced to colistin and tigecycline [6–8]. The high MDR shown by strains of *K. pneumoniae* does not fully explain its notable success as one of the most important agents of HAIs, suggesting the participation of other factors [9]. An increasing number of authors suggest that increased bacterial survival during infections is related to virulence factors [1,3,9–14].

**Citation:** Núñez, C.; Palavecino, A.; González, I.A.; Dreyse, P.; Palavecino, C.E. Effective Photodynamic Therapy with Ir(III) for Virulent Clinical Isolates of Extended-Spectrum Beta-Lactamase *Klebsiella pneumoniae*. *Pharmaceutics* **2021**, *13*, 603. https://doi.org/10.3390/ pharmaceutics13050603

Academic Editor: Udo Bakowsky

Received: 6 April 2021 Accepted: 15 April 2021 Published: 22 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Virulence factors shown by *Klebsiella pneumoniae* strains are common to enterobacteria, such as siderophores, enzymes, and capsules [9]. These factors may be part of genes conserved amongst all *K. pneumoniae*, called the core set of genes, but other genes vary in frequency between strains, and they are part of the accessory set of genes [9]. During colonization and infection, *K. pneumoniae* requires the expression of several core and accessory genes to deal with, for example, the nutritional stress due to nutrient sequestration or the immune response of the host [15]. For instance, during lung infection, the acquisition of iron is facilitated by the siderophores enterobactin (*ent*B) and yersiniabactin (*ybt*S) genes encoded on the bacterial chromosome [1,16]. Like iron, during infections of the lungs, urinary tract, and bloodstream, access to nitrogen is limited. The *all*S gene allows *K. pneumoniae* to use allantoin degradation as an alternative nitrogen source [17,18]. The polysaccharide capsule is involved, among others, in resistance to death by complement-mediated opsonophagocytosis [9], and inhibition of macrophages [19,20]. Some capsular varieties (such as K2) may produce hypervirulent strains with characteristic phenotypes such as the hypermucoviscous [19]. Some hypervirulent phenotypes are associated with the expression of the mucoviscosity-associated gene A (*mag*A) and the regulator of the mucoid phenotype A gene (*rmp*A) [21]. Moreover, the *rmp*A gene increases the ability of ESBL-producing strains to resist the bactericidal activity of serum and phagocytosis [22]. Since the virulence factors increase bacterial survival and may influence antibiotic resistance expression, the composition of the pool of the virulence gene of *K. pneumoniae* strains may be associated with the antibiotic susceptibility pattern [9,13].

Due to the emergence of multi-drug resistance (MDR), the deficit of new antibiotics is one of the most pressing threats to human health in the 21st century [23]. Given the high risk to public health caused by the deficiency of new antibiotics, the use of complementary antimicrobial therapies non-antibiotic-based, such as antibacterial photodynamic inactivation (aPDI), emerges as a promising alternative [24]. The aPDI may support the lack of antibiotic therapies against MDR and KPC strains [24,25]. The aPDI is a procedure based on the use of photosensitizer (PS) compounds to produce light-activated local cytotoxicity (photooxidative stress) [26]. The PSs work by energy absorbs of a specific wavelength in the UV–Vis range, which is then transferred to molecular oxygen in solution to produce reactive oxygen species (ROS) [27]. Molecular O<sup>2</sup> can accept this energy together with electrons, or it can only undergo a one-electron reduction to produce superoxide anion radical (O<sup>2</sup> •−), hydrogen peroxide (H2O2), and hydroxyl radical (HO• ) [28,29]. The energy transferred to the O<sup>2</sup> without one-electron reduction produces singlet oxygen (1O2) [27]. The ROS production generates photooxidative stress induced by aPDI, which occurs mainly due to the action of <sup>1</sup>O2. The oxidative action of <sup>1</sup>O<sup>2</sup> occurs on organic molecules close to the PS through concerted addition reactions of alkene groups [30]. These organic molecules can be structural proteins or lipids of the bacterial envelope; therefore, the damage occurs to the cell wall plasma membrane or other bacterial structures, leading to nonspecific cell death [27,31]. Previously, a PS compound based on a polypyridine Ir(III) complex (PSIR-3, see Figure 4) demonstrated aPDI activity, inhibiting the bacterial growth of CKP and synergism with imipenem [32,33]. In this work, we determine the frequency of the K2, *ent*B, *yb*tS, *all*S, *rmp*A, and *mag*A virulence genes, in a population of 118 clinical isolates of *K. pneumoniae* and evaluate their association to the susceptibility pattern to several antibiotics. Our data show that virulence factors K2<sup>+</sup> , *ybt*S + , and *all*S <sup>+</sup> were associated with a modification in the bacterial minimum inhibitory concentrations (MICs) and increased ESBL production probability. These virulence genes significantly increased the survival of the bacteria, to innate immunity. These more virulent ESBL bacteria were all susceptible to the aPDI treatment with PSIR-3 and demonstrate synergism with cephotaxime.

#### **2. Materials and Methods**

#### *2.1. Bacterial Isolates*

The ethics committee of the Faculty of Health Sciences, Central University of Chile, and the Central Metropolitan Health Service of Chile (MHSC), Act number: N 124/07, approved

the study protocol and the informed consent form. The clinical isolates of *K. pneumoniae* were obtained from 122 samples from unrelated patients, received in the bacteriology laboratory of "Hospital el Carmen" (HEC) for a period of six months (2017/2018). The HEC is a complex hospital with 412 beds and serving a population of more than 600,000 inhabitants. All clinical isolates were identified as *K. pneumoniae* following the protocols of the Institute of Clinical and Laboratory Standards (CLSI) [34]. As controls, the virulent strain (ATCC 43816 KPPR1) of susceptible *K. pneumoniae* (K2<sup>+</sup> , *ybt*S + , and *all*S + ) [35] and the MDR KPC<sup>+</sup> ST258 (KP35) (K2−, *ybt*S − and *all*S −) [36] strains were also included.

#### *2.2. Antimicrobial Susceptibility Testing*

The MIC of the antimicrobial agents were determined in 96-well plates by microdilution methodology in cations-adjusted Mueller-Hinton (ca-MHB) broth. Inoculum of <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>6</sup> colony forming units (CFUs)/mL of each clinical isolate was mixed with decreasing concentrations of each antibiotic and incubated overnight at 37 ◦C following the CLSI recommendations. The MIC for each antibiotic was determined as the last dilution in which no bacterial growth occurred, and the susceptibility intervals were assigned based on the cutoff points established by the CLSI (2018) for amikacin (Amk), cefotaxime (Cfx), ceftazidime (Cfz), imipenem (Imp), meropenem (Mer), and piperacillin–tazobactam (Pip–Taz). According to the CLSI protocols [34], clinical isolates were strains considered ESBL-producing when resistant to cefotaxime but susceptible to the combination of Cfx/clavulanic acid. Values are presented as the median in mg/L and interquartile range (IQR).

#### *2.3. DNA Extraction and PCR Amplification*

The total DNA was obtained from stationary bacterial cultures in LB broth using the phenol-chloroform methodology. In brief, pelleted bacteria were suspended in 300 µL of PBS and mixed with 300 µL of phenol: chloroform (25:24 vol:vol); the final mix was stirred well in a vortex and centrifuged at 13,000× *g* at 4 ◦C for 15 min. The aqueous phase was mixed 1:1 with chloroform and centrifuged as above. Genomic and plasmidial DNA contained in the aqueous phase was precipitated in 0.6 vol of 2-propanol and sedimented at 13,000× *g* at 4 ◦C for 20 min. The nucleic acids were washed twice with 70% ethanol and resuspended in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0). PCR reactions were performed, using 0.5 µM of each specific primer pair listed in Table 1, in 1× master mix GoTaq (Promega). The amplification was carried out at a final volume of 20 µL in a Veriti (Applied Biosystem) PCR machine with an initial denaturation step of 10 s at 95 ◦C, followed by 35 cycles of 10 s at 95 ◦C, 15 s at 58 ◦C, and 30 s of extension at 72 ◦C. A final extension step of 7 min at 72 ◦C was included, and PCR products were visualized on a 1.7% agarose gel.

#### *2.4. K. Pneumoniae Survival to Innate Immunity*

To evaluate the survival of *K. pneumoniae* to the innate immunity, the bactericidal activity of normal human serum (NHS), as well as phagocytosis by human macrophages (MΦ), and polymorphonuclear (PMN) cells was determined. Both serum and leukocytes were obtained from blood samples of healthy voluntary donors who had not taken any antibiotic or anti-inflammatory medication for at least ten days before the day of sampling.

#### 2.4.1. Susceptibility to Normal Human Serum

Serum susceptibility was carried out as before [37]; in brief, 75 µL of pooled NHS were mixed with 25 <sup>µ</sup>L suspension containing 2 <sup>×</sup> <sup>10</sup><sup>6</sup> CFUs of each isolate in a 96-well plate. As a control, bacterial cultures of each isolate were mixed with PBS. The mixtures were incubated at 37 ◦C for 3 h, and viable bacteria were enumerated by serial micro-dilution and colony counting on ca-MH agar plates. Serum resistance is expressed as viable bacteria in CFUs/mL compared to untreated isolates controls.


**Table 1.** Primers for genes encoding virulence factors of *Klebsiella pneumoniae.*

#### 2.4.2. Susceptibility to Phagocytosis by Macrophages and Polymorphonuclear Cells

The separation of mononuclear and polymorphonuclear cells was performed by centrifugation in a gradient density column of Histopaque (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer instructions. Monocytes were selected from other mononuclear cells by incubation in RPMI-1640 medium (Sigma-Aldrich) without fetal bovine serum (FBS) and differentiated into macrophages incubating during 7–9 days in RPMI-1640 10% FBS at 37 ◦C with 5% CO<sup>2</sup> [38]. The macrophage monolayer was infected with 2.5 <sup>×</sup> <sup>10</sup><sup>7</sup> CFUs with a multiplicity of infection (MOI) of 50:1 of each bacterial isolate and centrifuged at 200× *g* for 5 min, to synchronize phagocytosis. After 2 h of incubation, cells were washed and incubated for an additional 60 min in RPMI-1640 + 100 µg/mL gentamicin. Macrophages were lysed with 0.1% saponin for 10 min at room temperature, and viable bacteria were enumerated as above. For PMN assays [39], 5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells were combined with 5 <sup>×</sup> <sup>10</sup><sup>6</sup> CFUs of each isolate (MOI 10:1) in serum-free RPMI-1640 and synchronize by centrifugation at 524× *g* for 8 min at 4 ◦C. After 3 h, PMNs were lysed with 0.1% saponin, and viable bacteria were enumerated. Control groups of non-phagocyted bacteria were included.

#### *2.5. Synthesis of the PSIR-3 Compound*

The structural and photophysical characterization of the PSIR-3 compound was described previously [40]. The complex synthesized can be described by using the following general formula: [Ir(CˆN)2(NˆN)](PF6), where NˆN is the ancillary ligand; and CˆN corresponds to a cyclometalating ligand. In this study PSIR-3 is [Ir(ppy)2(ppdh)]PF<sup>6</sup> where ppdh is pteridino(7,6-f)(1,10)phenanthroline-1,13(10H,12H)-dihydroxy and ppy is 2-phenylpyridine [41]. The structure and purities of the compound were confirmed by nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR), and mass spectroscopy (MALDI–MS) measurements. The absorption spectra were measured in acetonitrile (ACN) solutions using a Shimadzu UV–Vis Spectrophotometer UV-1900. The molar extinction coefficients of the characteristic bands were determined from the absorption spectra. Photoluminescence spectra were taken on an Edinburgh Instrument spectrofluorimeter using ACN solutions of the compounds previously degassed with N<sup>2</sup> for approximately 20 min. The emission quantum yields (Φem) were calculated according to the description of the literature [42]. Fluorescence lifetimes were measured by using a timecorrelated single-photon counting (TC-SPC) apparatus (PicoQuant Picoharp 300) equipped with a sub-nanosecond LED source (excitation at 380 nm) powered by a PicoQuant PDL 800-B variable (2.5−40 MHz) pulsed power supply.

#### *2.6. Antimicrobial Activity of Photosensitizer Compounds*

Stock solutions of 2 g/L of the PSIR-3 compound solubilized in ACN were used to prepare working solutions in distilled water. For the antimicrobial assay, the collection of 118 clinical isolates of *K. pneumoniae* was used, and the control strains of susceptible *K. pneumoniae* KPPR1 and the MDR strain ST258 were also included. All bacteria were grown as axenic culture in Luria Bertani broth or agar medium as appropriate. PSIR-3 was mixed in 24-well plates at a final concentration of 4 mg/L for photodynamic experiments, with suspensions of 1 <sup>×</sup> <sup>10</sup><sup>7</sup> colony forming units (CFUs)/mL of each bacterium, in a final volume of 500 µL of cation-adjusted Mueller-Hinton (ca-MH) broth. Exposure to light was performed for 30 min in a chamber with a white LED lamp at a photon flux of 17 µW/cm<sup>2</sup> . After exposure to light, the CFUs of the viable bacteria were determined by broth-micro dilution and sub-cultured on ca-MH agar plates. Following the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2017) [34], the agar plates were incubated during 16–20 h at 37 ◦C, in the dark, and colony count was recorded, using a stereoscopic microscope. Control wells with bacteria culture with no photosensitizer or photosensitizer but not exposed to light were also included.

#### *2.7. Determination of the Synergy between PSIR-3 and Cfx*

The fractional inhibitory concentration index (FIC) value was determined using the following formula [43,44]. MICac is the MIC of a compound A, combined with a compound B, and MICbc is the MIC of the compound B combined with the compound A. The MICa and MICb are the MIC of the A and B compounds alone, respectively. Values in the FIC index ≤0.5 are considered synergistic, and values > 4 are considered antagonistic [44].

$$\text{FIC Index} = \frac{\text{MICac}}{\text{MIC}\_a} + \frac{\text{MICbc}}{\text{MIC}\_b}$$

To determine the MIC-Cfx combined with each PSs, 1 <sup>×</sup> <sup>10</sup><sup>7</sup> UFC/mL of ESBLproducing bacteria were aPDI treated for 30 min with 4 mg/L of each PS and mixed with serial dilution (32–0.125 mg/L) of Cfx in ca-MH broth, as above. To determine the MIC-PSs combined with Cfx, 1 <sup>×</sup> <sup>10</sup><sup>7</sup> UFC/mL of ESBL-producing bacteria was mixed with serial dilution of each PSs (32–0.125 mg/L) and a fixed concentration of 4 mg/L of Cfx, and then it was subjected to aPDI, as above.

#### *2.8. Statistical Analyses*

Statistical analyses were performed by using the Systat 13.2 software (Systat Software, Inc., San Jose, CA, USA) and GraphPad v6.01 (Prism) software. The X<sup>2</sup> test or Fisher's exact test for categorical variables and the Mann–Whitney *U* test for continuous non-parametric variables were used. The risk of virulence genes to modify the MIC of the antibiotic was established by determining the odds ratio with a CI: 95%.

#### **3. Results**

#### *3.1. Demographic Characterization*

This work seeks to demonstrate the capacity of aPDI to inhibiting the growth of clinical isolates of *K. pneumoniae*, which are diverse in antimicrobial susceptibility, genotype, and virulence. Phenotypic and genotypic characterization was conducted to determine the frequency of genes encoding virulence factors, the MIC values determined for various antibiotics, and susceptibility to innate immune components. From the clinical isolates of *K. pneumoniae* received in the laboratory, 118 were selected from different unrelated patients. The isolates were recovered mainly from urine samples, 113 (95.8%), and only five from respiratory samples (three endotracheal aspirates (2.5%) and two expectorations (1.7%)). As shown in Table 2, the samples were obtained from 78 (66%) females and 40 (34%) males, from 12 clinical services, with a higher contribution from the emergency room 41 (34.75%), secondly the unit of medicine 24 (20.34%), third ambulatory 16 (13.56%) and

tion.

in the fourth place urology 14 (11.86%). Of the 118 clinical isolates, 114 came from adults and 4 from pediatric patients. As shown in Figure 1A, the patient's age fluctuated between 7 months and 94 years, with a median (IQR: 25–75%) of 69.5 (54.8–84) years for females, and between 10 months and 92 years, with a median of 68.5 (60.3–80.8) years for males. There are no significant differences in age between genders (*p* = 0.279 Mann–Whitney *U* test). The results confirm the infections with MDR and not MDR *K. pneumoniae* mainly affects the elderly population. The elderly are the most susceptible population to present complications derived from infectious diseases [45].


**Table 2.** Frequency of *K. pneumoniae* isolation by hospital unit and genre.

**Figure 1.** Characterization of patients and clinical isolates of *K. pneumoniae*. (**A**) Box plot for the age of patients stratified by gender. Patients show a median of 69.5 years for females and 68.5 years for males. (**B**) Box plot showing the median in the population of the minimum inhibitory concentration (MIC) of the seven antibiotics commonly used to treat infections by Gram-negative bacteria. (**C**) Frequency of carrying of the six genes evaluated in the study population. (**D**) Frequency distribution of carriers of three or more virulence factors stratified by extended-spectrum beta-lactamase (ESBL) produc-**Figure 1.** Characterization of patients and clinical isolates of *K. pneumoniae*. (**A**) Box plot for the age of patients stratified by gender. Patients show a median of 69.5 years for females and 68.5 years for males. (**B**) Box plot showing the median in the population of the minimum inhibitory concentration (MIC) of the seven antibiotics commonly used to treat infections by Gram-negative bacteria. (**C**) Frequency of carrying of the six genes evaluated in the study population. (**D**) Frequency distribution of carriers of three or more virulence factors stratified by extended-spectrum beta-lactamase (ESBL) production.

producing bacteria.

*3.3. Virulence Gene Frequency* 

termined and expressed in mg/L in a log2 box plot. As shown in Figure 1B, the bacterial population was mainly susceptible to Amk with a median (IQR) of 8 (4–20). Similar to Amk, the population was mainly susceptible to carbapenem, Imp, and Mer with a median of 1 (0.5–1) and 1 (0.5–2), respectively. In contrast, most of the population was resistant to the cephalosporins, Cfx, and Cfz, with medians of 8 (8–8) and 32 (32–32). Finally, similar to cephalosporin, almost the entire population was resistant to the Pip–Taz combination with a median of 256 (64–256). From the population, 66 isolates resistant to Cfx were susceptible to the combination Cfx/clavulanic acid. Those isolates were identified as ESBL-

In this work, we select certain virulence genes belonging to families with different mechanisms of action. PCR determined the frequency of carrying genes encoding the virulence factors *rmp*A, *mag*A, K2, *ent*B, *all*S, and *ybt*S for each isolate using specific primers (Table 1). These genes were selected for being representatives of different families of virulence factors. As shown in Figure 1C, the harboring frequency was; *rmp*A [2.54% (3/118)], *mag*A [0% (0/118), K2 [39% (46/118)], *ent*B [83.9% (99)], *all*S [11.2% 13/118)] and *ybt*S [73.8% (87/118)]. The most frequent virulence factor was the *ent*B gene, followed by the *ybt*S gene. There were no isolates with the *mag*A gene, and only three isolates harbor the *rmp*A gene.

#### *3.2. Antibiotic Susceptibility*

In the *K. pneumoniae* population, the median and IQR (25–75%) of the MIC were determined and expressed in mg/L in a log<sup>2</sup> box plot. As shown in Figure 1B, the bacterial population was mainly susceptible to Amk with a median (IQR) of 8 (4–20). Similar to Amk, the population was mainly susceptible to carbapenem, Imp, and Mer with a median of 1 (0.5–1) and 1 (0.5–2), respectively. In contrast, most of the population was resistant to the cephalosporins, Cfx, and Cfz, with medians of 8 (8–8) and 32 (32–32). Finally, similar to cephalosporin, almost the entire population was resistant to the Pip–Taz combination with a median of 256 (64–256). From the population, 66 isolates resistant to Cfx were susceptible to the combination Cfx/clavulanic acid. Those isolates were identified as ESBL-producing bacteria.

#### *3.3. Virulence Gene Frequency*

In this work, we select certain virulence genes belonging to families with different mechanisms of action. PCR determined the frequency of carrying genes encoding the virulence factors *rmp*A, *mag*A, K2, *ent*B, *all*S, and *ybt*S for each isolate using specific primers (Table 1). These genes were selected for being representatives of different families of virulence factors. As shown in Figure 1C, the harboring frequency was; *rmp*A [2.54% (3/118)], *mag*A [0% (0/118)], K2 [39% (46/118)], *ent*B [83.9% (99)], *all*S [11.2% 13/118)] and *ybt*S [73.8% (87/118)]. The most frequent virulence factor was the *ent*B gene, followed by the *ybt*S gene. There were no isolates with the *mag*A gene, and only three isolates harbor the *rmp*A gene. No isolates showed the hypermucoviscosity phenotype (by string test) in agar plates in the population of 118 unrelated isolates.

### *3.4. Correlation of Virulence Factors with Antibiotic Resistance*

As shown in Figure 1D, there is a significantly higher frequency of ESBL strains that harbor three or more virulence factors compared to non-ESBL strains (Fisher's; *p* < 0.026). The non-parametric Mann–Whitney *U* test (Systat 13 software) was used to determine the association that each virulence gene has on the median MIC value assuming a null hypothesis α = 5%. As shown in Table 3, the *rmp*A gene was not significantly associated with a modification of the median-MIC of any of the antibiotics analyzed in this study (*p* > 0.05). On the other hand, the *all*S gene significantly influenced the median-MIC of Cfx, Cfz, and Pip–Taz (*p* < 0.05). Similarly, the K2 gene significantly influenced the median-MIC of Cfx and Pip–Taz (*p* < 0.05). The *ent*B gene significantly affected the median-MIC of Amk and Imp (*p* < 0.05). The *ybt*S gene significantly influenced the median-MIC of Cfx (*p* < 0.006). Finally, the *all*S gene significantly influenced the median-MIC of Cfx and Cfz antibiotics (*p* < 0.05). The MIC of the antibiotics more sensitive to the presence of virulence factors were the Cfx (sensitive to K2, *ybt*S, and *all*S genes) and Pip–Taz (sensitive to the K2 and *all*S genes). These data show that, regardless of the patient's conditions, the multi-virulence is effectively an independent risk factor that promotes ESBL-production of clinical populations of *K. pneumoniae* with a *p* < 0.026 Fisher's exact test.



Values in bold represent a *p* < 0.05, below the null hypothesis value with α = 5%, which means a significant difference in MIC due to the presence of the virulence factor. Pip–Taz, piperacillin–tazobactam.

As shown in Figure 2, the box plot for each antibiotic stratified by the presence or absence of virulence genes was constructed to verify if the influence is to increase or

decrease the median of the MIC. The presence of the *ent*B gene is associated with a decrease in the median-MIC for Amk and Imp. On the other hand, the K2 gene is associated with an increase in the median-MIC for Cfx and Pip–Taz. Similarly, the *ybt*S gene is associated with an increase in the median-MIC for Cfx. Finally, the *all*S gene is associated with an increased median-MIC for Cfz and decreased median-MIC for Cfx and Pip–Taz. Some of the virulence factors studied here were associated with the median- MIC modification for several antibiotics. In the *K. pneumoniae* population tested, the most influencing virulence factors were *ent*B, *ybt*S, and *all*S genes. The stratified MICs-box plot made it possible to distinguish how virulence genes modulate antibiotic susceptibility by increasing or decreasing. Remarkably, the sum of the genes of the K2<sup>+</sup> , *ybt*S + , and *all*S <sup>+</sup> genes contributes to increasing the median-MICs to values higher than the clinical susceptibility breakpoint established by the CLSI. These increased values occur similarly when efflux pumps are activated in other Gram-negative bacteria [46]. For example, in a population of *Pseudomonas aeruginosa*, the combined overexpression of the *mex*A and *mex*X efflux pump increased the median MIC for ciprofloxacin and cefepime above the cutoff points [46,47].

#### *3.5. Association of K2<sup>+</sup> , ybtS<sup>+</sup> , and allS<sup>+</sup> Virulence Genes to Survive the Innate Immunity*

Our results show that the K2 and *ybt*S virulence genes are risk factors for the production of ESBL and that the *all*S gene acts as a protective factor. Then, we selected two groups of clinical isolates of *K. pneumoniae*, which harbor or not these three genes, to assess their susceptibility to innate immunity components. Only three isolates, identified by the numbers 81, 92, and 111, are of the K2<sup>+</sup> , *ybt*S + , and *all*S <sup>+</sup> genotype, and unexpectedly all of them are ESBL-producers. From the genotype K2−, *ybt*S − and *all*S −, the isolates 13, 21, and 22 were selected as being non-ESBL. As controls, the susceptible but highly virulent *K. pneumoniae* KPPR1 (K2<sup>+</sup> , *ybt*S + , and *all*S + ) and the MDR but less virulent ST258 (K2−, *ybt*S − and *all*S −) strains were also included.

To determine the bacterial susceptibility to normal human serum (NHS), they were exposed for 3 h, and then the number of viable bacteria was determined by microdilution and plate counting. As shown in Figure 3A, the presence of all three virulence genes, K2<sup>+</sup> , *ybt*S + , and *all*S + , significantly (\*\* = *p* < 0.003; two-way ANOVA) increases the survival of ESBL-producing bacteria compared to non-ESBL bacteria lacking all three virulence genes. On average, the serum resistance was improved by four orders of magnitude (4log10). Similar to that observed with serum, ESBL-producing bacteria of the K2<sup>+</sup> , *ybt*S + , and *all*S + genotype show a significant (\*\*\*\* = *p* < 0.0001) increase, 4log10, in the survival to the MΦ activity, compared to no- ESBL bacteria that lacks these virulence genes (Figure 3B). Finally, comparable to MΦ, survival to the activity of PMNs of the bacteria of genotype K2<sup>+</sup> , *yb*tS<sup>+</sup> , and *all*S + increased significantly (\* = *p* < 0.02), at least one time on average, compared to non-ESBL strains that lack the virulence genes (Figure 3C). As shown in Figure 3, the virulent control KPPR1 strain was more resistant to serum (Figure 3D), macrophages (Figure 3E), and PMN (Figure 3F) compared to the MDR ST258 *K. pneumoniae* strain. Consistently the clinical control isolates have shown to be more susceptible to the bactericidal activity of the serum (3D) and the phagocytic activity of MΦ (3E) and PMN (3F). The co-occurrence of harboring multiple genes that encode virulence factors and the ESBL-production leads to enhanced virulence. The ESBL-producing strains of *K. pneumoniae* of the genotype K2<sup>+</sup> , *ybt*S + , and *all*S <sup>+</sup> were more resistant to innate immunity, consistently with studies over MDR populations of *K. pneumoniae* that increased their 30-day mortality over patients undergoing bloodstream infections [1,48]. Moreover, virulence genes, such as siderophores, which have an essential role in bacterial survival and virulence [49,50], have been previously associated with the MDR-*K. pneumoniae* [16,51]. In this study, ESBL-producer *K. pneumoniae* of the K2<sup>+</sup> , *ybt*S + , and *all*S <sup>+</sup> genotype shown a survival improvement for killing by PMNs. Previously, the PMN has demonstrated a limited binding and uptake capacity for MDR– *K. pneumoniae* [39]. The activity of the PMN is the most important cellular component of the innate immune response, essential as the first line of defense against bacterial infections [52].

**Figure 2.** Association of virulence genes in the measurement of median MIC. The box diagrams created with the Systat 13 software summarize the MIC measures for six commonly used antibiotics, stratified by the presence or absence of each of the six virulence genes. The data are presented as mg/L in a Log2 scale of MICs for each antibiotic stratified by the presence (+) or absence (−) of each virulence gene. The horizontal lines represent the median and the frequency limits between 25 and 75% of the individuals, the hollow dots represent single individuals, and the asterisks represent small groups of individuals. **Figure 2.** Association of virulence genes in the measurement of median MIC. The box diagrams created with the Systat 13 software summarize the MIC measures for six commonly used antibiotics, stratified by the presence or absence of each of the six virulence genes. The data are presented as mg/L in a Log<sup>2</sup> scale of MICs for each antibiotic stratified by the presence (+) or absence (−) of each virulence gene. The horizontal lines represent the median and the frequency limits between 25 and 75% of the individuals, the hollow dots represent single individuals, and the asterisks represent small groups of individuals.

**Figure 3.** Effect of virulence genes on the susceptibility of ESBL or non-ESBL strains to components of innate immunity. The susceptibility of ESBL-producing *K. pneumoniae* clinical isolates bearing the virulence genes *ybt*S+, K2+, and *all*S+ was determined and compared with non-ESBL producing bacteria that lack the virulence genes. (**A**,**D**) Susceptibility to normal human serum (NHS), (**B**,**E**) susceptibility to phagocytosis by macrophages (MΦ), and (**C**,**F**) susceptibility to phagocytosis by polymorphonuclear cells (PMN). The results of two independent experiments performed in triplicate are shown (n = 6). Viable bacteria were enumerated by colony count on ca-MH agar after serial micro-dilution. The colony forming units (CFUs)/mL values are presented as means +/− SD, on a log10 scale of treated bacteria (black bars) compared to untreated control bacteria (gray bars). \*\*\*\* = *p* < 0.0001, \*\* = *p* < 0.003 and \* = *p* < 0.02 of two-way ANOVA comparing the proportion of treated/untreated ESBL bacteria with non-ESBL bacteria. **Figure 3.** Effect of virulence genes on the susceptibility of ESBL or non-ESBL strains to components of innate immunity. The susceptibility of ESBL-producing *K. pneumoniae* clinical isolates bearing the virulence genes *ybt*S + , K2<sup>+</sup> , and *all*S <sup>+</sup> was determined and compared with non-ESBL producing bacteria that lack the virulence genes. (**A**,**D**) Susceptibility to normal human serum (NHS), (**B**,**E**) susceptibility to phagocytosis by macrophages (MΦ), and (**C**,**F**) susceptibility to phagocytosis by polymorphonuclear cells (PMN). The results of two independent experiments performed in triplicate are shown (n = 6). Viable bacteria were enumerated by colony count on ca-MH agar after serial micro-dilution. The colony forming units (CFUs)/mL values are presented as means +/− SD, on a log<sup>10</sup> scale of treated bacteria (black bars) compared to untreated control bacteria (gray bars). \*\*\*\* = *p* < 0.0001, \*\* = *p* < 0.003 and \* = *p* < 0.02 of two-way ANOVA comparing the proportion of treated/untreated ESBL bacteria with non-ESBL bacteria.

#### *3.6. Susceptibility of Clinical Isolates to aPDI with PSIR-3 3.6. Susceptibility of Clinical Isolates to aPDI with PSIR-3*

#### 3.6.1. Photophysical Properties of the PSIR-3 Compound 3.6.1. Photophysical Properties of the PSIR-3 Compound

We have previously shown that Ir(III)-based compounds, such as PSIR-3, have photodynamic antimicrobial activity against imipenem-resistant *Klebsiella pneumoniae* [32,33]. In this work, we tested a coordination compound characterized by a positive charge in the first coordination sphere (Figure 4B). The photophysical evaluation of the PSIR-3 performed in acetonitrile solution [40] revealed absorption processes at 375 and 392 nm attributable at the first instance of charge-transfer transitions (Figure 4C,D) [32]. When the We have previously shown that Ir(III)-based compounds, such as PSIR-3, have photodynamic antimicrobial activity against imipenem-resistant *Klebsiella pneumoniae* [32,33]. In this work, we tested a coordination compound characterized by a positive charge in the first coordination sphere (Figure 4B). The photophysical evaluation of the PSIR-3 performed in acetonitrile solution [40] revealed absorption processes at 375 and 392 nm attributable at the first instance of charge-transfer transitions (Figure 4C,D) [32]. When the PSIR-3 compound was excited with a wavelength corresponding to the lowest charge-transfer absorption energy, 375 nm, it showed maximum emission at 598 nm (Figure 4C,D). Figure 4C shows the recorded lifetimes of excited states in 0.32 µs and the calculated quantum yield (Φem) in

0.011 [40]. The aPDI activity of the PSIR-3 compound was compared with the positive PS control [Ru(bpy)3](PF6)<sup>2</sup> (bpy = 2,20 -bipyridine) called PS-Ru. According to the literature, the PS-Ru shows a charge-transfer absorption process at 450 nm with maximum emission at 600 nm (excited in 450 nm) in acetonitrile [42], with Φem of 0.095 [42], and a lifetime registered of its excited state of 0.855 µs (Figure 4C) [53]. The maximum absorption of PSIR-3 occurs at wavelengths below 400 nm, that although it is more energetic, it penetrates the tissues poorly. Therefore, this compound will activate better if it is directly irradiated, such as in superficial wounds. However, UTI is one of the most common diseases caused by *K. pneumoniae*, where probes that deliver the light dose within internal surfaces can irradiate the epithelial lining of the bladder [54]. Certain kinds of fiber arrays or inflatable balloons may provide a homogenous light power delivery [55,56]. This catheterization can be applied for inpatients suffering UTI that does not respond to antibiotic treatment. lated quantum yield (Φem) in 0.011 [40]. The aPDI activity of the PSIR-3 compound was compared with the positive PS control [Ru(bpy)3](PF6)2 (bpy = 2,2′-bipyridine) called PS-Ru. According to the literature, the PS-Ru shows a charge-transfer absorption process at 450 nm with maximum emission at 600 nm (excited in 450 nm) in acetonitrile [42], with Φem of 0.095 [42], and a lifetime registered of its excited state of 0.855 µs (Figure 4C) [53]. The maximum absorption of PSIR-3 occurs at wavelengths below 400 nm, that although it is more energetic, it penetrates the tissues poorly. Therefore, this compound will activate better if it is directly irradiated, such as in superficial wounds. However, UTI is one of the most common diseases caused by *K. pneumoniae*, where probes that deliver the light dose within internal surfaces can irradiate the epithelial lining of the bladder [54]. Certain kinds of fiber arrays or inflatable balloons may provide a homogenous light power delivery [55,56]. This catheterization can be applied for inpatients suffering UTI that does not respond to antibiotic treatment.

PSIR-3 compound was excited with a wavelength corresponding to the lowest chargetransfer absorption energy, 375 nm, it showed maximum emission at 598 nm (Figure 4C,D). Figure 4C shows the recorded lifetimes of excited states in 0.32 µs and the calcu-

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**Figure 4.** The photophysical properties of the PSIR-3 photosensitizer. (**A**) schematic representation of the absorption– emission process of photosensitizer molecules. Light excites external electrons to accede from a basal state S0 to a higher energetic state S1–Sn. Suppose the electrons return to its ground state; the energy is then released as fluorescence. However, when the excited electron enters an intersystem crossing process, the released energy excites molecular oxygen and converts it to reactive oxygen species (ROS). (**B**) chemical structure of the Ir(III) compound (PSIR-3, [Ir(ppy)2(ppdh)]PF6). (**C**) Summary of the photophysical properties of the PSIR-3 and PS-Ru compounds, where λabs = wavelength of absorbance, λem = wavelength of emission, Φem = emission quantum yield, and τ = time in the excited state. Data for PS-Ru were obtained from the literature. Adapted from [42], Elsevier, 2010; Adapted from [53], American Chemical Society, 1983. (**D**) The absorption and the emission spectra of the PSIR-3 in acetonitrile (ACN). **Figure 4.** The photophysical properties of the PSIR-3 photosensitizer. (**A**) schematic representation of the absorption– emission process of photosensitizer molecules. Light excites external electrons to accede from a basal state S<sup>0</sup> to a higher energetic state S1–Sn. Suppose the electrons return to its ground state; the energy is then released as fluorescence. However, when the excited electron enters an intersystem crossing process, the released energy excites molecular oxygen and converts it to reactive oxygen species (ROS). (**B**) chemical structure of the Ir(III) compound (PSIR-3, [Ir(ppy)<sup>2</sup> (ppdh)]PF<sup>6</sup> ). (**C**) Summary of the photophysical properties of the PSIR-3 and PS-Ru compounds, where λabs = wavelength of absorbance, λem = wavelength of emission, Φem = emission quantum yield, and τ = time in the excited state. Data for PS-Ru were obtained from the literature. Adapted from [42], Elsevier, 2010; Adapted from [53], American Chemical Society, 1983. (**D**) The absorption and the emission spectra of the PSIR-3 in acetonitrile (ACN).

#### 3.6.2. Antimicrobial Photodynamic Inhibition of the PSIR-3 over Clinical Isolates

Photodynamic treatment was verified to produce the observed growth inhibition of the 118 clinical isolates of *K. pneumoniae* compared to the untreated bacteria. The photodynamic activity of the PSIR-3 compound was compared to the activity of the PS-Ru reference compound as a positive control [41,57–59]. As seen in Figure 5, compared to the control of untreated bacteria, photodynamic treatment with 4 µg/mL PSIR-3 inhibits bacterial growth > 3 log<sup>10</sup> (>99.9%) of clinical isolates of *K. pneumoniae* (\*\*\*\* *p* < 0.0001; compared to untreated control). The results show that the bactericidal effect produced by PSIR-3 is light-dependent (ns = *p* > 0.05; compared to the untreated control). These results

are comparable with those obtained with the positive control compound PS-Ru, which has shown that bacterial growth inhibition is light-dependent (\*\*\*\* *p* < 0.0001; compared to the untreated control). are comparable with those obtained with the positive control compound PS-Ru, which has shown that bacterial growth inhibition is light-dependent (\*\*\*\* *p* < 0.0001; compared to the untreated control).

Photodynamic treatment was verified to produce the observed growth inhibition of the 118 clinical isolates of *K. pneumoniae* compared to the untreated bacteria. The photodynamic activity of the PSIR-3 compound was compared to the activity of the PS-Ru reference compound as a positive control [41,57–59]. As seen in Figure 5, compared to the control of untreated bacteria, photodynamic treatment with 4 µg/mL PSIR-3 inhibits bacterial growth > 3 log10 (>99.9%) of clinical isolates of *K. pneumoniae* (\*\*\*\* *p* < 0.0001; compared to untreated control). The results show that the bactericidal effect produced by PSIR-3 is light-dependent (ns = *p* > 0.05; compared to the untreated control). These results

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3.6.2. Antimicrobial Photodynamic Inhibition of the PSIR-3 over Clinical Isolates

**Figure 5.** Antimicrobial photodynamic inactivation of clinical isolates of *K. pneumoniae*. (**A**) Growth inhibition of 118 clinical isolates of *K. pneumoniae* subjected to antimicrobial photodynamic inactivation (aPDI) with PSIR-3. The bacteria were used at a concentration of 1 × 107 CFUs/mL and **Figure 5.** Antimicrobial photodynamic inactivation of clinical isolates of *K. pneumoniae*. (**A**) Growth inhibition of 118 clinical isolates of *K. pneumoniae* subjected to antimicrobial photodynamic inactivation (aPDI) with PSIR-3. The bacteria were used at a concentration of 1 <sup>×</sup> <sup>10</sup><sup>7</sup> CFUs/mL and mixed in triplicate with 4 mg/L of PSIR-3 or PSIR-4 compounds. For the aPDI, the mixture of bacteria with PS was exposed for 1 h at 17 µW/cm<sup>2</sup> of white light. As a control, bacteria combined with the photosensitizers (PSs) not exposed to light (PSIR-3 or PS-Ru) and bacteria not combined with the PSs (control) were included. Colony count enumerated of viable bacteria on ca-MH agar after serial micro-dilution. The CFUs/mL values are presented as means ± SD on a log<sup>10</sup> scale. (**B**) From the clinical isolates, 66 ESBL-producing bacteria were exposed to aPDI, using PSIR-3 or PS-Ru, and MIC for cefotaxime (Cfx) was performed in triplicate, in ca-MH broth, for 16–20 h. (**C**) For ESBL-producing bacteria, the MIC for PSIR-3 or PS-Ru, were determined in combination with 4 mg/L of Cfx, performed in triplicate in ca-MH agar for 16–20 h. The MIC values are presented as median ± SD of mg/mL on a log<sup>2</sup> scale. Not significant (ns) *p* > 0.05 by Student's *t*-test among treated bacteria compared to control; \*\*\*\* *p* < 0.0001 by Student's *t*-test among treated bacteria compared to control.

#### 3.6.3. Synergism between aPDI with PSIR-3 and Cefotaxime

Since PSIR-3 showed synergism combined with imipenem [32], we analyzed whether it shows synergism with cefotaxime in the population of clinical isolates. First, it was determined whether the combined treatment with Cfx increases the inhibition of bacterial growth of aPDI with PSIR-3. The 118 clinical isolates of *K. pneumoniae* were exposed to the preparation of 4 µg/mL of cefotaxime with 4 µg/mL of PSIR-3 (its MIC). Control bacteria without Cfx and exposure to light were included. As expected, the PSIR-3 compound mixed with cefotaxime significantly (\*\*\*\* *p* < 0.0001) increased the bactericidal effect on the clinical isolates population from 3 to 6 log<sup>10</sup> reduction (Figure 5A). As seen before, a significantly increased inhibitory effect was not observed when combining the cefotaxime with the PS-Ru control compound (ns *p* > 0.05). Secondly, the set of 66 clinical isolates characterized as ESBL-producing *K. pneumoniae* were treated with PSIR-3 aPDIfor 1 h, and serial dilutions determined the MIC for Cfx in ca-MH broth. As seen in Figure 5B, a significant reduction (\*\*\*\* *p* < 0.0001) from 8 µg/mL (8–8) to 0.17 µg/mL (0.17–0.333) on Cfx-MIC was observed compared to the untreated group. The combined treatment also reduced the PSIR-3-MIC, from 4 to 0.5 mg/L (Figure 5C). Similar to previously shown with imipenem [32], compound PSIR-3 produced a significant change in Cfx-susceptibility with a fractional inhibitory concentration (FIC) index of 0.15 (Table 4). Figure 5C shows the control compound, PS-Ru, did not significantly change Cfx-susceptibility with an FIC Index 1.58 (Table 4). Because synergy is defined as an FIC index of ≤0.5 [44], the increased inhibitory effect observed when combining PSIR-3 with Cfx is not summative but synergistic.

**Table 4.** Fractional inhibitory concentration (FIC) index calculation.


MIC values are de median for the ESBL-producing *K. pneumoniae*, n = 66. \* The MIC value modified by Cfx.

The behavior exhibited by PSIR-3 must be related, as mentioned in previous reports [32,33], to the external substituents bonded to polypyridine ligand structures and affinity to bacterial envelope [41]. This synergism is also comparable to other photosensitizers; for example, rose bengal showed an increase in the susceptibility of *Acinetobacter baumannii* for a range of antibiotics used along with aPDI [60]. In another example, conventional antibiotics and alternative compounds reported synergism in a murine model for pathogens of the ESKAPE group (*Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, and *Enterobacter* species) [61], using anti-biofilm peptides [62]. For now, it is difficult to accurately calculate the dose of light necessary to activate PSIR-3 effectively; however, we observed in vitro that with low doses of energy (17 µW/cm<sup>2</sup> of white light), it is bactericidal. Compounds with optimum absorbance at higher wavelengths, bordering the 630–750 nm, would improve the exposure of PS to light into the tissues, but being less energetic, the PSs must have a triplet excited state of easy access to promote energy transfer [63]. We, therefore, need to perform a better characterization of its antimicrobial activity when activated with defined wavelengths, before starting in vivo studies, in urinary infection models and to evaluate the need to deliver light through intraurethral catheters [64].

#### **4. Conclusions**

In this work, we saw that multi-drug resistance and virulence are significant factors in clinical isolates of *K. pneumoniae*. However, the increase in MICs can be neutralized by aPDI, turning resistant strains susceptible. APDI is effective in treating multidrug-resistant

bacteria and more virulent strains, as well as strains that combine both characteristics. The aPDI then becomes a great support to antimicrobial therapy in a shortage of new effective antibiotics. The photophysical characterization of PS indicates that its maximum absorption occurs at wavelengths lower than 400 nm, which could constitute a problem for its use in infections of internal organs due to low penetration. In UTI, optical fibers can be used through a catheter to deliver the dose of light [54,55]. Moreover, compounds with optimum absorbance at higher wavelengths, bordering the 630–750 nm, would improve the exposure of PS to light in the tissues.

**Author Contributions:** Conceptualization, C.E.P. and I.A.G.; methodology, C.E.P. and I.A.G.; software, C.E.P. and I.A.G.; validation, C.E.P., P.D. and I.A.G.; formal analysis, C.E.P., P.D. and I.A.G.; investigation, C.N. and A.P.; resources, C.E.P., P.D. and I.A.G.; data curation, C.E.P. and I.A.G.; writing—original draft preparation, C.E.P.; writing—review and editing, C.E.P.; visualization, C.E.P. and I.A.G.; supervision, C.E.P. and I.A.G.; project administration, C.E.P.; funding acquisition, C.E.P., P.D. and I.A.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are supported by UCEN grants CIP2019007 (awarded to C.E.P.), Fondecyt grants 1180673 and 1201173 (awarded to I.A.G. and P.D., respectively), Office of Naval Research (ONR) grant N62909-18-1-2180 (awarded to I.A.G.) and USM PM\_I\_2020\_31 project of P.D.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of the Faculty of Health Sciences, of the Universidad Central de Chile N: 01/2017 approved on 01-04-2017.

**Informed Consent Statement:** The informed consent form was approved by the ethics committee of the Faculty of Health Sciences of the Central University of Chile and by the Bioethics Committee of the Central Metropolitan Health Service of Chile (MHSC), Act number: N 124/07.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available because they are confidential data of patients protected by the informed consent protocol.

**Acknowledgments:** We would like to thank Myriam Pizarro, head of the "Hospital El Carmen de Maipú" laboratory, for her collaboration and provision of the clinical isolates of Klebsiella pneumoniae. We also would like to thank Susan Bueno from Pontificia Universidad Católica de Chile for providing the control strains of K. pneumoniae, KPPR1, and the typo strain ST258.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Susceptibility of Dental Caries Microcosm Biofilms to Photodynamic Therapy Mediated by Fotoenticine**

**Maíra Terra Garcia <sup>1</sup> , Rafael Araújo da Costa Ward <sup>1</sup> , Nathália Maria Ferreira Gonçalves <sup>1</sup> , Lara Luise Castro Pedroso <sup>1</sup> , José Vieira da Silva Neto <sup>2</sup> , Juliana Ferreira Strixino <sup>3</sup> and Juliana Campos Junqueira 1,\***


**Abstract:** Photodynamic therapy (PDT) mediated by Fotoenticine® (FTC), a new photosensitizer derived from chlorin e-6, has shown in vitro inhibitory activity against the cariogenic bacterium *Streptococcus mutans*. However, its antimicrobial effects must be investigated on biofilm models that represent the microbial complexity of caries. Thus, we evaluated the efficacy of FTC-mediated PDT on microcosm biofilms of dental caries. Decayed dentin samples were collected from different patients to form in vitro biofilms. Biofilms were treated with FTC associated with LED irradiation and analyzed by counting the colony forming units (log10 CFU) in selective and non-selective culture media. Furthermore, the biofilm structure and acid production by microorganisms were analyzed using microscopic and spectrophotometric analysis, respectively. The biofilms from different patients showed variations in microbial composition, being formed by streptococci, lactobacilli and yeasts. Altogether, PDT decreased up to 3.7 log10 CFU of total microorganisms, 2.8 log10 CFU of streptococci, 3.2 log10 CFU of lactobacilli and 3.2 log10 CFU of yeasts, and reached eradication of *mutans* streptococci. PDT was also capable of disaggregating the biofilms and reducing acid concentration in 1.1 to 1.9 mmol lactate/L. It was concluded that FTC was effective in PDT against the heterogeneous biofilms of dental caries.

**Keywords:** photodynamic therapy; dental caries; oral biofilms; Fotoenticine; chlorine; methylene blue

## **1. Introduction**

Dental caries is a multifactorial biofilm-mediated disease characterized by the mineral loss of tooth hard tissues [1,2]. Caries development is a result of dental biofilm acidification from carbohydrate consumption that alters the oral ecology, favoring the growth of acidogenic and acid-tolerating species [2–4]. Despite the restorative treatments available for dental caries, dentists commonly encounter failures in restorations associated with secondary caries [5]. Therefore, a proper disinfection of the dental cavity before the restorative procedure is essential to avoid the growth of bacteria under restorative material and caries recurrence [6,7]. In this context, alternative adjuvant methods to restorative treatment have been extensively investigated, such as antimicrobial photodynamic therapy (PDT).

The antimicrobial effect of PDT is based on a photophysical reaction through the association of a photosensitizer, light at an appropriate wavelength (usually from the visible to near infrared spectrum) and molecular oxygen [8–12]. In this process, the photosensitizer is activated by light to produce reactive oxygen species (ROS) that kill the microbial cells via an oxidative burst [8,13]. Therefore, the efficacy of PDT depends on the type of photosensitizer, parameters of irradiation, and application site [14]. In the last decades, different

**Citation:** Garcia, M.T.; Ward, R.A.d.C.; Gonçalves, N.M.F.; Pedroso, L.L.C.; Neto, J.V.d.S.; Strixino, J.F.; Junqueira, J.C. Susceptibility of Dental Caries Microcosm Biofilms to Photodynamic Therapy Mediated by Fotoenticine. *Pharmaceutics* **2021**, *13*, 1907. https://doi.org/10.3390/ pharmaceutics13111907

Academic Editor: Maria Nowakowska

Received: 9 October 2021 Accepted: 5 November 2021 Published: 10 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

photosensitizers have been investigated, including phenothiazines and chlorine derivatives [8,9,15,16]. Phenothiazines, such as methylene blue (MB), are widely studied and known, being already approved for clinical dental application in several countries around the world [17]. Chlorines are tetrapyrrolic substances derived from chlorophyll that have gained attention due to their photodynamic effects on a large number of pathogens [18]. Some derivatives of chlorines have been described as potential photosensitizers with high absorption in the red region of the visible spectrum and mechanism of action predominantly via type II reaction, producing singlet oxygen [19]. Among them, Fotoenticine (FTC), a new photosensitizer derived from chlorin e-6, has demonstrated strong photodynamic activity against the cariogenic bacterium *Streptococcus mutans* [9,19,20].

Recently, Terra-Garcia et al. [9] and Nie et al. [19] verified that PDT mediated by FTC was able to reduce the viability of biofilms formed by a reference *S. mutans* strain (UA159). In both studies, the antimicrobial effects of PDT with FTC were greater than MB, suggesting that FTC can be a useful photosensitizer in the control of *S. mutans*. Later, our research group reported that photodynamic inactivation by FTC was extended to several clinical strains of *S. mutans* isolated from patients with the active disease [20]. In all of these clinical strains, FTC-mediated PDT was able to eliminate the viable cells and disrupt the dense structure of *S. mutans* biofilms [20].

Although previous studies have suggested that PDT mediated by FTC can be a promising strategy to prevent or treat caries, these studies were limited to monospecies biofilms that do not represent the microbial complexity of dental biofilms. It is known that dental biofilms are composed of different microbial species that form a highly complex and organized structure on the tooth tissues, in which each microorganism has specific positions and functions. More than 700 bacterial species have been identified from dental biofilm, and approximately 40 species were already associated with caries [21]. In addition to *S. mutans*, other acidogenic and aciduric microorganisms in the biofilm may play an important role in dental caries, such as *Streptococcus sobrinus*, *Lactobacillus* spp., *Veillonella* spp., *Actinomyces* spp. and *Candida* spp. [22].

To overcome the limitations of in vitro biofilm studies with one or three selected species, many researchers have introduced different models of microcosm biofilms. Microcosms are more reliable models to simulate the in situ conditions of the oral cavity. Microcosm biofilms consist of a set of microorganisms from the natural oral microbiota that are formed in vitro using samples collected from the oral cavity, such as saliva or biofilms [23]. Thus, microcosm biofilms are capable of reproducing the biodiversity, ecological interactions, acid production and pH conditions of dental biofilms in the human oral cavity [24]. Using a microcosm model, the objective of this study was to evaluate the antimicrobial activity of PDT mediated by FTC on dental caries biofilm, investigating its effects on the microbial cells, biofilm structure and acid production.

#### **2. Materials and Methods**

#### *2.1. Collection of Dental Caries Samples*

This study was approved by the Ethics Committee for Research in Human Beings of the Institute of Science and Technology (ICT/UNESP) under protocol number 158617/2019, date of approval: 13 November 2020). Three patients of the dental clinics in the city of São José dos Campos were selected. Inclusion criteria were age between 20 and 40 years and presence of at least one dentin caries lesion in a molar tooth. The non-inclusion criteria were the use of antibiotics in the last 90 days and presence of caries lesions with pulp exposure.

The collection of samples from infected dentin was carried out after anesthesia and absolute isolation with a rubber dam. The material was collected with a sterile curette, transferred to brain heart infusion (BHI) broth with 20% glycerol and stored in a freezer at −80 ◦C. Teeth were then restored according to pre-determined treatment for each patient.

#### *2.2. Preparation of Photosensitizer and Light Source*

The photosensitizer Fotoenticine® (Nuevas Tecnologias Cientificas, NTC, Lianera Asturias, Spain) obtained at a concentration of 6.89 mg/mL was sterilized by filtration on membranes with pores of 0.22 µm (MFS, Dublin, Ireland) and stored in dark conditions. The chemical structure and absorption spectrum of the Fotoenticine® are showed in Figure 1. The light source was a LED at 660 nm (IrradLED®, Biopdi, São Carlos, Brazil) with a power density of 42.8 mW/cm<sup>2</sup> , energy density of 30 J/cm<sup>2</sup> and exposure time of 700 s.

**Figure 1.** Chemical structure [25] and absorption spectrum of Fotoenticine (200 µg/mL in 0.9% NaCl) determined with a spectrophotometer (DS-11, Denovix, DE, USA).

#### *2.3. Preparation of Bovine Tooth Specimens*

Bovine teeth were employed as substrate to form in vitro biofilms following the methodology described by Garcia et al. [9] with some modifications. Mandibular incisor teeth were used to prepare specimens measuring 4 mm in diameter and 2 mm in thickness. In summary, the crowns were separated from the roots using a straight handpiece with a diamond disc. The crowns were attached to a circular sample cutter with the buccal side facing upwards and cut with a trephine drill. Then, specimens were inserted into a metal matrix (with the dentin surface positioned in its interior) and polished with sandpaper discs of decreasing grain size in a low-speed water-cooling system (DP-10, Panambra Industrial e Técnica, São Paulo, Brazil) (Figure 2). The final thickness was confirmed with a digital caliper (Starrett, São Paulo, Brazil). Finally, the specimens were stored in a 0.1% (*v*/*v*) thymol solution at 4 ◦C.

**Figure 2.** (**A**) Representation of sample collection from infected dentin of patient. (**B**) Sample transferred to brain heart infusion (BHI) broth with 20% glycerol. (**C**) Separation of crown from the root of bovine tooth using a straight handpiece with a diamond disc. (**D**) Crown attached to a circular sample cutter with the buccal side facing upwards. (**E**) Tooth cut with a trephine drill. (**F**) Specimen inserted into a metal matrix for polishing. (**G**) Bovine specimen prepared. (**H**) Specimen positioned in the metal wire and submerged in BHI broth to form the biofilms. (**I**) Biofilm formed on bovine specimen. (**J**) Irradiation of biofilms with LED at 660 nm. (**K**) Colonies formed on BHI agar. (**L**) Scanning electron microscope. (**M**) Lactic dehydrogenase LDH UV K014 (Bioclin) to determine the acid production.

#### *2.4. Formation of Microcosm Biofilms on Bovine Tooth Specimens*

Microcosm biofilms were formed according to the methodology of Méndez et al. with some modifications [24]. Dental caries samples stored in freezer were thawed and homogenized. Then, 400 µL were transferred to 20 mL of BHI broth supplemented with 5% sucrose and incubated at 37 ◦C in 5% CO<sup>2</sup> for 48 h. The growth was centrifuged and washed twice in phosphate buffered saline (PBS). The pellet was resuspended in 20 mL of PBS. An inoculum of 225 µL of this microbial suspension was added in each well of 24-well microplates, containing the bovine tooth specimen suspended by a metal wire and submerged in 2 mL of BHI broth with 5% sucrose. The plates were incubated at 37 ◦C for 120 h to form the biofilms, under 5% CO<sup>2</sup> pressure since most cariogenic bacteria are capnophilic [26]. Every 24 h, the wells were washed (2×) with PBS, and 2 mL of a fresh BHI broth with 5% sucrose was replaced.

#### *2.5. Application of Photodynamic Therapy on Microcosm Biofilms*

After biofilm formation, the specimens were removed from the metal wire support, transferred to a new well and submerged in 200 µL of photosensitizer or PBS. To obtain an antimicrobial effect on biofilms, the photosensitizer was used at a concentration of 0.6 mg/mL that is considered able to diffuse through *S. mutans* biofilms [20]. The plates were shaken for 15 min (pre-irradiation time) in dark conditions. After that, the plates were taken to the IrradLED® and irradiated according to the parameters described above. The control groups not irradiated remained in dark conditions for the same period. According to these procedures, four groups of microcosm biofilms were established: treated with FTC and irradiated (FTC + L+), treated with PBS and irradiated (P − L+), treated with FTC in the dark (FTC + L−) and treated with PBS in the dark (P − L−).

#### *2.6. Analysis of Biofilms by Counting the Number of Viable Cells*

The specimens containing the treated biofilms (five per group) were transferred to Falcon tubes containing 4 mL of PBS. The adhered biofilms were detached using an ultrasonic homogenizer (Sonopuls HD2200, Bandelin Eletronic, Berlin, Germany) with a power of 7 W for 30 s. Serial dilutions of the biofilm suspension were performed and plated on BHI agar for counting the total number of microorganisms, as well as on the following selective culture media: Mitis Salivarius for counting streptococci, Mitis Salivarius Bacitracin Sucrose (MSBS) agar for mutans streptococci, Rogosa agar for lactobacilli, and Sabouraud Dextrose agar with chloramphenicol for yeasts. Next, the plates were incubated at 37 ◦C for 48 h to determine the number of colony forming units (CFU).

#### *2.7. Analysis of Biofilms by Scanning Electron Microscopy (SEM)*

After the treatments, the specimens with biofilms (two per group) were submerged in 1 mL of 2.5% glutaraldehyde (1 h) for cell fixation. After that, they were dehydrated with 1 mL of increasing ethanol concentrations (10, 25, 50, 75 and 90%), remaining for 20 min at each concentration. Finally, the biofilms were added to absolute ethanol for 1 h. The dehydrated biofilms were incubated at 37 ◦C for 24 h, and then placed in aluminum stubs and covered with gold for 160 s at 40 mA (Denton Vacuum Desk II, Denton Vacuum, Moorestown, NJ, USA). After metallization, the biofilms were analyzed in the scanning electron microscope (JEOL JSM-7900, JEOL, Peabody, MA, USA).

#### *2.8. Determination of Lactic Acid Production by the Microbial Cells in Biofilms*

The specimens with the treated biofilms (5 per group) were transferred to Falcon tubes containing 5 mL of PBS and incubated at 37 ◦C for 3 h. Then, 1 mL of each tube was placed in a freezer at −80 ◦C for 5 min to stop the acid production. To verify the concentrations of lactate, the enzymatic method of lactic dehydrogenase was used according to the manufacturer's instructions (Lactic dehydrogenase LDH UV K014, Bioclin, Belo Horizonte, Brazil). Absorbance was measured at 340 nm using a microplate spectrophotometer (Epoch, Biotek, Winooski, VT, USA), and the obtained values were expressed as mmol lactate/L.

#### *2.9. Statistical Analysis*

The results obtained in the viable cell counts (log10 CFU) and acid production were analyzed by ANOVA and Tukey test using the GraphPad Prism 8.4.3 program (GraphPad Software, Inc., San Diego, CA, USA). For statistical analysis, five biofilms per group were used. The assays were repeated twice. Level of significance of 5% was adopted in all the analyses.

#### **3. Results**

#### *3.1. Effects of FTC-Mediated PDT on Viable Cells of Microcosm Biofilms*

In the viable cell counts of the non-treated biofilms (control group P − L−), the microbial growth in non-selective culture media (BHI) was 7.21 log10 CFU for patient 1, 8.09 log10 CFU for patient 2 and 10.31 log10 CFU for patient 3. These data showed that the microcosm biofilm of patient 3 had the highest quantity of microorganisms, followed by patient 2 and 1. In the selective culture media, all patients showed cariogenic biofilms formed by streptococci (5 to 8 log10 CFU), mutans streptococci (5 to 7 log10 CFU) and lactobacilli (7 to 8 log10 CFU). However, the presence of yeasts was found only in microcosm biofilms of patients 2 and 3 (9 log10 CFU) (Figure 3).

**Figure 3.** Viable cell counts (log10 CFU) of microcosm biofilms from patients 1, 2 and 3 according to the following culture mediums: brain heart infusion (total number of microorganisms), Mitis Salivarius (streptococci), Mitis Salivarius Bacitracin Sucrose (mutans streptococci), Rogosa (lactobacilli), and Sabouraud Dextrose with chloramphenicol (yeasts).

In relation to the viable cell counts of the biofilms treated with PDT (FTC + L+), we observed statistically significant microbial reductions relative to the groups P − L− and P − L+. These microbial reductions occurred in both selective and non−selective culture media for all the patients studied. In the non-selective medium, the FTC + L+ caused reductions from 1.8 to 3.7 log10 CFU for the total microorganisms compared to the P − L− group. Significant microbial reductions were also found in the FTC + L+ group in all the selective culture media, indicating that the FTC-mediated PDT was effective in inhibiting the growth of different microorganisms associated with cariogenic biofilms, including streptococci, mutans streptococci, lactobacilli and yeasts. In addition, the group treated with FTC alone (FTC + L−) showed a reduction from 0.2 to 1.2 log10 CFU in comparison to the P − L− group, suggesting a slightly toxic effect of FTC in dark conditions (Figure 4).

Considering the biofilms of all patients, the mean of microbial reductions achieved by FTC-mediated PDT (in comparison to the P − L− group) ranged from 2.1 to 2.8 log10 CFU for streptococci, 1.8 to 3.2 log10 CFU for lactobacilli and 1.7 to 3.2 log10 CFU for yeasts. Interestingly, mutans streptococci had a greater susceptibility to PDT relative to the other microorganisms. FTC-mediated PDT caused a mutans streptococci reduction of 6.02 log10 CFU for the biofilm of patient 3 and a total eradication for patients 1 and 2 (Figure 5).

#### *3.2. Effects of FTC-Mediated PDT on Microcosm Biofilm Structures*

The SEM images confirmed that all the caries samples collected from patients were able to provide an in vitro formation of microcosm biofilms on the surface of bovine dentin specimens. However, the morphology and structure of the biofilms varied according to the experimental groups. In all the patient samples, the P − L− and P − L+ groups presented a dense and compact biofilm, forming a complex three-dimensional structure. These biofilms were composed of a large quantity of microbial aggregates embedded in an extracellular matrix, covering the entire dentin surface. Different morphologies of microorganisms were found, including coccus, coccobacilli, filamentous bacilli, fusiform bacilli and fungal hyphae, which represented the microbial diversity of cariogenic biofilms.

The FTC + L− group also showed a dense biofilm formed by a large number of microorganisms; however, the biofilm density was lower than that in the P − L− and P − L+ groups. When the microcosm biofilms were treated with PDT (FTC + L+ group), a substantial reduction in the amount of microorganisms adhered to tooth surface was clearly observed. The PDT was capable of disaggregating the biofilms, exposing the dentin surface formed by several dentinal tubules. The microorganisms on dentin surface exhibited

few cellular aggregates or isolated cells, allowing a more precise observation of microbial morphologies (Figure 6).

**Figure 4.** Means of log10 CFU and SD of microcosm biofilms (patients 1, 2 and 3) obtained in the following groups: treated with PBS in the dark (P − L−), treated with PBS and irradiated (P − L+), treated with FTC in the dark (FTC + L−) and treated with FTC and irradiated (FTC + L+). (**A**) Total microorganisms in brain heart infusion agar. (**B**) Streptococci in Mitis Salivarius agar. (**C**) Mutans streptococci in Mitis Salivarius Bacitracin Sucrose (MSBS) agar. (**D**) Lactobacilli in Rogosa agar. (**E**) Yeasts in Sabouraud Dextrose agar with chloramphenicol.

**Figure 5.** Microbial reductions of streptococci, mutans streptococci, lactobacilli and yeasts expressed in log10 CFU obtained in the group treated with FTC and irradiated (FTC + L+) in comparison to the control group treated with PBS in the dark (P − L−). (**A**) Microcosm biofilm from patient 1. (**B**) Microcosm biofilm from patient 2. (**C**) Microcosm biofilm from patient 3.

**Figure 6.** Scanning electron microscopy images according to the groups: treated with PBS in the dark (P − L−), treated with PBS and irradiated (P − L+), treated with FTC in the dark (FTC + L−) and treated with FTC and irradiated (FTC + L+). (**A**–**D**) Microcosm biofilm from patient 1. (**E**–**H**) Microcosm biofilm from patient 2. (**I**–**L**) Microcosm biofilm from patient 3.

## *3.3. Influence of FTC-Mediated PDT on Acid Production by the Microbial Cells of Microcosm Biofilms*

In the biofilms not treated with PDT (P − L−, P − L+ and FTC + L− groups), the results of acid production ranged from 1.95 to 1.96 mmol lactate/L for patient 1, from 2.79 to 2.82 mmol lactate/L for patient 2, and from 3.80 to 3.84 mmol lactate/L for patient 3. As with the results obtained in the viable cell counts, patient 3 showed the highest quantity of acid production, followed by patient 2 and 1. These results indicated that the acidogenic capacity of caries microcosm biofilms was related to the number of microbial cells.

The biofilms treated with PDT (FTC + L+ group) showed statistically significant reductions of acid production when compared to the P − L− group for all patients studied. The acid reductions caused by PDT were 1.28, 1.92 and 0.99 mmol lactate/L, respectively, for patients 1, 2 and 3 (Figure 7). In general, it was possible to observe that PDT mediated by FTC was able to kill the microbial cells with consequent disruption of biofilm structure and decreased acidogenicity.

**Figure 7.** Means and SD of lactic acid concentration (mmol lactate/L) for the groups: treated with PBS in the dark (P − L−), treated with PBS and irradiated (P − L+), treated with FTC in the dark (FTC + L−) and treated with FTC and irradiated (FTC + L+). (**A**) Microcosm biofilm from patient 1. (**B**) Microcosm biofilm from patient 2. (**C**) Microcosm biofilm from patient 3.

#### **4. Discussion**

Cariogenic biofilms are active and complex ecosystems rich in organic acid [27], which form a spatial and ordered organization of microbial communities on tooth surfaces [28]. The dental surfaces and the development of caries lesions provide different microenvironments that allow the colonization of adapted microbial communities, leading to variations

in the bacterial amount and microbial composition of cariogenic biofilms according to the caries stage [29,30]. In addition, several individual factors can influence the biofilm diversity, such as the host's diet, oral hygiene habits, tooth morphology, saliva composition, and discrepancies in the immune system [27,31]. Considering the variations in cariogenic biofilms among different individuals, in this study we collected samples from three patients with dentin caries to form in vitro microcosm biofilms and to investigate the potential of FTC-mediated PDT for the control of dental caries.

The microcosm biofilm obtained from patient 3 (10.31 log10 CFU) showed the highest total microbial count, followed by patient 2 (8.09 log10 CFU) and 1 (7.21 log10 CFU). In the three patients analyzed, the results were superior to those found by Mendéz et al. [24], who obtained a total microbial count of approximately 6.5 log10 CFU in the microcosm biofilms from dentin caries of children. Possibly, these differences can be attributed to the age of patients because in our study, only adults were included. However, Rudney et al. [32] did not observe differences in the quantity of microorganisms between adult and pediatric patients when microcosm biofilms were formed from saliva or dental plaque. The total microbial count ranged from 8 to 10 log10 CFU, independently of the inoculum source. Although the quantity of microorganisms formed in microcosm biofilms can vary depending on the study in the literature, it is evident that the microcosm model is able to provide very dense biofilms that can be useful for a large number of in vitro studies.

In this study, the microcosm biofilms from all patients were composed of streptococci, mutans streptococci and lactobacilli. In addition, the microcosm biofilms of patients 2 and 3 presented yeasts as well. Indeed, caries is closely associated with high counts of streptococci (particularly *Streptococcus mutans*), lactobacilli and yeasts of the genus *Candida* [2,33]. *Streptococcus* spp. and *Lactobacillus* spp. exhibit special properties that make them cariogenic bacteria, including the ability to adhere to dental surfaces, to produce a high quantity of acids from fermentable sugars, and to survive in an acid environment [34]. *Candida* species have been frequently isolated from dental caries lesions and also display acidogenic and aciduric properties. The presence of *Candida albicans* in cariogenic biofilms seems to enhance the virulence of *S. mutans* and, consequently, the severity of dental caries [35,36]. Many other bacterial genera have also been associated with cariogenic biofilms, such as *Actionomyces*, *Bifidobacteria*, *Veilonella*, *Prevotella* [2,34], and more recently, *Scardovia* [2]. However, the present study was limited to the identification of streptococci, mutans streptococci, lactobacilli and yeasts in the microcosm biofilms. Therefore, additional studies with a focus on other microbial groups are required, especially those that use molecular tools.

The viable cell counts in the microcosm biofilms of patients ranged from 5 to 8 log10 CFU for streptococci, 5 to 7 log10 CFU for mutans streptococci, 7 to 8 log10 CFU for lactobacilli and 0 to 9 log10 CFU for yeasts. Similar results were found by Vertuan et al. [37], Câmara et al. [38], and Mendéz et al. [24], who obtained bacterial counts between 6 to 7 log10 CFU for streptococci, 5 to 7 log10 CFU for mutans streptococci and 6 to 7 log10 CFU for lactobacilli in microcosm biofilms from oral samples. Nonetheless, these authors did not investigate the presence of yeasts in the microcosm biofilms formed in their studies.

When the FTC-mediated PDT was applied on microcosm biofilms, the viable cell numbers of all microbial groups studied were significantly decreased. In general, the microcosm biofilms from patients showed reductions between 2.1 to 2.8 log10 CFU for streptococci, 6.0 log10 CFU to total inhibition for mutans streptococci, 1.8 to 3.2 log10 CFU for lactobacilli and 1.7 to 3.2 log10 CFU for yeasts after the PDT treatment. Interestingly, the antimicrobial effects of FTC observed in our study were more pronounced than other photosensitizers cited in the literature. Using microcosm biofilms from dentin caries of patients, some authors found microbial reductions of 1.5, 1.3 and 1.9 log10 CFU with the photosensitizer methylene blue [24] and 1.5, 1.7 and 2.3 log10 CFU when curcumin was used as photosensitizer [39]. Probably, the greater photodynamic activity of FTC can be related to the positive charges of chlorins, action predominantly via type II mechanism

with singlet oxygen quantum yields from 0.5 to 0.8 [8], and high capacity to penetrate into the bacterial cells [13,19].

To our knowledge, this is the first study to investigate the effects of FTC-mediated PDT on microcosm biofilms. Until now, this new photosensitizer has been studied in single biofilms formed by *S. mutans*. Terra-Garcia [20] applied the FTC-mediated PDT on single biofilms of *S. mutans* formed by the reference strain UA159 or clinical strains isolated from dental caries, verifying microbial reductions between 5 to 6 log10 CFU for the clinical strains and total inhibition for the reference strain. Using a similar photosensitizer also derived from chlorin-e6, Nie et al. [19] found a bacterial reduction of 5 to 6 log10 CFU of *S. mutans* (UA159) in single biofilms treated with PDT. Promisingly, the results of the present study using microcosm models showed that *S. mutans* remained susceptible to PDT mediated by FTC even when mixed in a complex polymicrobial biofilm.

In addition to remaining susceptible to FTC-mediated PDT in microcosm biofilms, mutans streptococci were the microorganisms most susceptible to this therapy, being totally eradicated in the microcosm biofilms from patients 1 and 2. In relation to the biofilm of patient 3, mutans streptococci were also more susceptible to PDT than lactobacilli and yeasts. We hypothesized that the highest susceptibility of *S. mutans* to PDT mediated by FTC could be associated with the growth rate, microbial cell composition, or spatial organization of biofilms. The growth rates and generation times of *S. mutans* cells were more elevated than those of *Lactobacillus* cells [40], which could influence the structure and permeability of cell membrane. The cell composition of bacteria and yeast is also considered an important factor in the susceptibility to PDT. Due to the presence of a cell wall with porous layer of peptidoglycan, Gram-positive bacteria such as *S. mutans* are more susceptible to PDT than fungal cells such as *Candida* spp. that present a cell wall of chitin with a less porous layer of beta-glucan [41]. In addition, the spatial organization of *S. mutans* cells with other microorganisms in polymicrobial biofilms can affect their susceptibility to antimicrobial treatments [42]. Using dual-species model biofilms, previous studies showed that *S. mutans* use *Candida* cells as support for their adherence [43,44]. According to these authors, *S. mutans* cells have affinity for the *C. albicans* hyphae, appearing positioned over the hyphal surface in SEM images [43,44]. This fact may leave *S. mutans* more exposed to PDT action. However, future studies are required to unveil the spatial structure of cariogenic biofilms and to develop therapies targeting the biogeography of polymicrobial infections [28].

The SEM analysis performed in the present study confirmed the capacity of microcosm biofilms in reproducing the dental caries microbiome. The biofilms of non-treated groups showed a spatial structure formed by heterogeneous and complex communities embedded in an extracellular matrix. The FTC-mediated PDT was able to decrease the number of microbial cells and to disaggregate the biofilm structure, exposing the dentin surface. In a review article, Hu et al. [13] reported that PDT not only kills the bacterial and fungal cells present in biofilms, but the ROS generated can concomitantly degrade the matrix structure by attacking several biomolecules. During the PDT reaction, the burst of ROS causes oxidative damage in multiple non-specific targets, such as amino acids, nucleic acid bases, lipids, etc. Therefore, PDT can disaggregate the biofilms by a synergistic action on microbial cells and extracellular matrix. These factors make PDT an attractive approach for chronic biofilm infections [13].

Since the production of organic acids by microbial cells in biofilms is a determinant of the development of dental caries [34], in this study we also evaluated the influence of PDT on the acidogenicity of biofilms. The PDT mediated by FTC caused a substantial reduction in lactic acid production for the three biofilms analyzed in this study, probably as a consequence of the decrease in the number of acidogenic bacteria. On the other hand, Mendez et al. [24,39] did not observe reductions in lactic acid production when the dentin microcosm biofilms were treated with PDT mediated by methylene blue [24] or curcumin [39]. These authors did not find correlations between the reductions in CFU counts and lactic acid production, raising the hypothesis that the surviving cells could have increased their acid production capacity, which resulted in maintaining the acidogenicity of the biofilms. However, we think that the bacterial reductions caused by PDT with MB [24] or curcumin [39] may not have been enough to result in a decrease in acid production because the CFU reductions were lower than the microbial reductions found in our study using FTC.

In summary, we concluded that PDT mediated by FTC, a photosensitizer derived from chlorin e-6, has antimicrobial activity against dental caries microcosm biofilms. FTCmediated PDT led to significant reductions in total microorganisms, streptococci, mutans group streptococci, lactobacilli and yeasts. Mutans group streptococci were the microorganisms most susceptible to PDT, showing total eradication after this therapy. In addition, PDT with FTC was able to disaggregate the biofilm structure and to reduce the lactic acid concentration. Promisingly, FTC can be an attractive photosensitizer for PDT targeted to the control of cariogenic biofilms and dental caries.

**Author Contributions:** J.C.J. and J.F.S. conceived and designed the experiments. M.T.G., R.A.d.C.W., N.M.F.G. and L.L.C.P. performed the experiments. J.V.d.S.N. contributed to SEM analysis. M.T.G. and J.C.J. analyzed the data. M.T.G., J.V.d.S.N. and J.C.J. wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** National Council for Scientific Development (CNPq): Researcher on Productivity PQ-1C (306330/2018-0) and research grant (408369/2018-3) for JCJ. Coordination for the Improvement of Higher Education Personnel (CAPES): scholarship for MTG. São Paulo Research Foundation (FAPESP): scholarships for RACW (2020/08345-0) and NMFG (2020/06909-4).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee for Research in Human Beings of the Institute of Science and Technology (ICT/UNESP) (protocol code: 158617/2019, date of approval: 11/13/2020).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Communication* **Fluorescence Lifetime Imaging Microscopy of Porphyrins in** *Helicobacter pylori* **Biofilms**

**Antonella Battisti <sup>1</sup> , Paola Morici 1,2 and Antonella Sgarbossa 1,\***


**Abstract:** Bacterial biofilm constitutes a strong barrier against the penetration of drugs and against the action of the host immune system causing persistent infections hardly treatable by antibiotic therapy. *Helicobacter pylori* (Hp), the main causative agent for gastritis, peptic ulcer and gastric adenocarcinoma, can form a biofilm composed by an exopolysaccharide matrix layer covering the gastric surface where the bacterial cells become resistant and tolerant to the commonly used antibiotics clarithromycin, amoxicillin and metronidazole. Antimicrobial PhotoDynamic Therapy (aPDT) was proposed as an alternative treatment strategy for eradicating bacterial infections, particularly effective for Hp since this microorganism produces and stores up photosensitizing porphyrins. The knowledge of the photophysical characteristics of Hp porphyrins in their physiological biofilm microenvironment is crucial to implement and optimize the photodynamic treatment. Fluorescence lifetime imaging microscopy (FLIM) of intrinsic bacterial porphyrins was performed and data were analyzed by the 'fit-free' phasor approach in order to map the distribution of the different fluorescent species within Hp biofilm. Porphyrins inside bacteria were easily distinguished from those dispersed in the matrix suggesting FLIM-phasor technique as a sensitive and rapid tool to monitor the photosensitizer distribution inside bacterial biofilms and to better orientate the phototherapeutic strategy.

**Keywords:** porphyrins; fluorescence lifetime imaging miscroscopy (FLIM); antimicrobial photodynamic therapy (aPDT); *Helicobacter pylori* biofilm

### **1. Introduction**

Helicobacter pylori (Hp) can chronically colonize the human stomach where it plays an essential role in the development of gastritis, gastroduodenal ulcers and gastric cancer [1]. A key contribution to the persistence and the recurrence of Hp infections is provided by the bacterial ability to efficiently form a biofilm. Biofilms are matrix-enclosed bacterial populations in close proximity to each other and adherent to surfaces or interfaces. Hp biofilms are characterized by an extensive 3D network of highly hydrated exopolysaccharides where extracellular DNA, extracellular proteins, and outer membrane vesicles are embedded [2]. This extracellular matrix (ECM) protects the bacterial community from immune system cells and antimicrobial drugs. As a matter of fact, conventional pharmacological therapies have become less effective and new alternative strategies to fight bacterial infections are emerging. In this regard, one of the most promising approaches to overcome the antibiotic resistance problems is the photodynamic therapy (PDT). PDT combines the use of light irradiation with the presence of a photosensitizer molecule, able to generate cytotoxic reactive oxygen species (ROS) upon illumination. When this strategy is directed against microorganisms, the process is called antimicrobial photodynamic therapy (aPDT). The in situ-generated ROS are able to destroy biomacromolecules thus causing cell death. Typically, a photosensitizing drug is delivered to the area of interest before the treatment, but PDT can be particularly effective when the target microorganism naturally produces endogenous photosensitizers such as in the case of Hp porphyrins [3]. In previous studies

**Citation:** Battisti, A.; Morici, P.; Sgarbossa, A. Fluorescence Lifetime Imaging Microscopy of Porphyrins in *Helicobacter pylori* Biofilms. *Pharmaceutics* **2021**, *13*, 1674. https://doi.org/10.3390/ pharmaceutics13101674

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 3 September 2021 Accepted: 11 October 2021 Published: 13 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

we spectroscopically characterized the composition of Hp porphyrin extracts, showing that protoporphyrin IX (PPIX) and coproporphyrin I (CPI) are two of the main endogenous photosensitizing species [4,5]. Thanks to their presence, visible light irradiation conveyed by an intragastric fiberoptic laser system or by a LED-based illuminator has been shown to efficiently cause Hp photokilling without the administration of exogenous photosensitizers [6–8]. The correlation between photophysical properties and photosensitizing ability of porphyrins makes it mandatory to carefully analyze their fluorescence properties when they are closely associated with the biological target [9,10]. It has long been known that the vast majority of bacterial pathogens produce fluorescing porphyrins emitting in the red spectral region when excited by 405 nm violet light [11]. Thus, the assessment of red fluorescence in biological samples can be a signal of bacterial infection. Fluorescence imaging has recently emerged as a rapid and non-invasive technique for real-time visualization of the occurrence of bacteria and of their spatial distribution in several biological tissues and matrixes. The detection of the presence, location, and extent of fluorescent bacteria in wounds, for example, can be exploited by clinicians for targeted sampling during biopsies or to selectively treat the infected area [12,13]. Among the several fluorescence-based techniques, FLIM brings about several advantages because, in contrast to fluorescence intensity, the fluorescence decay time is independent of the local concentration of fluorophores and of the experimental set up. The fluorescence lifetime is a specific molecular feature of the fluorophore that can be sensitive to its surroundings. Fluorophores exhibiting the same spectral distribution in emission and in excitation as well may have different lifetimes, indicative of different molecular species or different conformations of the same molecule [14]. The analysis of FLIM data can be simplified by the phasor approach, a frequency domain technique that allows the transformation of the signal from every pixel in the image to a point in the phasor plot [15]. The FLIM-phasor approach has been used to create a metabolic fingerprint of individual bacteria and pop-ulations by monitoring the lifetime of the autofluorescent molecule reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H). While the fluorescence spectra of free and bound NAD(P)H are very similar, their lifetimes differ significantly and depend on the metabolic state of bacteria. Phasor fingerprints were generated for *Lactobacillus acidophilus* [16], *Escherichia coli*, *Salmonella enterica* serovar Typhimurium, *Pseudomonas aeruginosa*, *Bacillus subtilis*, and *Staphylococcus epidermidis* [17] in different metabolic state during the growth phase or under antibiotic stress as well.

In this work, the phasor analysis coupled to FLIM was applied to Hp biofilms in order to map the distribution of the bacterial porphyrins in different microenvironments. Our findings show that it is possible to discriminate and image different average lifetimes for porphyrins corresponding to different molecular states inside bacteria or dispersed in the ECM. Therefore, this method can be used to provide a real-time assessment and a rapid visualization of the photosensitizer distribution along with its degree of molecular packing not only in Hp biofilms but also in all the situations where it is important to know the uptake, localization pattern and interactions of the exogenously added dye to optimize the PDT.

#### **2. Materials and Methods**

#### *2.1. Bacterial Strains and Culture Conditions*

Bacterial strains Hp ATCC® 43504™ (laboratory-adapted) and Hp ATCC® 700824™ (J99, virulent, cagA+ and vacA+), supplied by LGC Standards S.r.l. (Milan, Italy), were selected for this study. Both strains were kept at −80 ◦C in Brucella Broth (BB, Thermo Fisher Scientific Remel Products, Lenexa, KS, USA) supplemented with 20% (*v*/*v*) glycerol and 10% (*v*/*v*) heat-inactivated fetal bovine serum (FBS, Gibco, Life Technologies, Carlsbad, CA, USA). Thawed bacteria were grown in BB supplemented with 10% (*v*/*v*) FBS and incubated in the dark at 37 ◦C in a microaerophilic atmosphere (CampyGen Compact, Oxoid Hampshire, UK) under shaking (170 rpm).

#### *2.2. Biofilm Formation Assay*

Hp strains from frozen stocks were cultured on Brucella agar plates supplemented with 7% laked horse blood (Oxoid, Hampshire, UK) and incubated for 3 days at 37 ◦C under microaerophilic conditions in accordance with previously published protocols [18]. A sterile cotton swab was used to harvest bacteria after incubation. Bacteria were then suspended in brain heart infusion (BHI) broth (Oxoid, Hampshire, UK) additioned with 0.5% β-cyclodextrin (Sigma-Aldrich, St. Louis, MO, USA) and 0.4% yeast extract (Oxoid, Hampshire, UK). Aliquots of 2 mL of this suspension, adjusted to 5 <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL concentration, were inoculated in the wells of 12-well culture plates (BD Falcon, Franklin Lakes, NJ, USA), where sterilized round glass coverslips were placed vertically in order to promote the adhesion of Hp cells at the air-liquid interface. BHI broth (without bacteria) was the negative control. Plates were incubated for 4 days at 37 ◦C under microaerophilic conditions. Biofilms grown on the glass coverslips were washed twice with phosphatebuffered saline (PBS, Gibco, Life Technologies, Carlsbad, CA, USA) to remove excess bacterial cells and immediately set for imaging at room temperature.

#### *2.3. Extraction of Porphyrins from Hp Strains*

According to a previously published protocol [19] with minor modifications, Hp cells of different ages (1–4 days) were centrifugated at 4 ◦C (7000× *g* for 10 min), washed in 20 mL pre-cooled buffer (0.05 M Tris, 2 mM EDTA, pH 8.2) and resuspended in 10 mL of the same buffer. An aliquot of 1.5 mL of a mixture of ethyl acetate and acetic acid (3:1, *v*/*v*) was added to the suspension and bacterial cells were lysed by sonication in ice (6 cycles as in the following: 30 s, stop, 60 s). Bacterial debris was removed by centrifugation at 4 ◦C (7000× *g* for 10 min), and the organic layer was washed twice with distilled water. Por-phyrins were extracted from the organic phase by adding 150 µL of HCl 3M. After fast vortexing, this solution was centrifuged for 5 min at 7000× *g* and then the bottom aqueous layer was collected for chromatographic analyses.

## *2.4. High Performance Liquid Chromatography*

The HPLC used for analyses was a Dionex Ultimate 3000 HPLC (ThermoFisher Scientific Inc., Waltham, MA, USA), equipped with a fraction collector, PDA detector and a (3 × 150 mm) Kinetex PFP column (Phenomenex Inc., Torrance, CA, USA) using water/formic acid 100/0.1 *v*/*v* (A) and acetonitrile/formic acid 100/0.1 *v*/*v* (B) as mobile phases, with a flow of 0.8 mL/min. Runs were performed in four steps as follows: 2 min at 25% B, 26 min with a linear gradient up to 95% B, 4 min purge step at 95% B and 8 min re-equilibration step.

### *2.5. Fluorescence Lifetime Imaging (FLIM)*

Standard porphyrins PPIX and CPI were either analyzed in methanol solution (about <sup>1</sup> <sup>×</sup> <sup>10</sup>−<sup>5</sup> M) or crystallized from solution on a glass coverslip by slow evaporation of the solvent. The measurements were performed using a Leica TCS SP5 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany). An external pulsed diode laser provided excitation at 405 nm and 640 nm, while a TCSPC acquisition card (PicoHarp 300, PicoQuant, Berlin, Germany) connected to internal spectral photomultipliers allowed for detection. On the basis on the lifetime values, laser repetition rate was fixed at 20 or 40 Hz. Image size was set to 256 × 256 pixels and scan speed was modulated between 200 and 400 Hz (lines per second). The detection wavelength range was set between 580 and 720 nm for λex= 405 nm and between 650 and 750 nm for λex= 640 nm thanks to the built-in AOBS detection system. About 200–300 photons per pixel were collected for each measurement, at a photon counting rate of 100–200 kHz. Data collected from different ROIs in the biofilms images were averaged. Globals for Images—SimFCS v.2 (Globals Software by LFD-UCI, Irvine, CA, USA, available at www.lfd.uci.edu, accessed on 1 September 2021) was used to calculate phasors from the FLIM images.

#### **3. Results** *3.1. Chromatographic Analysis of Hp Porphyrin Production*

**3. Results** 

ors from the FLIM images.

#### *3.1. Chromatographic Analysis of Hp Porphyrin Production* Bacterial extracts were obtained from Hp cultures of different ages (1–4 days) by

*Pharmaceutics* **2021**, *13*, 1674 4 of 7

Bacterial extracts were obtained from Hp cultures of different ages (1–4 days) by means of a specific protocol and characterized by HPLC analyses as described in Materials and Methods. The main peaks in the chromatograms were attributed to fluorescent porphyrins and flavins produced by bacteria, according to previously published results [5,6]. HPLC allowed for a comparison between the concentrations of the fluorescent species at different ages of the bacterial cultures in order to follow the production of these molecules with time (Figure 1A). As shown by the increasing height of the peaks, the fluorophores accumulate during the observed time span, suggesting a continuous production and accumulation. To visualize changes in the global fluorophores concentration, the main known peaks in the chromatograms (7.0–8.5 min and 15.7–16.7 min) were integrated and the total calculated area was reported in Figure 1B as a function of culture age. After a 2-day lag phase, where no significant change can be noticed, the global concentration of fluorophores starts to grow reaching almost a tenfold increase after four days. means of a specific protocol and characterized by HPLC analyses as described in Materials and Methods. The main peaks in the chromatograms were attributed to fluorescent porphyrins and flavins produced by bacteria, according to previously published results [5,6]. HPLC allowed for a comparison between the concentrations of the fluorescent species at different ages of the bacterial cultures in order to follow the production of these molecules with time (Figure 1A). As shown by the increasing height of the peaks, the fluorophores accumulate during the observed time span, suggesting a continuous production and accumulation. To visualize changes in the global fluorophores concentration, the main known peaks in the chromatograms (7.0–8.5 min and 15.7–16.7 min) were integrated and the total calculated area was reported in Figure 1B as a function of culture age. After a 2 day lag phase, where no significant change can be noticed, the global concentration of fluorophores starts to grow reaching almost a tenfold increase after four days.

the biofilms images were averaged. Globals for Images – SimFCS v.2 (Globals Software by LFD-UCI, Irvine, CA, USA, available at www.lfd.uci.edu) was used to calculate phas-

**Figure 1.** (**A**) Chromatograms of the extracts after 1 up to 4 days. Insets: expansion of peaks between 7.0 and 8.5 min and between 15.7 and 16.7 min (rescaled for better visualization). (**B**) Total area under the main peaks in the chromatograms (7.0–8.5 min and 15.7–16.7 min), corresponding to the global concentration of fluorescent species in the extracts. **Figure 1.** (**A**) Chromatograms of the extracts after 1 up to 4 days. Insets: expansion of peaks between 7.0 and 8.5 min and between 15.7 and 16.7 min (rescaled for better visualization). (**B**) Total area under the main peaks in the chromatograms (7.0–8.5 min and 15.7–16.7 min), corresponding to the global concentration of fluorescent species in the extracts.

#### *3.2. FLIM-phasor Analysis of Porphyrins in Hp Biofilms 3.2. FLIM-Phasor Analysis of Porphyrins in Hp Biofilms*

Fluorescence microscopy of Hp biofilms revealed that bacterial porphyrins are present both inside bacterial cells and in the ECM [4], as they confer red fluorescence to the whole system. Biofilms obtained from Hp after four days of incubation were then observed by confocal laser scanning microscopy and analyzed by the FLIM-phasor technique developed by Jameson *et al* [15]. Figure 2A shows a false-color image of a fluorescent Hp biofilm where bacteria can be easily distinguished from the matrix thanks to the higher fluorescence intensity. Conversion of the FLIM data to the phasor plot produced a cloud of pixels inside the universal circle (Figure 2B), revealing a multiexponential fluorescence lifetime decay. The corresponding phasor image in Figure 2C clearly shows that different areas of the biofilm (bacteria/ECM) are associated with different positions in the phasor plot. To inspect the behavior of porphyrins lifetime in different contexts, PPIX and CPI were also analyzed as monomers dissolved in methanol solutions and in their crystalline form. All these samples gave strictly monoexponential decays, which fall on the edge of the universal circle in the phasor plot (Figure 2B). As expected, crystalline porphyrins gave pixels clouds falling in the bottom-right area of the plot, corresponding to very short Fluorescence microscopy of Hp biofilms revealed that bacterial porphyrins are present both inside bacterial cells and in the ECM [4], as they confer red fluorescence to the whole system. Biofilms obtained from Hp after four days of incubation were then observed by confocal laser scanning microscopy and analyzed by the FLIM-phasor technique developed by Jameson et al. [15]. Figure 2A shows a false-color image of a fluorescent Hp biofilm where bacteria can be easily distinguished from the matrix thanks to the higher fluorescence intensity. Conversion of the FLIM data to the phasor plot produced a cloud of pixels inside the universal circle (Figure 2B), revealing a multiexponential fluorescence lifetime decay. The corresponding phasor image in Figure 2C clearly shows that different areas of the biofilm (bacteria/ECM) are associated with different positions in the phasor plot. To inspect the behavior of porphyrins lifetime in different contexts, PPIX and CPI were also analyzed as monomers dissolved in methanol solutions and in their crystalline form. All these samples gave strictly monoexponential decays, which fall on the edge of the universal circle in the phasor plot (Figure 2B). As expected, crystalline porphyrins gave pixels clouds falling in the bottom-right area of the plot, corresponding to very short lifetimes values (<1 ns), while monomeric porphyrins in methanol solution produced pixels in the left part of the phasor plot, with lifetimes exceeding 10 ns.

lifetimes values (<1 ns), while monomeric porphyrins in methanol solution produced pix-

els in the left part of the phasor plot, with lifetimes exceeding 10 ns.

*Pharmaceutics* **2021**, *13*, 1674 5 of 7

**Figure 2.** (**A**) False-color intensity-based image of bacterial biofilm. (**B** top) Overlap of the phasor plots of standard porphyrins PPIX (pink circle: methanol solution, green circle: crystal) and CPI (cyan circle: methanol solution, blue circle: crystal) and biofilm sample (red circle: bacteria, yellow circle: biofilm matrix). (**B** bottom) Phasor images of the standards, color code as in the plot. (**C**) Corresponding phasor image of the biofilm in A. Scale bar in A and C: 25 μm. **Figure 2.** (**A**) False-color intensity-based image of bacterial biofilm. (**B** top) Overlap of the phasor plots of standard porphyrins PPIX (pink circle: methanol solution, green circle: crystal) and CPI (cyan circle: methanol solution, blue circle: crystal) and biofilm sample (red circle: bacteria, yellow circle: biofilm matrix). (**B** bottom) Phasor images of the standards, color code as in the plot. (**C**) Corresponding phasor image of the biofilm in A. Scale bar in A and C: 25 µm.

#### **4. Discussion 4. Discussion**

Bacterial biofilms are composed of living cells embedded in an extracellular polymeric matrix along with several exocellular molecules excreted by the cells or captured from the environment. When bacterial cells die, they release substances in the ECM, mainly DNA along with other endogenous cytoplasmic components. Dead cells can be a supplier for the ECM because the leakage of cytoplasmic components due to bacterial cell lysis has been shown to favor biofilm formation [20]. On this basis, it is not surprising to find endogenous Hp porphyrins both inside the bacterial cells and dispersed in the ECM Bacterial biofilms are composed of living cells embedded in an extracellular polymeric matrix along with several exocellular molecules excreted by the cells or captured from the environment. When bacterial cells die, they release substances in the ECM, mainly DNA along with other endogenous cytoplasmic components. Dead cells can be a supplier for the ECM because the leakage of cytoplasmic components due to bacterial cell lysis has been shown to favor biofilm formation [20]. On this basis, it is not surprising to find endogenous Hp porphyrins both inside the bacterial cells and dispersed in the ECM of Hp biofilms.

of Hp biofilms. The chromatographic analysis performed on Hp extracts after different periods of culture revealed that porphyrins are produced from the first day, but their concentration drastically increases after 4 days of incubation. Although the planktonic and biofilm growing conditions are rather different, after 4 days fully formed biofilms likewise provided a sufficient concentration of porphyrins for analysis. Accordingly, Hp biofilms were grown The chromatographic analysis performed on Hp extracts after different periods of culture revealed that porphyrins are produced from the first day, but their concentration drastically increases after 4 days of incubation. Although the planktonic and biofilm growing conditions are rather different, after 4 days fully formed biofilms likewise provided a sufficient concentration of porphyrins for analysis. Accordingly, Hp biofilms were grown for 4 days and analyzed by FLIM coupled to the phasor approach.

for 4 days and analyzed by FLIM coupled to the phasor approach. It is known that intracellular lifetime measurements on porphyrins usually produce three lifetime values, generally attributed to the monomeric (>10 ns), dimeric (1.5-3.0 ns) and highly aggregated (<1 ns) forms [21], but these Hp biofilms do not show lifetimes >10 ns, as if no porphyrin monomers were present [4]. Nevertheless, the positioning of pixels inside the universal circle in the phasor plot matches with the presence of a combination of different lifetimes (Figure 2B). Monoexponential decays fall on the edge of the universal circle, as in the case of monomeric (pink and cyan circles) or crystalline (blue and green circles) porphyrins. The area where the biofilm pixels fall suggests that porphyrins are mostly present in several aggregated forms, likely in a mixture of dimers and higher oligomers, whose lifetimes are combined to produce the observed pixel cloud. Notably, the phasor analysis applied to FLIM acquisitions of Hp biofilms reveals different average lifetimes for porphyrins inside bacteria or dispersed in the ECM, as expressed by the slightly different position of the red and yellow circles in the plot of Figure 2B, corresponding to the red (bacteria) and yellow (matrix) areas in the phasor image (Figure 2C). This could be attributed either to a different composition between the intracellular and extracellular porphyrins mixture or to their different aggregation grade inside and outside the cells, along with the different environmental interactions. It is worth noting how the phasor It is known that intracellular lifetime measurements on porphyrins usually produce three lifetime values, generally attributed to the monomeric (>10 ns), dimeric (1.5–3.0 ns) and highly aggregated (<1 ns) forms [21], but these Hp biofilms do not show lifetimes >10 ns, as if no porphyrin monomers were present [4]. Nevertheless, the positioning of pixels inside the universal circle in the phasor plot matches with the presence of a combination of different lifetimes (Figure 2B). Monoexponential decays fall on the edge of the universal circle, as in the case of monomeric (pink and cyan circles) or crystalline (blue and green circles) porphyrins. The area where the biofilm pixels fall suggests that porphyrins are mostly present in several aggregated forms, likely in a mixture of dimers and higher oligomers, whose lifetimes are combined to produce the observed pixel cloud. Notably, the phasor analysis applied to FLIM acquisitions of Hp biofilms reveals different average lifetimes for porphyrins inside bacteria or dispersed in the ECM, as expressed by the slightly different position of the red and yellow circles in the plot of Figure 2B, corresponding to the red (bacteria) and yellow (matrix) areas in the phasor image (Figure 2C). This could be attributed either to a different composition between the intracellular and extracellular porphyrins mixture or to their different aggregation grade inside and outside the cells, along with the different environmental interactions. It is worth noting how the phasor approach allows for easy discrimination of the two different fluorescent areas.

#### approach allows for easy discrimination of the two different fluorescent areas. **5. Conclusions**

To the best of our knowledge, in this work the FLIM-phasor approach was employed for the first time to easily discriminate porphyrins located in different fluorescent areas of Hp biofilms. This could be exploited to assess the extent of bacterial contamination and the presence of a biofilm inside an infected tissue, suggestive of an advanced stage of infection,

thus simplifying the choice of the best treatment. Our findings indicate that porphyrins inside the cells and dispersed in the ECM show a slightly different global lifetime; likely, on the basis of the position of the relevant pixels cloud in the phasor plot, intracellular porphyrins may be organized in more packed structures. From the phototherapeutical point of view, this knowledge can be of great importance because, due to the short diffusion path of singlet oxygen and other cytotoxic reactive oxygen species, the more strictly the photosensitizer is bound to the cellular target, the more effective its photokilling activity will be. In addition, as the intracellular pigment concentration varies with the age of bacteria, the detection and the spatial distribution pattern of porphyrin fluorescence in Hp biofilm could provide some hints for the choice of the most appropriate therapeutical time window to perform aPDT. In general, this kind of analysis could be considered to be a useful and rapid tool for the exploration of fluorescent bacterial biofilms properties not only to obtain new insights into the biofilm structure and dynamics but also to develop and optimize novel antimicrobial strategies.

**Author Contributions:** Conceptualization, A.B., P.M. and A.S.; methodology, A.B., P.M. and A.S.; investigation, A.B., P.M. and A.S.; writing—review & editing, A.B., P.M. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Regione Toscana Bando FAS Salute 2014 Italy (Grant No. CUP B52I14005760002).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are available from the authors upon request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article*

## **Broad-Spectrum Antimicrobial ZnMintPc Encapsulated in Magnetic-Nanocomposites with Graphene Oxide/MWCNTs Based on Bimodal Action of Photodynamic and Photothermal Effects**

**Coralia Fabiola Cuadrado 1,\*, Antonio Díaz-Barrios <sup>2</sup> , Kleber Orlando Campaña <sup>1</sup> , Eric Cardona Romani <sup>3</sup> , Francisco Quiroz <sup>4</sup> , Stefania Nardecchia <sup>5</sup> , Alexis Debut <sup>6</sup> , Karla Vizuete <sup>6</sup> , Dario Niebieskikwiat <sup>7</sup> , Camilo Ernesto Ávila <sup>8</sup> , Mateo Alejandro Salazar <sup>8</sup> , Cristina Garzón-Romero <sup>8</sup> , Ailín Blasco-Zúñiga <sup>8</sup> , Miryan Rosita Rivera 8,\* and María Paulina Romero <sup>1</sup>**


**Abstract:** Microbial diseases have been declared one of the main threats to humanity, which is why, in recent years, great interest has been generated in the development of nanocomposites with antimicrobial capacity. The present work studied two magnetic nanocomposites based on graphene oxide (GO) and multiwall carbon nanotubes (MWCNTs). The synthesis of these magnetic nanocomposites consisted of three phases: first, the synthesis of iron magnetic nanoparticles (MNPs), second, the adsorption of the photosensitizer menthol-Zinc phthalocyanine (ZnMintPc) into MWCNTs and GO, and the third phase, encapsulation in poly (N-vinylcaprolactam-co-poly(ethylene glycol diacrylate)) poly (VCL-co-PEGDA) polymer VCL/PEGDA a biocompatible hydrogel, to obtain the magnetic nanocomposites VCL/PEGDA-MNPs-MWCNTs-ZnMintPc and VCL/PEGDA-MNPs-GO-ZnMintPc. In vitro studies were carried out using *Escherichia coli* and *Staphylococcus aureus* bacteria and the *Candida albicans* yeast based on the Photodynamic/Photothermal (PTT/PDT) effect. This research describes the nanocomposites' optical, morphological, magnetic, and photophysical characteristics and their application as antimicrobial agents. The antimicrobial effect of magnetics nanocomposites was evaluated based on the PDT/PTT effect. For this purpose, doses of 65 mW·cm−<sup>2</sup> with 630 nm light were used. The VCL/PEGDA-MNPs-GO-ZnMintPc nanocomposite eliminated *E. coli* and *S. aureus* colonies, while the VCL/PEGDA-MNPs-MWCNTs-ZnMintPc nanocomposite was able to kill the three types of microorganisms. Consequently, the latter is considered a broad-spectrum antimicrobial agent in PDT and PTT.

**Citation:** Cuadrado, C.F.;

Díaz-Barrios, A.; Campaña, K.O.; Romani, E.C.; Quiroz, F.; Nardecchia, S.; Debut, A.; Vizuete, K.; Niebieskikwiat, D.; Ávila, C.E.; et al. Broad-Spectrum Antimicrobial ZnMintPc Encapsulated in Magnetic-Nanocomposites with Graphene Oxide/MWCNTs Based on Bimodal Action of Photodynamic and Photothermal Effects. *Pharmaceutics* **2022**, *14*, 705. https://doi.org/ 10.3390/pharmaceutics14040705

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 31 December 2021 Accepted: 24 February 2022 Published: 26 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** antimicrobial nanomaterials; carbon nanotubes; graphene; magnetic nanoparticles; hydrogel; photodynamic therapy; photothermal therapy; nanocarrier

#### **1. Introduction**

In recent years, the study of microorganisms has increased substantially due to their ability to spread rapidly and adapt to different environments. For this reason, the World Health Organization has declared microbial diseases as one of the main threats for humanity [1]. Currently, there are antibiotic therapies that help fight infections caused by microorganisms such as *Klebsiella pneumoniae*, *Escherichia coli*, *Staphylococcus aureus* and *Neisseria gonorrhoeae*, among others. However, these have been losing their efficacy, which is why the development of new treatments is necessary [2]. The generation of new nanocomposites that help eliminate microorganisms provides a new alternative for the fight against infections caused by these pathogens [3,4].

Nanotechnology for the control of infectious diseases includes several strategies, such as the use of metal oxides for the generation of reactive oxygen species (ROS), nanocomposites capable of damaging membrane integrity or causing physical damage to the bacterial wall [5], inhibiting DNA replication and adenosine triphosphate (ATP) production in cells [6], use of graphene-family materials for the microbial elimination [7–10], and others. In this regard, Romero et al., 2020 showed the ability of GO as an antimicrobial agent to eliminate *E. coli* and *S. aureus* bacteria through the combination of Photodynamic Therapy (PDT) and Photothermic Therapy (PTT) [11]. Mei et al., 2021, synthesized a ZnPc-TEGMME@GO nanocomposite, which has a thickness between 1.47 and 2.61 nm, using concentrations <sup>≥</sup><sup>25</sup> <sup>µ</sup>g·mL−<sup>1</sup> of the nanocomposite and irradiation with dual light of 450 nm and 680 nm for 10 min. The nanocomposite increases its temperature to 100 ◦C and rapidly promotes singlet oxygen generation, causing cuts in the membranes of bacterial cells and, consequently, the death of Gram-positive and Gram-negative bacteria [12]. Yang et al., 2018, obtained the nonchemotherapeutic nanoagent Fe3O4-CNT-PNIPAM with a diameter of around 200 to 400 nm, which at a concentration of 0.1 mg·mL−<sup>1</sup> , and under 808 nm laser irradiation, 3 W·cm−<sup>2</sup> , for 5 min is capable of killing *S. aureus* and *E. coli* by PTT [13].

Photodynamic Therapy (PDT) is well known and studied for its use in cancer treatments, producing minimum side effects compared to other therapies used for cancer. PDT is an attractive operating modality based on the interaction of light with a photosensitizer (PS) and oxygen [14,15], thus producing ROS and free radicals capable of causing cell and microorganism death with high cytotoxicity [16–19]. This therapy has been used to treat skin diseases and cancers such as prostate, neck, lung, breasts and bladder cancers [20–23].

PS generally are classified by their activation wavelength, duration, and tissue penetration. One of the most widely used PS in PDT is the second generation PS, due to its photophysical and photochemical properties. Within this group are phthalocyanines, chlorins, and benzoporphyrins [24,25]. Phthalocyanines (Pcs) are hydrophobic compounds whose activation wavelengths of 650 and 700 nm allow deep tissue penetration. Pc reaches a high concentration in tumor tissue after 1 to 3 h of administration, and they are generally used to treat skin and subcutaneous lesions [26].

Zinc Menthol-Phthalocyanine (ZnMintPc) is a hydrophobic drug derived from phthalocyanine, whose structure is based on porphyrins but with a central Zinc atom with four methoxy groups around it that allow PS to be soluble in certain organic solvents [27]. The use of ZnMintPc has been limited due to its hydrophobic nature. When encapsulated by hydrogels, Pcs is soluble in aqueous media [28–30]. Pcs can also be combined with nanoparticles (Np) to create hybrid nanostructures that increase the quantum yield of singlet oxygen, cell uptake, and their therapeutic effectiveness [31,32].

Due to the hydrophobic nature of PS, several types of nanocarriers have been studied that prevent them from adding to each other and losing their physicochemical characteristics [20,33,34]. Among these nanocarriers are carbon nanostructures such as carbon nanotubes [35–37], graphene [33] and fullerenes [20,38,39], among others. These functionalized nanocarriers have excellent optical and mechanical properties. These compounds have cytotoxicity in biological systems, depending on their concentration, size, surface property and functional groups [40]. The surface modification of these nanocarriers with biomolecules such as polyethyleneimine, polyethylene glycol, and human proteins, improves the cytotoxicity and biocompatibility for biological applications [41], in addition to having other applications such as immunotherapy, imaging, and the development of vaccines and antimicrobial agents [42–46].

Photothermal treatment (PTT) is a type of phototherapy that works by turning light energy into heat through the use of photothermal agents (PTAs) [47]. There has been great interest in applying PTT to eliminate pathogenic bacteria in recent years. PTAs, when irradiated with near-infrared (NIR) light, generate much heat, causing protein denaturation, rupture of the bacterial cell membrane, and death of microorganisms [13,48,49]. An ideal PTA must meet specific requirements, such as high photothermal conversion efficiency, biocompatibility, ease of synthesis, photostability, and rapid elimination [50,51]. The best known PTAs are carbon-based nanomaterials, conjugated polymer-based nanomaterials, inorganic, and small molecule-based nanomaterials [48,52].

PTT/PTT synergies are considered among the most effective methods for microbe killing due to high specificity, minimal invasiveness, low risk of developing drug resistance, and selectivity [53,54]. These phototherapy techniques have a dual mode of action in which the PTT facilitates the absorption of PS because the increase in temperature will causes an increase in the permeability of the microorganism. At the same time, by PDT, the PS produces ROS, which can destroy proteins, lipids, and microbial DNA, causing the death of the bacteria, and increasing the effectiveness of the PTT since they are capable of reducing the heat resistance of bacteria [12,48].

CNTs were discovered by Iijima in 1991 [55] and consist of quasi-one-dimensional structures formed by several layers of graphene rolled up coaxially to form tubes. They are classified into two types: those with a single layer known as single-wall carbon nanotubes (SWCNTs), and those with several layers known as multiwall carbon nanotubes (MWCNTs) [56,57]. CNTs possess excellent optical, electrical, thermal, physical, and kinetic properties, and excellent cell permeability [58,59]. For this reason, there is much interest in their application as drug transport systems, electrochemical biosensors, and biological markers, among others [60,61].

CNTs have been used as biosensors, biomarkers, and drug transporters with a high carrying capacity [62,63]. Hybrid compounds have been created to improve the effectiveness of their action. Proteins, polymers, cell recognition ligands, nanoparticles, hydrophilic coatings have been incorporated into these structures, which provide them with new functions such as cell recognition, and controlled drug release, avoiding aggregations in aqueous media during targeted transport [64–66].

Graphene is a two-dimensional nanostructure with sp<sup>2</sup> hybridization and strongly cohesive carbons, which gives the structure excellent optical, electronic, mechanical, and chemical properties, which vary depending on its lateral size [67]. Graphene is a material with very high resistance and hardness. It is light and has low toxicity and increased flexibility, making it an innovative material in construction, technology, medicine, and other industries [43,68,69]. Because of its excellent physicochemical characteristics, graphene has been used in the biomedical field to make biosensors, drug delivery systems, antibacterial agents for early detection of cancer, gene therapy, and for cancer cell imaging/mapping, among other uses [70].

In recent years, great interest has arisen in the synthesis of magnetic nanoparticles due to their unique physicochemical properties. They can be used in PPT/PDT [71], drug delivery, theranostics, and others [72–74]. Magnetic nanoparticles (MNPs), such as magnetite and hematite obtained from iron oxides, are widely used in biomedicine because they are biocompatible and have no cytotoxic effects at concentrations below

<sup>100</sup> <sup>µ</sup>g·mL−<sup>1</sup> . For breast, glia, and normal cells with cancer [75], at higher concentrations, an interaction between cell membrane phospholipids and iron nanoparticles occurs, resulting in membrane failure [76–78]. MNPs can be anchored to carbon structures to obtain drug nanocarriers and directed by an external magnetic field [79,80].

VCL/PEGDA is a biocompatible hydrogel that can be obtained by emulsion polymerization. This hydrogel is very promising since it responds to physiological changes in the temperature of the human body [81,82]. Therefore, it can be used as a drug delivery system to encapsulate hydrophobic and hydrophilic agents. A study carried out by Romero et al., 2021 showed the sustained release capacity of the drug colchicine encapsulated in VCL/PEGDA [83].

PS must meet specific requirements for PDT strategy. They must be selective, disperse well in tissue, and their photostability time must be adequate for the treatment. Due to these needs, we developed two magnetic nanocarriers based on MWCNTs and GO materials in this work. Both nanocarriers are decorated with Fe-MNPs, giving the compounds superparamagnetic properties. These nanocarriers were functionalized with PS ZnMintPc, a drug used in Photodynamic Therapy. A VCL/PEGDA hydrogel was used to help with dispersion of the hydrophobic compounds ZnMintPc and MWCNTs in aqueous media by providing them with a lipophilic envelope. Finally, the antimicrobial effect of these nanocomposites was evaluated to eliminate the bacteria *S. aureus*, *E. coli*, *C. albicans* using the PDT/PTT strategy.

#### **2. Materials and Methods**

#### *2.1. Materials*

For the synthesis and functionalization process, MWCNTs and GO were provided by the Van de Graff Laboratory, Department of Physics PUC-RIO, Rio de Janeiro, Brazil. Zinc Menthol-Phthalocyanine was provided by the Federal University of São Carlos, São Carlos, Brazil; Fe(SO4), H2SO4·7H2O from MERCK, Rio de Janeiro, Brazil; NH4OH, 14.8N and N,N Dimethylformamide from Fisher Scientific, Princeton, NJ, USA; Fe2(SO4)3·H2O from Fisher Scientific. HNO<sup>3</sup> from the Fermont, Monterrey, Mexico; saline solution Fisiol UB (pH = 7) from Lamosan, Quito, Ecuador and Tween80 from La casa del Químico, Quito, Ecuador.

For VCL/PEGDA hydrogel synthesis by emulsion polymerization, the following reagents were used. N-vinylcaprolactam (VCL; Sigma Aldrich, Darmstadt, Germany, 98%), and Poly(ethylene glycol) diacrylate (PEGDA; Sigma Aldrich, Mn 250), the initiator ammonium persulfate (APS; FMC Corporation, Philadelphia, PA, USA, >99%), the emulsifier sodium dodecyl sulfate (SDS; STEOL®CS-230 Stepan, Northbrook, IL, USA) and a buffer of sodium hydrogen carbonate (Sigma Aldrich, ≥99.7%) were used as provided.

Characterizations of MNPs-MWCNTs and MNPs-GO were carried out by FT-IR spectroscopy analysis in a JASCO FT/IR-4100 spectrometer, JASCO International Co., Ltd., Tokyo, Japan. ES (wavenumber range 7800 to 350 cm−<sup>1</sup> , resolution of 0.7 cm−<sup>1</sup> ) and Raman spectroscopy analysis in a HORIBA Raman spectrometer LabRAM HR Evolution (Horiba, Kyoto, Japan), where the samples were excited with a 2.33 eV (532 nm).

Magnetization (*M*) measurements were carried out using a Quantum Design Versalab VSM, Quantum Design, Darmstadt, Germany. FR, magnetometer in the temperature range between −210 and +60 ◦C with applied magnetic fields, µ0H, up to 3 T.

Stability over time of the PS magnetic nanocomposites was characterized by UV-VIS spectroscopy analysis at a wavelength range of 280 to 780 nm (resolution better than 1.8 Å), using a UV-VIS Spectrophotometer model Evolution 220 from Thermo Fisher Scientific, Waltham, MA, USA. DPBF photobleaching and thermic studies were carried out with homemade equipment using an LED red light at 635 nm, 65.5 mW·cm−<sup>2</sup> . A DPBF photobleaching study was characterized in a UV-VIS Specord 210 Plus. XRD analysis was performed on a PANalytical brand EMPYREAN Diffractometer (Malvern Panalytical, Malvern, UK) operating in a θ–2θ configuration (Bragg-Brentano geometry) equipped with a copper X-ray tube (Kα radiation λ = 1.54056 Å) at 45 kV and 40 mA.

#### *2.2. Methods*

#### 2.2.1. Morphological Studies

Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) evaluated morphology and semiquantitative elemental composition. For this, an aliquot of the sample was fixed to an aluminum sample holder using a double layer of carbon tape. The analyses were carried out on an SEM Tescan Mira 3 (Tescan, Brno, Czech Republic). equipped with a Schottky field emitter (JEOL Ltd., Tokyo, Japan). EDS was performed in the SEM chamber using a Bruker, X-Flash 6|30 detector (Bruker, Billerica, MA, USA) with a resolution of 123 eV at Mn Kα. Tapping-mode atomic force microscopy was used to determine the shape and thickness of GO (Bruker Dimension Icon AFM).

For Transmission Electron Microscopy (TEM), the samples were dispersed in a BRAN-SON 1510 ultrasonic (Transcat, Rochester, NY, USA) bath for 30 min. Next, approximately 5 µL of the sample was placed on a TEM grid (formvar/carbon, 300 mesh), and the solvent was removed with filter paper. The samples were observed in a TEM FEI, Tecnai G2 Spirit Twin (ELECMI, Zaragoza, Spain) equipped with an Eagle 4k HR camera (Tronic Extreme, Coventry, UK) at 80 kV.

For scanning transmission electron microscopy (STEM), 5 µL of the sample was placed on a TEM grid (formvar/carbon, 300 mesh), and the solvent was removed with filter paper. Staining was performed with 1% Phosphotungstic Acid for 1 s for *S. aureus* and 1 min for *E. coli*, the solvent was removed, and the sample was observed in an SEM Tescan, Mira 3 in transmission mode.

#### 2.2.2. Synthesis of Graphene Oxide

GO was prepared according to Hummers' method [84]. Graphite powder with 99% purity was used for the synthesis of GO. Chemical products, including NaNO3, KMnO4, H2SO4, and aqueous solutions of H2O2, and HCl, were purchased from Sigma Aldrich. GO powder was obtained after lyophilization of the suspected GO in deionized water.

#### 2.2.3. Purification of MWCNTs

The purification of MWCNTs was carried out using an acid attack. A 5.3 mg sample of MWCNTs was dispersed with 5% Tween 80 in 10 mL of distilled water stirred at 200 rpm for 24 h to ensure that the MWCNTs were well dispersed. Then, in a solution of 4 mL of HNO<sup>3</sup> and 12 mL of H2SO<sup>4</sup> (1:3 ratio), the solution of MWCNTs previously treated was allowed to cool, and then stirred magnetically for 3 h. Several washes of the MWCNTs were performed using 0.22 µm pore micropore filtration until a neutral pH was obtained.

#### 2.2.4. Synthesis of MNPs-MWCNTs and MNPs-GO

In 18 mL of pure water, the following were dispersed: purified MWCNTs, 225 mg of FeSO4, 450 mg of (Fe<sup>2</sup> (SO4)3), and 5% Tween 80. The sample was placed in a magnetic stirrer for 3 h, then was added carefully to 150 mL of NH4OH. The mixture was exposed to magnetic stirring in an inert atmosphere for 1 h at 200 rpm. Finally, several magnetic purifications of the MNPs-MWCNTs nanocarrier were carried out, until the pH was neutralized, and allowed to dry.

The same process was used to synthesize of MNPs-GO, using 5.3 mg of GO instead of MWCNTs.

As a control sample for the magnetic measurements, free-standing iron nanoparticles were prepared using the same co-precipitation method but without GO or MWCNTs.

### 2.2.5. Synthesis of Polyethylene Glycol Diacrylate-Vinylcaprolactam (VCL/PEGDA)

Hydrogel synthesis was carried out by emulsion polymerization of 2 g of VCL, 0.08 g of PEGDA, STEOL CS-230, and 0.08 of NaHCO<sup>3</sup> dispersed in 235 mL deionized water. The mixture was slowly placed in the chemical reactor with stirring at 350 rpm and a temperature of 70 ◦C, maintaining a stream of Nitrogen for one hour. Then, the initiator (0.03 g of Ammonium Persulfate dissolved in 15 mL of distilled water) was added to the

solution, and the reaction was kept at 70 ◦C for 7 h. After this time, the mixture was allowed to cool under stirring at 200 rpm and 25 ◦C to avoid aggregation for 12 h. Finally, the hydrogel was dialyzed against DDI water to remove impurities and unreacted reagents [83].

2.2.6. Functionalization of MNPs-MWCNTs; MNPs-GO with ZnMintPc in the Presence of VCL/PEGDA

In 10 mL of pure water, 2 mg of MNPs-MWCNTs were dispersed with 5% Tween, and the sample stirred magnetically for 24 h at 200 rpm, then sonicated for 30 min. A volume of 10 mL of VCL/PEGDA was added to the mixture, and it was stirred magnetically for 4 h at 200 rpm.

To 20 mL of the VCL/PEGDA-MNPs-MWCNTs solution, 0.67 mL of ZnMintPc solution (0.25 mM) were added, after which the mixture was sonicated for 4 h at 250 rpm. The solution was covered with aluminum foil to avoid ZnMintPc photodegradation.

The same process was carried out with MNPs-GO, obtaining the VCL/PEGDA-MNPs-GO nanocomposite.

#### 2.2.7. Quenching Experiment of 1,3-Diphenyl Isobenzofuran

The fluorescence and absorption characteristics of 1,3-diphenylisobenzofuran (DPBF), a singlet oxygen trapping chemical, are widely employed to detect and quantify singlet oxygen [85]. This agent has an absorption range between 410–420 nm, emitting blue fluorescence. When DPBF interacts with oxygen, it produces o-dibenzoylbenzene, without absorbing visible light. The amount of <sup>1</sup>O<sup>2</sup> produced is shown by a reduction in DPBF absorbance [11]. A sample of 5 mg of DPBF was dispersed in 1 mL of DMF to distribute in the solutions with nanocomposites later.

For this experiment, a UV-VIS SPECORD 210 Plus spectrophotometer (resolution of 2.3–2.5 nm) in a range of 300 to 800 nm was used. A reference solution was prepared with 10 mL of deionized water and 10% Tween 80. Then, in 3 mL of this reference solution, 100 µL of VCL/PEGDA-MNPs-GO (0.1 mg/mL) was added. The same process was repeated with the nanocomposites VCL/PEGDA-MNPs-MWCNTs (0.1 mg/mL), VCL/PEGDA-MNPs-MWCNTs-ZnMintPc (0.1 mg/mL) and VCL/PEGDA-MNPs-GO-ZnMintPc (0.1 mg/mL). After that, 20 µL of the DPBF (18.5 mM) solution was placed in each of them, and they were irradiated at different times while observing a decrease in absorbance of 418 nm due to DPBF.

#### 2.2.8. Thermal Studies

Thermal studies were carried out by irradiating, in deionized water, VCL/PEGDA, MNPs, GO, MWCNTs, VCL/PEGDA-MNPs-GO, and VCL/PEGDA-MNPs-MWCNTs, with a red light of 630 nm and 65.5 mW·cm−<sup>2</sup> and taking the temperature every 5 to 10 min until it did not change.

#### 2.2.9. Antimicrobial Studies

The antimicrobial study used cryovials with the microorganisms *Staphylococcus aureus* ATCC: 25923, *Escherichia coli* ATCC: 25922, and *Candida albicans* ATCC: 10231 as test subjects. Cryovials were thawed at room temperature, and the contents were inoculated in Mueller-Hinton broth (DifcoTM). The culture medium was incubated overnight at 37 ◦C. After this period, the absorbance of each medium was determined by spectrophotometry (Thermo Scientific TM Orion TM AquaMate 8000 UV-Vis, Thermo Fisher Scientific, Waltham, MA, USA) and diluted in Mueller-Hinton broth to reach the concentration established for the bioassays: 10<sup>7</sup> CFUs mL−<sup>1</sup> for *E. coli*, 10<sup>6</sup> CFUs mL−<sup>1</sup> for *S. aureus*, and 10<sup>5</sup> CFUs mL−<sup>1</sup> for *C. albicans*.

Upon reaching the indicated concentration, each medium was dispensed into microtubes: six aliquots of 1 mL per magnetic nanocomposites (VCL/PEGDA-MNPs-GO-ZnMintPc, VCL/PEGDA-MNPs-MWCNTs-ZnMintPc, VCL/PEGDA-MNPs-GO, and VCL/ PEGDA-MNPs-MWCNTs) and six aliquots of 1 mL as control. Half of the aliquots were

used to evaluate the activity of the magnetic nanocomposites under light irradiation, while the other half was not exposed to these conditions.

The microtubes were centrifuged at 3000 rpm for 10 min. The supernatant was discarded, and 1 mL of PBS was added to wash the cells and remove the remains of the culture medium. The pellet was resuspended and recentrifuged under the same conditions. PBS was discarded and 1 mL of the compound was dispensed into each microtube. In the control microtubes, the cells were resuspended again with PBS. Each tube was vortexed to dissolve the pellet again and incubated at 37 ◦C in the dark for 45 min.

At the end of the incubation period, three aliquots of each compound and three controls were subjected to light irradiation using red light of 630 nm, 65.5 mW·cm−<sup>2</sup> . The remaining tubes were not irradiated.

Serial dilutions of each aliquot were made in PBS. A 4 µL aliquot of each dilution was inoculated into an 8-part Petri dish with Mueller-Hinton agar (DifcoTM). Each inoculum was streaked for colony isolation. Petri dishes were incubated for 24 h at 37 ◦C, and after this period the number of colonies was counted per dilution.

### **3. Results**

#### *3.1. Composition and Structure Characterization of Magnetic Nanocomposites*

FT-IR spectroscopy was performed on the magnetic nanocomposites to study the difference in oxygen-related functional groups (Figure 1). Figure 1a shows the FT-IR spectra of MNPs-MWCNTs, which presents characteristic bands. As the literature indicates, the band at 3373 cm−<sup>1</sup> is attributed to the vibration of the hydroxyl group (OH), the 1636 cm−<sup>1</sup> band corresponding to the FeOO and shows the successful decoration of MWCNTs with iron nanoparticles through hydrogen bonds [86–88]. Other bands appear in the range 1400–1730 cm−<sup>1</sup> , corresponding to the OH–C=O, –COO, –COOH groups added due to acid treatment and functionalization with MNPs. Finally, the characteristic bands of the MNPs appear at 709 and 623 cm−<sup>1</sup> , which indicate the stretching vibration of Fe–O–Fe characteristic of Fe-MNPs, which agrees with Abrinaei, Kimiagar and Zolghadr 2019 [89].

**Figure 1.** FT-IR spectra: (**a**) MNPs-MWCNTs; (**b**) MNPs-GO.

Figure 1b presents the FT-IR spectra of the MNPs-GO nanocarrier where characteristic bands of GO are identified. There is a band around 3327 cm−<sup>1</sup> corresponding to the bending and vibration of the OH stretching of the C-groups, the band at 1636 cm−<sup>1</sup> corresponds to the vibration of the C=C, the bands around 800 cm−<sup>1</sup> and 1269 cm−<sup>1</sup> represent the epoxy group, and bands at 1342 cm−<sup>1</sup> , 1247 cm−<sup>1</sup> , 1049 cm−<sup>1</sup> show the stretching vibration of the carboxy groups C−O, C−C−O, and alkoxy C−O, respectively; as shown in the work of Al-Ruqeishi et al., 2020 [90].

Figure 2a shows the Raman spectra of non-purified MWCNTs (Figure 2a), purified MWCNTs (Figure 2b), and MNPs-MWCNTs (Figure 2c). These spectra show characteristic bands of the MWCNTs: band D or band of defects and the first and second-order bands G and 2D, respectively. The D-band is at 1337 cm−<sup>1</sup> and indicates the presence of defects in the graphite, resulting from the presence of multiple carbon sheets that are not directly aligned sheet to sheet, which induces a loss of translational symmetry in the two-dimensional network. Due to the same effect, a secondary phonon is produced that gives rise to the presence of the G-band at 1566 cm−<sup>1</sup> . The fundamental band G is a tangential elongation band attributed to the in-plane vibration of the C-C bond, is typical of carbon-derived materials, and is consistent with reports in the literature [91–94].

Figure 2d shows the Raman spectra of Iron-MNPs with its characteristic bands, two A1g modes at 226, 502 cm−<sup>1</sup> , five E<sup>g</sup> modes at 248, 291, 300, 407 and 615 cm−<sup>1</sup> , and the characteristic two-magnon scattering band at 1320 cm−<sup>1</sup> , which agree with the results presented by Soler and Qu 2012 [95]. Some of these bands can be observed in the spectra shown in Figure 2c,f.

Figure 2a,b show Raman spectra of unpurified MWCNTs and purified MWCNTs. The *<sup>I</sup><sup>D</sup> IG* ratio of the purified MWCNTs is higher than that of unpurified MWCNTs because treatment with acids causes some bonds to break and form functional groups, generating defects in the structure of MWCNTs.

In the Raman spectrum of MNPs-MWCNTs (Figure 2c), the increase in the *<sup>I</sup><sup>D</sup> IG* ratio is due to charge transfer effects between MNPs and MWCNTs; the result of the functionalization is a structure of MWCNTs with defects. In addition, extra bands that correspond to the MNPs are observed.

**Figure 2.** Raman Spectra: (**a**) non-purified MWCNTs, (**b**) purified MWCNTs, (**c**) MNPs-MWCNTs; (**d**) Fe-MNPs; (**e**) GO; (**f**) MNPs-GO; Eláser = 2.33 eV, λ = 532 nm.

Figure 2e shows the characteristic bands of GO, an intense D band at 1340 cm−<sup>1</sup> , a G-phase vibration band at 1589 cm−1, and the D + D' band located around 2900 cm−1, which is activated by defects and appears with a combination of phonons with different linear moments around points K and Γ in the Brilloüin zone and agrees with Cançado et al., 2011, and Muhammad Hafiz et al., 2014 [96,97]. The ratio of the bands *<sup>I</sup><sup>D</sup> IG* = 1.036 results from the degree of disorder in GO due to the presence of Carboxylic acid functional groups at its ends.

Figure 2f shows the spectra of the MNPs-GO nanocomposite where there is a shift of D band, since the nature of iron-MNPs when combined with carbon nanostructures affects the spectral amplification of the phonon peaks, agreeing with the information presented by Ramirez et al., 2017 and Satheesh et al., 2018 [98,99].

#### *3.2. Magnetic Properties*

We studied the magnetic properties of three samples: the nanocomposites VCL/PEGDA-MNPs-MWCNTs and VCL/PEGDA-MNPs-GO and, for comparison, the free-standing iron nanoparticles (MNPs). All the studied samples showed Fe nanoparticles' typical ferromagnetic (FM) behavior, with a very small coercivity of less than 2 mT at room temperature (less than 5 mT at T = −210 ◦C). This small coercivity indicates that the MNPs were not directly attached to the carbon structures, since directly attached MNPs have large coercivities of hundreds of mT [100,101]. This was expected because the hydrogen bonds present between MNPs and carbon structures mentioned in the results of the FT-IR spectroscopy in Section 3.1. are weak and therefore generate a reduction of the Fe–C magnetic coupling. Figure 3a shows some representative *M* vs. *H* loops measured at two different temperatures, −210 ◦C and +20 ◦C, for the three samples. From these curves we obtained saturation magnetization (*MS*) as a function of temperature using the usual law of approach to saturation (LAS) [102–104] given by:

$$M(H) = M\_S \left(1 - \frac{a}{H} - \frac{b}{H^2}\right) + \chi H\_\prime \tag{1}$$

$$\text{Tr}\,\mathsf{f}\mathbb{C}\mathbb{I}$$

**Figure 3.** Magnetic properties: (**a**) Magnetization vs. applied magnetic field: (**i**) the free-standing nanoparticles, (**ii**) VCL-PEGDA-MNPs-MWCNTs, and (**iii**) VCL-PEGDA-MNPs-GO. The curves shown reflect measurements at two different temperatures, T = −210 ◦C (circles) and T = 20 ◦C (triangles). The solid lines are examples of the fit of the data using the law of approach to saturation (LAS) of Equation (1). (**b**) Saturation magnetization as a function of temperature: (**iv**) the free-standing nanoparticles, (**v**) VCL-PEGDA-MNPs-MWCNTs and (**vi**) VCL-PEGDA-MNPs-GO. The solid lines match the data showing that MS follows Bloch's law given Equation (2).

In this equation, *a* and *b* are constants, and the last term accounts for the nonferromagnetic contributions, such as the disordered shell of the nanoparticles. The latter is valid only at high fields, close to saturation; thus, we used it to fit our *M*(*H*) curves for applied fields between 1 and 3 T, as shown by the solid lines in Figure 3a for T = −210 ◦C. From these fits we obtained *M<sup>S</sup>* at several temperatures between −210 ◦C and +60 ◦C (see

Figure 3b). In the case of the VCL-PEGDA-MNPs-MWCNTs and VCL-PEGDA-MNPs-GO samples, the magnetization value appeared substantially reduced since most of the mass (>98%) corresponds to the hydrogel. Therefore, to directly compare the measured magnetic moment with that of pure iron, the saturation magnetization value was corrected by the mass fraction of nanoparticles, x = 1.43% for the VCL-PEGDA-MNPs-MWCNTs and x = 1.37% for the VCL-PEGDA-MNPs-GO sample.

The saturation magnetization as a function of temperature is presented in Figure 3b, where we also show that, for all three samples, *M<sup>S</sup>* closely follows the well-known Bloch's law for FM nanoparticles [105–107]:

$$M\_S(T) = M\_0 \times \left[1 - B\left(T - T\_0\right)^{\frac{3}{2}}\right],\tag{2}$$

where *M*<sup>0</sup> is the saturation magnetization at the absolute zero degrees Kelvin (*T*<sup>0</sup> = −273.15 ◦C), and *B* is the so-called spin-wave constant. The results for these magnetic parameters are presented in Table 1. The values obtained for *B* are very similar to those measured in other Fe-nanoparticles systems [104,105] (B ~ 10−5–10−<sup>4</sup> ◦C <sup>−</sup>3/2) implying the existence of comparable thermally induced magnetic field excitations within the FM volume. On the other hand, our results show that the saturation magnetization is, in all cases, much smaller than that of pure iron, *MFe* = 222 emu·g −1 . However, in the case of the free-standing nanoparticles, *M<sup>S</sup>* remains larger than for the nanoparticles submerged within the hydrogel, indicating that the non-FM (disordered) shell increases in the presence of the gel medium. Moreover, the difference between the samples becomes even greater at room temperature, where the saturation magnetization decreases due to thermal effects (see Table 1). From this analysis, the effective FM volume (the size of the FM core of the nanoparticles) can be estimated by comparing *M*<sup>0</sup> with *MFe*, such that the fraction of the nanoparticle volume that remains, FM, can be estimated as:

$$f = \frac{M\_0}{M\_{Fe}} \,\prime \tag{3}$$

The very small FM volume fraction in the case of the VCL-PEGDA-MNPs-MWCNTs and VCL-PEGDA-MNPs-GO samples likely indicates that the presence of the hydrogel induces strong oxidation of the surface of the nanoparticles or some interdiffusion that corrodes the surface and reduces the magnetic moment. Moreover, the larger value of the spin-wave constant in these two samples is consistent with a more significant influence of surface effects [107], which produce a faster decrease in saturation magnetization when the temperature increases.

**Table 1.** Magnetic parameters for the three studied samples: saturation magnetization at *T*<sup>0</sup> = −273.15 ◦C (*M*<sup>0</sup> ), saturation magnetization *M<sup>S</sup>* at 20 ◦C, a fraction of the volume of the nanoparticles that remain ferromagnetic (f), and spin-wave constant (B).


#### *3.3. Optical Properties of Magnetic Nanocomposites*

UV-VIS absorbance spectra and photoluminescence emission were obtained. In the Supplementary Material (Figure S1a,b,d), we show the ZnMintPc-DMF and the nanocomposites VCL/PEGDA-ZnMintPc and VCL/PEGDA-MNPs-MWCNTs-ZnMintPc, respectively. We observed the presence of bands at 354 nm (Band of B or Soret), 616 nm, and 684 nm (B and Q), characteristic of the absorption spectra of ZnMintPc.

Furthermore, it can be seen that as the concentration of ZnMintPc increases, the absorption of the B and Q bands increases proportionally without their dislocation. This allows us to conclude that the Beer-Lambert law is fulfilled and there is no drug aggregation, indicating that N,N-Dimetilformamida (DMF) is a suitable solvent for this PS [108,109]. Furthermore, it is also concluded that the VCL-PEGDA hydrogel disperses PS similarly to DMF. In the Supporting Information (Figure S1c,d), the UV-VIS spectra of nanocomposites based on MNPs-MWCNTs (VCL-PEGDA-MNPs-MWCNTs and VCL-PEGDA-MNPs-MWCNTs-ZnMintPc) are presented, showing the band corresponding to MWCNTS at 265 nm, which is mentioned in the research by Wang et al., 2012 [110].

Figure S2a in the Supporting Information shows UV-VIS spectra of MNPs-GO, where GO shows a π→π\* transition of aromatic C–C bonds in the 230 nm band, which cannot be displayed, and a shoulder around 315 nm attributed to the *n*→π\* transitions of C=O bonds, as in Bera et al., 2017 [111]. The last band has undergone a hypsochromic shift due to functionalization with both MWCNTs and GO in the presence of an organic solvent (ammonium hydroxide used for MNPs syntheses), as described in the literature [112,113].

The calibration curve shown in Figure S3a was obtained from the Q band at 684 nm related to ZnMintPc and described in Figures S1 and S2, which is in a region of the spectra of interest for treatment in PDT. It can be observed that the higher the concentration of ZnMintPc in the nanocomposites (VCL-PEGDA-ZnMintPc, VCL-PEGDA-MNPs-GO-ZnMintPc, and VCL- PEGDA-MNPs-MWCNTs-ZnMintPc), the greater the intensity of the 684 nm band; but when comparing with the ZnMintPc dispersed in DMF, the latter shows the highest intensity in relation to all nanocomposites. This indicates that the VCL-PEGDA hydrogel adequately disperses the hydrophobic ZnMintPc PS in an aqueous solution, as shown in the literature [114–116]. It can be seen that the 684 nm absorption band of ZnMintPc (0.52 µM) decreases as the number of functionalized components increases, as presented in Figure S3b. The percentage of decrease for VCL/PEGDA-MNPs-MWCNTs-ZnMintPc nanocomposites is 38.2%, and for VCL/PEGDA-MNPs-GO-ZnMintPc it is 35.54%.

The temporal stability study of the VCL/PEGDA-ZnMintPc nanocomposites VCL/PEGDA-MNPs-MWCNTs-ZnMintPc and VCL/PEGDA-MNPs-GO-ZnMintPc, observed in Figure 4, was conducted at one, two and 24 h. In all three cases, as time passed, the intensity of the B band increased while the Q band decreased. In all three cases, as time passes, the intensity of the B band increased while the Q band decreased due to PS photobleaching without aggregation in 24 h.

The curves showing stability of the magnetics nanocomposites vs. time (Figure 5) indicates that the intensity of PS decays exponentially. The nanocomposites for 24 h were evaluated, and the nanocomposites based on GO and MWCNTs decreased the photobleaching rate of the nanocomposite. Table 2 shows the percentage decay of PS over time that is related to PS release. After 24 h, nanocomposites based on GO and MWCNT only allowed PS decays of 45.67% and 43.33% while, in the hydrogel, the PS decayed 56.24%. Photobleaching in VCL/PEGDA-MNPs-GO-ZnMintPc and VCL/PEGDA-MNPs-MWCNTs-ZnMintPc nanocomposites can be considered very successful.

#### *3.4. Photodynamic Analyses*

DPBF photobleaching was performed in VCL/PEGDA-MNPs-GO, VCL/PEGDA-MNPs-MWCNTs, VCL/PEGDA-MNPs-GO-ZnMintPc, and VCL/PEGDA-MNPs-MWCNTs-ZnMintPc, to evaluate the efficiency of singlet oxygen production of the nanocomposites using 630 nm light with an intensity of 65.5 mW·cm−<sup>2</sup> . In the Supporting Information (Figures S4 and S5), the decrease of each nanocomposite's 418 nm DPBF band as a function of time is shown. For the VCL/PEGDA-MNPs-GO (Figure S4a) and VCL/PEGDA-MNPs-MWCNTs (Figure S4b) nanocomposites, a slight decrease in the 418 nm band at an irradiation time of 29 and 35 min, respectively, was observed. On the other hand, for nanocomposites with the presence of PS (VCL/PEGDA-MNPs-GO-ZnMintPc, Figure S5a and VCL/PEGDA-MNPs-MWCNTs-ZnMintPc Figure S5b), rapid decay of the 418 nm

band was observed until all the DPBF present was photobleached in solution. Total photobleaching after 9 and 11 min of irradiation, respectively, was observed.

**Figure 4.** Stability curves over time for (**a**) VCL-PEGDA-ZnMintPc; (**b**) VCL/PEGDA-MNPs-MWCNTs-ZnMintPc and (**c**) VCL/PEGDA-MNPs-GO-ZnMintPc; ZnMintPc = 0.27 µM.

**Figure 5.** Decay curves of: VCL-PEGDA-ZnMintPc, VCL/PEGDA-MNPs-MWCNTs-ZnMintPc and VCL/PEGDA-MNPs-GO-ZnMintPc. ZnMintPc = 0.27 µM.


**Table 2.** Decay of ZnMintPc in nanocomposites in 24 h.

For each nanocomposite, absorbance vs. photoirradiation curves were constructed. Taking the 418 nm band of DPBF, an exponential decay fit to obtain the decay time presented in Figure 6a,b was made.

Figure 6a shows that the nanocomposites without PS (VCL/PEGDA-MNPs-GO and VCL/PEGDA-MNPs-MWCNTs) have long DPBF photobleaching times. The VCL/PEGDA-MNPs-GO photobleaching time was less than in VCL/PEGDA-MNPs-MWCNTs, indicating that GO has a better capacity to generate <sup>1</sup>O<sup>2</sup> by photooxidation of DBPF, as corroborated by the work of Romero et al., 2020 [11]. For nanocomposites with PS (VCL/PEGDA-MNPs-GO-ZnMintPc and VCL/PEGDA-MNPs-MWCNTs-ZnMintPc), curve fitting show photobleaching over two times. The first one indicates a rapid decay in the first 60 s of photoirradiation, and the second one indicates a slow decay from 60 s to 700 s of photoirradiation. Once again, slow decay results in longer photobleaching time, as seen in the purple curve in Table 3 for the MWCNT-based nanocomposite (1140 ± 260 s).

The presence of PS in the nanocomposites generates a rapid photooxidation of DPBF. Therefore, the results indicate that the photobleaching of DPBF was mainly due to PDT effects mediated by VCL/PEGDA-MNPs-GO-ZnMintPc and VCL/PEGDA-MNPs-MWCNTs-ZnMintPc nanocomposites, with the nanocomposite containing GO and the photosensitizing ZnMintPc exhibiting more significant singlet oxygen generation.

**Figure 6.** Decay curve of DPBF from (**a**) VCL/PEGDA-MNPs-GO and VCL/PEGDA-MNPs-MWCNTs; (**b**) VCL/PEGDA-MNPs-GO-ZnMintPc and VCL/PEGDA-MNPs-MWCNTs-ZnMintPc. DPBF = 18.5 mM, GO = 3.47 <sup>µ</sup>g·mL−<sup>1</sup> , MWCNTs = 3.47 <sup>µ</sup>g·mL−<sup>1</sup> , MNPs = 93.3 <sup>µ</sup>g·mL−<sup>1</sup> and ZnMintPc = 8.1 µM.


**Table 3.** Decay times of nanocomposites.

#### *3.5. Thermal Studies*

The thermal study curves in Figure 7 show that the solutions of MNPs, GO and MWCNTs (blue, violet and light blue, respectively) act as photothermal materials when irradiated with red light for about 100 min. They can reach temperatures between 50.8 and 54.8 ◦C, making them suitable for use in Photothermal Therapy due to their ability to convert red and near-infrared (NIR) light into heat, and to transport drugs, as mentioned in the literature [71,117–120]. In the thermal curves of Figure 7, it can also be observed that VCL/PEGDA, VCL/PEGDA-MNP-GO, and VCL/PEGDA-MNP-MWCNTs nanocomposites irradiated with red light for about 100 min show a slight decrease in temperature compared to the thermal curve of deionized water (reference), and around 80 min of irradiation, curves representing the presence of hydrogel reach temperatures close to deionized water (the light doses are shown in the Table 4).

**Figure 7.** Thermal studies. deionized water (control black line), VCL/PEGDA (red line), MNPs (blue line), GO (violet line), MWCNTs (light blue line), VCL/PEGDA-MNPs-GO (yellow line) and VCL/PEGDA-MNPs-MWCNTs (brown line). GO = 3.47 <sup>µ</sup>g·mL−<sup>1</sup> , MWCNTs = 3.47 <sup>µ</sup>g·mL−<sup>1</sup> , MNPs = 93.3 <sup>µ</sup>g·mL−<sup>1</sup> .

MNPs, GO, and MWCNTs solutions raised their temperature around 10 ◦C, relative to the control sample (deionized water) and VCL/PEGDA-MNP-GO and VCL/PEGDA-MNP-MWCNTs nanocomposites. It can be concluded that the VCL/PEGDA hydrogel can absorb a large amount of energy without increasing its temperature. This would be expected because its main components, VCL and PEGDA, have good calorific capacities [121–123]. Therefore the hydrogel would inherit this property, which explains why the nanocomposites covered by hydrogel maintain a temperature similar to the temperature of the control sample (deionized water) at an irradiation time of more than 100 min with red light. Around 30 to 45 min, all the nanocomposites reach a threshold temperature that did not change when irradiated for a longer time.


**Table 4.** Light dose applied in the nanocomposites.

## *3.6. Morphological Studies of Magnetic Nanocomposites*

The functionalized VCL/PEGDA-MNPs-GO-ZnMintPc and VCL/PEGDA-MNPs-MWCNTs-ZnMintPc nanocomposites were characterized by SEM, TEM, EDS, and XRD, and the results are presented in Figures 8b,e,f , S6, S9, S11 and S13 for GO-based nanocomposites, in Figures 8a,d, S7, S10, S12 and S14 for MWCNTs-based nanocomposites and in Figures 8c and S8 for free-standing MNPs.

**Figure 8.** (**a**) SEM and TEM images of purified MWCNTs; (**b**) TEM of GO; (**c**) SEM of MNPs. (**d**) TEM of VCL/PEGDA-MNPs-MWCNTs-ZnMintPc. (**e**) TEM of VCL/PEGDA-MNPs-GO-ZnMintPc; (**f**) XRD analysis of MNPs-GO.

The SEM and TEM images in Figure 8 show the morphology of (a) carbon nanotubes, that have the shape of fibers and in TEM their internal structure and walls, (b) the structure of large sheets of GO. Figure S6d presents the height profile of GO and indicates that

the thickness of the GO is roughly 2.8 nm. According to Sun et al., 2010 study [124], this indicates that the sheet is four-layered. Various GO sheet sizes are depicted, but the most common is roughly 2 µm. In Figure 8c, we can observe the MNPs with a spherical shape with an average size of ~72 nm. Figure 8d,e shows the morphology of the hydrogel coating MNPs-GO-ZnMintPc and MNPs-MWCNTs-ZnMintPc, so it is difficult to differentiate the structures covered by the hydrogel. In the Figure 8f, we present the XRD pattern for the MNPs-GO sample, where the diffraction peaks of the iron nanoparticles decorating the GO are observed at 2θ = 30.27◦ , 35.6◦ , 43.3◦ , 53.7◦ , 57.1◦ and 63.0◦ , indicating that the MNPs retain their original crystalline structure after functionalization, agreeing with the results of Amiri, Baghayeri, and Sedighi 2018; Cao et al., 2016 [125,126].

#### *3.7. Morphological Studies of Bacteria in Magnetic Nanocomposites*

To understand the interaction of magnetic nanocomposites with microorganisms, STEM images of *S. aureus* and *E. coli* bacteria were obtained in the presence of VCL/PEGDA-MNPs-GO-ZnMintPc and VCL/PEGDA-MNPs-MWCNT-ZnMintPc nanocomposites (Figure 9b,c,e,f). In the STEM image of *S. aureus* and *E. coli* pure (Figure 9a,d), their structure and morphology are not altered, and the membrane covers them without ruptures. When interacting with the VCL/PEGDA-MNPs-GO-ZnMintPc and VCL/PEGDA-MNPs-MWCNT-ZnMintPc nanocomposites, no changes were observed in the morphology of the bacteria (images taken 24 h after the bacterial-nanocomposite solutions were prepared) [127–129]. It was possible to observe how the nanocomposite can completely cover the microorganism, allowing the nanocomposites dispersed in the hydrogel to be photoexcited with the red-light source and cause microbial elimination.

**Figure 9.** STEM of (**a**) *S. aureus*; (**b**) *S. aureus* + C1 (VCL/PEGDA-MNPs-GO-ZnMintPc); (**c**) *S. aureus* + C2(VCL/PEGDA-MNPs-MWCNTs-ZnMintPc); (**d**) *E. coli*; (**e**) *E. coli* + C1; (**f**) *E. coli* + C2.

#### *3.8. Antimicrobial Effect of Magnetic Nanocomposites*

The antimicrobial effect of the VCL/PEGDA-MNPs-GO-ZnMintPc (C1), VCL/PEGDA-MNPs-MWCNTs-ZnMintPc (C2), MNPs-GO (C3), and MNPs-MWCNTs (C4) nanocomposites were evaluated while irradiating with 630 nm red light at 65 mW cm−<sup>2</sup> in the presence of the microorganisms *S. aureus*, *E. coli* and *C. albicans*.

The results obtained for each colony are shown in Figure 10. The histograms present the LOG (CFU mL−<sup>1</sup> ) evaluation standardized to 1. A concentration of 10<sup>7</sup> CFU mL−<sup>1</sup> for *S. aureus*, 10<sup>6</sup> CFU mL−<sup>1</sup> for *E. coli*, and 10<sup>5</sup> CFU mL−<sup>1</sup> for *C. albicans* was used. Results show that light alone cannot eliminate microorganisms (control+light samples), as in Figure 11 with *C. albicans*. To eliminate the microorganism, it was necessary to irradiate it in the presence of a nanocomposite (C1 + light or C2 + light).

**Figure 10.** PDT/PTT antimicrobial effect. Standardized result of LOG(UFC/mL) by (**a**) *S. aureus;* (**b**) *E. coli* and (**c**) *C. albicans* based in C1, C2, C3 and C4 nanocomposites. The concentration of MNPs in C1, C2, C3 and C4 was 93.3 <sup>µ</sup>g·mL−<sup>1</sup> . The concentration of ZnMintPc in C1 and C2 was 8.1 µM. The concentration of GO in C1 and C3 was 3.47 <sup>µ</sup>g·mL−<sup>1</sup> . The concentration of MWCNTs in C2 and C4 was 3.47 <sup>µ</sup>g·mL−<sup>1</sup> . Time of irradiation of C1 and C2 nanocomposites was 30 min and for C3 and C4 was 40 min. Significant differences in means according to the Tukey test (\*\*\* *p* ≤ 0.001).

**Figure 11.** Antimicrobial effect of C1 and C2 nanocomposites at *C. albicans* in vitro. C1: GO-MNPs-VGLPEGDA- ZnMintPc, C2: MWCNT-MNPs-VGLPEGDA- ZnMintPc, C3: GO-MNPs-VGLPEGDA, C4: MWCNT-MNPs-VGLPEGDA. The concentration of MNPs in C1, C2, C3 and C4 was 93.3 μg·mL−1. The concentration of ZnMintPc in C1 and C2 was 8.1 μM. The concentration of GO in C1 and C3 was 3.47 μg·mL−1. The concentration of MWCNTs in C2 and C4 was 3.47 μg·mL−1. **Figure 11.** Antimicrobial effect of C1 and C2 nanocomposites at *C. albicans* in vitro. C1: GO-MNPs-VGLPEGDA- ZnMintPc, C2: MWCNT-MNPs-VGLPEGDA- ZnMintPc, C3: GO-MNPs-VGLPEGDA, C4: MWCNT-MNPs-VGLPEGDA. The concentration of MNPs in C1, C2, C3 and C4 was 93.3 <sup>µ</sup>g·mL−<sup>1</sup> . The concentration of ZnMintPc in C1 and C2 was 8.1 µM. The concentration of GO in C1 and C3 was 3.47 <sup>µ</sup>g·mL−<sup>1</sup> . The concentration of MWCNTs in C2 and C4 was 3.47 <sup>µ</sup>g·mL−<sup>1</sup> .

**Figure 10.** PDT/PTT antimicrobial effect. Standardized result of LOG(UFC/mL) by (**a**) *S. aureus;* (**b**) *E coli* and (**c**) *C. albicans* based in C1, C2, C3 and C4 nanocomposites. The concentration of MNPs in C1, C2, C3 and C4 was 93.3 μg·mL−1,. The concentration of ZnMintPc in C1 and C2 was 8.1 μM. The concentration of GO in C1 and C3 was 3.47 μg·mL−1. The concentration of MWCNTs in C2 and C4 was 3.47 μg·mL−1. Time of irradiation of C1 and C2 nanocomposites was 30 min and for C3 and C4

was 40 min. Significant differences in means according to the Tukey test (\*\*\* *p* ≤ 0.001).

In Figure 10c, the samples without hydrogel C3 + light and C4 + light only had a complete response with *C. albicans,* and it could be considered that the photothermic effect allowed microbial elimination. This is in agreement with dos Santos et al., 2019 [132]. This conclusion is based on the results shown in Figure 7 of the photothermal effect for com-The quantitative result of colony counts of *S. aureus*, *E. coli*, and *C. albicans* after being irradiated with a red LED (630 nm 65 mW cm−<sup>2</sup> ) indicates that for the colonies of *S. aureus* and *E. coli* (Figure 10a,b), samples C3, C4 in light and dark, were not different from the control group. In contrast, the count in samples C1 + light and C2 + light was significantly lower than the control group (\*\*\* *p* ≤ 0.001). The colony count of *C. albicans* in the samples + dark was not different from the control group, but the count in the samples C1 + light, C2 + light, C3 + light, and C4 + light was significantly lower than the control group (\*\*\* *p* ≤ 0.001). This means that after irradiation, the C1 nanocomposite eliminated all *E. coli*, and *C. albicans*, and some *S. aureus*. C2 nanocomposite eliminated *S. aureus*, *E. coli*, and *C. albicans*. C3 and C4 nanocomposites eliminated all *C. albicans*. Therefore, all nanocomposites can eliminate some of the microorganisms used in this study, with C2 being the best due to its ability to eliminate the three types of microorganisms. This agrees with the results obtained by Huo et al., 2021; Liu et al., 2021 and Ren et al., 2020 [129–131].

In Figure 10c, the samples without hydrogel C3 + light and C4 + light only had a complete response with *C. albicans*, and it could be considered that the photothermic effect allowed microbial elimination. This is in agreement with dos Santos et al., 2019 [132]. This conclusion is based on the results shown in Figure 7 of the photothermal effect for compounds based on MWCNT, MNP, GO, where it is observed that their temperature increased as the irradiation time increased. For the samples with hydrogel and photosensitizer (C1 + light and C2 + light), it can be concluded that the elimination of the microorganisms *S. aureus* (Figure 10a), *E. coli* (Figure 10b), and *C. albicans* (Figure 10c) was produced by the photodynamic effect, as supported by the research of Mei et al., 2021 [12] as well as the data reported by Xu, Yao and Xu 2019 [52]. This conclusion is based on the results in Table 3 that show that compounds containing ZnMintPc have a longer decay period than those which do not.

According to the results obtained, the VCL/PEGDA-MNPs-GO-ZnMintPc and VCL/ PEGDA-MNPs-MWCNTs-ZnMintPc nanocomposites have a ferromagnetic character, typical of nanocomposites with iron nanoparticles and with low saturation magnetization, due to being covered by a diamagnetic hydrogel layer, in agreement with the studies carried out by Donadel et al., 2008, Mahdavi et al., 2013, and Qu et al., 2010 [133–135]; in which MNPs were synthesized for bioapplications and it was demonstrated that the surface modification caused a reduction in saturation magnetization, with values between 67 to 22 emu·g −1 depending on the type of biopolymer to be used.

Due to the physical and magnetic properties of these nanocomposites, it was shown that they could avoid the early extinction of the fluorescence of PS ZnMintPc, thus improving their photodynamic effect, as mentioned in the work of Huang et al., 2011 and Xiao et al., 2021. The thermodynamic studies carried out by Kou et al., 2019, Srivastava and Kumar, 2010, and Tager et al., 1993 indicate that VCL and PEGDA have good heat capacities of around 258 and 94 J·mol−<sup>1</sup> ·K−<sup>1</sup> , which is why they are usually used to synthesize

cryogels. This explains that the nanocomposites covered by the VCL/PEGDA hydrogel are capable of being maintained up to a maximum temperature of 40 ◦C receiving doses of red light of up to 393 J·cm−<sup>2</sup> , which in comparison to nanocomposites without hydrogel, raise their temperature to around 55 ◦C, as described in the literature [71,117–120].

The internalization mechanism of these nanocomposites occurs through direct contact between the microorganism-nanocomposite and the PTT/PDT effect, as seen in Figure 9. Several authors have discussed the mechanism of programmed cell death due to the PDT/PTT effect, and as mentioned by Buzzá et al., 2021, Patil et al., 2021, among others in their scientific articles, the ability of PS, GO, and MWCNT to locate in various organelles and the action of the PTT/PDT effect promotes ROS generation followed by physical damage to the membrane. Oxidative stress leads to changes in calcium and lipid metabolism, generating cytokines and stress response mediators that lead to induction of apoptosis by the mitochondrial pathway and specific protein oxidation [136–140]. It can be concluded that this mechanism is the cause of the death of the microorganisms *S. aureus*, *E. coli*, and *C. albicans* in the present study. The results shown in Figure 10c indicate the elimination of *C. albicans* by the PTT effect and, as mentioned by Mocan et al., 2014, and Pérez-Hernández et al., 2015 in their research, PTT causes apoptosis, or programmed cell death, rather than necrotic cell death, by activating the intrinsic route. Inflammatory reactions are triggered by necrotic cell death, carbon-based nanomaterials in the photothermal treatment activate the flux of free radicals within the cell, and the oxidative state mediates cellular damage in PC cells via apoptotic pathway [141,142]. Therefore, it can be concluded that this is the mechanism that causes the death of *C. albicans* when there is no presence of PS.

The excretion pathway of these nanocomposites in living systems has been investigated by authors such as Dias et al., 2021 and Liu et al., 2008, who indicate that these nanocomposites based on carbon materials and MNPs can be excreted through the biliary and urinary tracts [143,144].

#### **4. Conclusions**

The present study involved synthesis of magnetic nanocomposites VCL/PEGDA-MNPs-MWCNTs-ZnMintPc and VCL/PEGDA-MNPs-GO-ZnMintPc to eliminate three types of microbial colonies: *S. aureus*, *E. coli* and *C. albicans*.

After these nanocomposites were synthesized, optical, magnetic, and morphological characterizations showed that GO, MWCNTS, iron MNPS and ZnMintPc are covered by VCL/PEGDA hydrogel.

The optical properties of these nanocomposites allow them to prevent the rapid disintegration of PS, which is essential in PDT.

Using photodynamic analysis, the nanocomposites the applicability in PDT of VCL/ PEGDA-MNPs-MWCNTs-ZnMintPc and VCL/PEGDA-MNPs-GO-ZnMintPc were with low dose red light. It was observed that the nanocomposite VCL/PEGDA-MNPs-GO- Zn-MintPc had higher efficiency than the nanocomposite based on MWCNTs since it produced faster photobleaching of DPBF because it is capable of transporting a more significant amount of ZnMintPc and MNPs due to its large specific area. In addition, GO contributed to PS in the formation of <sup>1</sup>O2. Since the nanocomposites are coated with a hydrogel, they are also suitable for controlled PS release systems, with promising applications for PDT.

The nanocomposite that contains GO and PS ZnMintPc had higher efficiency since it produced faster photobleaching of DPBF because it is capable of transporting a more significant amount of ZnMintPc and MNPs due to its large specific area. In addition, the GO contributed to PS in the formation of <sup>1</sup>O2.

Finally, we demonstrated that the VCL/PEGDA-MNPs-GO-ZnMintPc nanocomposite was able to eliminate colonies of *E. coli* and *C. albicans*, and the VCL/PEGDA-MNPs-MWCNTs-ZnMintPc nanocomposite eliminated the three types of microorganisms, which can therefore be considered as a broad-spectrum antimicrobial agent in PDT and PTT.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/pharmaceutics14040705/s1, Figure S1. Optical properties: (a) UV-VIS spectra of DMF(3mL) with different concentrations of ZnMintPc, (b) UV-VIS spectra of VCL/PEGDA(3mL) with different concentrations of ZnMintPc, (c). UV-VIS spectra of VCL/PEGDA (3mL) with MNPs-MWCNTs at different concentrations. (d). VCL/PEGDA with MNPs-MWCNTs and ZnMintPc at different concentrations; Figure S2. Optical properties of Magnetic Nanocomposites: (a) UV-VIS spectra of VCL/PEGDA (3mL) with different concentrations of MNPs-GO, (b) UV-VIS spectra of VCL/PEGDA (3mL) with MNPs-GO-ZnMintPc at different concentrations; Figure S3. (a) Composites calibration curve of ZnMintPc (0.52 µM) from: PS in DMF, VCL/PEGDA, VCL/PEGDA-MNPs-GO and VCL/PEGDA-MNPs-MWCNTs; (b) Absorbance of ZnMintPc-DMF and nanocomposites; Figure S4. Photodynamic Analyses of: (a) UV-VIS spectra of VCL/PEGDA-MNPs-GO and (b) UV-VIS spectra of VCL/PEGDA-MNPs-MWCNTs; Figure S5. Photodynamic Analyses of: (a) UV-VIS spectra of VCL/PEGDA-MNPs-GO-ZnMintPc and (b) UV-VIS spectra of VCL/PEGDA-MNPs-MWCNTs-ZnMintPc; Figure S6: (a) SEM analysis, (b) TEM analysis, (c) Atomic force microscopy (AFM) image (d)Height profile along the line of the panel from GO (e) EDS analysis and (f) XRD of GO; Figure S7. (a) SEM, (b) TEM, (c) EDS and (d) XRD analysis of MWCNTs; Figure S8. (a) SEM, (b) TEM, (c) EDS and (d) XRD analysis of MNPs; Figure S9. (a) SEM, (b) TEM, (c) EDS and (d) XRD analysis of MNPs-GO; Figure S10. (a) SEM, (b) TEM, (c) EDS and (d) XRD analysis of MNPs-MWCNTs; Figure S11: (a) SEM, (b) TEM, (c) EDS analysis of VCL/PEGDA-MNPs-GO; Figure S12: (a) SEM, (b) TEM, (c) EDS analysis of VCL/PEGDA-MNPs-MWCNTs; Figure S13. (a) SEM, (b) TEM, (c) EDS and (d) XRD analysis of VCL/PEGDA-MNPs-GO-ZnMintPc; Figure S14. (a) SEM, (b) TEM, (c) EDS and (d) XRD analysis of VCL/PEGDA-MNPs-MWCNTs-ZnMintPc.

**Author Contributions:** Validation, C.F.C.; formal analysis, C.F.C. and M.P.R.; investigation, C.F.C., A.D.-B., K.O.C., E.C.R., F.Q., S.N., A.D., K.V., D.N., C.E.Á., M.A.S., C.G.-R., A.B.-Z., M.R.R. and M.P.R.; resources, F.Q., M.R.R. and M.P.R.; data curation, C.F.C.; writing—original draft preparation, C.F.C.; writing—review and editing, C.F.C., A.D.-B., E.C.R., S.N., A.D., D.N., C.G.-R. and M.P.R.; visualization, C.F.C.; supervision, M.P.R.; project administration, M.P.R.; funding acquisition, M.R.R. and M.P.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** National Polytechnic School through the PIIF-20-05 project. Research Office of the Pontificia Universidad Católica del Ecuador project code QINV0324-IINV529010100.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** Zinc Menthol-Phthalocyanine was given by the Federal University of São Carlos-Brazil. To student Carlos Moscoso for helping with experiments in micro-organisms. To the National Polytechnic School through the PIIF-20-05 project. To the Research Office of the Pontificia Universidad Católica del Ecuador project code QINV0324-IINV529010100.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Parietin Cyclodextrin-Inclusion Complex as an Effective Formulation for Bacterial Photoinactivation**

**Abdallah Mohamed Ayoub 1,2,†, Bernd Gutberlet 1,†, Eduard Preis <sup>1</sup> , Ahmed Mohamed Abdelsalam 1,3 , Alice Abu Dayyih <sup>1</sup> , Ayat Abdelkader 1,4, Amir Balash <sup>5</sup> , Jens Schäfer <sup>1</sup> and Udo Bakowsky 1,\***


**Abstract:** Multidrug resistance in pathogenic bacteria has become a significant public health concern. As an alternative therapeutic option, antimicrobial photodynamic therapy (aPDT) can successfully eradicate antibiotic-resistant bacteria with a lower probability of developing resistance or systemic toxicity commonly associated with the standard antibiotic treatment. Parietin (PTN), also termed physcion, a natural anthraquinone, is a promising photosensitizer somewhat underrepresented in aPDT because of its poor water solubility and potential to aggregate in the biological environment. This study investigated whether the complexation of PTN with (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) could increase its solubility, enhance its photophysical properties, and improve its phototoxicity against bacteria. At first, the solubilization behavior and complexation constant of the PTN/HP-β-CD inclusion complexes were evaluated by the phase solubility method. Then, the formation and physicochemical properties of PTN/HP-β-CD complexes were analyzed and confirmed in various ways. At the same time, the photodynamic activity was assessed by the uric acid method. The blue light-mediated photodegradation of PTN in its free and complexed forms were compared. Complexation of PTN increased the aqueous solubility 28-fold and the photostability compared to free PTN. PTN/HP-β-CD complexes reduce the bacterial viability of *Staphylococcus saprophyticus* and *Escherichia coli* by > 4.8 log and > 1.0 log after irradiation, respectively. Overall, the low solubility, aggregation potential, and photoinstability of PTN were overcome by its complexation in HP-β-CD, potentially opening up new opportunities for treating infections caused by multidrug-resistant bacteria.

**Keywords:** physcion; hydroxypropyl-β-cyclodextrin; photosensitizer; antimicrobial photodynamic therapy

## **1. Introduction**

The overuse of antibiotics has resulted in inexorable antibiotic resistance, which worsens day by day, extending hospital admissions, raising healthcare costs, and eventually leading to increased death rates. In 2020, more than 670,000 people were infected in Europe with antibiotic-resistant bacteria, with approximately 33,000 deaths. The total economic burden of antibiotic resistance in Europe is expected to be 1.1 billion Euros [1]. The loss

**Citation:** Ayoub, A.M.; Gutberlet, B.; Preis, E.; Abdelsalam, A.M.; Abu Dayyih, A.; Abdelkader, A.; Balash, A.; Schäfer, J.; Bakowsky, U. Parietin Cyclodextrin-Inclusion Complex as an Effective Formulation for Bacterial Photoinactivation. *Pharmaceutics* **2022**, *14*, 357. https://doi.org/ 10.3390/pharmaceutics14020357

Academic Editor: Nejat Düzgüne¸s

Received: 20 December 2021 Accepted: 1 February 2022 Published: 4 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in economic output due to illness or death-related infection was estimated to surpass 1 trillion USD per year after 2030 and approach 2 trillion USD annually by 2050, accounting for a total of 100 trillion USD decline in global production between 2014 and 2050 [2]. Even worse, the number of deaths caused by worldwide antibiotic resistance is expected to increase to 10 million per year by 2050, up from 700,000 in 2014 [3]. Development of antibiotic resistance results in higher costs because of switching to more expensive antibiotics [4]. Multiple reasons, scientific, economic, and regulatory, hinder the development of new antibiotics [5]. Moreover, other drugs for chronic diseases are more appealing to pharmaceutical companies than short-course antibiotics. Consequently, approvals of new systemic antibiotics by the US Food and Drug Administration have dropped by 90% in the last 30 years [5]. Thus, alternative treatment strategies against resistant bacteria are urgently sought.

Photodynamic therapy (PDT) against cancer cells or antimicrobial photodynamic therapy (aPDT) have advantages over traditional treatments, e.g., minimal invasiveness and excellent safety profiles [6]. PDT and aPDT for selective damage to the area of interest depend on three nontoxic components: a photosensitizer (PS), light, and oxygen [7]. They are two-step procedures in which a photosensitizer (PS) is administered to patients, followed by light irradiation at a specific wavelength matching the absorbance spectra of the PS. Once exposed to light, the PS is excited from its low-energy ground state into a high-energy singlet state which undergoes intersystem crossing to the longer-lived triplet state [6]. Via type I and type II mechanisms, the PS in the triplet state can generate reactive oxygen species (ROS) such as singlet oxygen superoxide anions and hydroxyl radicals, which are the leading cause of cell death [8].

Unlike traditional antibiotics, which have a particular target known as the key-hole principle, this mechanism singles out aPDT as a multi-target procedure leaving no room for resistance development [9]. Additionally, aPDT does not necessitate a specific extracellular or intracellular localization of the PS to exert its damaging effect [9]. As a result, bacteria are unlikely to establish antibiotic resistance against this multi-targeted approach even after repeated administration [8]. Furthermore, such a broad-spectrum activity against gram-positive and gram-negative bacteria, fungi, viruses, and protozoa is beneficial in the empirical therapy of undiagnosed infections. Another advantage of aPDT is that fastgrowing cells such as bacteria can accumulate PSs at a higher rate, resulting in increased selectivity [10]. This is especially harmful to bacteria, as their DNA is not isolated from the cytoplasm like that of eukaryotic cells [11]. Additionally, the photodestructive effect of aPDT can be physically controlled in practice by local light applications. The photoantimicrobial effect is much faster than traditional therapies that can take days to be effective [12]. Moreover, the generated ROS during PDT can chemically oxidize the virulence factors such as lipopolysaccharide, protein toxins, proteases, and α-hemolysin [11]. Although extensive work has been performed in aPDT, only three photosensitizers (methylene blue, toluidine blue O, and indocyanine green) have received clinical approval in dentistry so far [12].

Parietin (PTN), also termed physcion, is an anthraquinone naturally present as a secondary metabolite in lichens (e.g., *Xanthoria parietina* [13]), in other fungi (e.g., *Aspergillus*, *Penicillium* [14]), and also in plants (e.g., *Rheum*, *Rumex*, and *Ventilago* [14]). The UV protectant role of PTN has been detected in lichens adapted to UV stressed environments, whereby the mycobiont synthesizes PTN to protect the photobiont against oxidation mediated by excessive solar radiation [14,15]. *Xanthoria parietina* has been used in traditional medicine for kidney problems and menstrual disorders and as a painkiller [16]. Although PTN offers several activities, including antibacterial and antifungal [14], and has antitumoral [17] and other photosensitizing properties [18,19], it was studied only in organic solvents and is not applicable in clinical use. Like many lipophilic PSs, PTN aggregates unduly in the biological environment, losing its singlet oxygen quantum yield and limiting its routine use. To solve these drawbacks, we recently reported the encapsulation of PTN into liposomes to promote its aqueous solubility and stability in the biological milieu and enhance selectivity and delivery to the targeted cells [19]. However, its high hydrophobicity

does not favor entrapping a higher amount in the lipid bilayer. Such a problem can be addressed with cyclodextrin (CD) complexation to increase the inclusion capacity of PTN in the delivery system.

CD is widely utilized as a typical solubilizer for hydrophobic drugs to enhance their aqueous solubility and chemical stability with a high loading capacity and a straightforward procedure [20]. CD can enclose the hydrophobic drug in its central cavity to form a host-guest complex or supramolecular species without altering its framework structure, while the outer surface is still hydrophilic to ensure water solubility [11]. CD is a cyclic biocompatible oligosaccharide structurally composed of 6–8 D-glucopyranose monomers connected by α-1,4-glucose bonds [11]. According to the number of glucose units, the natural CD may differ in water solubility and hydrophobic cone dimension available for drug accommodation. There are different varieties of CD, such as α-CD (six glucose units with a cavity volume of 0.174 nm<sup>3</sup> ), β-CD (seven glucose units with a cavity volume of 0.262 nm<sup>3</sup> ), or γ-CD (eight glucose units with a cavity volume of 0.427 nm<sup>3</sup> ) [21]. However, natural CD, particularly β-CD, can damage the renal tubule either by microcrystalline precipitation in the kidney because of lower water solubility or as a cyclodextrin/cholesterol complex [20]. Therefore, the natural CDs were chemically modified to enhance their aqueous solubility and decrease nephrotoxicity. Among them, hydroxypropyl-β-CD (HP-β-CD) is the most notable derivative, with a greater water solubility of 60% (*w/w*) compared to 2% for its parent form, β-CD [20]. Owing to its biocompatibility and minimal toxicity, HP-β-CD has long been used as a delivery system in PDT for many photosensitizers [11] such as hypericin [10], chlorophyll a [22], aminolevulinic acid [23], temoporfin [24], chlorin e6 [25], and curcumin [26]. These studies showed enhanced water solubility and delivery to the targeted tissues without significantly changing their photophysical properties. To the best of our knowledge, no work has investigated the inclusion of PTN in any cyclodextrin complex.

Various mechanisms of aPDT have already been investigated, but its success depends mainly on the radiation strength and radiant exposure, or the overall dosimetry [27]. Differences among the used photosensitizers require a multifactorial concept in dosimetry that should always be considered [28]. PTN by itself shows a concentration-dependent antimicrobial effect during irradiation and in the dark [17]. Free PTN is not suitable for therapy because of its low water solubility and poor bioavailability, which can be overcome by water-soluble PTN/HP-β-CD complexes. Hegge et al. showed an advantage of their cyclodextrin conjugate in terms of thermal stability, photostability, and easier solubilization than the ethanolic solution [29]. The conjugation to cyclodextrin results in a slightly reduced affinity of photosensitizers for gram-negative bacteria, which is compensated by the improved solubility, availability, and efficiency of singlet oxygen generation [30]. Furthermore, targeting moieties can easily be incorporated into the complex to improve selectivity [31,32].

This study aimed to improve the aqueous solubility of PTN, simultaneously maintaining its photoactivity, to obtain a promising candidate for further use in PDT. The solubility behavior was studied at different concentrations of HP-β-CD. Additionally, the complex formation (PTN/HP-β-CD) was evidenced by various characterizations, such as proton nuclear magnetic resonance (1H NMR), Fourier-transform infrared spectroscopy (FT-IR), powder x-ray diffraction (PXRD), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). PTN delivery with minimal loss of photoactivity was evaluated by the uric acid method assessing the photodynamic activity of PTN/HP-β-CD complexes in water. Furthermore, the photodegradation profile of PTN/HP-β-CD complexes upon exposure to blue LED irradiation was monitored and compared to that of free PTN in ethanol. The photodynamic activity of PTN/HP-β-CD complexes was tested for the first time against gram-positive and gram-negative bacterial strains.

#### **2. Materials and Methods**

### *2.1. Materials*

Parietin (PTN, purity >98%) was purchased from Cayman (Hamburg, Germany). 2-hydroxypropyl-β-cyclodextrin (HP-β-CD, average MW = 1483 g/mol) was obtained from Sigma-Aldrich (Taufkirchen, Germany). Ultrapure water was generated by PURELAB flex 4 (ELGA LabWater, High Wycombe, UK) and used for all experiments in this study. All other chemicals and solvents were of analytical grades and used as received.

#### *2.2. Bacterial Strains and Media*

Glycerol stock cultures of *Staphylococcus saprophyticus* subsp. *bovis* (*S. saprophyticus*, DSM 18669, DSMZ, Braunschweig, Germany) and *Escherichia coli* DH5 alpha (*E. coli*, DSM 6897, DSMZ, Braunschweig, Germany) were prepared and stored at −80 ◦C. The stocks were thawed one day before the bacterial viability assay and cultured in Mueller Hinton broth (MHB, Sigma Aldrich Chemie) on an orbital shaker (Compact Shaker KS 15 A, equipped with Incubator Hood TH 15, Edmund Bühler, Bodelshausen, Germany) set at 200 rpm and 37 ◦C.

#### *2.3. Light Source*

The irradiation experiment was performed with a prototype low-power LED device consisting of an array of light-emitting diodes custom-made by Lumundus GmbH (Eisenach, Germany). The device can emit light at wavelengths of 457 nm and 652 nm for blue and red regions, respectively. The actual light dose (J/cm<sup>2</sup> ) = Irradiance (W/cm<sup>2</sup> ) × irradiation time (in sec) [33].

#### *2.4. Stoichiometry: Job's Plot*

The continuous variation technique (Job's plot) was employed to determine the complex stoichiometry [34]. Briefly, equimolar stock solutions of PTN (100 µM in ethanol) and HP-β-CD (100 µM in water) were mixed at different ratios (1:9; 2:8; 3:7, and so on), maintaining a final volume of 10 mL to get different mole fractions of PTN from 0 to 1, while the total concentration of PTN and HP-β-CD remained constant. The suspensions were then stirred overnight on a magnetic stirrer at 200 rpm (IKA RT 15, IKA-Werke, Staufen, Germany). Any insoluble materials were removed by centrifugation of the suspension (16,800× *g* for 15 min) (Centrifuge 5418, Eppendorf, Hamburg, Germany) and filtration of the supernatant (0.45 µm nylon filter, Pall Corporation, New York, NY, USA). The amount of PTN solubilized in the complex was estimated spectrophotometrically at λ = 434 nm and was plotted (∆*A* × *R*) against the mole fraction (*R*) of PTN, where ∆*A* denotes the difference in absorbance in the absence and presence of HP-β-CD:

$$R = \frac{[PTN]}{[PTN] + [HP \cdot \beta \text{-CD}]} \tag{1}$$

### *2.5. Phase Solubility Study*

A phase solubility study was performed in water at 25 ◦C according to the method described by Higuchi and Connors [35]. A known excess of PTN was added to vials containing different concentrations of HP-β-CD (0.28 mM-35.7 mM), and the suspensions were stirred at 25 ◦C for 48 h on a magnetic stirrer at 200 rpm (IKA RT 15, IKA-Werke, Staufen, Germany). Uncomplexed PTN was removed by centrifugation at 16,800× *g* for 15 min (Centrifuge 5418, Eppendorf, Hamburg, Germany), and PTN concentration was measured spectrophotometrically at λ = 434 nm (UV mini-1240, Shimadzu, Kyoto, Japan). ¯ The phase solubility graph was constructed by plotting PTN concentration (mM) against HP-β-CD concentration, and the apparent stability constant (KS) was calculated according to the following equation.

$$K\_S = \frac{Slope}{\left[S\_0(1 - Slope)\right]} \tag{2}$$

*S*<sup>0</sup> is the intrinsic solubility of PTN in water as measured in our study to be 0.17 µg/mL.

#### *2.6. Preparation of PTN/HP-β-CD Complexes and Physical Mixture*

PTN/HP-β-CD inclusion complexes were prepared by the freeze-drying method [10,34]. Briefly, PTN and HP-β-CD (at a molar rate of 1:1) were dissolved in ethanol and ultrapure water, respectively. PTN was added stepwise to the HP-β-CD aqueous solution placed on a magnetic stirrer at 200 rpm and 25 ◦C for 48 h (IKA RT 15, IKA-Werke, Staufen, Germany). Insoluble PTN was removed by centrifugation at 16,800× *g* for 15 min and filtration (0.45 µm nylon filter, Pall Corporation, New York, USA). The obtained solution was then lyophilized in a freeze-dryer (Christ Alpha 1-4 LSC, Martin Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany). The physical mixture was prepared by mixing PTN and HP-β-CD (in a 1:1 molar ratio) in a porcelain mortar with grinding for 15 min and then stored in a desiccator until further analysis.

### *2.7. Characterization of PTN/HP-β-CD Complexes*

## 2.7.1. <sup>1</sup>H NMR Spectroscopy and 2D ROESY

<sup>1</sup>H NMR spectroscopic measurements were recorded by an NMR spectrometer equipped with an auto-tune sample head (JEOL ECX-400 Nuclear Magnetic Resonance Instrument, JEOL, Akishima, Japan). Samples were prepared by dissolving an equivalent amount of free and complexed PTN in a suitable NMR solvent (DMSO-d6). Samples were then transferred into NMR tubes and assessed through several scanning cycles fixed to a minimum of 64 scans. The results were processed by MNOVA software (version 14.2.1, Mestrelab Research S.L., Santiago de Compostela, Spain).

#### 2.7.2. FT-IR Analysis

The FT-IR measurements were performed by a Brucker α-alpha FT-IR instrument (Bruker Optic, Ettlingen, Germany) with an attenuated total internal reflectance diamond crystal. The neat solid samples were placed directly on the diamond crystal, after which the adjustable pressure arm was positioned over the sample to press it gently. FT-IR spectra were recorded in the transmission mode from 4000 to 400 cm−<sup>1</sup> [36].

#### 2.7.3. Powder X-ray Diffraction (PXRD)

The formation of PTN/HP-β-CD complexes was confirmed by XRD, determining its crystallinity after inclusion. XRD patterns of PTN/HP-β-CD complexes, the corresponding physical mixture, and the individual solid components were recorded with CuK α radiation (λ = 1.7903 Å) at a voltage of 40 kV and 35 mA current (X'Pert Pro MDP X-ray powder diffractometer, PANalytical, Almelo, Netherlands). Samples were scanned at room temperature from 2θ = 10◦ to 2θ = 60◦ with a step of 0.03◦/min.

#### 2.7.4. Scanning Electron Microscopy (SEM)

The surface morphology of the PTN/HP-β-CD inclusion complexes was investigated by SEM (Hitachi S-510, Hitachi-High Technologies Europe, Krefeld, Germany) equipped with a secondary electron detector. Briefly, the individual components (pure PTN, HP-β-CD), their physical mixture, and PTN/HP-β-CD inclusion complexes were mounted on an aluminum pin stub by conductive double-sided adhesive carbon tabs. The powders were sputter-coated thrice with a thin layer of gold (10 mA for 1 min) in an Edwards S150 Sputter Coater (Edwards Vacuum, Crawley, UK). The samples were visualized at an acceleration voltage of 10 kV.

#### 2.7.5. Differential Scanning Calorimetry (DSC)

The thermal behavior of the obtained complex and its pure substances were assessed by DSC measurements (DSC-7, Perkin Elmer, Rodgau, Germany). Briefly, accurately weighted amounts of the solid materials were filled into aluminum pans and heated over the temperature range 20–300 ◦C at a rate of 10 ◦C min−<sup>1</sup> under a nitrogen purge for cooling. In parallel, an empty pan sealed in the same way was served as a reference.

#### 2.7.6. UV/Vis Absorption Spectroscopy

The absorption spectrum of PTN/HP-β-CD inclusion complexes (80 µg/mL) was obtained from λ = 200 to 700 nm by a UV/Vis spectrophotometer (Multiskan GO, Thermo Scientific, Waltham, MA, USA) and compared with that of free PTN in ethanol. The absorbance spectra were recorded using an aqueous solution of HP-β-CD or pure ethanol as background references for PTN/HP-β-CD complexes and free PTN, respectively.

#### 2.7.7. Singlet Oxygen Quantum Yield

The photodynamic activity of PTN/HP-β-CD complexes was evaluated by analyzing the singlet oxygen generation (1O2) using uric acid (UA), and as previously mentioned, using rose bengal (RB) as a standard photosensitizer with a reported singlet oxygen quantum yield of 0.75 in water [19]. Briefly, uric acid (100 µM) was mixed with PTN (10 µg/mL) and irradiated 5 times for 1 min by a blue LED (λirr = 457 nm, irradiance = 220.2 W/m<sup>2</sup> , Lumundus, Eisenach, Germany). The absorption spectra of UA were recorded after each irradiation procedure by a UV-Vis spectrophotometer (Multiskan GO, Thermo Scientific, Waltham, MA, USA). The normalized UA absorbance at λ = 296 nm was plotted versus the irradiation time (in seconds), and the decomposition rate constant of uric acid was calculated to quantify the singlet oxygen quantum yield using the following equation.

$$
\Phi[^{1}\text{O}\_{2}]\_{PTN} = \Phi[^{1}\text{O}\_{2}]\_{RB} \xrightarrow[K\_{RB}\text{F}\_{PTN}]{K\_{PTN}} \tag{3}
$$

Φ[ <sup>1</sup>*O*2]*PTN* and <sup>Φ</sup>[ <sup>1</sup>*O*2]*RB* are the singlet oxygen quantum yields of PTN and RB, respectively. kPTN and kRB are the rate constants of uric acid degradation by PTN and RB, respectively. F is the absorption correction factor given by F = 1 <sup>−</sup> <sup>10</sup>–OD (OD at the irradiation wavelength).

#### 2.7.8. Photostability of Inclusion Complexes

The effect of complexation on photodegradation of PTN was studied, and the degradation profile was compared with that of free PTN in ethanol. Solutions of either PTN/HPβ-CD complexes in water or free PTN in ethanol (100 µg/mL) were irradiated by an LED device (λirr = 457 nm, irradiance = 220.2 W/m<sup>2</sup> ) at 5 min interval with a total irradiation time of 30 min. After each irradiation time, the absorbance spectra were measured at λmax = 434 nm (Multiskan GO, Thermo Scientific, Waltham, MA, USA). The absorbance spectra were normalized for better comparison, and PTN's remaining percentage was calculated and compared with free PTN [37].

#### *2.8. Bacterial Viability Assay*

The antibacterial activity was determined by incubating the formulations with the bacterial suspensions and irradiating the samples. Both microorganisms (*S. saprophyticus* and *E. coli*) were treated equally, and similar bacterial densities were used. The overnight cultures were diluted to an optical density (OD600) of 0.025 measured by a spectrophotometer (Shimadzu UV mini-1240, Kyoto, Japan). These suspensions were placed in an orbital ¯ shaker (Compact Shaker KS 15 A, equipped with Incubator Hood TH 15, Edmund Bühler) set at 300 rpm and 37 ◦C. The growth of the bacterial suspensions was stopped by placing them on ice at an OD<sup>600</sup> of 0.4. A total of 150 uL of each bacterial suspension was incubated with an equal volume of PTN/HP-β-CD complexes (containing 200 µM PTN) in 12-well

cell culture plates (TC plate standard, Sarstedt, Nümbrecht, Germany) for 30 min at 37 ◦C and 100 rpm, so that the final PTN concentration was set to 100 µM. These solutions were irradiated with blue-LED (λirr = 457 nm) for 30 min at a radiant exposure of 39.6 J/cm<sup>2</sup> . After irradiation, the suspensions were serially diluted with MHB and plated onto Mueller Hinton II Agar plates (BD, Heidelberg, Germany). After incubating the plates for 18 h at 37 ◦C and 90% relative humidity, the viable colonies were counted and the colony-forming units per milliliter were calculated [CFU/mL]. Filter-sterilized phosphate-buffered saline (PBS) (pH 7.4) was used as a control. The experiments were performed in triplicates.

### *2.9. Statistical Analysis*

Unless otherwise stated, all experiments were performed in triplicates, and the results are expressed as means ± standard deviations. A two-tailed Student's t-test was performed to identify statistically significant differences. Probability values of *p* < 0.05 were considered significant.

#### **3. Results and Discussion**

#### *3.1. Stoichiometry: Job's Plot*

Job's method was employed to determine the stoichiometry of PTN and HP-β-CD, which was indicated by a maximum on Job's plot at a specific molar ratio. As shown in Figure 1A, (∆Abs x R) is greatest at a mole fraction of 0.5, indicating that the stoichiometry between PTN and HP-β-CD is 1:1, where a single PTN molecule can be included in the cavity of one HP-β-CD molecule. Qiu et al. dealt with the complexation of anthraquinone, emodin, in HP-β-CD [34], and the authors presented a similar profile with 1:1 stoichiometry between emodin and HP-β-CD.

**Figure 1.** (**A**) Job's plot for different mole fractions of PTN (R). (**B**) Phase solubility diagram of PTN/HP-β-CD complex system at 25 ◦C. The concentration of HP-β-CD was in the range of 0–35 mM. All measurements were performed in triplicate, and the values were expressed as means ± SDs (*n* = 3).

#### *3.2. Phase Solubility Study*

The phase solubility was conducted by evaluating the change in PTN solubility as a function of HP-β-CD concentration to determine the stoichiometric ratio and stability constant of PTN in HP-β-CD. The inclusion complex formation was visually observed by the typical yellow color of PTN, indicating its solubilization in HP-β-CD solution. As depicted in Figure 1B, PTN solubility increased proportionally with an increase in HP-β-CD concentration, resulting in a correlation coefficient of 0.975 over the studied concentration

range. PTN solubility in water rose considerably from 0.00059 mM in the absence of HPβ-CD to 0.017 mM in the presence of 35 mM HP-β-CD because of the inclusion of PTN within the hydrophobic cavity of HP-β-CD. This host-guest system is a typical A<sup>L</sup> type revealing soluble complex formation according to Higuchi and Connors [35] and showing a 1:1 stoichiometry between PTN and HP-β-CD. The slope in Figure 1B is below unity, implying the formation of an inclusion complex at a molar ratio of 1:1 consistent with Job's plot. The stability constant is K<sup>S</sup> = 733.46 M−<sup>1</sup> , and according to the literature, it is considered optimal in the range of 50 to 2000 M−<sup>1</sup> [34]. Smaller values suggest poor interactions between the guest and the CD, whereas higher values indicate difficult complex dissociation, leading to incomplete guest release from the inclusion complex. As a result, a 1:1 molar ratio of PTN and HP-β-CD was employed for the inclusion complex formation for further characterization.

### *3.3. Characterization of PTN/HP-β-CD Complexes*

## 3.3.1. <sup>1</sup>H NMR Spectroscopy and 2D ROESY

The <sup>1</sup>H NMR investigations for free and complexed PTN were performed to explore the possible inclusion mode of the PTN/HP-β-CD complexes [38]. Accordingly, we compared the NMR spectra of free PTN, pure HP-β-CD, and the PTN/HP-β-CD complexes. As illustrated in Figure 2A, pure PTN in DMSO-d6 showed peaks corresponding to the methyl and methoxy protons at 2.4 and 3.91 ppm, respectively. In addition, the aromatic ring protons exhibited their corresponding peaks at 6.8, 7.1, and 7.5 ppm. The intense peaks at 11.9 and 12.1 ppm corresponded to the phenolic protons on each side of the anthraquinone structure [39]. Figure 2B indicated the prominent peaks of pure HP-β-CD in DMSO-d6, showing the primary chemical shifts for H-1 to H-6 protons between 3 and 6 ppm except for the methyl protons that appeared at 1 ppm. The inclusion of PTN into HP-β-CD resulted in the disappearance of the PTN's aromatic and hydroxyl protons between 6.5 and 12 ppm, as annotated in Figure 2C. Moreover, the methyl and methoxy protons entirely vanished at 2.4 and 3.9 ppm.

For further explanation of the inclusion pattern, the chemical shifts in the presence and absence of PTN were recorded (Table 1). The inclusion of PTN into the HP-β-CD had a negligible influence on the H-4, H-5, and H-6 protons (0.01 ppm). In contrast, the values for H-2 and H-3 indicated a slightly significant change (0.02–0.05 ppm), which might be brought about by the spatial interaction of these protons with the hydroxyl protons of PTN. It is worth noting that the H-3 and H-5 protons are on the interior side of the CD cavity, with the H-3 near the wider opening and the H-5 protons near the narrower side of HP-β-CD [40]. Because of the minor change in the chemical shift of the H-5 (about 0.01 ppm) compared to the more considerable change in the H-3 (about 0.02 ppm), we could presume that PTN might interact with the HP-β-CD through its wider side. According to these findings, PTN is suspected of penetrating the HP-β-CD cavity by its methyl or methoxy substituted rings. The disappearance of their corresponding protons supported this after inclusion with HP-β-CD. Therefore, we hypothesized that PTN could have two inclusion possibilities, as illustrated in Figure 3A,B.

Furthermore, the 2D ROESY of the PTN/HP-β-CD complexes was obtained to attain more conformational details of the spatial configuration of the complexed PTN [41]. The ROESY spectrum of the PTN/HP-β-CD complexes (Figure 3C) showed a relative correlation between the hydroxyl protons of PTN and the H-3 protons of the CD molecule, which is likely driven by hydrogen bonding forces. Moreover, the aromatic protons of PTN at positions 2, 4, 8, and 10 had a spatial correlation with the H-2 and H-5 protons of HP-β-CD, which explains the significant proton shifting of the H-2 (0.05 ppm) and strengthens the hypothesis that PTN was introduced through the broader side of the CD cavity.

**Figure 2.** <sup>1</sup>H NMR spectra of (**A**) PTN, (**B**) HP-β-CD, and (**C**) the PTN/HP-β-CD complexes in DMSO-d<sup>6</sup> .



**Figure 3.** The possible penetration patterns of PTN through the HP-β-CD cavity by either the methyl (**A**) or the methoxy (**B**) substituted rings. (**C**) ROESY spectrum of the PTN/HP-β-CD complexes in DMSO-d6.

#### 3.3.2. FT-IR Analysis

The possible interactions between PTN and HP-β-CD in the solid-state were assessed by comparing the FT-IR spectra of pure PTN, HP-β-CD, their physical mixture, and PTN/HP-β-CD complexes. The FT-IR spectrum of pure PTN (Figure 4A) shows many absorption bands: at 2936 cm−<sup>1</sup> (CH<sup>3</sup> asymmetric), 2844 cm−<sup>1</sup> (CH<sup>3</sup> symmetric), 1674–1613 cm−<sup>1</sup> (C=O free and conjugated), 1557 cm−<sup>1</sup> (C=C aromatic), and 1383–1364 cm−<sup>1</sup> (C–O phenyl) as previously reported [39]. The pure HP-β-CD spectrum (Figure 4B) showed a broad band at 3331 cm−<sup>1</sup> corresponding to OH stretching vibrations of various hydroxyl groups. The absorption bands at 2922 cm−<sup>1</sup> , 1640 cm−<sup>1</sup> , and 1149 cm−<sup>1</sup> are also related to CH<sup>2</sup> stretching vibrations, O-H bending vibrations, and C-O-C stretching vibrations, respectively. The absorption bands of the valence vibrations of the C–O bonds in the ether and hydroxyl groups of HP-β-CD (1082 and 1035 cm−<sup>1</sup> ) were noticed as previously reported [42]. Figure 4C depicts that the physical mixture spectrum was equivalent to a simple combination of PTN and HP-β-CD. However, the characteristic carbonyl peaks of

pure PTN were present in the physical mixture but absent in PTN/HP-β-CD complexes (Figure 4D). Also, there is neither a band at 1557 cm−<sup>1</sup> nor at 1383–1364 cm−<sup>1</sup> assigned to C=C aromatic and C–O phenyl, respectively. The band of the valence vibration of the O–H bond in PTN/HP-β-CD complexes was shifted to 3353 cm−1, and this was related to water release upon host-guest interaction as observed previously in the literature [42]. Overall, the inclusion complex did not display any new IR peaks signifying that no chemical bonds are formed with the obtained complex.

**Figure 4.** FT-IR spectra of (**A**) PTN, (**B**) HP-β-CD, (**C**) physical mixture (1:1 molar ratio of PTN and HP-β-CD), and (**D**) PTN/HP-β-CD complexes over the wavenumber range 4000–400 cm−<sup>1</sup> .

#### 3.3.3. Powder X-ray Diffraction (PXRD)

PXRD was used to identify any change in the crystallinity of PTN upon complexation. The PXRD pattern of pure PTN (Figure 5A) displayed well-resolved characteristic diffraction peaks at different 2*θ*, revealing its crystalline character. The position of peaks corresponds to what has previously been described in the literature [43]. In the case of HP-β-CD (Figure 5B), a diffuse pattern without any sharp peaks was obtained, indicating its amorphous nature. The physical mixture (Figure 5C) exhibited almost all of the distinctive peaks of PTN and HP-β-CD but with lower intensity confirming the presence of both components as isolated species while preserving the crystalline nature of PTN.

**Figure 5.** Powder X-ray diffraction patterns of (**A**) PTN, (**B**) HP-β-CD, (**C**) physical mixture (1:1 molar ratio of PTN and HP-β-CD), and (**D**) PTN/HP-β-CD complexes. Scanning angle of 2θ = 10–60; step width of 0.03◦/min.

On the other hand, the diffractogram of PTN/HP-β-CD complexes (Figure 5D) exhibited the broad peak of pure HP-β-CD with the absence of peaks assigned to pure PTN, which may be due to the presence of the drug in a molecularly dispersed form in HP-β-CD. According to the literature, the amorphous structure is attributed in part to the HP-β-CD structure and in part to the lyophilization step during preparation [44]. These results confirm those obtained by FT-IR, which imply the disappearance of drug crystallinity in the obtained complex.

#### 3.3.4. Scanning Electron Microscopy (SEM)

SEM was used to demonstrate the morphological changes in PTN upon formation of PTN/HP-β-CD complexes, and the results are presented in Figure 6. Pure PTN appeared as irregularly sized needle-shaped crystals (Figure 6A), while pure HP-β-CD existed as spherical crystals, well separated from each other with a porous surface (Figure 6B). The shape of the physical mixture and the inclusion complex was completely different. SEM pictures of the physical mixture indicated no change in the crystal state regarding the original morphology of individual components. The needle-shaped PTN crystals are distributed between HP-β-CD crystals (Figure 6C). However, PTN/HP-β-CD complexes appeared as roughly rectangular homogeneous particles without the spherical shape of HP-β-CD or the needle shape of PTN. This morphological alteration to a single solid phase can indicate a successful formation of PTN/HP-β-CD complexes and is consistent with the data obtained in the FT-IR and XRPD studies.

**Figure 6.** SEM micrographs recorded at an acceleration voltage of 10 kV of (**A**) PTN, (**B**) HP-β-CD, (**C**) physical mixture (1:1 molar ratio of PTN and HP-β-CD), and (**D**) PTN/HP-β-CD complexes with the respective magnification (**E**–**H**) corresponding to the white rectangle. Scale bars represent 200 µm in (**A**–**D**) and 30 µm in (**E**–**H**).

#### 3.3.5. Differential Scanning Calorimetry (DSC)

DSC is widely used to characterize the inclusion of active moieties in the CD cavity by a shift or disappearance in the melting point of the guest molecules [45]. Figure 7 displays that pure PTN exhibits a sharp endothermic peak at 210 ◦C corresponding to its melting point, indicating its crystalline nature, which is well-matched with previous reports [39,46]. As reported before, the pure HP-β-CD showed a broad peak at 187 ◦C because of water loss from the cyclodextrin cavity at higher temperatures [47]. As clearly evidenced in Figure 7, the physical mixture thermogram was quite different from that of the pure components. In the physical mixture, the PTN peak was reduced in intensity and shifted to a higher temperature (218 ◦C), while HP-β-CD shifted to a lower temperature (183 ◦C) in comparison to the pure HP-β-CD (187 ◦C). This may be due to interactions during the DSC run, at least within the ~100–180 ◦C temperature range, and agrees with a previous study attributing these slight peak shifts to the weak interaction between the cyclodextrin and drug during the physical mixture preparation [48].

**Figure 7.** DSC curves of HP-β-CD, PTN, physical mixture (1:1 molar ratio of PTN and HP-β-CD), and PTN/HP-β-CD complexes. The heating rate was 10 ◦C min−<sup>1</sup> . The thermograms were adjusted for better visualization.

On the contrary, Figure 7 depicts a notable difference in the thermal profile of PTN/HPβ-CD complexes in comparison to the parent material and their physical mixture. The endothermic peak of PTN melting was no longer visible, revealing the amorphous state of the drug because of an interaction between PTN and HP-β-CD as a result of guest– host inclusion complex formation. The absence of drug and cyclodextrin peaks in the complex thermogram indicated successful inclusion complex formation. Moreover, the dehydration peak of HP-β-CD appeared only in the pure HP-β-CD and in the physical mixture thermograms but was unseen in those of PTN/HP-β-CD complexes, confirming the complex formation. The plausible explanation of this phenomenon may be that the cyclodextrin cavity was occupied with the hydrophobic drug and no space was available for water molecules [48].

#### 3.3.6. UV/Vis Absorption Spectroscopy

Figure 8A shows that HP-β-CD has no absorption peak within the recorded spectrum as it does not have any double bond (π-electrons) to absorb UV energy, as previously reported [49]. As depicted in Figure 8A, the UV-Vis absorption spectra of free and complexed PTN have two characteristic absorption peaks at λmax = 290 nm and 434 nm, respectively, similar to those reported before [17]. The UV-Vis absorption spectra of free PTN and PTN/HP-β-CD complexes were comparable along the scanned wavelength. The peak position is similar to that of free PTN in ethanol, revealing that the complexation had no significant effect on the PTN absorption spectrum. Moreover, it indicates that the solubility of PTN in water is enhanced without aggregations. However, a slightly hypochromic shift was observed in PTN/HP-β-CD complexes, which could be attributed to the more hydrophilic microenvironment surrounding PTN [50]. Similar behavior was reported by Cannavà et al. [51], who studied the effect of sulfobutyl ether β-cyclodextrin on idebenone and reported a significant hypochromic shift because of the complexation within the cyclodextrin cavity [51]. The absorption spectrum of nifedipine was also almost identical to the free form but with lower absorbance intensity at some wavelengths [52].

#### 3.3.7. Singlet Oxygen Quantum Yield

Since the phototoxicity of the photosensitizer involves the production of cytotoxic species such as singlet oxygen, it is useful to investigate the singlet oxygen generation efficiency. This can be performed by monitoring the decay curves of UA absorbance as a function of irradiation time. Following the established measuring procedure [19], the decomposition of UA upon exposure to generated singlet oxygen was employed to measure singlet oxygen generation. UA was selected because it does not absorb light in the blue region, specifically at the PTN irradiation wavelength. A similar irradiation experiment was also performed in parallel as a negative control, where UA solution was irradiated with a blue LED. In the absence of PTN, no photobleaching of UA was observed after irradiation (data not shown), and therefore, any loss in UA absorbance upon irradiation is due to the photodynamic activity of PTN. Figure 8B,C depicts that the decrease in UA absorbance is a function of light exposure time, indicating the irradiation time-dependent singlet oxygen production, with the lowest UA absorbance at 5 min irradiation time. A good linear relation between Ln normalized absorbance of UA at λ = 296 nm and the irradiation time denotes that the decay kinetics is first order.

A comparison of UA decays in Figure 8B revealed that free PTN induced significantly faster UA degradation than observed in the case of complexed PTN (Figure 8C). The rate constants for UA decomposition were 7.04 <sup>×</sup> <sup>10</sup>−<sup>4</sup> and 2.28 <sup>×</sup> <sup>10</sup>−<sup>4</sup> s −1 , while the singlet oxygen quantum yields were 0.49 and 0.35 for free and complexed PTN, respectively. This decrease in singlet oxygen generation upon complexation with HP-β-CD may be due to the photosensitizers' environment affecting the quantum yield. It is well-established that singlet oxygen has a longer lifetime in organic solvents than in water, where fast quenching of singlet oxygen between water molecules reduces the singlet oxygen lifetime [53]. Moreover, PTN/HP-β-CD complexes may be more stable and less susceptible to photodegradation

than ethanolic PTN solutions. According to the literature, cyclodextrin complexation changes the photochemical characteristics of the guest molecules and has been long used to protect drugs against photodegradation [54]. Previous research suggests that β-cyclodextrin complexation has a negligible effect on the UV spectra, but it causes significant changes in the emissions spectra and fluorescence quantum yields as it usually impacts the ground and the excited states [50]. These results further confirm that in water, HP-β-CD maintains the PTN molecules mainly as monomeric species, which are the photoactive form for ROS generation.

**Figure 8.** (**A**) UV/Vis spectra of PTN in ethanol (80 µg/mL), HP-β-CD in water (0.5 mg/mL) and PTN/HP-β-CD inclusion complexes in water (80 µg/mL). (**B**,**C**) Absorption spectra of uric acid upon irradiation for different times in the presence of (**B**) free PTN and (**C**) PTN/HP-β-CD complexes. The insets represent the plot of Ln normalized absorbance of uric acid at λ = 296 nm versus irradiation time in s (λirr = 457 nm, irradiance = 220.2 W/m<sup>2</sup> ). All measurements were performed in triplicate, and the values were expressed as means ± SDs (*n* = 3).

#### 3.3.8. Photostability of Inclusion Complex

Considering the practical use of PTN/HP-β-CD complexes in aPDT, it is better to investigate the influence of irradiation time on the degradation of free and complexed PTN. Figure 9A,B show that the decrease in absorbance measured at λmax = 434 nm is light-dependent, with the lowest absorbance detected after 30 min irradiation. This may be due to several photoactivations of the photosensitizer with successive irradiation, leading to photodecomposition or photobleaching. As shown in Figure 9C, less than 28% of free PTN remained after 30 min irradiation compared to about 70% in the case of the complexed form, indicating a better photostability of HP-β-CD. Figure 9D presents that complexed PTN could retain its characteristic yellow color even after 30 min irradiation. The slight color change of free PTN to yellowish-orange is most probably due to the formation of photodegradation products evidenced by a decrease in the peak intensity at 434 and 287 nm and a simultaneous increase of the new small peak at 488 nm, which appeared only after irradiation with the highest intensity after 30 min (Figure 9A). This photostability can be beneficial in PDT since PS molecules are still available for further photoactivation leading to more ROS generation with additional irradiation.

**Figure 9.** Effect of HP-β-CD on the photodegradation of PTN after blue LED irradiation at different time intervals (λirr = 457 nm, irradiance = 220.2 W/m<sup>2</sup> ). (**A**) The absorption spectra of PTN in ethanol and (**B**) the absorption spectra of PTN/HP-β-CD complexes in ultrapure water. (**C**) Decrease in the PTN content of free and complexed PTN at different irradiation intervals with a blue LED. (**D**) Photomicrograph of PTN/HP-β-CD complexes and PTN before and after 30 min irradiation. All measurements were performed in triplicate, and the values were expressed as means ± SDs (*n* = 3).

Additionally, the photobleaching of free PTN could limit the irradiation time and reduce its photosensitizing efficacy, especially when a longer irradiation time is required. This is consistent with a previous study, where HP-β-CD complexed cilnidipine exhibited lower photodegradation than free cilnidipine in ethanol, and the authors related this to the protective effect conferred by the host-guest system [55]. This effect was also reported before for curcumin after complexation in cyclodextrin [56]. According to the literature [57], the host-guest structure can reduce the hydrolytic or photolytic degradation of a drug in inclusion complexes by providing a molecular shield against reactive substances. This shielding effect can presumably result in lower availability and interaction of complexed PTN molecules with the light source, leading to lower singlet oxygen generation as stated before compared to the free PTN.

#### *3.4. Bacterial Viability Assay*

In this study, antibacterial activity was examined by irradiating bacteria treated with the prepared PTN/HP-β-CD complexes, and the results were presented in Figure 10. For the used concentration (100 µM PTN), no significant dark toxicity could be observed toward either microorganism when comparing the non-irradiated control sample and the non-irradiated formulation. In addition, the effect of the light source was negligible as it did not influence bacterial viability as seen in the irradiated control sample [9,58,59]. Furthermore, the unirradiated and irradiated HP-β-CD also showed no significant change in bacterial viability. In contrast, the irradiated PTN/HP-β-CD complexes significantly reduced the bacterial viability of the gram-positive *S. saprophyticus* by > 4.8 log. Therefore, it can be assumed that the antibacterial effect originated exclusively from the irradiated PTN/HP-β-CD complexes. According to the American Society for Microbiology, a bacterial reduction of 99.9986% qualifies the formulation as antibacterial [60]. The viability of the gram-negative *E. coli* was reduced by > 1.0 log after incubation with PTN/HP-β-CD complexes and irradiation. As described in the literature, the antibacterial effect of PTN is lower with gram-negative germs, and higher concentrations are needed for the same effect [14]. In addition, the affinity of the cyclodextrin complex to gram-negative bacteria can be slightly reduced because of their unique cellular structure. The difference in the cell wall structure between gram-negative and gram-positive bacteria may account for their different response to aPDT. The accumulation of PSs inside gram-negative bacteria is limited because of two mechanisms. Firstly, the lipid-rich membrane bilayer enclosing the cell wall reduces the inward penetration of PTN/HP-β-CD complexes into gramnegative bacteria [5]. Secondly, gram-negative bacteria widely use porins and efflux pumps to regulate nutrient and toxin influx and efflux, and hence they act as natural resistance mechanisms [5]. However, compared with antibacterial studies on pure PTN, the concentration used here is reduced almost tenfold. Comini et al. showed no dark toxicity of PTN against *E. coli* up to 320 µg/mL, and an antibacterial activity of irradiated PTN only above a concentration of 250 µg/mL [17]. This underlines the superiority of the cyclodextrin complex at a used PTN concentration of just 28.45 µg/mL. Overall, the complexation of PTN not only improved the water solubility and photostability of PTN but also increased the antibacterial effect, enabling the therapeutic application of PTN.

**Figure 10.** Bacterial viability of *Staphylococcus saprophyticus* subsp. *bovis* (*S. saprophyticus*) and *Escherichia coli* DH5 alpha (*E. coli*) treated with PTN/HP-β-CD complexes (100 µM PTN) or only with HP-β-CD for 30 min at 37 ◦C and then irradiated (IR) with a blue LED (λirr = 457 nm, 39.6 J/cm<sup>2</sup> ) or kept in the dark. Control represents bacterial suspension treated with filter-sterilized phosphatebuffered saline (PBS) (pH 7.4). The results are expressed as means ± SDs (*n* = 3). Statistical differences are denoted as "\*\*\*" *p* < 0.001.

#### **4. Conclusions**

The efficacy of parietin (PTN) as a natural photosensitizer is poor because of its hydrophobic structure and π–π stacking, which consequently results in aggregation-induced quenched fluorescence, limited ROS production, and a diminished photodynamic effect. In this study, HP-β-CD was employed to enhance the water solubility of PTN and decrease its aggregation in aqueous vehicles. The water solubility of PTN was enhanced up to 28-fold after complexation with HP-β-CD. Various characterization methods confirmed that PTN was adequately included in the cyclodextrin cage. Even though PTN/HP-β-CD complexes have a polar environment, they are still photoactive because HP-β-CD provides a protecting hydrophobic cavity for PTN, resulting in a shielding effect from water molecules. This can be a critical factor in maintaining its photodynamic activity, as confirmed by uric acid degradation due to singlet oxygen generation. Additionally, the complexation was shown to slow down the photodegradation of PTN. Although PTN is reported to have a dark antimicrobial effect, it is presumed that aPDT could considerably reduce the required dose to give a comparable effect and lower toxic effects. In the long-term prospect, the limited efficacy of PTN/HP-β-CD against gram-negative bacteria may be overcome by the delivery of PTN in cationic formulations.

**Author Contributions:** Conceptualization, A.M.A. (Abdallah Mohamed Ayoub), B.G. and U.B.; investigation, A.M.A. (Abdallah Mohamed Ayoub), B.G., A.M.A. (Ahmed Mohamed Abdelsalam), E.P., A.A.D., A.A. and A.B.; writing—original draft preparation, A.M.A. (Abdallah Mohamed Ayoub), B.G. and E.P.; writing—review and editing, A.M.A. (Abdallah Mohamed Ayoub), B.G., E.P., J.S. and U.B.; supervision, U.B.; project administration, U.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** Abdallah Mohamed Ayoub and Ahmed Mohamed Abdelsalam would like to express their gratitude to Yousef Jameel Foundation, the German Academic Exchange Service and the Egyptian Ministry of Higher Education and Scientific Research (DAAD/MHESR), respectively for providing the scholarship funding for the doctoral scholarships.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Hypericin and Pheophorbide a Mediated Photodynamic Therapy Fighting MRSA Wound Infections: A Translational Study from In Vitro to In Vivo**

**Ben Chung Lap Chan 1,† , Priyanga Dharmaratne 2,† , Baiyan Wang <sup>3</sup> , Kit Man Lau <sup>1</sup> , Ching Ching Lee 1,2 , David Wing Shing Cheung 1,3, Judy Yuet Wa Chan <sup>1</sup> , Grace Gar Lee Yue <sup>1</sup> , Clara Bik San Lau <sup>1</sup> , Chun Kwok Wong 1,4 , Kwok Pui Fung 1,3 and Margaret Ip 2,\***


**Abstract:** High prevalence rates of methicillin-resistant *Staphylococcus aureus* (MRSA) and lack of effective antibacterial treatments urge discovery of alternative therapeutic modalities. The advent of antibacterial photodynamic therapy (aPDT) is a promising alternative, composing rapid, nonselective cell destruction without generating resistance. We used a panel of clinically relevant MRSA to evaluate hypericin (Hy) and pheophobide a (Pa)-mediated PDT with clinically approved methylene blue (MB). We translated the promising in vitro anti-MRSA activity of selected compounds to a full-thick MRSA wound infection model in mice (in vivo) and the interaction of aPDT innate immune system (cytotoxicity towards neutrophils). Hy-PDT consistently displayed lower minimum bactericidal concentration (MBC) values (0.625–10 µM) against ATCC RN4220/pUL5054 and a whole panel of community-associated (CA)-MRSA compared to Pa or MB. Interestingly, Pa-PDT and Hy-PDT topical application demonstrated encouraging in vivo anti-MRSA activity (>1 log<sup>10</sup> CFU reduction). Furthermore, histological analysis showed wound healing via re-epithelization was best in the Hy-PDT group. Importantly, the dark toxicity of Hy was significantly lower (*p* < 0.05) on neutrophils compared to Pa or MB. Overall, Hy-mediated PDT is a promising alternative to treat MRSA wound infections, and further rigorous mechanistic studies are warranted.

**Keywords:** photodynamic therapy; methicillin-resistant *Staphylococcus aureus*; hypericin; wound infection model

## **1. Introduction**

Infections caused by antimicrobial resistant (AMR) bacteria are serious global health concerns and are exacerbated with prior asymptomatic carriage [1–3]. Methicillin-resistant *Staphylococcus aureus* (MRSA) is one of the commonest AMR bacteria that confers illnesses ranging from localized skin infections to systemic diseases, including toxic shock syndrome [4]. The prevalence of hospital-associated MRSA (HA-MRSA) infection varies geographically, and Hong Kong is one of the high-prevalence regions in Asia. According to the Asian Network for Surveillance of Resistant Pathogens (ANSORP) study, 57% of all

**Citation:** Chan, B.C.L.; Dharmaratne, P.; Wang, B.; Lau, K.M.; Lee, C.C.; Cheung, D.W.S.; Chan, J.Y.W.; Yue, G.G.L.; Lau, C.B.S.; Wong, C.K.; et al. Hypericin and Pheophorbide a Mediated Photodynamic Therapy Fighting MRSA Wound Infections: A Translational Study from In Vitro to In Vivo. *Pharmaceutics* **2021**, *13*, 1399. https://doi.org/10.3390/ pharmaceutics13091399

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis, Gerhard Litscher and Maria Nowakowska

Received: 20 July 2021 Accepted: 27 August 2021 Published: 3 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

inpatient isolates of *S. aureus* from Hong Kong hospitals were confirmed as methicillinresistant [5]. Additionally, in Hong Kong, the prevalence of nasal carriage of *S. aureus* and MRSA were 27.6% and 1.3%, respectively, among children in daycare centers and kindergartens [6,7].

Microbes by their nature continually adapt to survive the antimicrobial treatments we use to combat them, resulting in an ever increasing level of antimicrobial resistance [8], and the development of nonantimicrobial treatments may be beneficial concerning resistance development. Photodynamic therapy (PDT) consists of the administration of a nontoxic drug or dye known as a photosensitizer (PS) either systemically, locally, or topically applied to a patient, followed by illumination with visible or near-infrared (NIR) light in the presence of oxygen, leading to the generation of cytotoxic reactive oxygen species (ROS) in the proximate environment causing cell death/ tissue damage [9,10]. The advantages of PDT over conventional therapies include rapid bacterial killing, applicability over a broad spectrum (Gram-negative or Gram-positive) [11,12], and efficacy against biofilms [13,14], fungi [15,16], parasites [17] and viruses [18]. To date, the clinical applications of PDT have been confined mainly to localized infections in dermatology and dentistry [19,20], wound healing [21,22], and for surface disinfection including medical devices [23].

It was reported that PDT for localized microbial infections exerts its therapeutic effect both by direct bacterial killing and the activation of the host immune response, particularly innate immunity [24]. Neutrophils are among the first line of defence recruited to the site of infection to release enzymes for killing infectious organisms and to secrete cytokines that promote inflammation. The importance of neutrophils against microbial infections is reflected by the observation that Photofrin ®-PDT exhibited significant cytotoxicity for cultured MRSA, but the therapy had a low efficacy in a murine model of MRSA arthritis, even though Photofrin® accumulated well in the infected joint. It was discovered that 30% of intra-articular leukocytes, mainly neutrophils, were killed immediately during or following Photofrin-PDT [25]. Therefore, we assume it is important to examine specific PS-PDTs cytotoxicity towards human neutrophils. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 3 of 15 **Table 1.** Electronic absorption and basic photophysical data for 3 photosensitizers used. **Compound λmax/nm λem/nm ΦF a ΦΔ b Ref** 

Hypericin (Hy) is a naturally occurring polycyclic quinine (Figure 1a) extracted from plant species of the genus *Hypericium* including the species *Hypericum perforatum* L. (St John's Wort) [26]. Recent reports showed that Hy has the potential to treat several types of cancer and some benign skin disorders [27,28]. Interestingly, Yow et al. reported Hy could induce a significant cytotoxic effect on clinically isolated methicillin-sensitive *S. aureus* (MSSA) and MRSA [29]. In the aspect of wound healing, *H. perforatum*, which is a popular folk remedy for the treatment of wounds in Turkey, has been shown to possess remarkable in vivo wound healing activity, and Hy was found in the active fractions [30]. Methylene Blue 664 (monomer in aqueous medium) 709 0.04 0.5 [38,39] Hypericin 598 (DMSO) 651 0.2 0.73 [40,41] Pheophobide a 667 (DMSO) 677 0.26 0.62 [42,43] a Fluorescence quantum yield; b Singlet oxygen quantum yield. The compelling evidence of Hy and Pa led us to investigate their PDT effects in vitro and in vivo against a broad spectrum of clinically relevant MRSA panels along with their toxicity towards neutrophils, in view of depicting their overall anti-MRSA efficacy.

(**a**) Structure of Hypericin (**b**) Structure of Pheophobide a (**c**) Structure of Methylene blue

**Figure 1.** Chemical structures of 3 PSs used in the current investigation*.* **Figure 1.** Chemical structures of 3 PSs used in the current investigation.

**2. Materials and Methods**  *2.1. General*  Pheophorbide a was purchased from Frontier Scientific Inc. (Logan, UT, USA) and hypericin and methylene blue were purchased from Sigma-Aldrich Co. (St Louis MA,USA). The PS solution for in vitro PDT study was prepared freshly by dissolving Pa Pheobhobide a (Pa) is also a natural compound, derived from the breakdown of chlorophyll a [31]. The extended π-π conjugated system (Figure 1b) and stability of the compound in various solvents make it suitable as a photosensitizing agent. Studies have revealed that Pa-PDT is effective in eradicating a variety of tumors, including pigmented melanoma, colonic cancer, Jurkat leukemia, and pancreatic carcinoma [32–35]. Besides the

and Hy in DMSO to make a 10 mM stock solution. It was then diluted in Tween 80 and MHB to set the desired stock solution. A serial two-fold dilution procedure was employed to obtain final working concentrations. Tween 80 and DMSO concentrations were

BAA-44, two mutant strains [AAC(6)′ APH(2)′′ and RN4220/pUL5054], five communityacquired (CA-MRSA) and five hospital-acquired MRSA (HA-MRSA) clinical strains were obtained from the Department of Microbiology, Faculty of Medicine, The Chinese

Minimal bactericidal concentrations (MBCs) of Pa-PDT, Hy-PDT and MB-PDT for sixteen MRSA strains were determined according to the modified method adopted by Clinical and Laboratory Standards Institute (CLSI) guidelines [12,44,45]. Briefly, an overnight bacterial culture suspension was adjusted to McFarland Standard 0.5 and suspended in Mueller Hinton Broth (MHB) to make a final concentration of 1.0×106 colony forming unit (CFU)/mL. Photosensitizers at different concentrations (100 µL) and MRSA suspension (100 µL) were added into 96-well plate and incubated at 37°C for 2 h under dark condition as a pre-irradiation step. After incubation, the mixed solutions were irradiated from above at a light intensity of 40 mW/cm2 using a 300 W quartz-halogen lamp attenuated by a 5 cm layer of water as a heat buffer and a color filter cut-on at 610 nm (for MB and Pa, λ > 610 nm) or 590 nm (for Hy, λ > 590 nm) for 20 min, i.e., 48 J/cm2. Dark control group and a solvent control group were included. All experiments were repeated three times. The MBCs were determined as the minimum concentration of the photosensitizers required for complete inhibition of bacterial growth on a blood agar

*2.2. In Vitro Photodynamic Minimal Bactericidal Concentration (PD-MBC) Studies* 

University of Hong Kong.

plate.

anticancer activity of Pa, it has also been tested for its photodynamic activity against MRSA with modification to its structure (Na salt of Pa) [36].

Photophysical properties are of paramount importance when selecting a photosensitizer. The absorption spectrum of the compound plays a pivotal role during in vivo applications, and it has to be within the therapeutic window (550–950 nm) [37]. The key photophysical properties of Hy and Pa are summarized in Table 1 along with the gold standard of PDT studies (Methylene blue, MB, Figure 1c).


**Table 1.** Electronic absorption and basic photophysical data for 3 photosensitizers used.

<sup>a</sup> Fluorescence quantum yield; <sup>b</sup> Singlet oxygen quantum yield.

The compelling evidence of Hy and Pa led us to investigate their PDT effects in vitro and in vivo against a broad spectrum of clinically relevant MRSA panels along with their toxicity towards neutrophils, in view of depicting their overall anti-MRSA efficacy.

#### **2. Materials and Methods**

#### *2.1. General*

Pheophorbide a was purchased from Frontier Scientific Inc. (Logan, UT, USA) and hypericin and methylene blue were purchased from Sigma-Aldrich Co. (St Louis, MA, USA). The PS solution for in vitro PDT study was prepared freshly by dissolving Pa and Hy in DMSO to make a 10 mM stock solution. It was then diluted in Tween 80 and MHB to set the desired stock solution. A serial two-fold dilution procedure was employed to obtain final working concentrations. Tween 80 and DMSO concentrations were maintained ≤ 0.1% and ≤1% (*v*/*v*), respectively, in each test group.

The bacterial strains MRSA, ATCC 43300, ATCC BAA-42, ATCC BAA-43, ATCC BAA-44, two mutant strains [AAC(6)0 APH(2)00 and RN4220/pUL5054], five communityacquired (CA-MRSA) and five hospital-acquired MRSA (HA-MRSA) clinical strains were obtained from the Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong.

#### *2.2. In Vitro Photodynamic Minimal Bactericidal Concentration (PD-MBC) Studies*

Minimal bactericidal concentrations (MBCs) of Pa-PDT, Hy-PDT and MB-PDT for sixteen MRSA strains were determined according to the modified method adopted by Clinical and Laboratory Standards Institute (CLSI) guidelines [12,44,45]. Briefly, an overnight bacterial culture suspension was adjusted to McFarland Standard 0.5 and suspended in Mueller Hinton Broth (MHB) to make a final concentration of 1.0×10<sup>6</sup> colony forming unit (CFU)/mL. Photosensitizers at different concentrations (100 µL) and MRSA suspension (100 µL) were added into 96-well plate and incubated at 37◦C for 2 h under dark condition as a pre-irradiation step. After incubation, the mixed solutions were irradiated from above at a light intensity of 40 mW/cm<sup>2</sup> using a 300 W quartz-halogen lamp attenuated by a 5 cm layer of water as a heat buffer and a color filter cut-on at 610 nm (for MB and Pa, <sup>λ</sup> <sup>≥</sup> 610 nm) or 590 nm (for Hy, <sup>λ</sup> <sup>≥</sup> 590 nm) for 20 min, i.e., 48 J/cm<sup>2</sup> . Dark control group and a solvent control group were included. All experiments were repeated three times. The MBCs were determined as the minimum concentration of the photosensitizers required for complete inhibition of bacterial growth on a blood agar plate.

#### *2.3. Animal Studies-Mouse Model of MRSA-Infected Wound* Previously published murine skin infection models [44–47] were used to validate the

*2.3. Animal Studies-Mouse Model of MRSA-Infected Wound* 

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 4 of 15

#### 2.3.1. Animal Model in vivo efficacy of the PS-PDT treatment against MRSA. All animal experiments were

2.3.1. Animal Model

Previously published murine skin infection models [44–47] were used to validate the in vivo efficacy of the PS-PDT treatment against MRSA. All animal experiments were conformed to the university guidelines and approved by the Animal Experimentation Ethics Committee (Ref. no.12/076/MIS, 8 February 2013) of The Chinese University of Hong Kong. Female Balb/c mice (25–30 g) were supplied by Laboratory Animal Services Centre (LASEC), The Chinese University of Hong Kong. They were housed in individually ventilated cages (IVC) under the conditions of 22–25 ◦C and a 12-h light-dark cycle, with free access to chow and tap water. conformed to the university guidelines and approved by the Animal Experimentation Ethics Committee (Ref. no.12/076/MIS, 8 February 2013) of The Chinese University of Hong Kong. Female Balb/c mice (25–30 g) were supplied by Laboratory Animal Services Centre (LASEC), The Chinese University of Hong Kong. They were housed in individually ventilated cages (IVC) under the conditions of 22–25 °C and a 12-h light-dark cycle, with free access to chow and tap water. It is apparent from the in vitro results that, MRSA ATCC RN4220/pUL5054 strain was susceptible for all three PSs with comparatively lower MBC values. Hence, this

It is apparent from the in vitro results that, MRSA ATCC RN4220/pUL5054 strain was susceptible for all three PSs with comparatively lower MBC values. Hence, this selected to establish infection on a full-thick wound in mice. Mice were anesthetized by an intraperitoneal (i.p.) injection of ketamine (40 mg/kg) and xylazine (8 mg/kg), with the hair of the back shaved and the skin cleansed with 10% povidone-iodine solution. A circular full-thickness wound (4 mm in diameter) was established through a disposable skin puncher on the back subcutaneous tissue of each animal. The lesion, overlaid with gauze, was dressed with an adhesive bandage. selected to establish infection on a full-thick wound in mice. Mice were anesthetized by an intraperitoneal (i.p.) injection of ketamine (40 mg/kg) and xylazine (8 mg/kg), with the hair of the back shaved and the skin cleansed with 10% povidone-iodine solution. A circular full-thickness wound (4 mm in diameter) was established through a disposable skin puncher on the back subcutaneous tissue of each animal. The lesion, overlaid with gauze, was dressed with an adhesive bandage. For the in vivo studies, Pa and Hy were prepared according to our previously

For the in vivo studies, Pa and Hy were prepared according to our previously published protocol [48]. Briefly, 1% DMSO, 4% ethanol and 95% PBS constituted the final test solution. published protocol [48]. Briefly, 1% DMSO, 4% ethanol and 95% PBS constituted the final test solution. 2.3.2. Intravenous Treatment

#### 2.3.2. Intravenous Treatment Our research group previously investigated Pa-PDT-mediated anticancer activity

Our research group previously investigated Pa-PDT-mediated anticancer activity (against MCF-7 tumors) in vivo [48]. So, the dosage and optimum therapeutic window for these kinds of compounds were established (2.5 mg/Kg) for intravenous injection. Three days after wound induction, mice were anesthetized with a ketamine/ xylazine cocktail, and 50 <sup>µ</sup>L of MRSA (1 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/ mL) was inoculated onto the wound. One day later, 20 µL of photosensitizers (Pa or Hy) at 2.5 mg/kg were intravenously injected into the mice via the tail vein as for the stratified groups. Ten minutes after the application of photosensitizers, PDT illumination at 1 W was performed for either 30 s or 10 min for all PDT groups, corresponding to 30 J/wound and 600 J/wound, respectively. A continuouswave laser was generated from the Ceralas medical laser system with excitation at 670 nm (Biolitec group, Bonn, Germany). The treatments were repeated every other day and lasted until Day 8 (three treatment cycles, Figure 2). Wound sizes were recorded before and after treatment. At the end of the experiment, animals were euthanized with an overdose of terminal pentobarbital solution. The wound (5 × 10 mm) was then excised aseptically. (against MCF-7 tumors) in vivo [48]. So, the dosage and optimum therapeutic window for these kinds of compounds were established (2.5 mg/Kg) for intravenous injection. Three days after wound induction, mice were anesthetized with a ketamine/ xylazine cocktail, and 50 µL of MRSA (1 × 108 CFU/ mL) was inoculated onto the wound. One day later, 20 µL of photosensitizers (Pa or Hy) at 2.5 mg/kg were intravenously injected into the mice via the tail vein as for the stratified groups. Ten minutes after the application of photosensitizers, PDT illumination at 1 W was performed for either 30 s or 10 min for all PDT groups, corresponding to 30 J/wound and 600 J/wound, respectively. A continuouswave laser was generated from the Ceralas medical laser system with excitation at 670 nm (Biolitec group, Bonn, Germany). The treatments were repeated every other day and lasted until Day 8 (three treatment cycles, Figure 2). Wound sizes were recorded before and after treatment. At the end of the experiment, animals were euthanized with an overdose of terminal pentobarbital solution. The wound (5 × 10 mm) was then excised aseptically.

**Figure 2.** Timeline for the intravenous treatment.

#### **Figure 2.** Timeline for the intravenous treatment*.*  2.3.3. Topical Treatment

Three days after wound induction, mice were anesthetized with a ketamine/ xylazine cocktail and the adhesive bandage was removed. A 50 µL of MRSA suspension

(1 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/ mL) was dropped onto each wound. A dressing (Tegaderm™ film, 3M, Company, St. Louis, MA, USA) was applied to cover the wound immediately to maintain wound moisture. Thirty minutes after bacterial inoculation, 50 µL of 800 µM PS solutions or Fucidin® cream was injected under the dressing by syringe and allowed to spread over the wound. Photoactivation (Biolitec group, Bonn, Germany) was initiated immediately. A single dosage of laser at 0.5 W for 60 s was delivered by an optical fiber 2 mm in diameter, corresponding to 30 J/wound. The dark control (PS alone) groups and the Fucidin® cream (2% fucidic acid) group (positive control) did not receive any laser irradiation but were sham-irradiated under visible light. The animals were returned to individually ventilated cages (IVC) after treatment and thoroughly examined daily. To avoid any possible phototoxicity, all mice were kept in a dark room for 4 h after PDT/sham irradiation. After 2 days of treatment, once daily (Figure 3), the dressings were removed and the wounds were exposed. The wound sizes were recorded every two days. On Day 10, animals were euthanized with an overdose of dorminal pentobarbital solution. The wound (5 × 10 mm) was then excised aseptically. Fucidin® cream (2% fucidic acid) group (positive control) did not receive any laser irradiation but were sham-irradiated under visible light. The animals were returned to individually ventilated cages (IVC) after treatment and thoroughly examined daily. To avoid any possible phototoxicity, all mice were kept in a dark room for 4 h after PDT/sham irradiation. After 2 days of treatment, once daily (Figure 3), the dressings were removed and the wounds were exposed. The wound sizes were recorded every two days. On Day 10, animals were euthanized with an overdose of dorminal pentobarbital solution. The wound (5 × 10 mm) was then excised aseptically. Each skin sample was divided into two portions. One-piece was used for histological examination to determine the maturity of wound repair, and the second was weighed and homogenized in 0.5 mL of PBS solution for bacterial viability counts. Quantification of viable bacteria was performed by culturing serial dilutions (10 µL) of the bacterial suspension on blood agar plates. For this purpose, all plates were incubated at 37 0C for 24 h and evaluated for the presence of the staphylococcal strain. The bacteria were quantified by counting the number of CFU per plate.

Three days after wound induction, mice were anesthetized with a ketamine/ xylazine cocktail and the adhesive bandage was removed. A 50 µL of MRSA suspension (1 × 108 CFU/ mL) was dropped onto each wound. A dressing (TegadermTM film, 3M, Company, St. Louis, MA,USA) was applied to cover the wound immediately to maintain wound moisture. Thirty minutes after bacterial inoculation, 50 µL of 800 µM PS solutions or Fucidin® cream was injected under the dressing by syringe and allowed to spread over the wound. Photoactivation (Biolitec group, Bonn, Germany) was initiated immediately. A single dosage of laser at 0.5 W for 60 s was delivered by an optical fiber 2 mm in diameter, corresponding to 30 J/wound. The dark control (PS alone) groups and the

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 5 of 15

2.3.3. Topical Treatment

**Figure 3.** Timeline for the topical treatment*.*  **Figure 3.** Timeline for the topical treatment.

2.3.4. Histological Evaluation Wound tissues collected from the animal study were initially fixed in 10% buffered formalin, followed by dehydration and paraffin-embedding. Paraffin blocks were cut into 5 µm tissue sections including the epidermis, the dermis, and the subcutaneous panniculus. The sections were stained with hematoxylin and eosin (H&E) and assessed by light microscopy for wound healing. Each skin sample was divided into two portions. One-piece was used for histological examination to determine the maturity of wound repair, and the second was weighed and homogenized in 0.5 mL of PBS solution for bacterial viability counts. Quantification of viable bacteria was performed by culturing serial dilutions (10 µL) of the bacterial suspension on blood agar plates. For this purpose, all plates were incubated at 37 ◦C for 24 h and evaluated for the presence of the staphylococcal strain. The bacteria were quantified by counting the number of CFU per plate.

#### *2.4. In Vitro Cytotoxic MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide)*  2.3.4. Histological Evaluation

*Assay on Human Neutrophils*  Human neutrophils were purified from the fresh buffy coat fraction of blood from adult volunteers at the Hong Kong Red Cross Blood Transfusion Service, Hong Kong and separated by the Percoll method which was routinely performed in our laboratory [49]. Wound tissues collected from the animal study were initially fixed in 10% buffered formalin, followed by dehydration and paraffin-embedding. Paraffin blocks were cut into 5 µm tissue sections including the epidermis, the dermis, and the subcutaneous panniculus. The sections were stained with hematoxylin and eosin (H&E) and assessed by light microscopy for wound healing.

#### *2.4. In Vitro Cytotoxic MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) Assay on Human Neutrophils*

Human neutrophils were purified from the fresh buffy coat fraction of blood from adult volunteers at the Hong Kong Red Cross Blood Transfusion Service, Hong Kong and separated by the Percoll method which was routinely performed in our laboratory [49].

Our studies showed that human neutrophils exhibited a short lifespan after isolating from buffy coats. Most human neutrophils did not survive 48 h after isolation. Therefore, freshly isolated human neutrophils were used in the present study, and the experiments were done within 24 h after isolation. Freshly isolated human neutrophils were plated in

96-well plates at 10<sup>5</sup> cells/well. Serial dilutions of three photosensitizers, Pa, MB, and Hy were added to the wells. After 24 h at 37 ◦C incubation, MTT solution (50 µL, 5 mg/mL) were added to each well. Then, the plates were incubated at 37 ◦C for 3 h. After incubation, 150 µL of DMSO was added to each well. The OD of the wells was determined by a spectrophotometer at 590 nm. Toxicity was represented by the ratio of OD of a well in the presence of compounds with the OD of control wells in the presence of a medium containing DMSO.

#### **3. Results**

#### *3.1. Bactericidal Activity Assay on MRSA Strains*

It is apparent from Table 1 that the Pa-PDT group showed significantly higher (*p* < 0.05) anti-MRSA activity against MRSA ATCC RN4220/pUL5054, W44, and W46-47 (MBC; 3.125–12.5 µM) than the positive control MB (MBC; 120->160 µM). Similarly, the Hy-PDT group demonstrated significantly higher (*p* < 0.05) anti-MRSA activity against MRSA ATCC RN4220/pUL5054 and a whole panel of CA-MRSA strains (MBC; 0.625–10 µM) compared to MB. However, HA-MRSA was more resistant towards HY-PDT, except HA-232 (MBC; 2.5 µM). Interestingly, Hy-PDT showed the lowest MBC values compared to Pa-PDT or MB-PDT, indicating the importance of further investigations. The dark toxicities of all three PSs were 4-8 times lower than their PDT counterparts (Table 2). Out of these sixteen MRSA strains tested, RN4220/pUL5054 was sensitive to three photosensitizers, especially to Hy and Pa. Therefore, it was selected to establish the in vivo model.

**Table 2.** The Minimal Bactericidal Concentrations (MBCs) of Pa, Hy and MB against sixteen MRSA strains.


<sup>a</sup> CA: community associated; <sup>b</sup> HA: hospital associated.

#### *3.2. Animal Studies-Mouse Model of MRSA-Infected Wound*

#### 3.2.1. Effect of PDT of Pa and Hy with Intravenous Injection in MRSA-Infected Wound Model

Neither of the PDTs (30 s or 10 min) upon Pa and Hy intravenous (i.v.) injection (2.5 mg/kg) significantly (*p* < 0.05) reduced bacterial load at Day 8 (Figure 4). It was observed that mice receiving 30 s of Pa-PDT and Hy-PDT treatments resulted in slight but insignificant promotion of wound closure. However, this trend could not be observed in the 10 min PDT-treated groups (Figure 5). There was no body weight loss in treatment groups when compared with the control group, implying that the treatments did not cause distress in the mice (Figure 6).

*3.2. Animal Studies-Mouse Model of MRSA-Infected Wound* 

*3.2. Animal Studies-Mouse Model of MRSA-Infected Wound* 

*3.2. Animal Studies-Mouse Model of MRSA-Infected Wound* 

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Wound Model

Wound Model

Wound Model

cause distress in the mice (Figure 6).

cause distress in the mice (Figure 6).

cause distress in the mice (Figure 6).

3.2.1. Effect of PDT of Pa and Hy with Intravenous Injection in MRSA-Infected

3.2.1. Effect of PDT of Pa and Hy with Intravenous Injection in MRSA-Infected

3.2.1. Effect of PDT of Pa and Hy with Intravenous Injection in MRSA-Infected

Neither of the PDTs (30 s or 10 min) upon Pa and Hy intravenous (i.v.) injection (2.5 mg/kg) significantly (*p* < 0.05) reduced bacterial load at Day 8 (Figure 4). It was observed that mice receiving 30 s of Pa-PDT and Hy-PDT treatments resulted in slight but insignificant promotion of wound closure. However, this trend could not be observed in the 10 min PDT-treated groups (Figure 5). There was no body weight loss in treatment groups when compared with the control group, implying that the treatments did not

Neither of the PDTs (30 s or 10 min) upon Pa and Hy intravenous (i.v.) injection (2.5 mg/kg) significantly (*p* < 0.05) reduced bacterial load at Day 8 (Figure 4). It was observed that mice receiving 30 s of Pa-PDT and Hy-PDT treatments resulted in slight but insignificant promotion of wound closure. However, this trend could not be observed in the 10 min PDT-treated groups (Figure 5). There was no body weight loss in treatment groups when compared with the control group, implying that the treatments did not

Neither of the PDTs (30 s or 10 min) upon Pa and Hy intravenous (i.v.) injection (2.5 mg/kg) significantly (*p* < 0.05) reduced bacterial load at Day 8 (Figure 4). It was observed that mice receiving 30 s of Pa-PDT and Hy-PDT treatments resulted in slight but insignificant promotion of wound closure. However, this trend could not be observed in the 10 min PDT-treated groups (Figure 5). There was no body weight loss in treatment groups when compared with the control group, implying that the treatments did not

**Figure 4.** Bacterial load of wounds in different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg, irradiation for 30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n* = 5). **Figure 4.** Bacterial load of wounds in different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg, irradiation for 30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n* = 5). **Figure 4.** Bacterial load of wounds in different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg, irradiation for 30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n* = 5). **Figure 4.** Bacterial load of wounds in different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg, irradiation for 30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n* = 5).

**Figure 5.** Wound areas in different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg irradiation for 30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n* = 5). **Figure 5.** Wound areas in different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg irradiation for 30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n* = 5). **Figure 5.** Wound areas in different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg irradiation for 30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n* = 5). **Figure 5.** Wound areas in different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg irradiation for 30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n* = 5).

**Figure 6.** Body weight of different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg, irradiation for30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n*=5). **Figure 6.** Body weight of different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg, irradiation for30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n*=5). **Figure 6.** Body weight of different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg, irradiation for30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n*=5). **Figure 6.** Body weight of different groups of mice after intravenous injection treatment with or without PDT. (**A**) Pa and Hy 2.5 mg/kg, irradiation for30 s for PDT groups; (**B**) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (*n*=5).

#### 3.2.2. Effect of PDT of Topically Applied MB, Pa or Hy in MRSA-Infected Wound Model

Topical application of Fucidin cream eradicated MRSA in the wound. Pa-PDT and Hy-PDT treatment groups showed significant antibacterial effects against MRSA when compared with the no treatment group (1 log decrease of CFU, *p* < 0.05) (Figure 7). The size of Fucidin cream-treated wounds was slightly larger and there was no great difference in wound sizes among all other groups after treatment (Figure 8). There was no body weight loss in the treatment groups when compared with the control group (Figure 9), implying that the treatments did not cause distress in the mice.

9), implying that the treatments did not cause distress in the mice

9), implying that the treatments did not cause distress in the mice

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 8 of 15

**Figure 7.** Bacterial load of wounds in different groups of mice after topical treatment with or without PDT. Data are mean ± SEM (*n* = 6–10).\* *p* < 0.05 and \*\*\* *p* < 0.001 indicated significant bacterial load difference between No treatment and treatment groups by Student's *t*-test. **Figure 7.** Bacterial load of wounds in different groups of mice after topical treatment with or without PDT. Data are mean ± SEM (*n* = 6–10).\* *p* < 0.05 and \*\*\* *p* < 0.001 indicated significant bacterial load difference between No treatment and treatment groups by Student's *t*-test. **Figure 7.** Bacterial load of wounds in different groups of mice after topical treatment with or without PDT. Data are mean ± SEM (*n* = 6–10).\* *p* < 0.05 and \*\*\* *p* < 0.001 indicated significant bacterial load difference between No treatment and treatment groups by Student's *t*-test.

3.2.2. Effect of PDT of Topically Applied MB, Pa or Hy in MRSA-Infected Wound Model

Topical application of Fucidin cream eradicated MRSA in the wound. Pa-PDT and Hy-PDT treatment groups showed significant antibacterial effects against MRSA when compared with the no treatment group (1 log decrease of CFU, *p* < 0.05) (Figure 7). The size of Fucidin cream-treated wounds was slightly larger and there was no great difference in wound sizes among all other groups after treatment (Figure 8). There was no body weight loss in the treatment groups when compared with the control group (Figure

3.2.2. Effect of PDT of Topically Applied MB, Pa or Hy in MRSA-Infected Wound Model

Topical application of Fucidin cream eradicated MRSA in the wound. Pa-PDT and Hy-PDT treatment groups showed significant antibacterial effects against MRSA when compared with the no treatment group (1 log decrease of CFU, *p* < 0.05) (Figure 7). The size of Fucidin cream-treated wounds was slightly larger and there was no great difference in wound sizes among all other groups after treatment (Figure 8). There was no body weight loss in the treatment groups when compared with the control group (Figure

**Figure 8.** Wound areas of mice in different groups of mice after topical treatment with or without PDT. Data are mean ± SEM (*n* = 6–10). **Figure 8.** Wound areas of mice in different groups of mice after topical treatment with or without **Figure 8.** Wound areas of mice in different groups of mice after topical treatment with or without PDT. Data are mean ± SEM (*n* = 6–10). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 9 of 15

**Figure 9.** Body weight of different groups of mice before and after treatment with or without PDT. Data are mean ± SEM (*n* = 6–10). **Figure 9.** Body weight of different groups of mice before and after treatment with or without PDT. Data are mean ± SEM (*n* = 6–10).

3.2.3 Histological Evaluation 3.2.3. Histological Evaluation

the no treatment group (Figure 10).

×100 magnification. Scale bar, 200 µm.

PDT. Data are mean ± SEM (*n* = 6–10).

Histopathological assessment of untreated wounds on day 8 (Figure 10) indicated incomplete epithelialization, loose granulation tissue with areas of poorly stained extracellular matrix where collagen fibers were either immature or lacking as a sign of Histopathological assessment of untreated wounds on day 8 (Figure 10) indicated incomplete epithelialization, loose granulation tissue with areas of poorly stained extracellular matrix where collagen fibers were either immature or lacking as a sign of obvious

obvious ulcer formation. It is apparent from Figure 10, Hy and Hy-PDT mediated groups

epithelial cells and fibrous tissue proliferation. MB-PDT had a minor healing effect in granulation and collagen formation and its re-epithelialization was worse than that of the no treatment group. Wound healing of the Fucidin cream-treated group was worse than

**Figure 10.** Representative wound section with haematoxylin and eosin stained and examined at

ulcer formation. It is apparent from Figure 10, Hy and Hy-PDT mediated groups showed rather good wound healing compared to the treatment naïve group by showing epithelial cells and fibrous tissue proliferation. MB-PDT had a minor healing effect in granulation and collagen formation and its re-epithelialization was worse than that of the no treatment group. Wound healing of the Fucidin cream-treated group was worse than the no treatment group (Figure 10). 3.2.3 Histological Evaluation Histopathological assessment of untreated wounds on day 8 (Figure 10) indicated incomplete epithelialization, loose granulation tissue with areas of poorly stained extracellular matrix where collagen fibers were either immature or lacking as a sign of obvious ulcer formation. It is apparent from Figure 10, Hy and Hy-PDT mediated groups

**Figure 9.** Body weight of different groups of mice before and after treatment with or without PDT.

#### *3.3. Cytotoxicity Effect of Pa-PDT, Hy-PDT or MB-PDT on Human Neutrophils* showed rather good wound healing compared to the treatment naïve group by showing

Data are mean ± SEM (*n* = 6–10).

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 9 of 15

Three photosensitizers, Pa, MB and Hy, were incubated with human neutrophils for 24 h. No light irradiation was applied to the photosensitizers. Viability of human neutrophils was determined by MTT assay. As shown in Figure 11, Pa and MB were more cytotoxic to human neutrophils at 24-h incubation with LC<sup>50</sup> at ~10.16 µM and ~11.22 µM, respectively, whereas Hy showed a LC<sup>50</sup> higher than 50 µM. epithelial cells and fibrous tissue proliferation. MB-PDT had a minor healing effect in granulation and collagen formation and its re-epithelialization was worse than that of the no treatment group. Wound healing of the Fucidin cream-treated group was worse than the no treatment group (Figure 10).

**Figure 10.** Representative wound section with haematoxylin and eosin stained and examined at ×100 magnification. Scale bar, 200 µm. **Figure 10.** Representative wound section with haematoxylin and eosin stained and examined at ×100 magnification. Scale bar, 200 µm.

Cytotoxicity of the three photosensitizers with light irradiation for 20 min (48 J/cm<sup>2</sup> ) on human neutrophils was also examined. As shown in Figure 12, Hy-PDT possessed the strongest cytotoxicity with LC<sup>50</sup> less than 3 µM, whereas LC<sup>50</sup> of Pa-PDT and MB-PDT were ~4.44 µM and ~4.23 µM, respectively. As predicted, drugs with light irradiation exhibited significantly higher cytotoxicity than drugs without light irradiation. Given the high cytotoxic properties of photosensitizers with light irradiation, we expected that no cytokines would be produced in human neutrophils, as well as in the photosensitizeradministered wound sites when light irradiation was applied.

**Figure 11.** Cytotoxicity of Pa, MB and Hy, without light irradiation on human neutrophils isolated from buffy coat. Data are mean ± SEM (*n* = 3). **Figure 11.** Cytotoxicity of Pa, MB and Hy, without light irradiation on human neutrophils isolated from buffy coat. Data are mean ± SEM (*n* = 3).

Cytotoxicity of the three photosensitizers with light irradiation for 20 min (48 J/cm2)

on human neutrophils was also examined. As shown in Figure 12, Hy-PDT possessed the strongest cytotoxicity with LC50 less than 3 µM, whereas LC50 of Pa-PDT and MB-PDT

exhibited significantly higher cytotoxicity than drugs without light irradiation. Given the

*3.3. Cytotoxicity Effect of Pa-PDT, Hy-PDT or MB-PDT on Human Neutrophils* 

respectively, whereas Hy showed a LC50 higher than 50 µM.

Three photosensitizers, Pa, MB and Hy, were incubated with human neutrophils for

24 h. No light irradiation was applied to the photosensitizers. Viability of human neutrophils was determined by MTT assay. As shown in Figure 11, Pa and MB were more cytotoxic to human neutrophils at 24-h incubation with LC50 at ~10.16 µM and ~11.22 µM,

high cytotoxic properties of photosensitizers with light irradiation, we expected that no cytokines would be produced in human neutrophils, as well as in the photosensitizer-

administered wound sites when light irradiation was applied.

**Figure 12.** Cytotoxicity of Pa, MB and Hy, with light irradiation, on human neutrophils isolated from buffy coat with. Data are mean ± SEM (*n* = 3). **Figure 12.** Cytotoxicity of Pa, MB and Hy, with light irradiation, on human neutrophils isolated from buffy coat with. Data are mean ± SEM (*n* = 3).

including MRSA, and acts as the gold standard for efficacy comparison for PDTs with Pa and Hy. In vitro results showed that Hy-PDT and Pa-PDT against some MRSA strains had better bactericidal activity than MB-PDT. Among the sixteen MRSA strains tested, the

**4. Discussion** 

### **4. Discussion**

PDT with MB is widely tested to be effective against Gram-positive bacteria, including MRSA, and acts as the gold standard for efficacy comparison for PDTs with Pa and Hy. In vitro results showed that Hy-PDT and Pa-PDT against some MRSA strains had better bactericidal activity than MB-PDT. Among the sixteen MRSA strains tested, the lowest MBC for PDTs with Hy, Pa, and MB were 0.625 µM (0.5 µg/mL), 3.125 µM (2 µg/mL), and 80 µM (32 µg/mL) respectively. Therefore, Hy-PDT and Pa-PDT exhibited potent bactericidal activity on MRSA strains. Hy possesses appropriate photochemical and photobiological properties, such as a high singlet oxygen quantum yield and cytoplasmic membrane localization, which makes it suitable for use as a PS in PDT [29,50]. Furthermore, its absorption maxima (λmax) 570 nm at longer wavelength [51] makes Hy suitable for PDT because of its high penetration ability. The obtained in vitro results for Hy are comparable with the previously published data in Yow et al. [29] for two CA-MRSA strains (W45 and W47, Table 2) where a > 6 log<sup>10</sup> CFU reduction of MRSA was obtained at an 8 µM concentration and 30 Jcm−<sup>2</sup> light dose. However, MBC values for RN4220/pUL5054, W44, W46 and W48 showed significantly lower (*p* < 0.05) MBC values compared to the published report.

In addition, it was found that Pa-PDT inhibited P-glycoprotein-mediated multidrug resistance via c-Jun N-terminal kinase (JNK) activation in human hepatocellular carcinoma [52]. As P-glycoprotein is an important class of efflux pumps that is always associated with a high prevalence of antibiotic resistance, it is hypothesized that Pa can circumvent drug resistance in MRSA as well.

To evaluate the efficacies of photodynamic therapy mediated by Pa and Hy, an MRSAinfected wound-bearing mice model was used. We found that MRSA was far more resistant to PDT in the more complicated environment of the murine dorsal wound than in transparent Petri dishes in vitro. It is possible because the wound tissue provided more layers of organic material to scatter light and to host the bacteria. The open wound of skin tissue, as the natural colony of Staphylococci, might provide a more nutritious matrix for bacterial survival and prosperity, and the aqua dependency of the cytotoxicity of photosensitizers might also partly be impeded in the relatively lower moistures microenvironment of the wounds.

However, the bodyweight of all mice was weighed before and after 8 days of treatment and there was no difference between groups and there was also no significant behavioral change in the mice, indicating that wound induction, MRSA infection, and our treatments had little effect on the general health status of the mice.

It was observed that intravenous injection of photosensitizers with PDT treatments had no antibacterial effect. This may be because none of the photosensitizers had an affinity to wound tissue, so the circulating photosensitizers had little opportunity to aggregate at the wound to generate an adequate amount of ROS by light illumination to kill MRSA. We found that 10 min of PDT resulted in burnt scab formation shortly after light illumination in some cases, while 30 s of PDT did not. The 30 s of PDT treatment also resulted in better wound healing.

The in vivo antibacterial effect of Hy-PDT and Pa-PDT was found to be significant and also stronger than MB-PDT at the same concentration, suggesting encouraging antibacterial effects of them against MRSA wound infection. However, it is interesting that the complete elimination of MRSA by Fucidin cream treatment was accompanied by worse wound healing as reflected by large open wound areas. It was also surprising that the histological appearance of wound healing in MRSA-infected wounds receiving only water and light illumination was significantly higher than that of No treatment group. We also observed more abundant vessel formation in the dermis of some samples than in any other group without significant improvement in open wound area. It has been reported that red light illumination promoted wound healing by promoting ATP release from mitochondria, activating the lymphatic system, increasing blood circulation and forming new capillaries [53]. Since we did not do further cellular nor molecular analyses, we are not in

a position to explain the exact reason behind the varied wound closures among different treatment groups.

We tested the wound healing effects of several commercially available antibacterialagent-free moisturizer skin creams and none of them promoted nor inhibited skin wound healing (data not shown). Pa-PDT, MB-PDT, and Hy-PDT were all toxic to neutrophils in vitro but the neutrophils were more resistant to Pa, MB, and Hy treatments without light illumination. Hy and MB were less toxic, while Pa showed some toxicity even without light illumination. Since our treatments did not include whole body illumination, the topically applied photosensitizers and locally irradiated light illumination could hardly have any toxic effects on the immune system inside the body. In addition, insignificant body weight differences among all groups also indicated the minimum effect of our treatments on the general health of animals.

#### **5. Conclusions**

Hy-PDT, Pa-PDT and MB-PDT were all capable of killing MRSA in vitro, and Hy-PDT showed highest efficacy against panel of MRSAs. Both Hy-PDT and Pa-PDT showed better efficacy than MB-PDT in antibacterial and wound healing effects against MRSAinfected wounds in our murine model, and the efficacy of Hy-PDT was the best among all treatments tested.

**Author Contributions:** B.C.L.C., P.D., B.W., K.M.L., C.C.L., D.W.S.C., J.Y.W.C. and G.G.L.Y. were responsible for conducting the experimental work; C.B.S.L. and C.K.W. were responsible for the study design, K.P.F. and M.I. were responsible for supervision, administration and writing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant from the Health and Medical Research Fund (HMRF 13120302).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong (Ref No.12/076/MIS and 8 February 2013).

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


## *Article* **In Vivo Quantification of the Effectiveness of Topical Low-Dose Photodynamic Therapy in Wound Healing Using Two-Photon Microscopy**

**Hala Zuhayri <sup>1</sup> , Viktor V. Nikolaev <sup>1</sup> , Anastasia I. Knyazkova <sup>1</sup> , Tatiana B. Lepekhina <sup>1</sup> , Natalya A. Krivova <sup>1</sup> , Valery V. Tuchin 1,2 and Yury V. Kistenev 1,\***


**Abstract:** The effect of low-dose photodynamic therapy on in vivo wound healing with topical application of 5-aminolevulinic acid and methylene blue was investigated using an animal model for two laser radiation doses (1 and 4 J/cm<sup>2</sup> ). A second-harmonic-generation-to-auto-fluorescence aging index of the dermis (SAAID) was analyzed by two-photon microscopy. SAAID measured at 60–80 µm depths was shown to be a suitable quantitative parameter to monitor wound healing. A comparison of SAAID in healthy and wound tissues during phototherapy showed that both light doses were effective for wound healing; however, healing was better at a dose of 4 J/cm<sup>2</sup> .

**Keywords:** wound healing; low-dose photodynamic therapy; photosensitizer; two-photon microscopy; second-harmonic-generation-to-auto-fluorescence aging index of the dermis

## **1. Introduction**

Wound healing is a complex physiological and dynamic process that occurs in the skin at the cellular level. It involves different overlapping phases of cellular activity that occur in the proper sequence, at a definite time, and for a specific duration [1,2]. Hemostasis begins within minutes of a wound occurring with vascular constriction and ends in the formation of a fibrin clot. The latter is considered essential in promoting the onset of the inflammatory and repair phases [3]. As soon as the clot is formed, proinflammatory cytokines and growth factors are released, such as fibroblasts, plateletderived growth factor and epidermal growth factor. Inflammatory cells migrate into the wound and promote the inflammatory phase, characterized by the sequential infiltration of neutrophils, macrophages, and lymphocytes [3,4]. Neutrophils play an important role in clearing microbes and cellular debris in the wound area. Macrophages are responsible for inducing and removing apoptotic cells, thus paving the way for the resolution of inflammation. As macrophages clear apoptotic cells, they undergo a phenotypic transition to a reparative state that stimulates keratinocytes, fibroblasts, and angiogenesis to promote tissue regeneration. In this way, macrophages promote a transition to the proliferative phase of healing [5]. The latter generally follows and overlaps with the inflammatory phase, beginning approximately on the third or fourth day after injury. It includes granulation tissue formation, re-epithelialization, neoangiogenesis, regeneration of the extracellular matrix (ECM), and wound contraction [6,7].

Within the wound bed, fibroblasts produce collagen, glycosaminoglycans, and proteoglycans, major ECM components. Following proliferation and ECM synthesis, wound healing enters the most prolonged and final remodeling phase, typically beginning one

**Citation:** Zuhayri, H.; Nikolaev, V.V.; Knyazkova, A.I.; Lepekhina, T.B.; Krivova, N.A.; Tuchin, V.V.; Kistenev, Y.V. In Vivo Quantification of the Effectiveness of Topical Low-Dose Photodynamic Therapy in Wound Healing Using Two-Photon Microscopy. *Pharmaceutics* **2022**, *14*, 287. https://doi.org/10.3390/ pharmaceutics14020287

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 28 December 2021 Accepted: 21 January 2022 Published: 26 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

week after injury following collagen deposition in the wound [8]. Collagen, including collagen types I and III, is one of the major skin components [9]. During the wound healing process, type III collagen is replaced by type I collagen, which provides tensile stiffness. Collagen fibers remodel by aligning with the body's tension lines and gaining strength through cross-linking [7–10]. In this final stage of healing, an attempt to recover the normal tissue structure occurs, and the granulation tissue is gradually remodeled, forming scar tissue [10,11].

Photo processes induced by external radiation mediated by photoactive compounds in tissues contribute to skin disease curing, skin rejuvenation, and wound healing [12,13]. Photodynamic therapy (PDT) is widely used to treat skin diseases like acne, viral warts, and skin cancers [14–17]. PDT enables a reduction in treatment time, accelerated tissue repair, and the promotion of wound healing [18,19].

PDT is based on using a photosensitizer (PS), which is accumulated in tissues, followed by irradiation of the tissue with a light source of an appropriate wavelength. The latter causes the generation of reactive oxygen species (ROS) [14,20]. ROS stimulates various cell administration pathways: apoptosis, necrosis, autophagy, depending on the type of cell treated, the concentration of the PS used, the dose of light energy supplied, and PS intracellular location [20–22]. High concentrations of ROS cause cell death, while low concentrations can cause the triggering of cellular repair processes, including proliferation and autophagy, and offer treatment by promoting healing [20].

The first PS generation was mainly constituted by hematoporphyrin and its derivatives, which have low selectivity and high cutaneous phototoxicity [23,24]. The second PS generation included porphyrin, phthalocyanine, benzoporphyrin, thiopurine derivatives, chlorin, and phenothiazines. It is characterized by greater deep light penetration capability due to its maximum absorption in the 630–800 nm spectral range corresponding to the tissue's highest transparency, good solubility, and higher chemical purity [14,25]. For skin treatments, 5-aminolevulinic acid (5-ALA), which is converted into the endogenous PS protoporphyrin IX (PpIX), has an absorption maximum at 630 nm and is widely used [22,26,27]. Methylene blue (MB) is a well-known phenothiazine derivative with great importance in medicine, including antimicrobial and antiviral light-induced activity [25,28,29]. The third PS generation corresponds to enhanced forms of the first and second generations. It is often combined with a carrier such as a lipoprotein, liposomes, nanoparticles, or conjugated antibodies to increase selectivity and contrast in affected areas [30–33].

According to the Arndt–Schultz law, no tissue response will occur when low-level laser light is applied with a low dose. If used with a high dose, it can inhibit tissue response and even induce the proliferation of cancer cells or microorganisms [34,35]. Therefore, there is an optimal dose where a maximal response is obtained [36]. Some studies suggest that open wound healing stimulation occurs in the 0.5–1 J/cm<sup>2</sup> light dose ranges and the 2–4 J/cm<sup>2</sup> range for superficial wound healing stimulation through the skin [37,38]. Alternatively, doses were proposed in the region of 4 J/cm<sup>2</sup> with a range of 1–10 J/cm<sup>2</sup> for superficial targets [38]. However, according to most previous studies, the optimal light doses are in the 1–5 J/cm<sup>2</sup> interval [39–41]. Byrnes et al. established that 632 nm light at an energy density of 4 J/cm<sup>2</sup> activates collagen formation [41]. Prabhu et al. studied the effect of a 632.8 nm laser with different energy doses, including 1 J/cm<sup>2</sup> and 2 J/cm<sup>2</sup> , and achieved good results [42]. Another study demonstrates the utility of photobiomodulation (PBM) therapy (810 nm with a total energy of 3 J/cm<sup>2</sup> ) in mitigating burn injury and provides the biological rationale for its clinical application in wound healing [43]. PBM with various light parameters has been used widely in skincare but can cause certain types of unwanted cells to proliferate in the skin; this can lead to skin tumors, such as papillomas and cancers. H. Goo and his colleagues confirmed that LEDs with a wavelength of 642 nm with a total fluence of 21.6 J/cm<sup>2</sup> , increased tumor size, epidermal thickness, and systemic proinflammatory cytokine levels [34]. In an infrared neural stimulation (INS) study, Throckmorton and colleagues evaluated the parameters of light such as wavelength, radiant exposure, and optical spot size using three commonly used wavelengths of INS,

1450 nm, 1875 nm, and 2120 nm. The pulsed diode lasers at 1450 nm and 1875 nm had a consistently higher (∼1.0 J/cm<sup>2</sup> ) stimulation threshold than that of the Ho:YAG laser at 2120 nm (∼0.7 J/cm<sup>2</sup> ). An acute histological evaluation of diode-irradiated nerves revealed a safe range of radiant exposures for stimulation [44].

Since two optical methods are mainly used for wound healing—PDT based on exogenous PSs and low-level light therapy (LLLT) without the use of exogenous PSs—it is crucial to find an optimal technology that combines the advantages of both methods—using low doses of radiation, correcting the effects that stimulate healing wounds, and using selective staining of wounds with exogenous photosensitizers with an optimal (relatively low) concentration. This approach is called low-dose PDT (LDPDT).

Traditionally, wounds are observed invasively with a histochemical assessment of biopsies. Similar methods are non-quantitative and may cause tissue damage and healing delay [45]. Recent studies have turned to optical imaging methods. Two-photon microscopy (TPM) is a modern molecular imaging method that enables noninvasive evaluation and monitoring of skin morphological structure and functions at the cellular and subcellular levels with a high spatial resolution [46–48]. TPM, including autofluorescence (AF) and second harmonic generation (SHG), can provide functional and structural imaging of biological tissue and assess cells' in vivo metabolic status [48]. Type I collagen induces SHG due to its noncentrosymmetric molecular structure. Therefore, TPM can be used for collagen disordering control, which occurs in wound healing [49–51]. The SHG-to AF aging index of the dermis (SAAID) is defined as the difference between SHG and AF intensity signals, indicative of type I collagen and elastin, respectively, and normalized to the sum of both signals as follows [52–56]:

$$\text{SAAID} = \text{(SHG} - \text{AF)} / \text{(SHG} + \text{AF)}.\tag{1}$$

SAAID is a suitable quantitative parameter for characterizing a skin condition, which, as a specific parameter for monitoring wound healing, was proposed in [54], but has not been studied in detail.

This study aims to investigate the dynamics of SAAID in vivo using TPM during LDPDT wound healing by employing the topical administration of two different photosensitizers, 5-ALA and MB, and two laser fluences, 1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> .

#### **2. Materials and Methods**

#### *2.1. An Animal Model of a Wound*

In vivo experiments were performed using fifteen male CD1 mice aged 6–7 weeks and weighing 25–30 g according to experimental protocol No.4, 10.02.2021, registration No. 6, as approved by the Bioethical Committee of Tomsk State University. Animals were obtained from the Department of Experimental Biological Models of the Research Institute of Pharmacology, TSC SB RAMS. Before the experiment, the mice were kept for 7 days in the standard conditions of a conventional vivarium with free access to water and food, and a 12/12 light regime in a ventilated room at a temperature of 20 ± 2 ◦C and a humidity of 60%.

The mice were anesthetized by isoflurane using the Ugo Basile gas anesthesia system, where the mice were put in a glass chamber connected to isoflurane. The parameter on the isoflurane cylinder was set to 3–4%. It is recommended that the mouse be placed inside for 10–15 min. The wound area was depilated by Veet cream (France), rinsed with saline solution, and sterilized with chlorhexidine 20%. No additional drugs were used. The paws' skin was folded and raised using forceps. Then, the mouse was placed in a lateral position and using medical scissors the skin layers were completely removed and excisional circular wounds (diameter 5 mm) were created, as shown in Figure 1. This procedure was repeated on both the hind paws of each animal. The operations were carried out entirely under the influence of isoflurane gas attached via a mask to the mouse's nose, but here the isoflurane cylinder was set to 1–1.5%. The experiment was performed in a time-lapsed schedule for wound aging on days 1, 3, 7, and 14. The day of wound formation was denoted as day 0.

**Figure 1.** A full-thickness cutaneous wound.

#### *2.2. Wound Healing Assay*

The photos for illustrations were taken using a 16 MP camera with a 26 mm focal length, a 2x magnification lens, and an f/1.9 aperture at observational time points until the wounds healed; examples are presented in Figure 2. The wound size was calculated with a digital caliper by measuring its larger A and minor B diameters on every measurement day. The wound area was determined by the formula S = (A × B × π)/4. The digital caliper accuracy was 0.02 mm. Thus, this value makes an insignificant contribution to the measurement error when calculating the area and it was not considered in our analysis. The percentage of wound closure was calculated as follows:

$$\left[ (\text{S}\_0 - \text{S}) / \text{S}\_0 \right] \times 100,\tag{2}$$

where S<sup>0</sup> is the area of the original wound and S is the area of the current wound.

#### *2.3. Low Dose Photodynamic Therapy Protocol*

The photosensitizers were prepared by dissolving the powder 5-ALA and MB in saline solution. The 0.1–0.2 mL of 5-ALA 20%/MB 0.01% saline solutions were topically administered for 30 min of incubation, dripping directly on the wound when the mouse was placed on a base under isoflurane. The wounds were irradiated by an AlGalnP laser (λ = 630 nm, P = 5 mW) with two fluences: 1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> for 3 min 45 s and 15 min, respectively. The animals were randomly divided into three basic groups; each group included 5 mice: the control group, LDPDT/1 J/cm<sup>2</sup> group, and LDPDT/4 J/cm<sup>2</sup> group.

In the LDPDT groups, 5-ALA was applied on the wounds on the right hind paws, MB was applied on the left ones. Thus, in total, there were 5 groups: the control, LDPDT-5- ALA/1 J/cm<sup>2</sup> , LDPDT-MB/1 J/cm<sup>2</sup> , LDPDT-5-ALA/4 J/cm<sup>2</sup> , and LDPDT-MB/4 J/cm<sup>2</sup> groups. The LDPDT procedure was repeated once immediately after wound formation.

**Figure 2.** Digital photograph assessment of healing progression from day 0 to day 14: (**a**) control group; (**b**) LDPDT–5-ALA/1 J/cm<sup>2</sup> ; (**c**) LDPDT–5-ALA/4 J/cm<sup>2</sup> ; (**d**) LDPDT–MB/1 J/cm<sup>2</sup> ; (**e**) LDPDT–MB/4 J/cm<sup>2</sup> .

### *2.4. Two-Photon Microscope*

The wound was analyzed using a two-photon microscope, MPTflex (Jenlab GmbH, Jena, Germany). The pump laser wavelength was 760 nm and filters at bands 373–387 nm for a SHG signal and at 406–610 nm for an AF signal were used. The repetition frequency was 80 MHz with laser pulse width ~200 fs. The manufacturer's declared spatial resolution <0.5 µm (horizontal); <2 µm (vertical) with focusing optics: magnification 40× NA 1.3. The object was placed directly under the cover glass of a 100–170 µm thickness. A special metal ring was used as a cover glass holder. The space between the glass and the lens was filled with Carl Zeiss™ Immersol™ immersion oil to obtain a better signal. Skin structure was studied at 0–80 µm depth with a 4 µm step, while the pump laser power was varied from 5 mW at a depth of 0 µm (the beginning of the stratum corneum) to 40 mW at a depth of 80 µm. SHG and AF images were recorded on a 512 × 512-pixel matrix; the image size was 70 × 70 µm. The AF and SHG channels were electronically separated

using appropriate spectral filters and recorded in digital matrices in "\*.tiff" format in two independent channels. RGB color space was used for visualization, where the SHG and AF signals were shown in the red and green channels, respectively. Five stacks for different skin areas were scanned for each mouse during measurement. AF and SHG images were processed using SPCImage and Python software. This study did not perform data preprocessing as areas with acceptable magnification and image quality were selected. The SAAID index was chosen as the main characteristics of the AF and SHG signals. SAAID had been calculated depending on depth for all groups.

#### *2.5. Statistical Analysis*

All calculated parameters were expressed as mean ± standard deviation of the mean (SD). The Mann–Whitney U test was used to analyze the differences between two independent datasets. The level of significance was set at *p* < 0.05.

#### **3. Results**

#### *3.1. Visual Observation*

The digital photographs of the wounds at different time points were taken for the control and LDPDT groups on the surgery day (0) and successively on days 1, 3, 7, and 14 (Figure 2). The presented photos were only shown to illustrate the process of wound healing in the groups.

During healing, the wound sizes were evaluated according to Equation (2) for five mice from each group (Table 1). The values are presented as mean ± standard deviation. Figure 3 illustrates the wound healing rate for all groups.

**Table 1.** The wound size in mm<sup>2</sup> during wound healing (mean <sup>±</sup> standard deviation).


**Figure 3.** Wound healing rate.

#### *3.2. In Vivo TPM Imaging*

In vivo TPM imaging was used to assess differences in wound healing. Figure 4 shows the SHG and AF images of all groups at a depth of 60 µm. Type I collagen fibers indicated by the SHG signal are shown in red and the elastin fibers indicated by AF signals are in green. The white scale bar shows a length of 10 µm.

**Figure 4.** SHG (red) and AF (green) images of wound healing. (**a**) Control group; (**b**) LDPDT–5- ALA/1 J/cm<sup>2</sup> ; (**c**) LDPDT–5-ALA/4 J/cm<sup>2</sup> ; (**d**) LDPDT–MB/1 J/cm<sup>2</sup> ; (**e**) LDPDT–MB/4 J/cm<sup>2</sup> .

Different forms of collagen structure could be observed between days 1 and 14: on day 1, the collagen was disorganized and dispersed in a blurry form. Over the following days, organized collagen gradually started to form, which was confirmed by the increase in SHG signal intensity. On day 14, the collagen fibers were rearranged, cross-linked, and aligned (Figure 4). On the seventh day, the SHG signal intensity in LDPDT groups was higher so the collagen formation was better in LDPDT groups. Figure 5 shows the difference between the organized collagen in healthy tissue and the disorganized collagen in a wound at depths from 4 to 80 µm.

**Figure 5.** (**a**) SHG (red) and AF (green) of healthy skin at depths from 4 to 80µm; (**b**) SHG (red) and AF (green) of a wound at depths from 4 to 80 µm.

#### *3.3. The SAAID Estimation*

For quantification of relative amounts of elastin and collagen, the SAAID was estimated for various depths. The corresponding SAAID curve for healthy skin (comparison area) is shown in Figure 6. The SAAID index for the control group is shown in Figure 7a. The SAAID curve started at slightly negative values and was still negative on day 1. On day 3, the curve did not differ much as the values remained negative. On day 7, the SAAID had a minimum value of −0.4 (a depth of 40 µm). On day 14, the curve shape changed compared to day 1 and day 3, the value of SAAID at 70 µm increased to zero, and then the SAAID reached its maximum of 0.2 at a depth of 80 µm. Figure 7b shows the SAAID index for the LDPDT 5-ALA 4 J/cm<sup>2</sup> group. It was different from the control group; in general the values were larger and on days 7 and 14 they were approximately close to healthy skin values, contrary to the control group. On day 7, the SAAID index started from zero and reached a minimum value of −0.4 (at a depth of 35 µm), rising to zero at 75 µm and a maximum of +0.2 at a depth of 80 µm. With a slight change and larger values, the curve for day 14 is shown. The SAAID reached its minimum at a value of −0.35 (a depth of 25 µm), after which the SAAID curve reached a maximum of +0.3 at a depth of 80 µm. Therefore, the SAAID was found to be substantially affected by the day of the wound healing process. The SAAID of healthy skin vs. control group and LDPDT 5-ALA (1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> ) groups on day 14 are shown in Figure 8a. The difference was evident, especially in the range from 40 µm to 80 µm, with higher values for LDPDT 5-ALA/4 J/cm<sup>2</sup> . Additionally, Figure 8b shows the SAAID for healthy skin, the control group, and LDPDT–MB (1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> ) groups on day 14.

**Figure 6.** SAAID for healthy skin.

**Figure 7.** (**a**) SAAID for control group; (**b**) SAAID for LDPDT–5-ALA/4 J/cm<sup>2</sup> group.

**Figure 8.** (**a**) SAAID for LDPDT–5-ALA (4 J/cm<sup>2</sup> and 1 J/cm<sup>2</sup> ) groups; (**b**) SAAID for LDPDT–MB (4 J/cm<sup>2</sup> and 1 J/cm<sup>2</sup> ) groups.

A comparison of LDPDT 5-ALA and MB/4 J/cm<sup>2</sup> vs. healthy skin and the control group on day 14 is presented in Figure 9. When MB was employed, the values were closer to those of healthy skin. The specificity of the SAAID for healthy skin, the control group of wound healing, and the LDPDT groups with 5-ALA and MB (1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> ) was analyzed. The SAAID differences for the studied groups at various depths are shown in Figure 10 and these differences were more evident at depths of 60 µm and 80 µm.

**Figure 9.** SAAID for LDPDT–5-ALA and LDPDT–MB/4 J/cm<sup>2</sup> groups.

**Figure 10.** Index SAAID on day 14 at 40 µm, 60 µm and 80 µm for healthy skin, control, LDPDT–5- ALA/1 J/cm<sup>2</sup> , LDPDT–5-ALA/4 J/cm<sup>2</sup> , LDPDT–MB/1 J/cm<sup>2</sup> , and LDPDT–MB/4 J/cm<sup>2</sup> .

The SAAID index at depths from 0 to 40 µm on day 14 described a healthy and regenerated skin epidermis layer. The index values for healthy skin, the control area, and skin after LDPDT exposure did not differ significantly, as demonstrated in Figures 7–10. It indicates that the index did not show significant differences in the epidermis after recovery or that these changes were minor. There were differences in SAAID values deeper than 60 µm (papillary dermis layer) between the control and LDPDT groups. These differences were most pronounced at a depth of 80 µm. It indicates a significant recovery of the papillary layer during LDPDT so it could be concluded that on day 14 after exposure to LDPDT/4 J/cm<sup>2</sup> , both 5-ALA and MB showed accelerated skin regeneration.

#### *3.4. Mann–Whitney U Test*

The Mann–Whitney test was applied to assess differences in between the wound skin group and the healthy skin group. SAAID variation *p*-values for the five groups, depths from 40 to 80 µm, in all days are shown in Tables 2–4. Statistical power was set at 0.95. The higher the *p*-value, the more minor the difference between a wound skin group and the healthy skin group. Thus, Figure 11 shows the *p*-value on day 14 by depth. This dependence was calculated with a smaller step in depths than the data presented in Figure 10. Indeed, we see that SAAID measured in the 47–50 µm depth interval is the most sensitive to the wound state.



\* *p* < 0.05.

**Table 3.** The *p*-value for the SAAID in LDPDT–5-ALA/4 J/cm<sup>2</sup> and MB/4 J/cm<sup>2</sup> relative to healthy skin.


\* *p* < 0.05.

**Table 4.** The *p*-value for the SAAID in LDPDT 5ALA/1 J/cm<sup>2</sup> and MB/1 J/cm<sup>2</sup> relative to healthy skin.


\* *p* < 0.05.

**Figure 11.** *p*-values of SAAID for all groups at depths from 40 to 60 µm and 80 µm on day 14.

It is worth noting that the *p*-value for LDPDT/4 J/cm<sup>2</sup> was much higher than for the control group, indicating better healing.

#### **4. Discussion**

The most common collagens are fibrils (collagens of types I, II, III, V, and XI) and reticular structures (collagens IV, VIII, and X) in the intercellular matrix. Since the type of collagen is directly related to the amino acid sequence underlying its structure, the optical properties of collagens vary depending on its type. As mentioned in the Introduction, collagen I, typical for skin, can be visualized through SHG-microscopy. According to our data, during the wound healing process, especially in the first three days, the collagen was disorganized and disordered. However, after a time, a newly laid collagen matrix gradually formed to fill the wound gap, which was detected by the increase in SHG signal intensity. The disorganized collagen is rearranged, cross-linked, and aligned, which can be observed on day 14 (see Figure 4).

From studying and comparing the five groups (control, LDPDT–5-ALA/4 J/cm<sup>2</sup> , LDPDT–5-ALA/1 J/cm<sup>2</sup> , LDPDT–MB/4 J/cm<sup>2</sup> , and LDPDT–MB/1 J/cm<sup>2</sup> ), it has been demonstrated that the 4 J/cm<sup>2</sup> laser dose is better in comparison with 1 J/cm<sup>2</sup> . For the 4 J/cm<sup>2</sup> dose, MB enables better healing compared to 5-ALA (see Figure 3). The benefits of MB are its low cost and wide availability. In any case, a fourfold fluence increase did not cause a substantial increase in efficiency, which suggests that at the used concentrations of photosensitizers, it is quite acceptable to choose a fluence between 1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> .

SAAID values have previously been demonstrated for the papillary dermis during skin aging [53]. In [51], SAAID was applied to estimate skin damage by curettage. Every phase of wound healing was studied, as in our research, and similar values were obtained for different depths. Later, the same group of researchers [54] showed similar results for chronic wounds, consistent with our research. The results for estimating wound healing during the photodynamic therapy process using SAAID have not previously been studied in the literature. In this study, variations of the SAAID value during the wound healing process were established. On day 14, in the LDPDT 4 J/cm<sup>2</sup> group, the SAAID curve was practically the same as for healthy skin (see Figures 7 and 8).

SAAID variance during wound healing demonstrates that the healing efficiency does not increase significantly with a fourfold fluence increase. Moreover, the healing process with low fluence is more uniform across the dermis (see Figure 8).

The *p*-value of the difference of SAAID measured in wounds relative to the healthy skin group was calculated using the Mann–Whitney test. On day 14, the *p*-values were higher, so the differences are smaller relative to healthy skin (see Figure 11). This appears significantly and more clearly in both LDPDT groups. Consequently, SAAID is a suitable quantitative parameter for monitoring the wound healing process.

Interestingly, the most essential SAAID variance between healthy and wounded skin was in the 47–55 µm depth interval (Figure 11). It is an area of the papillary dermis with a large amount of collagen and elastin, but there are practically no capillary blood vessels yet. Recent studies showed that liquid or small molecules preferentially colocalize with elastin fibers [57], affecting its fluorescence in an area of inflammation. Therefore, SAAID change during wound healing can be associated with both collagen and elastin transformation.

#### **5. Conclusions**

Two-photon microscopy is becoming a conventional tool for noninvasive medical diagnostics of superficial tissues and dynamic monitoring of skin pathology treatment. The latter requires the discovery of specific quantitative criteria of the tissue state. The SAAID index was shown to be a suitable variant of the quantitative criterium for wound healing supervision, including optimal regimes of wound healing low-dose photodynamic therapy.

The development of this approach can be as follows. First of all, fluorescence lifetime imaging combined with SGH and AF channels will give additional information about cell metabolism in a wound area [58]. Other optical modalities, for example, optical coherence tomography, will allow data to be obtained about wound tissue morphology at a lower spatial resolution (the typical value is about 5–7 µm) but with a much larger dimension of tissue area visualization compared to TPM.

The phototherapy of wounds is closely related to the problem of infectious disease treatment, especially in the presence of antibiotic-resistant bacteria in the bacterial biofilms covering the wound surface [35,59]. Since the development of antibacterial drugs is not keeping pace with microbial resistance, it is necessary to use alternative antibacterial approaches, including antimicrobial PDT. In this sense, the effectiveness of wound healing is determined mainly by the combined effect of light on the bacterial flora hidden in the depth of the wound and the restoration of the main protein components of the skin collagen and elastin. Both processes require efficient staining of pathogens and dermal cells and a homogeneous distribution of excitation light for the effective production of ROS throughout the entire thickness of the wound.

In this case, it is necessary to overcome the limited depth of light penetration into the tissue in order to enhance the therapeutic effect. It can be done by partially suppressing tissue light scattering when exposed to immersion agents using the optical clearing method [60]. This enables not only an increase in the efficiency of therapy (a decrease in fluence) but also makes it possible to improve optical monitoring of the healing process, providing a greater depth of probing [61], which is vital for deep wounds. It is also important that many optical clearing agents, such as glycerol, are good solvents for photodynamic dyes, ensuring the stability of their absorption spectra [15] and possessing bactericidal properties, which can give synergy in the light treatment of wounds. Deeper photodynamic wound therapy can be achieved by using indocyanine green (ICG) as a PDT dye that is effective in the near-infrared range [16].

**Author Contributions:** Conceptualization, Y.V.K. and V.V.T.; methodology, H.Z. and N.A.K.; software, V.V.N.; validation, H.Z. and V.V.N.; investigation, H.Z., V.V.N., A.I.K., T.B.L. and N.A.K.; writing—original draft preparation, H.Z. and A.I.K.; writing—review and editing, Y.V.K. and V.V.T.; visualization, H.Z.; supervision, Y.V.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a grant under the Decree of the Government of the Russian Federation No. 220 of 9 April 2010 (Agreement No. 075-15-2021-615 of 4 June 2021).

**Institutional Review Board Statement:** The study was approved by the Ethics Committee of Tomsk State University (Protocol No. 4, 10.02.2021), registration No. 6.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

**Acknowledgments:** The authors thank Peter Mitchell, full member of the Institute of Linguists of Great Britain, for his professional editing.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


## *Article* **The In Vivo Quantitative Assessment of the Effectiveness of Low-Dose Photodynamic Therapy on Wound Healing Using Optical Coherence Tomography**

**Hala Zuhayri , Viktor V. Nikolaev , Tatiana B. Lepekhina, Ekaterina A. Sandykova, Natalya A. Krivova and Yury V. Kistenev \***

> Laboratory of Laser Molecular Imaging and Machine Learning, Tomsk State University, Lenin Ave. 36, 634050 Tomsk, Russia; zuhayri.n.hala@gmail.com (H.Z.); vik-nikol@bk.ru (V.V.N.); tatiana\_lepekhina@mail.ru (T.B.L.); katrin\_nemchenko@mail.ru (E.A.S.); nakri@res.tsu.ru (N.A.K.)

**\*** Correspondence: yuk@iao.ru

**Abstract:** The effect of low-dose photodynamic therapy on in vivo wound healing was investigated using optical coherence tomography. This work aims to develop an approach to quantitative assessment of the wound's state during wound healing including the effect of low-dose photodynamic therapy using topical application of two different photosensitizers, 5-aminolevulinic acid and methylene blue, and two laser doses of 1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> . It was concluded that the laser dose of 4 J/cm<sup>2</sup> was better compared to 1 J/cm<sup>2</sup> and allowed the wound healing process to accelerate.

**Keywords:** low dose photodynamic therapy; wound healing; 5-aminolevulinic acid; methylene blue; optical coherence tomography

### **1. Introduction**

According to the World Health Organization (WHO), burn wounds result in approximately 180,000 deaths every year and nearly 11 million injuries that require medical treatment worldwide [1]. Cutaneous wounds are widespread and differentiated into acute and chronic wounds [2].

Wound healing is a complex physiological process at the cellular and molecular levels including the extracellular matrix synthesis, the replacement of type III collagen with type I collagen, and scar tissue formation [3–6]. These processes are divided into four overlapping stages: coagulation (hemostasis), inflammation, proliferation, and remodeling [7,8]. Some underlying diseases affect the wound healing process including peripheral arterial and venous disease or diabetes; acute wounds may have impaired healing, which can lead to a chronic stage [9–11]. In developed countries, 1–6% of the population suffers from chronic wounds [12–14].

It is known that a low dose photo process with photoactive compounds promotes the healing of skin diseases and leads to results in rejuvenation and wound healing [15,16]. Low-dose photodynamic therapy (LDPDT) is widely used to treat skin diseases and wound healing where it reduces the treatment time, accelerates tissue repair, and promotes healing [17,18]. The method is based on using a photosensitizer (PS), which accumulates in tissues, followed by irradiation of the tissue with a light source with an appropriate wavelength. The latter causes the formation of reactive oxygen species (ROS) [19,20]. Low concentrations of ROS can trigger cell repair processes including proliferation and offer promising treatments to accelerate healing. Different PSs have been studied in the wound healing process, which has a relevant role in ensuring PDT effectiveness in skin wound healing [21] such as 5-aminolevulinic acid (5-ALA) and methylene blue (MB) [22–25]. MB is a popular PS among the phenothiazinium derivatives that have attracted the attention of different research groups working and achieving good results in wound healing [25–27].

**Citation:** Zuhayri, H.; Nikolaev, V.V.; Lepekhina, T.B.; Sandykova, E.A.; Krivova, N.A.; Kistenev, Y.V. The In Vivo Quantitative Assessment of the Effectiveness of Low-Dose Photodynamic Therapy on Wound Healing Using Optical Coherence Tomography. *Pharmaceutics* **2022**, *14*, 399. https://doi.org/10.3390/ pharmaceutics14020399

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 18 December 2021 Accepted: 9 February 2022 Published: 11 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Recently, MB was shown to have an antioxidant role [28]. Additionally, 5-ALA is among the most effective photosensitizers and is widely used to present a better achievement concerning wound healing [17,22,29].

The Arndt–Schultz Law is an appropriate model to demonstrate that low levels of light have a better effect in wound healing than higher levels, which may have an inhibitory or cytotoxic effect [30,31]. Hawkins and Abrahamse studied the behavior in vitro of human skin fibroblasts using different irradiation doses of 0.5, 2.5, 5, 10, and 16 J/cm<sup>2</sup> . They demonstrated that higher laser doses (10 and 16 J/cm<sup>2</sup> ) resulted in increased cellular damage as well as decreased cell viability and proliferation [32]. Results for different energy doses were described for 4 J/cm<sup>2</sup> [33] and for 1 J/cm<sup>2</sup> and 2 J/cm<sup>2</sup> [34]. Basso et al. demonstrated that irradiation of cultured human gingival fibroblasts with energy doses of 0.5 and 3 J/cm<sup>2</sup> resulted in a significant increase in cellular metabolism compared with the non-irradiated control group and the cells irradiated with higher energy doses of 5 and 7 J/cm<sup>2</sup> [35]. The most significant biological effects were seen with predominant dose values (i.e., up to 5 J/cm<sup>2</sup> ), which were within the Arndt–Schultz curve [36].

Traditionally, wounds have been observed invasively with a histochemical assessment of the biopsies [3,8]. Visual observation is a common tool for wound assessment. Additionally, clinical wound evaluation is a widely used and the least expensive method of assessing wound depth. This method relies on a subjective evaluation of the external features of the wound such as wound appearance, capillary refill, and burn wound sensibility to touch and pinprick, providing diagnostic accuracy at the level of 60–75% [37]. These methods are not quantitative and can lead to additional tissue damage and impair healing. Accordingly, the development of noninvasive and accurate methods of wound analysis is relevant.

Optical coherence tomography (OCT) is a noninvasive 3D imaging method of biological tissues with a spatial resolution of 5–10 µm and a penetration depth of 1–2 mm [38,39]. Epidermal thickness is a critical parameter for assessing epithelialization during wound healing [40,41].

OCT could detect essential morphological changes during wound healing (e.g., epidermis, dermis, adipose tissue, and granulation) that was based primarily on their backscattering characteristics [42–44]. The use of polarization-sensitive OCT revealed higher birefringence in scars compared to healthy skin [45]. OCT-based angiography provides in vivo, three-dimensional vascular information by using flowing red blood cells as intrinsic contrast agents, allowing visualization of functional vessel networks within microcirculatory tissue beds non-invasively, without needing dye injection [46].

This work aims to develop a method for quantitative in vivo evaluation of wounds using OCT during the wound healing process including a quantitative assessment of the effect of LDPDT using the topical application of two different photosensitizers (5-ALA and MB) and two laser doses of 1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> .

#### **2. Materials and Methods**

#### *2.1. Wound Model Protocol*

This study used 15 male laboratory CD1 mice, weighing 25–30 g and aged 6–7 weeks, obtained from the Department of Experimental Biological Models of the Research Institute of Pharmacology, TSC SB RAMS. Before the experiment, the mice were kept seven days in the standard conditions of a conventional vivarium with free access to water and food, and a 12/12 light regime, in a ventilated room at a temperature of 20 ± 2 ◦C and a humidity of 60%. The experimental protocol of this research was approved by the Bioethical Committee of Tomsk State University (Protocol No. 4, 10.02.2021), registration No. 6.

The mice were anesthetized by isoflurane using the Ugo Basile gas anesthesia system, where the mice were put in a glass chamber connected to isoflurane (Figure 1). The wound area was prepared through depilation using Veet cream (made in France), rinsed with saline solution, and sterilized using chlorhexidine 20%. A full-thickness cutaneous wound (diameter 5 mm) was formed by cutting out a whole layer skin flap with scissors on both of the hind paws of each animal under isoflurane anesthesia. The experiment was performed

in a time-lapsed schedule for the wound aging on days 1, 3, 7, and 14. Additionally, the day of wound formation was defined as day 0. performed in a time-lapsed schedule for the wound aging on days 1, 3, 7, and 14. Additionally, the day of wound formation was defined as day 0.

wound area was prepared through depilation using Veet cream (made in France), rinsed with saline solution, and sterilized using chlorhexidine 20%. A full-thickness cutaneous wound (diameter 5 mm) was formed by cutting out a whole layer skin flap with scissors on both of the hind paws of each animal under isoflurane anesthesia. The experiment was

*Pharmaceutics* **2022**, *14*, x 3 of 13

**Figure 1.** Anesthetized mice by isoflurane using the Ugo Basile gas anesthesia system. **Figure 1.** Anesthetized mice by isoflurane using the Ugo Basile gas anesthesia system.

#### *2.2. Low Dose Photodynamic Therapy Protocol*

*2.2. Low Dose Photodynamic Therapy Protocol*  Both of the photosensitizers 5-ALA 20% and MB 0.01% in saline solution were topically administered directly on the wound; after 30 min, the irradiation was started by an AlGalnP laser (λ = 630 nm, P = 5 mW) with two doses: 1 J/cm2 and 4 J/cm2, and the procedure was carried out under the influence of isoflurane. 5-ALA was applied on the wounds on the right hind paws, MB was applied on the left ones. The animals were divided according to the laser dose and photosensitizer into five groups: the control group, Both of the photosensitizers 5-ALA 20% and MB 0.01% in saline solution were topically administered directly on the wound; after 30 min, the irradiation was started by an AlGalnP laser (λ = 630 nm, P = 5 mW) with two doses: 1 J/cm<sup>2</sup> and 4 J/cm<sup>2</sup> , and the procedure was carried out under the influence of isoflurane. 5-ALA was applied on the wounds on the right hind paws, MB was applied on the left ones. The animals were divided according to the laser dose and photosensitizer into five groups: the control group, the LDPDT–5-ALA 1 J/cm<sup>2</sup> , LDPDT–MB 1 J/cm<sup>2</sup> , LDPDT–5-ALA 4 J/cm<sup>2</sup> , and LDPDT–MB 4 J/cm<sup>2</sup> . LDPDT procedure was repeated once immediately after wound formation.

#### the LDPDT–5-ALA 1 J/cm2, LDPDT–MB 1 J/cm2, LDPDT–5-ALA 4 J/cm2, and LDPDT–MB *2.3. Optical Coherence Tomography Protocol*

4 J/cm2. LDPDT procedure was repeated once immediately after wound formation. *2.3. Optical Coherence Tomography Protocol*  The experiments were carried out using optical coherence tomography (OCT) on the GANYMEDE−II system (Thorlabs, USA) with the basic scanning module OCTG-900. It is possible to obtain information on the optical characteristics, morphology, and elastic properties of biological tissues using OCT. The GANYMEDE-II system uses a superluminescent diode with an operating wavelength of 930 ± 50 nm. The superluminescent diode allows one to reach a signal penetration depth up to 2.9 mm with an axial resolu-The experiments were carried out using optical coherence tomography (OCT) on the GANYMEDE−II system (Thorlabs, USA) with the basic scanning module OCTG-900. It is possible to obtain information on the optical characteristics, morphology, and elastic properties of biological tissues using OCT. The GANYMEDE-II system uses a superluminescent diode with an operating wavelength of 930 ± 50 nm. The superluminescent diode allows one to reach a signal penetration depth up to 2.9 mm with an axial resolution of up to 6.0 microns (air/tissue). The width of the spectral band was 100 nm. Figure 2 shows an example of placing a mouse on the substrate of OCT. As a result, B-scans were obtained—two-dimensional images. Data processing was carried out using ThorImageOCT 5.0.1., with the following parameters: size 2469 × 675 pixel, FOV 4.66 × 1.94 mm, and pixel size 1.89 × 2.88 µm, with 20 frames. The experiment was repeated with a 30◦ rotation around the previous position.

tion of up to 6.0 microns (air/tissue). The width of the spectral band was 100 nm. Figure 2 shows an example of placing a mouse on the substrate of OCT. As a result, B-scans were obtained—two-dimensional images. Data processing was carried out using ThorImageOCT 5.0.1., with the following parameters: size 2469 × 675 pixel, FOV 4.66 × 1.94 mm, and pixel size 1.89 × 2.88 µm, with 20 frames. The experiment was repeated with a 30°

rotation around the previous position.

**Figure 2.** The mouse positioning during OCT imaging. **Figure 2.** The mouse positioning during OCT imaging.

#### *2.4. Statistical Analysis*

*2.4. Statistical Analysis*  The OCT data were exported using the ThorImageOCT 5.0.1 program to files. txt. Statistical analysis and data processing were carried out in Python 3.6 using libraries (numpy, scipy, matplotlib). All calculated parameters were expressed as the mean ± standard deviation. The Pearson test was applied to assess the level of statistically sig-The OCT data were exported using the ThorImageOCT 5.0.1 program to files. txt. Statistical analysis and data processing were carried out in Python 3.6 using libraries (numpy, scipy, matplotlib). All calculated parameters were expressed as the mean ± standard deviation. The Pearson test was applied to assess the level of statistically significant differences among groups under study. Statistical power was used at 0.95 and 0.99. The *p*-values were calculated for all groups on all days.

#### nificant differences among groups under study. Statistical power was used at 0.95 and **3. Results**

#### 0.99. The *p*-values were calculated for all groups on all days. *3.1. OCT Imaging*

**3. Results**  *3.1. OCT Imaging*  Figure 3a shows a photo of healthy skin, and an OCT B-scan for the area, marked with a red arrow, is shown in Figure 3b. The B-scan data were normalized so that the stratum corneum, corresponding to the region with the highest pixel intensity, was lo-Figure 3a shows a photo of healthy skin, and an OCT B-scan for the area, marked with a red arrow, is shown in Figure 3b. The B-scan data were normalized so that the stratum corneum, corresponding to the region with the highest pixel intensity, was located at the top of the image. After normalization, the signal intensity was calculated at different tissue depths from 0 to 0.8 mm (A-scan) and visualized as shown in Figure 4. The maximum intensity values were at a depth of 0 to 2–4 µm, which corresponded to the stratum corneum, and then the signal intensity gradually decreased to ~20 µm, which corresponded to the epidermis. The dermis starts from 30–40 µm, which was accompanied by a decrease in intensity to the minimum values at a depth of 0.8 mm.

cated at the top of the image. After normalization, the signal intensity was calculated at different tissue depths from 0 to 0.8 mm (A-scan) and visualized as shown in Figure 4. Photos of the observation area and B-scans of the wound at different time points on days 1, 3, 7, and 14 for the control (without LDPDT) are shown in Figure 5.

The maximum intensity values were at a depth of 0 to 2–4 µm, which corresponded to the stratum corneum, and then the signal intensity gradually decreased to ~20 µm, which corresponded to the epidermis. The dermis starts from 30–40 µm, which was accompanied by a decrease in intensity to the minimum values at a depth of 0.8 mm. The wound healed typically without pathologies, and the injury was close to healing on day 14. The difference in signal intensities at different stages of wound healing on different days is shown in Figure 6. On day 1 after the wound forming procedure, the signal had a low intensity, while the signal of the dermis started decreasing from 0.3 µm, so the signal from 0 to 0.3 corresponded to the formed wound scab. The signal intensity increased on days 3 and 7. On day 14, the signal intensity values were close to healthy skin.

OCT images for the LDPDT-5-ALA groups are shown for a laser dose of 1 J/cm<sup>2</sup> in Figure 7 and 4 J/cm<sup>2</sup> in Figure 8. The intensity signal for LDPDT-5-ALA 4 J/cm<sup>2</sup> had the same behavior as the control. On day 1 for LDPDT-5-ALA 1 J/cm<sup>2</sup> , the attenuation signal corresponding to the dermis started from ~0.3 mm. Similar to the control, the signal

**Figure 3.** (**a**) Visual observation for healthy skin, (**b**) OCT imaging.

(**a**) (**b**)

(**a**)

(**b**)

intensity increased on days 3 and 7, and on day 14, the signal intensity values were close to healthy skin, as shown in Figure 9a. In the same way for LDPDT-5-ALA 4 J/cm<sup>2</sup> , the intensity signal started decreasing from ~0.2 mm on day 1. On days 3 and 7, the signal intensity values increased to close to the value of healthy skin on day 14 more than the LDPDT-5-ALA 1 J/cm<sup>2</sup> group, as shown in Figure 9b. Figure 3a shows a photo of healthy skin, and an OCT B-scan for the area, marked with a red arrow, is shown in Figure 3b. The B-scan data were normalized so that the stratum corneum, corresponding to the region with the highest pixel intensity, was located at the top of the image. After normalization, the signal intensity was calculated at different tissue depths from 0 to 0.8 mm (A-scan) and visualized as shown in Figure 4.

The OCT data were exported using the ThorImageOCT 5.0.1 program to files. txt. Statistical analysis and data processing were carried out in Python 3.6 using libraries (numpy, scipy, matplotlib). All calculated parameters were expressed as the mean ± standard deviation. The Pearson test was applied to assess the level of statistically significant differences among groups under study. Statistical power was used at 0.95 and

Measurements were similarly carried out for the MB photosensitizer with two laser doses of 1 J/cm<sup>2</sup> (Figure 10) and 4 J/cm<sup>2</sup> (Figure 11). The intensity signal for LDPDT-MB on day 14 was close to the values for healthy skin for different exposure doses, as shown in Figure 12. The maximum intensity values were at a depth of 0 to 2–4 µm, which corresponded to the stratum corneum, and then the signal intensity gradually decreased to ~20 µm, which corresponded to the epidermis. The dermis starts from 30–40 µm, which was accompanied by a decrease in intensity to the minimum values at a depth of 0.8 mm.

**Figure 3. Figure 3.** (**a** (**a**) Visual observation for healthy skin, ( ) Visual observation for healthy skin, ( **b**) OCT imaging. **b**) OCT imaging.

*Pharmaceutics* **2022**, *14*, x 4 of 13

**Figure 2.** The mouse positioning during OCT imaging.

0.99. The *p*-values were calculated for all groups on all days.

*2.4. Statistical Analysis* 

**3. Results** 

*3.1. OCT Imaging* 

**Figure 4.** Dependence of signal intensity on depth for healthy skin: S1 start point of the stratum corneum; S2—the endpoint of epidermis; S3—start point of the dermis. **Figure 4.** Dependence of signal intensity on depth for healthy skin: S<sup>1</sup> start point of the stratum corneum; S2—the endpoint of epidermis; S3—start point of the dermis.

days 1, 3, 7, and 14 for the control (without LDPDT) are shown in Figure 5.

skin.

 **Figure 5.** (**a**) Visual observation and (**b**) the corresponding B-scans for the control group.

The wound healed typically without pathologies, and the injury was close to healing

on day 14. The difference in signal intensities at different stages of wound healing on different days is shown in Figure 6. On day 1 after the wound forming procedure, the signal had a low intensity, while the signal of the dermis started decreasing from 0.3 µm, so the signal from 0 to 0.3 corresponded to the formed wound scab. The signal intensity increased on days 3 and 7. On day 14, the signal intensity values were close to healthy

Photos of the observation area and B-scans of the wound at different time points on

**Figure 5.** (**a**) Visual observation and (**b**) the corresponding B-scans for the control group.

**Figure 6.** Dependence of signal intensity on depth for the control group during wound healing.

**Figure 7.** (**a**) Visual observation and (**b**) the corresponding B-scans for the LDPDT-5-ALA 1 J/cm<sup>2</sup> group.

**Figure 8.** (**a**) Visual observation and (**b**) the corresponding B-scans for the LDPDT-5-ALA 4 J/cm<sup>2</sup> group.

**Figure 9.** Dependence of signal intensity on depth for the (**a**) LDPDT-5-ALA 1 J/cm<sup>2</sup> group and (**b**) LDPDT-5-ALA 4 J/cm<sup>2</sup> group.

**Figure 10.** (**a**) Visual observation and (**b**) the corresponding B-scans for the LDPDT-MB 1 J/cm<sup>2</sup> group.

**Figure 11.** (**a**) Visual observation and (**b**) the corresponding B-scans for the LDPDT-MB 4 J/cm<sup>2</sup> group.

**Figure 12.** Dependence of signal intensity on depth for the (**a**) LDPDT-MB 1 J/cm<sup>2</sup> group and (**b**) LDPDT-MB 4 J/cm<sup>2</sup> group.

#### *3.2. Quantitative Comparison of the Spatial Proximity of the OCT Signal Intensity*

For a quantitative comparison of spatial profiles, we used the curve proximity factor (CPF) *S*, similar to the Pearson's correlation coefficient, to compare healthy skin and wound curves in all days to all groups [47]:

$$S = \frac{\sum\_{i} |X\_{i} - Y\_{i}|}{\frac{1}{2} \sum\_{i} |X\_{i} + Y\_{i}|},\tag{1}$$

where *X<sup>i</sup>* , *Y<sup>i</sup>* is the intensity of the OCT signal for a definite depth from the wound and healthy skin, respectively. The higher the CPF value, the closer the wound state to healthy skin. The 0.01 and 0.05 significance levels were used to assess the statistical differences between the wound and healthy skin groups. The CPF values for all groups are shown in Table 1.


**Table 1.** CPF values for the studied groups (mean ± standard deviation).

\* *p* > 0.01,\*\* *p* > 0.05.

The CPF (1) was also used to estimate the effectiveness of different laser doses for each photosensitizer on day 14. The CPF values calculated for the OCT signal intensity curves corresponding to 4 J/cm<sup>2</sup> and 1 J/cm<sup>2</sup> for 5-ALA (Figure 9) and MB (Figure 12) are shown in Table 2. These quantitative estimations demonstrated a "proximity" between the curves corresponding to 4 J/cm<sup>2</sup> laser dose and the curves corresponding to the 1 J/cm<sup>2</sup> laser dose for the same photosensitizer.

**Table 2.** CPF values for the studied groups (mean ± standard deviation).


## **4. Discussion**

The proposed method of wound state quantitative evaluation is based on the OCT visualization of tissue structure transformation. The averaged scatter A-line intensity profile obtained from the horizontal rectangle in the OCT B-scan image of healthy skin is shown in Figure 4. Three areas are highlighted in the figure, representing changes in the attenuation coefficient. The red (S1), green (S2), and blue (S3) lines correspond to the beginning of the stratum corneum, the end of the epidermis, and the beginning of the dermis, respectively. After inflicting a wound in the first days, there are no surface layers of the skin (horny, epidermis); instead, a scab forms on the surface. These changes in the skin are visible on A-scans. Over time, the skin recovers, and on A-scans, we can see the appearance of areas characteristic of the epidermis's end and the dermis's beginning. These changes are reflected in the OCT signal attenuation curve (see Figures 6, 8, 9 and 12).

For wound state quantitative estimation, we used the curve proximity factor, introduced by us earlier [47]. According to Table 1, in the control group, the CPF mean value on day 1 was about 0.053, and decreased to 0.021 on day 14, while in the LDPDT groups, the mean values of this coefficient on day 14 ranged from 0.017 to 0.021. The Pearson test demonstrated that for LDPDT groups on day 14, *p*-value did not exceed 0.01, while for the control group, this value was equal to 0.03. Therefore, for all LPDT groups, the wound state had no statistically significant difference compared to healthy skin for the used statistical power levels.

The CPF value for LDPDT groups for the 4 J/cm<sup>2</sup> laser dose was smaller than the LDPDT groups for 1 J/cm<sup>2</sup> . We also calculated the CPF values for the OCT signal intensity curves corresponding to 4 J/cm<sup>2</sup> and 1 J/cm<sup>2</sup> for 5-ALA (the first column in Table 1) and MB (the second column in Table 2) and conducted a Pearson test to check the statistical significance of these differences. *p*-value was shown to be larger for 5-ALA.

Therefore, after comparing the CPF parameter for five groups: control, LDPDT 5-ALA 4 J/cm<sup>2</sup> , LDPDT 5-ALA 1 J/cm<sup>2</sup> , LDPDT-MB 4 J/cm2, and LDPDT-MB 1 J/cm<sup>2</sup> , it was concluded that the laser dose of 4 J/cm<sup>2</sup> for LDPDT 5-ALA was definitely better compared to 1 J/cm<sup>2</sup> and probably better for LDPDT MB. It should be noted that the conclusion depends on the volume and quality of the dataset.

A possible reason for the 4 J/cm<sup>2</sup> dose preference relative to the 1 J/cm<sup>2</sup> dose is as follows. According to previous works [33–35], when low-level laser light is applied and a dose is too low, no tissue response will occur. If too a high dose is applied, it can inhibit a tissue response. It has been seen in studies of wound healing where too low a dose did not have an impact, and too high a dose (above 5 J/cm<sup>2</sup> ) prolonged wound healing while the optimal dose resulted in faster healing. In this interval, according to the Arndt–Schultz curve, the larger dose causes a stronger biological effect.

In any case, LDPDT allows for accelerating of the wound healing process, which is consistent with the literature data [32–34,48,49].

#### **5. Conclusions**

In this paper, a study of quantitative in vivo evaluation of wounds using OCT during the wound healing process was carried out. 5-ALA and MB were used as photosensitizers for LDPDT, with two laser doses of 1 and 4 J/cm<sup>2</sup> .

An approach to quantitative estimation of wound state based on the CPF, Equation (1) [47] was proposed. The method was used to quantify the effectiveness of LDPDT to accelerate the wound healing process. CPF parameter estimation allowed us to compare LDPDT regimes quantitatively and to obtain objective arguments about the superiority of one regime over another.

Therefore, the proposed CPF parameter, estimated from OCT data, has demonstrated its feasibility for the quantitative estimation of the human wound state during healing. This approach is noninvasive, simple in implementation, and suitable for continuous monitoring throughout the wound healing process and sufficient resolution to assess both anatomy and pathology. It makes it a promising technique for applications in wound healing and the evaluation of novel therapeutics.

Another approach, which was proven to be effective in monitoring wound healing is two-photon microscopy. Previously, our group analyzed the two-photon microscopy images of the wound healing process and succeeded in quantitatively assessing the state of the wound and studying the effect of low-dose photodynamic therapy using the techniques of two-photon microscopy. The results of this study are completely consistent with the results obtained earlier [50].

**Author Contributions:** Conceptualization, Y.V.K.; Methodology, H.Z. and E.A.S.; Software, V.V.N.; Validation, H.Z. and V.V.N.; Investigation, H.Z., V.V.N., T.B.L. and N.A.K.; Writing—original draft preparation, E.A.S.; Writing—review and editing, Y.V.K.; Visualization, H.Z.; Supervision, Y.V.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a grant under the Decree of the Government of the Russian Federation No. 220 of 9 April 2010 (Agreement No. 075-15-2021-615 of 4 June 2021).

**Institutional Review Board Statement:** The study was approved by the Ethics Committee of Tomsk State University (Protocol No.4, 10.02.2021), registration No. 6.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **In Vitro Effect of Photodynamic Therapy with Different Lights and Combined or Uncombined with Chlorhexidine on** *Candida* **spp.**

**Vanesa Pérez-Laguna 1,\* , Yolanda Barrena-López <sup>2</sup> , Yolanda Gilaberte 3,4,† and Antonio Rezusta 2,3,5,†**


**Citation:** Pérez-Laguna, V.; Barrena-López, Y.; Gilaberte, Y.; Rezusta, A. In Vitro Effect of Photodynamic Therapy with Different Lights and Combined or Uncombined with Chlorhexidine on *Candida* spp. *Pharmaceutics* **2021**, *13*, 1176. https://doi.org/10.3390/ pharmaceutics13081176

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 18 July 2021 Accepted: 28 July 2021 Published: 30 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** Candidiasis is very common and complicated to treat in some cases due to increased resistance to antifungals. Antimicrobial photodynamic therapy (aPDT) is a promising alternative treatment. It is based on the principle that light of a specific wavelength activates a photosensitizer molecule resulting in the generation of reactive oxygen species that are able to kill pathogens. The aim here is the in vitro photoinactivation of three strains of *Candida* spp., *Candida albicans* ATCC 10231, *Candida parapsilosis* ATCC 22019 and *Candida krusei* ATCC 6258, using aPDT with different sources of irradiation and the photosensitizer methylene blue (MB), alone or in combination with chlorhexidine (CHX). Irradiation was carried out at a fluence of 18 J/cm<sup>2</sup> with a light-emitting diode (LED) lamp emitting in red (625 nm) or a white metal halide lamp (WMH) that emits at broad-spectrum white light (420–700 nm). After the photodynamic treatment, the antimicrobial effect is evaluated by counting colony forming units (CFU). MB-aPDT produces a 6 log<sup>10</sup> reduction in the number of CFU/100 µL of *Candida* spp., and the combination with CHX enhances the effect of photoinactivation (effect achieved with lower concentration of MB). Both lamps have similar efficiencies, but the WMH lamp is slightly more efficient. This work opens the doors to a possible clinical application of the combination for resistant or persistent forms of Candida infections.

**Keywords:** candidiasis; *C. albicans*; antimicrobial photodynamic therapy; methylene blue

#### **1. Introduction**

*Candida* spp. are commensal fungal species commonly colonizing human mucosal and skin surfaces, but they may become pathogenic in some particular scenarios such as treatment with antibiotics, immunocompromised patients, etc., producing in these cases infections that range from superficial to severe skin and mucosal lesions, to even systemic invasion at its worst degree [1]. For example, oral candidiasis is the most common opportunistic infection affecting the human oral cavity. It is caused by an overgrowth of *Candida* spp., being the most prevalent *Candida albicans* [2,3].

Due to the recurrence of *Candida* spp. infections, high systemic antifungal therapy have been widely used, thereby antifungal resistances are increasing. Moreover, patientdependent, interactions with other medical regimens and organ toxicity can happen [4].

Therefore, it is necessary to develop new treatments such as antimicrobial photodynamic therapy (aPDT). It is based on the use of photosensitizing molecules that are excited with visible light of the appropriate wavelength and reacts with the oxygen, generating reactive species of oxygen to destroy the target pathogen [5–7].

Superficial wound infections are potentially suitable for treatment by aPDT because of the ready accessibility of these wounds for both topical delivery of the photosensitizer and light, and because of the exposure to oxygen [6,8,9].

Different aPDT studies have demonstrated that *Candida* spp. can be effectively photoinactivated in vitro and in vivo [5,10–13].

Future directions of aPDT include the combination with antimicrobials in order to enhance the microbial inactivation and prevent the regrowth when the light from aPDT is turned off and the photoinactivation ends. This original approach has already shown significant potential. It could help to implement the use of aPDT and reduce the amount of antimicrobials used and, thus, the multidrug resistance problem [14–16].

Chlorhexidine (CHX) is an antiseptic drug, mainly available in over-the-counter products as routine hand hygiene in healthcare personnel, to clean and prepare the skin before surgery, and before injections in order to help reduce the amount of microorganisms that potentially can cause skin infections [17–20]. CHX gluconate is also available as a prescription mouthwash to treat gingivitis and as a prescription oral chip to treat periodontal disease [21–23] and recently against COVID-19 in dentistry [24].

Here, we investigate the aPDT and the CHX uncombined or in combination against *Candida* spp. As a photosensitizing molecule, we use methylene blue (MB), the main member of the phenothiazine family, well known for its ability to produce singlet oxygen when it is irradiated by red light and react with molecular oxygen [6,8]. As a source of irradiation, we use a light-emitting diode (LED) lamp emitting in red or a white metal halide lamp (WMH) that emits at broad-spectrum white light which is comparable to the emission spectrum of daylight.

The aim is to compare the antimicrobial effect of MB-aPDT when a specific irradiation source or a non-specific broad-spectrum source is used to excite different concentrations of MB. Furthermore, the effects of the combination of aPDT with CHX are evaluated.

#### **2. Materials and Methods**

The procedure used tried to follow the materials and methods of our previous works and was adapted as follows [25–27]:

#### *2.1. Chemicals, Media, Strains and Light Sources*


fluence corresponds to a 42.86 min (≈43 min) irradiation time for the samples using the red-LED lamp and 3 min and 25 sec for the samples irradiated with WMH lamp.

#### *2.2. In Vitro Photodynamic Treatment of Yeast Suspension*

*C. albicans*, *C. parapsilosis* or *C. krusei* seeded on Sabouraud dextrose agar were cultured aerobically overnight at 35 ◦C. The inoculum was prepared in distilled water and adjusted to 5 <sup>±</sup> 0.03 on the McFarland scale (concentrations in the range of >1 <sup>×</sup> <sup>10</sup><sup>6</sup> colony-forming units (CFU) per 100 µL and was deposited into 96-well microtiter plates. Two-fold serial dilutions concentrations from 640 µg/mL to 0.03 µg/mL of the MB were added, in absence or presence of 10 µg/mL of CHX (MB+/CHX−/light+) (MB+/CHX+/light+). The final volume in each well was 100 µL. Irradiation proceeded with no preincubation period; the suspensions were immediately subjected to irradiation with fluence of 18 J/cm<sup>2</sup> using the red-LED lamp or the broad spectrum-WMH lamp. Control samples were subjected to identical treatment, in the absence or presence of the photosensitizer, and were either kept in darkness or irradiated to evaluate the effect of each parameter: negative or initial control (MB−/CHX−/light−), irradiation control (MB−/CHX−/light+), control of photosensitizer in darkness (MB+/CHX−/light−) and antiseptic controls (MB−/CHX+/light−) (MB−/CHX+/light+). After completing the aPDT protocol, samples and controls were assessed in serial dilutions of each suspension and were cultured on blood agar and incubated overnight at 35 ◦C. The dilutions were made and aliquots were cultured to have blood agar plates with a number of CFUs in the range of 0 to 200 per plate in order to be able to count them reliably.

#### *2.3. Efficacy*

The efficacy of aPDT treatment was assessed by counting the number of CFU/100 µL using a Flash and Go automatic colony counter (IUL S.A., Barcelona, Spain). A reduction of 6 log<sup>10</sup> in the number of CFU/100 µL was considered indicative of fungicidal activity. The minimum concentration of MB that reduced yeast survival by 3 log<sup>10</sup> was also evaluated. All experiments were carried out at least five times. The results are expressed as mean and standard deviation.

#### **3. Results**

## *3.1. Photoinactivation of Yeasts by MB-aPDT (MB+/CHX*−*/Light+)*

MB-aPDT effectively inactivated *Candida* spp. achieving a reduction of 6 log<sup>10</sup> in the number of CFU/100 µL in all the studied strains (Figure 1). The minimum concentration of MB required to achieve this effect was 320 µg/mL in all cases except in those irradiated with a WMH lamp in *C. parapsilosis* that required 80 µg/mL and in *C. krusei* between 320–640 µg/mL (Table 1). Analyzing in more detail the sensitivity of each strain to MBaPDT, *C. krusei* is the most resistant and *C. parapsilosis* and *C. albicans* show a very similar ratio of response, although *C. parapsilosis* is slightly more sensitive to white light than *C. albicans* (Figure 1 and Supplementary Material Figure S2I).

**Figure 1.** Photoinactivation by antimicrobial photodynamic therapy with methylene blue alone or in combination with chlorhexidine of *C. albicans* (**A**,**D**) *C. parapsilosis* (**B**,**E**) and *C. krusei* (**C**,**F**) using the 625 nm lamp-LED (**A**–**C**) or the WMH lamp (**D**–**F**). The error bars represent the standard deviation calculated for five measurements. C0, initial inoculum control; **Figure 1.** Photoinactivation by antimicrobial photodynamic therapy with methylene blue alone or in combination with chlorhexidine of *C. albicans* (**A**,**D**) *C. parapsilosis* (**B**,**E**) and *C. krusei* (**C**,**F**) using the 625 nm lamp-LED (**A**–**C**) or the WMH lamp (**D**–**F**). The error bars represent the standard deviation calculated for five measurements. C<sup>0</sup> , initial inoculum control; CHX, chlorhexidine; LEDs, light-emitting diodes; MB, methylene blue; WMH, white metal halide.

CHX, chlorhexidine; LEDs, light-emitting diodes; MB, methylene blue; WMH, white metal halide.


**Table 1.** Minimum concentrations of methylene blue (µg/mL) required to reduce the number of *Candida* spp. 3 or 6 log<sup>10</sup> in the number of colony forming units using the 625 nm-LED lamp or the WMH lamp.

aPDT: antimicrobial photodynamic therapy; CHX: chlorhexidine; CFU: colony forming units; LED: light-emitting diodes; MB: methylene blue; WMH: white metal halide.

#### *3.2. Fungicidal Effect of MB-aPDT Combined with CHX (MB+/CHX+/Light+)*

The antimicrobial effect of MB-aPDT on *Candida* spp. was maintained in the presence of CHX, as evidenced by the 6 log<sup>10</sup> reduction in the number of CFU/100 µL in all experiments. Moreover, the combination of MB-aPDT using the WMH lamp + CHX achieves this degree of reduction on *C. albicans* decreasing 4-fold the required photosensitizer concentration (the necessary concentration is 1/4) (Figure 1 and Table 1).

To achieve a 3 log<sup>10</sup> reduction in the number of CFU/100 µL when MB-aPDT is used in combination with CHX, the required concentration of MB needed is at least half compared to the concentration needed using MB-aPDT alone. The greatest reduction of the photosensitizer concentration (8-fold) is achieved against *C. albicans* using the red-LED lamp. On the other hand, the greatest reduction against *C. parapsilosis* occurs with the WMH irradiation (1/4–1/8 of the initial photosensitizer concentration) (Figure 1 and Table 1).

## *3.3. Control of Inoculum and Toxic Effects of MB (MB+/CHX*−*/Light*−*), CHX (MB*−*/CHX+/Light*−*) and Irradiation (MB*−*/CHX*−*/Light+)*

No reduction in the number of CFU/100 µL from the initial inoculums (MB−/CHX−/ light−) was observed.

Samples with the different MB concentrations evaluated under the same conditions used in irradiation but keeping it in darkness (MB+/CHX−/light−) (dark MB in Figure 1) show significant reductions at the highest concentrations tested as follows: reductions of up to a maximum of 3.5 log<sup>10</sup> in *C. albicans*, 4 log<sup>10</sup> in *C. parapsilosis* and 4.5 log<sup>10</sup> in *C. krusei* were achieved by 640 µg/mL of MB. In all experiments, the effects of keeping the microbial suspension with the different MB concentrations in dark (light−) for 43 min or 3 min and 25 sec (using the time of the irradiation with the red-LED or the WMH lamp respectively) is similar, except for *C. krusei* (reduction of 4.5 log<sup>10</sup> after 43 min vs. 2.5 log<sup>10</sup> after 3 min and 25 sec) (Figure 1).

The irradiation with the red-LED lamp or with WMH lamp in the absence of photosensitizer and antimicrobial (MB−/CHX−/light+) did no significantly reduce the number of yeasts (reduction ≤0.3 log10, Figure 1).

In the absence of photosensitizer and irradiation, the tested concentration of CHX (10 µg/mL) (MB−/CHX+/light−) (dark MB-CHX with the value of 0 MB concentration in Figure 1) failed to effectively inactivate the yeast. A maximum reduction of 1 log<sup>10</sup> was observed against *C. parapsilosis.*

The cumulative effect of CHX and irradiation (MB−/CHX+/light+) (Figure 1) reaches a maximum reduction in the number of CFU/100 µL of 1.3 log<sup>10</sup> against *C. parapsilosis* being the most sensitive strain to this effect.

#### **4. Discussion**

MB-aPDT is effective in eradicating *Candida* spp. (>6 log<sup>10</sup> reduction in the number of CFU/100 µL of *C. albicans*, *C. parapsilosis* or *C. krusei*) and the combination with CHX enhances the photoinactivation, i.e., the effect is achieved with lower aPDT-dose (Figure 1 and Table 1).

Regarding the comparison of MB-aPDT results obtained with those reported by other authors, many variables should be considered. Table 2 summarizes different studies against *Candida* spp. in suspension, specifying the methodology and results. Daliri et al. reported a reduction of 3.43 log<sup>10</sup> of *C. albicans* using 200 µg/mL of MB which is notably lower than the one reached in the present study (MB concentration range of 80–160 µg/mL is able to inhibit 4 log10). They used a bigger number of CFU in the inoculum and this could affect but the mismatch may be because they use a laser irradiation source [28]. Application times are usually short when lasers are used and it does not always guarantee adequate oxygenation [6]. Valkov et al. report the absence of effect of MB-aPDT using very low MB concentration (<2 µg/mL) [13]. Ferreira et al. and de Oliveira-Silva et al. achieved different reductions of *C. albicans*, 0.5 log<sup>10</sup> and 6 log<sup>10</sup> respectively, with 32 µg/mL of MB and a fluence of 30 J/cm<sup>2</sup> for 3–4 min. This shows the variability between experiments [29,30]. The results of Ferreira et al. are closer to those of this work (32 µg/mL of MB does not produce complete photoinactivation) (Table 2).

Focusing on *C. parapsilosis*, Güzel Tunçcan et al. achieved a reduction of 4 log<sup>10</sup> with 25 µg/mL of MB. The comparison with our data and the possible explanation is very difficult because the methodology used is dissimilar [31]. Cern ˇ áková et al. demonstrated that using 9.6 µg/mL of MB inhibited between 1.13–1.27 log<sup>10</sup> of *C. parapsilosis* in suspension, similar results to this work (this concentration does not generate complete photoinactivation) [32]. Finally, Ahmed et al. used 100 µg/mL of MB and achieved reductions of 0.58–0.85 log<sup>10</sup> at fluences of 90–180 J/cm<sup>2</sup> respectively [33]. Again, the difference may be due to the fact that they used a laser irradiation source and therefore it would be less effective (Table 2).

Against *C. krusei*, concentrations of 16 µg/mL [34] or150 µg/mL of MB [35] even at high fluences only achieves a maximum reduction of 0.65 log10. More similar result to ours was obtained by Souza et al. using 100 µg/mL with a reduction of 1.54 log<sup>10</sup> using a fluence of 28 J/cm<sup>2</sup> [36]. All MB-aPDT studies together lead us to conclude that *C. krusei* is the most resistant *Candida* spp. to MB-aPDT as well as it is more resistant to antifungals in general mainly due to the characteristics of its membrane [37] (Table 2).

Regarding the MB-aPDT combination with CHX, it stands out that >6 log<sup>10</sup> reduction in the number of CFU/100 µL of *C. albicans* was achieved reducing the concentration of photosensitizer needed from 320 to 80 µg/mL when WMH lamp was used (Figure 1, Table 1 and Supplementary Figure S2). Furthermore, the addition of CHX halved the concentration of MB required to reach a reduction of 3 log<sup>10</sup> in *C. albicans* and *C parapsilosis*, and slightly less than half against *C. krusei*. Therefore, a synergistic effect is seen between MB at concentrations unable to achieve complete photoinactivation and CHX. These results are relevant because the presence of CHX could help to avoid the microbial regrowth of those microorganisms not completely destroyed when PDT is finished. This is one of the disadvantages of using aPDT for infections in the clinic, the risk of microbial regrowth after its application. The combination with antimicrobials could play a crucial role to overcome this limitation of aPDT in this context [14,15].

To our knowledge, there are not studies combining aPDT plus CHX in vivo against *Candida* spp. Recently, the effectiveness of MB-aPDT combined with CHX and zinc oxide ointment has been studied on wound healing process after rumenostomy. This study in cattle ratifies the use of aPDT and suggests that it could be performed for other surgical procedures as a complementary approach or an alternative for topical administration of antibiotics [38]. The combination of CHX plus aPDT has been tried against other microorganisms such as *Porphyromonas gingivalis* biofilm on a titanium surface in a dental framework. The application of CHX and subsequent aPDT using toluidine blue O was

shown to be an efficient method to reduce *P. gingivalis* in titanium surfaces [39]. Regarding other studies of antimicrobials plus aPDT against *Candida* spp., Giroldo et al. demonstrated that yeasts, both in suspension and in biofilms, were much more susceptible to antifungal treatments after MB-aPDT, explained by the increase of membrane permeability caused by aPDT [40]. Regarding the in vitro combination of MB-aPDT with fluconazole against resistant strains of *C. albicans, C. glabrata* and *C. krusei*, a synergistic effect was found in fluconazole resistant strains of *C. albicans* and *C. glabrata*, but not against *C. krusei*. [34]. These results do not agree with those found by Snell et al. They showed that fluconazole did not increase the aPDT inactivation of *C. albicans* using MB or another photosensitizer of the protoporphyrin family. However, miconazole did enhance the fungicidal activity of aPDT [41]. Moreover, to our knowledge, there are no studies using aPDT in combination with antimicrobials that report antagonistic effects, which support the use of aPDT in combination with CXH due to the possible advantages [15].

Considering clinical practice, MB-aPDT (660 nm and 7.5 J/cm<sup>2</sup> ) has been tried in HIV patients diagnosed with oral candidiasis comparing it with an antifungal commonly used in candidiasis. After 30 days, the antimicrobial was effective, but there were recurrences except when 450 µg/mL of MB was used [42].

All together indicates that aPDT or antimicrobial alone may not be entirely effective against *Candida* spp. that is characterized for causing highly recurrent infection especially in predisposed or immunosuppressed patients. On the other hand, combined treatments such as aPDT plus antimicrobials may prevent recurrent infections and avoid resistance. In addition, the combination in terms of clinical application would decrease the intensity of blue staining caused when the MB is applied on the skin or mucous membranes, making the aPDT procedure more cosmetically appealing.

Regarding the concentration of 10 µg/mL CHX used for this study, it was chosen by taking into account other protocols for the combination of antimicrobials plus aPDT and considering that by itself produces no effect under experimental conditions [25,27], Figure 1 C0-CHX. Other studies using other conditions report very different results: e.g., Azizi et al. using 1000 µg/mL of CHX achieved a reduction of 0.71 log<sup>10</sup> [43]; Do Vale et al. calculated that the minimum inhibitory concentration for *C. albicans* was 3.74 µg/mL using an exposure time of 12–48 h [44]; Ellepola et al. proved that with 50 µg/mL of CHX applied for 30 min achieved 0.38 and 0.5 log<sup>10</sup> of *C. albicans* and *C. krusei* reduction respectively [45].

Regarding the use of the red-LED lamp or the WHM lamp as a source of irradiation for aPDT, the second proved to be more effective in photoinactivating *Candida* spp. with the exception of against *C. krusei* (Figure 1, Table 1 and Supplementary Material Figure S2II). The use of LED lamp emitting in red matching the absorption spectra peak of the MB tends to be more efficient in the sense of not wasting irradiation energy and therefore red emission sources are usually the ones chosen for MB-aPDT studies (e.g., shown in Table 2). In addition, red LEDs lamps have added advantages at the time of transferring the use of aPDT to clinical application because they are available in all the PDT clinical units; in addition, these lamps stimulate cellular repair mechanisms in fibroblasts [46] and are already used to treat acne vulgaris, herpes simplex virus infection, shingles, or severe wound healing [6,47].

It is also worth noting the time factor to facilitate the use in the clinic since the WMH lamp needs 3 min and 25 seconds to photoinactivate *Candida* spp. compared to 43 min for the LED lamp, due to the greater irradiance of the former compare to the latter (90 mW/cm<sup>2</sup> vs. 7 mW/cm<sup>2</sup> respectively). Furthermore, the experiments were performed without preincubation of the photosensitizer MB with *Candida* spp. prior to irradiation. Andrade et al. and Soria-Lozano et al. demonstrated that a pre-incubation time did not produce greater inactivation of the microorganism, so it is not necessary to add more time to the aPDT procedure [12,48].

On the other hand, a broad-spectrum WMH lamp could be a model of daylight, i.e., which could be used as a source of irradiation for aPDT instead of this lamp. The advantages are that the treatment could be carried out at home and it would require less equipment and personnel (cheaper). However, it also has disadvantages, such as the imprecision in the quantification of the dose of light or duration of exposure, considering that intensity of daylight depends on the season of the year, weather conditions, or geographic location [9,49–51]. Another limitation for the use of daylight is the limitation to treat Candida infections not accessible for this light, such as the mouth or the genitalia, which otherwise are the most frequent. Nevertheless, the WMH lamp achieves better results than the LED lamp in this work, demonstrating its efficacy.



Overall, our study aims to open the way for the application of this alternative therapy, MB-aPDT alone or better in combination with CHX, either using lamps with a specific or broad- emission spectrum or even daylight as an irradiation source, to deal with cutaneous and mucosal candidiasis. However, it should be borne in mind that the present findings were obtained following in vitro irradiation of *Candida* spp., therefore clinical studies are required to confirm these results.

#### **5. Conclusions**


**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics13081176/s1, Figure S1: Photos and emission spectra of the lamps used in the photoinactivation experiments. A: graph of the emission spectrum of the red-LED lamp and B: emission spectrum of the white metal halide lamp, Figure S2: Photoinactivation of *Candida* spp. using MB-aPDT. I: Comparison of the response of yeast when they are irradiated with the red-LED lamp (left A) or with the WMH lamp (right B). II: Comparison of the response of each strain to irradiation with the two lamps (A: *C. albicans;* B: *C. parapsilosis*; C: *C. krusei*). The error bars represent the standard deviation calculated for five measurements. C<sup>0</sup> , initial inoculum control; LEDs, light-emitting diodes; MB, methylene blue; WMH, white metal halide.

**Author Contributions:** Conceptualization, V.P.-L., Y.G. and A.R.; methodology, validation and figures, V.P.-L. and Y.B.-L.; analysis, V.P.-L.; resources and funding acquisition, V.P.-L., Y.G. and A.R.; writing—original draft preparation, V.P.-L.; writing—review and editing, V.P.-L., Y.B.-L., Y.G. and A.R.; supervision, V.P.-L. and A.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grant CTQ2013-48767-C3-2-R from the Spanish Ministerio de Economía y Competitividad. Antonio Rezusta and Yolanda Gilaberte were funded by the Aragón Government: B10\_17R Infectious Diseases of Difficult Diagnosis and Treatment research group and B18\_17D Dermatology and Photobiology research group, respectively as recognized by the Government of Aragon.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank the IIS Aragon for the GIIS-023.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Study of Viral Photoinactivation by UV-C Light and Photosensitizer Using a Pseudotyped Model**

**Mohammad Sadraeian 1,2,\* , Fabio Francisco Pinto Junior <sup>1</sup> , Marcela Miranda <sup>1</sup> , Juliana Galinskas <sup>3</sup> , Rafaela Sachetto Fernandes <sup>1</sup> , Edgar Ferreira da Cruz <sup>3</sup> , Libing Fu <sup>2</sup> , Le Zhang <sup>2</sup> , Ricardo Sobhie Diaz <sup>3</sup> , Gustavo Cabral-Miranda 4,5 and Francisco Eduardo Gontijo Guimarães 1,\* ,†**


**Abstract:** Different light-based strategies have been investigated to inactivate viruses. Herein, we developed an HIV-based pseudotyped model of SARS-CoV-2 (SC2) to study the mechanisms of virus inactivation by using two different strategies; photoinactivation (PI) by UV-C light and photodynamic inactivation (PDI) by Photodithazine photosensitizer (PDZ). We used two pseudoviral particles harboring the Luciferase-IRES-ZsGreen reporter gene with either a SC2 spike on the membrane or without a spike as a naked control pseudovirus. The mechanism of viral inactivation by UV-C and PDZ-based PDI were studied via biochemical characterizations and quantitative PCR on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDZ treatments could destroy single stranded RNA (ssRNA) and the spike protein of the virus, with different ratios. However, the virus was still capable of binding and entering into the HEK 293T cells expressing angiotensin-converting enzyme 2 (ACE-2). A dose-dependent manner of UV-C irradiation mostly damages the ssRNA, while PDZ-based PDI mostly destroys the spike and viral membrane in concentration and dosedependent manners. We observed that the cells infected by the virus and treated with either UV-C or PDZ-based PDI could not express the luciferase reporter gene, signifying the viral inactivation, despite the presence of RNA and DNA intact genes.

**Keywords:** viral inactivation; photodynamic inactivation; SARS-CoV-2 pseudovirus; enveloped virus; UV-C light; photosensitizer

### **1. Introduction**

Novel coronavirus disease (COVID-19), caused by the SC2 virus, was first detected in December 2019 in the Hubei province of China, and has since sparked a global health crisis, with 5.1 million deaths reported by the World Health Organization (WHO) as of November 20 in 2021 [1]. This pandemic situation demands urgent attention toward finding novel strategies that might contribute to the prevention of viral spread via the inactivation of virions on surfaces, aerosols, and the human body.

**Citation:** Sadraeian, M.; Junior, F.F.P.; Miranda, M.; Galinskas, J.; Fernandes, R.S.; da Cruz, E.F.; Fu, L.; Zhang, L.; Diaz, R.S.; Cabral-Miranda, G.; et al. Study of Viral Photoinactivation by UV-C Light and Photosensitizer Using a Pseudotyped Model. *Pharmaceutics* **2022**, *14*, 683. https://doi.org/10.3390/ pharmaceutics14030683

Academic Editor: Maria Nowakowska

Received: 26 February 2022 Accepted: 15 March 2022 Published: 21 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The UV-C light has been used in healthcare facilities for environmental disinfection (air, liquid, and solid surfaces) [2]. The efficacy of this inactivation may depend not only on the wavelength but also on factors such as the pathogens (e.g., bacterial or viral species), light output, and environmental conditions [3]. UV-C light at 254 nm radiation enables the deposit of the energetic photons during interaction with the coronavirus, damaging the viral genome, and, consequently, the virus replication and proliferation can theoretically be abrupted [4]. In the case of RNA viruses like SC2, UV irradiation forms several RNA photoproducts, primarily from adjacent pyrimidine nucleotides, such as uracil dimers, as well as RNA–protein cross-links [3]. The formation of the uracil dimer potentially leads to frameshift or point mutations in the genome, known as UV-signature mutations of virus [5]. Hence, we should remain vigilant about the long-term effects of irradiation-mediated strategies for viral inactivation. There are several studies on the effects of UV-C for LD90 viral inactivation based on the time and dose of irradiation [2,6,7]; however, the mechanism of action of how UV-C inactivates viruses is still unclear [7,8].

Photodynamic therapy (PDT) is another light-based strategy that has been proposed to treat infections by damaging microorganisms, fungi, parasites, and viral particles. PDT is based on the use of photo-sensitive agents named photosensitizers (PS) which, in light-excited conditions and the presence of molecular oxygen, produce reactive oxygen species (ROS) [9–15]. PDT may damage cells via ROS generation, causing necrosis and apoptosis without harming the neighboring tissues. The advantages of utilizing photosensitizers for photodynamic inactivation (PDI) include its short-term toxicity, the absence of cell genome alterations, and avoiding the development of viral-induced resistance. Hence, the antiviral potential therapeutic effects of PDT and PDI on SC2 have been investigated with promising results [16,17]. Photoditazine photosensitizer (PDZ) is a porphyrin derivative with a chlorine core which allows it a high absorption in the red light spectrum with λmax of between 650–670 nm, as an advantage compared to the first generation of photosensitizers which are porphyrin core-based and which absorbs wavelengths too short for superior tissue penetration [18]. Understanding the mechanism of viral photoinactivation is important in finding and optimizing light-based strategies to battle viral infection. There are several reports on the mechanisms of viral photoinactivation with limited experiments on virion damage and viral propagation [2,19,20] due to the restriction of working with highly pathogenic viruses like HIV and SC2 viruses. Addressing these containment issues, the setting up of pseudotyped models in BSL2 labs can speed up studying the viral–cell mechanism and neutralizing assay towards in vivo studies [21,22]. Herein, we introduced the application of a pseudotyped model for studying the viral mechanism on four levels; virion damage; viral-cell entry; DNA integration; and expression of reporter genes. In this study, we followed the effects of UV-C irradiation and PDI on viral spike proteins and ssRNA in a HIV-based pseudotyped model of SC2 containing the Luciferase-IRES-ZsGreen reporter gene. Finally, we aimed to study the pseudovirus during cell internalization, genome integration, and reporter gene expression, after undergoing treatments by UV-C and PDZ photosensitizer under different concentrations and conditions (Figure 1).

**Figure 1.** Schematic picture of the mechanism of SARS-CoV-2 pseudovirus infectivity. Unlike SC2 ssRNA virus, which has viral reproduction independent of the host genome, this counterpart pseudovirus carries on reporter ssRNA with LTR, which causes integration into the genome. In this study, the pseudovirus has been treated with either UV-C irradiation or photodynamic inactivation (PDI) by Photodithazine photosensitizer. The mechanism of infectivity of photo-inactivated pseudovirus particles has been compared on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. The figure was created with BioRender software. **2. Materials and Methods Figure 1.** Schematic picture of the mechanism of SARS-CoV-2 pseudovirus infectivity. Unlike SC2 ssRNA virus, which has viral reproduction independent of the host genome, this counterpart pseudovirus carries on reporter ssRNA with LTR, which causes integration into the genome. In this study, the pseudovirus has been treated with either UV-C irradiation or photodynamic inactivation (PDI) by Photodithazine photosensitizer. The mechanism of infectivity of photo-inactivated pseudovirus particles has been compared on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. The figure was created with BioRender software.

#### *2.1. Chemical Reagents*  **2. Materials and Methods**

#### *2.1. Chemical Reagents*

All reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA), unless otherwise stated. All reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA), unless otherwise stated.

#### *2.2. Cells and Viruses 2.2. Cells and Viruses*

*2.3. Plasmids* 

The HEK 293T cells expressing ACE-2 receptor were gifted from BEI Resources as catalog number NR-52511. ACE-2 enzyme is a critical receptor for virus entrance into the host cell. The HEK 293T cells were used as control cells for assays, and for pseudovirus generation. The cells were maintained at 37 °C in 5% CO2 in DMEM medium-high glucose (DMEM-HG) with 10% fetal bovine serum (Gibco Invitrogen, Grand Island, NY, USA). HEK 293T is a derivative human cell line isolated from human embryonic kidneys (HEK) and expresses a mutant version of the SV40 large T antigen. The HEK 293T cells expressing ACE-2 receptor were gifted from BEI Resources as catalog number NR-52511. ACE-2 enzyme is a critical receptor for virus entrance into the host cell. The HEK 293T cells were used as control cells for assays, and for pseudovirus generation. The cells were maintained at 37 ◦C in 5% CO<sup>2</sup> in DMEM medium-high glucose (DMEM-HG) with 10% fetal bovine serum (Gibco Invitrogen, Grand Island, NY, USA). HEK 293T is a derivative human cell line isolated from human embryonic kidneys (HEK) and expresses a mutant version of the SV40 large T antigen.

#### *2.3. Plasmids*

Vector backbone pMD2.G and Vector backbone psPAX2 were gifts from Didier Trono (Addgene plasmid # 12259 and # 12260, respectively). The other plasmids were donated by BEI Resources and their sequences are available at (https://www.beiresources.org/ (accessed on 11 February 2022)) with the following catalog numbers [23]:

HDM-IDTSpike-fixK (BEI catalog number NR-52514): Plasmid expressing under a CMV promoter the Spike viral entry from SARS-CoV-2 strain Wuhan-Hu-1 (Genbank NC\_045512);

pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI catalog number NR-52516): Lentiviral backbone plasmid that uses a CMV promoter to express luciferase followed by an IRES and ZsGreen;

HDM-Hgpm2 (BEI catalog number NR-52517): lentiviral helper plasmid expressing HIV Gag-Pol under a CMV promoter;

HDM-tat1b (NR-52518): Lentiviral helper plasmid expressing HIV Tat under a CMV promoter.

pRC-CMV-Rev1b (NR-52519): Lentiviral helper plasmid expressing HIV Rev under a CMV promoter.

#### *2.4. UV-Vis Spectroscopy*

UV-Vis spectroscopy was used to determine plasmid and protein concentrations by using Nanodrop 1000 UV-Visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [24].

#### *2.5. Dynamic Light Scattering*

Hydrodynamic radii, electrophoretic mobility, and polydispersity of SC2 Spikepseudovirus were measured before and after photo inactivation. For UV-C inactivation and PDI inactivation, we followed the inactivation protocols as explained in Sections 2.7 and 2.8. Then, samples with 70 µL volume at 1 mg/mL in UV-transparent 96-well plates were measured using a DLS Wyatt Möbius (Wyatt Technologies, Dernbach, Germany) with incident light at 532 nm, at an angle of 163.5◦ . Samples were equilibrated at 25 ± 0.1 ◦C for 600 s before the measurements, and this temperature was held constant throughout the experiments. All samples were measured in triplicate with 10 acquisitions and a 5 s acquisition time. The change in the cumulant-fitted hydrodynamic radius in nanometers was monitored during the storage period. Results were calculated using the Dynamics 7.1.7 software (Wyatt Technologies, Santa Barbara, CA, USA).

#### *2.6. Generation of Pseudovirus with SARS-CoV-2 Spike and Naked Control*

SC2 Spike pseudotyped lentiviruses were generated by transfecting 293T cells, adjusted with the protocol explained by Thermo Fisher Scientific (Waltham, MA, USA). Briefly, seed 293T cells to be 95–99% confluent at transfection. At 16–24 h after seeding, the cells were transfected with the plasmids required for lentiviral production by using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions and using the following plasmid with 1 mL total volume per well of a six-well plate. The 293T cells were transfected with a lentiviral backbone plasmid encoding Firefly luciferase and ZsGreen reporter proteins, a plasmid expressing SC2 Spike, and plasmids expressing HIV-1 *gag*, *pol*, and *tat* proteins, to assemble the membrane of viral particles. The same protocol was followed to generate naked control pseudovirus without adding the viral entry plasmid encoding SC2 Spike. At 8 h post-transfection, the packaging medium was removed and replaced. At 24 h post-transfection, the entire volume of cell supernatant was harvested and stored at 4 ◦C. Then, 1 mL of fresh medium was replaced. At 52 h post-transfection, the entire volume of the cell supernatant was harvested. The pseudovirus product was aliquoted in small volumes of 400 µL and stored at −80 ◦C prior to use and underwent a single freeze-thaw.

#### *2.7. Viral Inactivation Using UV-C Irradiation*

A total of 40 µL of pseudovirus were diluted in 60 µL of DMEM-HG without supplementation in each well of a 96-well plate, which were exposed to the UV-C lamp 254 nm (HNS G5, OSRAM Germicidal Puritec, Munich, Germany) placed 1 cm above the plate to allow a uniform irradiance over the plate (10 <sup>±</sup> 2 mW/cm<sup>2</sup> ). Light was delivered by 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm<sup>2</sup> , respectively. Controls were not submitted to irradiation. After irradiation, aliquots of 80 µL were placed into the plates containing the previously seeded 293T/ACE2 cells and incubated for 8 h at 37 ◦C with 5% CO<sup>2</sup> for viral adsorption. Then, 120 µL of DMEM-HG medium containing 12% fetal bovine serum was added, and the plate was incubated for 48 h at 37 ◦C with 5% CO2. Afterward, the cells were placed into a lysis buffer solution to proceed with either Firefly luciferase assay or proviral DNA assay. Results were normalized in relation to controls for the calculation of viral inhibition rates of each sample.

#### *2.8. Photosensitizer-Based Photodynamic Inactivation*

A total of 40 µL of pseudovirus were diluted in 60 µL of DMEM-HG without supplementation in each well of a 96-well plate. The Photodithazine photosensitizer (PDZ) (Photodithazine® Company, Moscow, Russia) with a serial dilution of 10, 50, and 250 µg/mL was added, and incubated in the dark at RT (22 ◦C) for 15 min, then were irradiated using a homemade LED device at 670 nm (red light). All irradiations were performed with an irradiance rate of 30 mW/cm<sup>2</sup> in a time-dependent manner of 1, 10, and 20 min which equal the light doses of 1.8, 18, and 36 J/cm<sup>2</sup> , respectively. Afterwards, the treated ssRNA viruses were either harvested for the viral RNA load and DLS characterization or were incubated with the 293T/ACE-2 cells, as described in Section 2.7. After that, the cells were harvested for previral DNA load and luciferase activity measurement.

### *2.9. Quantification of Viral RNA and Proviral DNA*

The total ssRNA pseudovirus, before and after irradiation, was extracted and purified using the RNeasy Lipid Tissue Mini Kit, according to the manufacturer's (QIAGEN, Hilden, Germany) protocol.

The pseudovirus was treated with either 36 s UV-C irradiation or 10 min PDI in the presence of 10 µg/mL PDZ. The viral RNA load refers to the virus genome of freecell pseudovirus, before and after treatment. Viral load measurement was carried out using one-step reverse transcriptase (RT) and real-time PCR in a single buffer system using the Abbott Real Time on the automated m2000, over the dynamic range of detection of 40 to 10,000,000 copies/mL (Abbott, IL, USA) [25]. The protocol was followed as described by Kumar et al. for the TaqMan One-Step RT and PCR Master Mix Reagents Kit (Thermo Fisher Scientific, Waltham, MA, USA) with primers and probes for long terminal repeat (LTR) region of 640 bp, with two identical regions located at both ends of the either proviral DNA or RNA viral of SC2 pseudovirus. Briefly, a volume of 5 µL RNA sample and 20 µL Master Mix were used for a one-step RT-qPCR reaction with 20 µM forward primer (5-GCCTCAATAAAGCTTGCCTTGA-3); 20 µM reverse primer (5- GGGCGCCACTGCTAGAGA-3); 10 µM Taqman probe (5-FAM-CCAGAGTCACACAACAG ACGGGCACA-TAMRA-3); and an Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA), as reported previously [25–27].

Quantification of Proviral DNA were completed with the TaqMan Real-Time PCR Assay. The cells were infected with the treated virus (Sections 2.7 and 2.8). The cells were harvested three days after infection, centrifuged, and separated the pellets. The number of infected cells containing proviral DNA of pseudovirus was measured using qPCR. The quantification was executed based on the previously published protocol [27,28] for amplification of proviral DNA of pseudovirus (region LTR) with the primers described in Section 2.9.

#### *2.10. Flow Cytometry*

Direct fluorescence detections were applied using flow cytometry (Becton-Dickson Accuri C6, Mountain View, CA, USA) to analyze the expression of ZsGreen in the 293T/ACE-2 cells incubated with treated pseudovirus, as explained before (Sections 2.7 and 2.8). After 48 h, the ZsGreen-positive cells were harvested, fixed by 2% paraformaldehyde (PFA), quantified by blue laser (20 mW) irradiation at 488 nm and analyzed in the channel FL1: 533/30. The acquired data were analyzed by Flow-Jo software version 7.5 (Tree Star Inc., Ashland, OR, USA).

## *2.11. Luciferase Assay*

The infected cells which were harvested before (Sections 2.7 and 2.8) were lysed with 20 µL of Luciferase Cell Culture Lysis Reagent (Promega, Madison, WI, USA), then mixed with 100 µL of Luciferase Assay Reagent (Promega, Madison, WI, USA), and the light emission was measured.

#### *2.12. Titration of Pseudovirus*

The pseudovirus particles were titrated using a method similar to SC2 pseudovirus generation. Virus titers were determined by measuring relative luciferase units (RLUs). The HEK293T cells expressing human ACE-2 (293T-ACE-2) were produced to test the correlation between ACE-2 expression and SC2 pseudovirus susceptibility. Particles were generated in two forms; with a SC2 spike and a negative control without a viral entry protein. Both pseudo-typed particles harbored a Luciferase-IRES-ZsGreen backbone. In a mirror plate, the percentage of cell viability was measured during the viral infection with a serial dilution of the virus starting at 50 µL pseudovirus in a total volume of 100 µL (0.5) for the spike pseudovirus. After 8 h of pseudovirus incubation, the media were replaced with 150 µL fresh media. After 48 h incubation, the wells containing 50 µL pseudovirus were studied for cell confluency. Afterwards, the titers of pseudotyped particles were quantified by a Luciferase assay expressed in RLU, to determine the number of transducing particles per mL.

#### *2.13. Confocal Microscopy*

One day before UV-C or PDI treatment, 2 × 104 cells per well of 293T/ACE-2 cells were seeded on a multiple-chamber slide (Nalge-Nunc International, Naperville, Ill, USA). The next day, cells were incubated with treated pseudovirus, as explained before (Sections 2.7 and 2.8). After 48 h, the ZsGreen-positive cells were washed four times with PBS, fixed by 2% PFA. Images were obtained with an inverted LSM 780 multiphoton laser scanning confocal microscope (Zeiss, Jena, Germany), a 63 × 1.2 water immersion objective to couple with the bottom side of the cover slip, and the Zeiss LSM software was used to treat the images. The wavelength of Argon ion laser at 488 nm was used to excite the expressed ZsGreen protein compared to cell autofluorescence. The molecular localization of ZsGreen was analyzed for each image pixel in spectral and channel modes in the ranges 492–700 nm and 492–537 nm, respectively. The cells' autofluorescence were analyzed from 585 to 692 nm.

Each pixel was associated with an emission spectrum which allowed the spatial separation of the expressed ZsGreen fluorescence (bright blue-greenish color) and the cell auto-fluorescence (yellow-orange color). Considering the spectral of the cell autofluorescence (maximum at 575 nm) is almost constant, the expression of the ZsGreen by the active pseudovirus internalization would be promptly signaled by the spectral superposition of the protein emission at around 515 nm.

#### *2.14. Statistical Analyses*

Statistical analyses were performed using the GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Data are shown as mean and SEM of the indicated number of replicate values. If no error bar appears present, the error bars are smaller than, and obscured by, the symbol. The method for statistical comparison used was unpaired twotailed Student's *t*-test, unless specifically indicated otherwise. was unpaired two-tailed Student's *t*-test, unless specifically indicated otherwise. **3. Results and Discussion** 

indicated number of replicate values. If no error bar appears present, the error bars are smaller than, and obscured by, the symbol. The method for statistical comparison used

#### **3. Results and Discussion** *3.1. Generation of Pseudovirus with SARS-CoV-2 Spike and Naked Control*

#### *3.1. Generation of Pseudovirus with SARS-CoV-2 Spike and Naked Control* The spike-pseudotyped lentiviral particles were generated, which can infect 293T

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 7 of 15

The spike-pseudotyped lentiviral particles were generated, which can infect 293T cells expressing the human ACE-2 receptor. In parallel, the naked control pseudovirus was generated, which harbors a backbone plasmid-encoding luciferase-IRES-ZsGreen reporter, but without the SC2 Spike on the membrane (Figure 1). cells expressing the human ACE-2 receptor. In parallel, the naked control pseudovirus was generated, which harbors a backbone plasmid-encoding luciferase-IRES-ZsGreen reporter, but without the SC2 Spike on the membrane (Figure 1). *3.2. Titration of Pseudovirus* 

#### *3.2. Titration of Pseudovirus* The pseudovirus particles were titrated in two forms; particles with a SC2 spike and

The pseudovirus particles were titrated in two forms; particles with a SC2 spike and a negative control without a viral entry protein. Both pseudo-typed particles harbored a Luciferase-IRES-ZsGreen backbone. After 48 h incubation, the cell confluence reached 100% for all wells in a mirror plate containing non-transduced cells, however, the wells containing 50 µL pseudovirus showed 90% cell confluence (Figure 2A). The titers of pseudotyped particles were quantified by a Luciferase assay. Titers of >10<sup>5</sup> RLUs per mL were measured in a 96-well plate (Figure 2B). Unsurprisingly, the ACE2-expressing cells incubated with naked pseudovirus without a spike did not show the expression of luciferase (Figure 2C). The other negative control was the incubation of 293T non-ACE2 control cells with Spikepseudovirus, which did not show the luciferase expression, as expected (Figure 2D). In previous reports, researchers used polybrene to facilitate the lentiviral infection through minimizing charge-repulsion between the virus and cells [29], but we found this SC2 pseudovirus no need to polybrene for binding to the ACE-2 receptor. a negative control without a viral entry protein. Both pseudo-typed particles harbored a Luciferase-IRES-ZsGreen backbone. After 48 h incubation, the cell confluence reached 100% for all wells in a mirror plate containing non-transduced cells, however, the wells containing 50 μL pseudovirus showed 90% cell confluence (Figure 2A). The titers of pseudotyped particles were quantified by a Luciferase assay. Titers of >105 RLUs per mL were measured in a 96-well plate (Figure 2B). Unsurprisingly, the ACE2-expressing cells incubated with naked pseudovirus without a spike did not show the expression of luciferase (Figure 2C). The other negative control was the incubation of 293T non-ACE2 control cells with Spike-pseudovirus, which did not show the luciferase expression, as expected (Figure 2D). In previous reports, researchers used polybrene to facilitate the lentiviral infection through minimizing charge-repulsion between the virus and cells [29], but we found this SC2 pseudovirus no need to polybrene for binding to the ACE-2 receptor.

**Figure 2.** Titration of SARS-CoV-2 spike-pseudovirus particles in 293T cells expressing ACE-2. (**A**) Study of the percentage of cell viability during the viral infection respecting a serial dilution starting at 1:2 (0.5); (**B**,**C**) The graph shows the titers of the expression of Luciferase reporter as determined by measuring relative luciferase units (RLUs). The RLU data are the average of three-fold serial dilution of virus starting at 50 μL virus in a total volume of 100 μL (0.5) for the Spike-pseudovirus (**B**); naked pseudovirus without spike (**C**); or the Spike-pseudovirus with 293T cells without ACE2 receptor (**D**). After 8 h of pseudovirus incubation, the media were replaced with 150 μL fresh media. *3.3. Viral Inactivation using UV-C Irradiation or Photosensitizer-Based PDI*  **Figure 2.** Titration of SARS-CoV-2 spike-pseudovirus particles in 293T cells expressing ACE-2. (**A**) Study of the percentage of cell viability during the viral infection respecting a serial dilution starting at 1:2 (0.5); (**B**,**C**) The graph shows the titers of the expression of Luciferase reporter as determined by measuring relative luciferase units (RLUs). The RLU data are the average of threefold serial dilution of virus starting at 50 µL virus in a total volume of 100 µL (0.5) for the Spikepseudovirus (**B**); naked pseudovirus without spike (**C**); or the Spike-pseudovirus with 293T cells without ACE2 receptor (**D**). After 8 h of pseudovirus incubation, the media were replaced with 150 µL fresh media.

#### A volume of 40 μL of pseudovirus was diluted in 60 μL of DMEM-HG without *3.3. Viral Inactivation Using UV-C Irradiation or Photosensitizer-Based PDI*

supplementation in each well of a 96-well plate, which were exposed to the UV-C lamp 254 nm for 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm2, respectively. Figure 3A represents the effect of UV-C irradiation on the photo-inactivation of ssRNA pseudovirus. The results showed that UV-irradiation may inactivate 74%, 93%, and A volume of 40 µL of pseudovirus was diluted in 60 µL of DMEM-HG without supplementation in each well of a 96-well plate, which were exposed to the UV-C lamp 254 nm for 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm<sup>2</sup> , respectively. Figure 3A represents the effect of UV-C irradiation on the photo-inactivation of ssRNA pseudovirus. The results showed that UV-irradiation may inactivate 74%, 93%, and 99.99% of SC2 Spike-pseudovirus particles during 1, 6, and 36 s irradiation, respectively. These

results are comparable with the published results on SC2 elsewhere [20], due to the discrepancies of the cell-entry mechanism among virus and pseudovirus. Furthermore, a time-dependent manner of PDI was performed to find the maximum viral inactivation with the minimum time and concentration of Photogem PS (PDZ). As Figure 3B demonstrates, the viral inactivation depended on both time and the PS concentration. We observed that 99.8% of the pseudovirus were inactivated in the presence of 50 µg/mL PDZ with 10 min irradiation. Hence, we selected this time and concentration for further studies. These results indicate that both UV-C irradiation and PDI, as two distinct strategies, are highly effective in inactivating pseudovirus replication, while there could be some differences in the mechanism of infectivity between UV-C irradiation and PDI. Hence, we extended our studies focusing on the viral RNA and proviral DNA loads, as described in Section 3.4. discrepancies of the cell-entry mechanism among virus and pseudovirus. Furthermore, a time-dependent manner of PDI was performed to find the maximum viral inactivation with the minimum time and concentration of Photogem PS (PDZ). As Figure 3B demonstrates, the viral inactivation depended on both time and the PS concentration. We observed that 99.8% of the pseudovirus were inactivated in the presence of 50 μg/mL PDZ with 10 min irradiation. Hence, we selected this time and concentration for further studies. These results indicate that both UV-C irradiation and PDI, as two distinct strategies, are highly effective in inactivating pseudovirus replication, while there could be some differences in the mechanism of infectivity between UV-C irradiation and PDI. Hence, we extended our studies focusing on the viral RNA and proviral DNA loads, as described in Section 3.4.

99.99% of SC2 Spike-pseudovirus particles during 1, 6, and 36 s irradiation, respectively. These results are comparable with the published results on SC2 elsewhere [20], due to the

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 8 of 15

**Figure 3.** Study of the effect of photo-inactivation of ssRNA pseudovirus considering the relative luciferase units with a time-dependent manner of UV-C irradiation at 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm2, respectively (**A**); and PDZ-based PDI in a serial dilution of 10, 50 and 250 μg/mL in a time-dependent manner of 1, 10 and 20 min which equal the light doses of 1.8, 18 and 36 J/cm2, respectively (**B**). Data are ± means S.E.M. (*n* = 3). **Figure 3.** Study of the effect of photo-inactivation of ssRNA pseudovirus considering the relative luciferase units with a time-dependent manner of UV-C irradiation at 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm<sup>2</sup> , respectively (**A**); and PDZ-based PDI in a serial dilution of 10, 50 and 250 µg/mL in a time-dependent manner of 1, 10 and 20 min which equal the light doses of 1.8, 18 and 36 J/cm<sup>2</sup> , respectively (**B**). Data are ± means S.E.M. (*n* = 3).

#### *3.4. Study the Infectivity Mechanism of UV-C Irradiation and PDI Using qPCR 3.4. Study the Infectivity Mechanism of UV-C Irradiation and PDI Using qPCR*

Viral inactivation could be due to either viral protein or viral genome damage [8,30,31]. We suppose that any damage to the virus spike may lead to loss of the virus binding ability and neutralization of the virus infectivity, while damaging the viral genome may affect the viral and proviral loads of pseudovirus. The results of the viral RNA load showed that both 36 s UV-C irradiation and PDZ-based PDI (10 min irradiation, 50 μg/mL PDZ) can damage ssRNA by 83% and 74%, respectively. The RNA of both control (naked pseudovirus without spike) and spike-positive viral particles were destroyed during irradiation (Figure 4A). By comparing the PDZ-based PDI in two forms of enveloped and non-enveloped (naked) viruses, we found out that PDI may damage the viral genome independently from the virus type. Viral inactivation could be due to either viral protein or viral genome damage [8,30,31]. We suppose that any damage to the virus spike may lead to loss of the virus binding ability and neutralization of the virus infectivity, while damaging the viral genome may affect the viral and proviral loads of pseudovirus. The results of the viral RNA load showed that both 36 s UV-C irradiation and PDZ-based PDI (10 min irradiation, 50 µg/mL PDZ) can damage ssRNA by 83% and 74%, respectively. The RNA of both control (naked pseudovirus without spike) and spike-positive viral particles were destroyed during irradiation (Figure 4A). By comparing the PDZ-based PDI in two forms of enveloped and non-enveloped (naked)viruses, we found out that PDI may damage the viral genome independently from the virus type.

The results of the proviral DNA assay may interpret the virus's ability to complete the subsequent steps of cell binding, internalization, and genome integration after reverse transcription. The proviral DNA load of 36 s UV-C irradiation was as much as the RNA viral load, signifying that the UV-C based viral inactivation is independent of damaging the spike protein. In parallel, the proviral DNA load of PDZ-based PDI (10 min irradiation, 50 μg/mL PDZ) was decreased by 13%, which is half of the RNA viral load (26%), signifying the PDI-treated pseudovirus may lose cell infectivity due to damaging the spike The results of the proviral DNA assay may interpret the virus's ability to complete the subsequent steps of cell binding, internalization, and genome integration after reverse transcription. The proviral DNA load of 36 s UV-C irradiation was as much as the RNA viral load, signifying that the UV-C based viral inactivation is independent of damaging the spike protein. In parallel, the proviral DNA load of PDZ-based PDI (10 min irradiation, 50 µg/mL PDZ) was decreased by 13%, which is half of the RNA viral load (26%), signifying the PDI-treated pseudovirus may lose cell infectivity due to damaging the spike (Figure 4A,B). Presumably, PDZ-based PDI destroys more of the spike than the viral genome, which leads to losing the binding ability of the virus. Unsurprisingly, the naked control particle

showed no DNA load, as the control particle lacks a spike for cell binding. Furthermore, we observed that the cells infected by either UV-C or PDI-treated pseudovirus could not express the luciferase reporter gene (Figure 4C), signifying the total viral inactivation despite the presence of RNA and DNA intact genes. control particle showed no DNA load, as the control particle lacks a spike for cell binding. Furthermore, we observed that the cells infected by either UV-C or PDI-treated pseudovirus could not express the luciferase reporter gene (Figure 4C), signifying the total viral inactivation despite the presence of RNA and DNA intact genes.

(Figure 4A,B). Presumably, PDZ-based PDI destroys more of the spike than the viral genome, which leads to losing the binding ability of the virus. Unsurprisingly, the naked

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 9 of 15

**Figure 4.** The pseudovirus has been treated with either 36 s UV-C irradiation or 10 min Photodynamic Inactivation in the presence of 50 μg/mL Photodithazine. The mechanism of infectivity of photo-inactivated pseudovirus particles has been compared on three levels; (**A**) viral RNA load referring to the free-cell virus; (**B**) proviral DNA load referring to the ability of the treated virus to complete the subsequent steps of cell internalization, reverse transcription and genome integration; (**C**) luciferase activity referring to the expression of luciferase reporter after DNA integration. nt/n0 represents the fraction of the targeted genome region that remained intact after treatment. **Figure 4.** The pseudovirus has been treated with either 36 s UV-C irradiation or 10 min Photodynamic Inactivation in the presence of 50 µg/mL Photodithazine. The mechanism of infectivity of photoinactivated pseudovirus particles has been compared on three levels; (**A**) viral RNA load referring to the free-cell virus; (**B**) proviral DNA load referring to the ability of the treated virus to complete the subsequent steps of cell internalization, reverse transcription and genome integration; (**C**) luciferase activity referring to the expression of luciferase reporter after DNA integration. nt/n<sup>0</sup> represents the fraction of the targeted genome region that remained intact after treatment.

In this study, nt/n0 represents the fraction of the targeted genome region that remained intact after treatment. In the viral RNA load, the targeted genome region is ssRNA of pseudovirus with LTR sequences. In the proviral DNA load, the targeted genome region is the integrated DNA of pseudovirus genome after reverse transcriptase. Unlike the SC2 virus, the mechanism of infectivity of the SC2 pseudovirus includes DNA integration, which is one of the advantages of utilizing the pseudotyped model. Therefore, we could follow a simple protocol for calculation of RNA and DNA load and compare the qPCR data with luciferase assay results, otherwise to estimate the infectivity based on qPCR data, the infectivity of virus should be assessed by estimation from the qPCR results, according to the protocol published by Sabino et al. [20,32]. In this study, nt/n<sup>0</sup> represents the fraction of the targeted genome region that remained intact after treatment. In the viral RNA load, the targeted genome region is ssRNA of pseudovirus with LTR sequences. In the proviral DNA load, the targeted genome region is the integrated DNA of pseudovirus genome after reverse transcriptase. Unlike the SC2 virus, the mechanism of infectivity of the SC2 pseudovirus includes DNA integration,which is one of the advantages of utilizing the pseudotyped model. Therefore, we could follow a simple protocol for calculation of RNA and DNA load and compare the qPCR data with luciferase assay results, otherwise to estimate the infectivity based on qPCR data, the infectivity of virus should be assessed by estimation from the qPCR results, according to the protocol published by Sabino et al. [20,32].

#### *3.5. DLS Measurements before and after Irradiation 3.5. DLS Measurements before and after Irradiation*

DLS measurement demonstrated that 36 s UV-C irradiation on pseudovirus with 18 J/cm2 resulted in a slight decrease in the size distribution compared to the non-irradiated pseudovirus (Figure 5A). On the other hand, in the PDI study, the increase of PDZ concentration from 10 to 50 μg/mL had a significant effect on the size and polydispersity of the virus, and yielded significant aggregated particles (Figure 5B). We assumed that this aggregation may interrupt our results on the cell toxicity therefore we found that PDZ with 10 μg/mL was an appropriate concentration for further studies on flow cytometry and microscopy observations. DLS measurement demonstrated that 36 s UV-C irradiation on pseudovirus with 18 J/cm<sup>2</sup> resulted in a slight decrease in the size distribution compared to the non-irradiated pseudovirus (Figure 5A). On the other hand, in the PDI study, the increase of PDZ concentration from 10 to 50 µg/mL had a significant effect on the size and polydispersity of the virus, and yielded significant aggregated particles (Figure 5B). We assumed that this aggregation may interrupt our results on the cell toxicity therefore we found that PDZ with 10 µg/mL was an appropriate concentration for further studies on flow cytometry and microscopy observations.

#### *3.6. Green Fluorescent Measurement by Flow Cytometry*

Furthermore, the cells were infected with viruses, which were treated with either 36 s of UV-C or PDZ (10 µg/mL) of PDI, to measure the expression of the ZsGreen protein. The flow cytometry results showed that 46.3% of the virus-infected cells were emitting green fluorescence, while the cells treated with UV-C or the PDI-treated viruses were not able to express the ZsGreen (Figure 5C).

**Figure 5.** (**A**) Dynamic Light Scattering histograms of hydrodynamic radius (Rh) for pseudovirus showed optimal polydispersity with Rh of 100 nm. During 36 s of UV-C irradiation (360 mJ/cm2), a slight decrease in the size of pseudovirus was observed (Blue arrow) with no significant aggregation; (**B**) PDZ-based PDI significantly affected the size and polydispersity of the virus, resulting in major aggregated particles in a higher concentration of PDZ (50 μg/mL); (**C**) Flow cytometric diagram on the left demonstrated the percentage of pseudovirus-infected cells expressing ZsGreen Fluorescent protein. The middle and right diagrams represent the cells infected by UV irradiated-virus and PDZ-based PDI virus, which do not express ZsGreen protein. FITC rate indicates the green fluorescent emission from ZsGreen. Data are ± means S.E.M. (*n* = 2). **Figure 5.** (**A**) Dynamic Light Scattering histograms of hydrodynamic radius (Rh) for pseudovirus showed optimal polydispersity with R<sup>h</sup> of 100 nm. During 36 s of UV-C irradiation (360 mJ/cm<sup>2</sup> ), a slight decrease in the size of pseudovirus was observed (Blue arrow) with no significant aggregation; (**B**) PDZ-based PDI significantly affected the size and polydispersity of the virus, resulting in major aggregated particles in a higher concentration of PDZ (50 µg/mL); (**C**) Flow cytometric diagram on the left demonstrated the percentage of pseudovirus-infected cells expressing ZsGreen Fluorescent protein. The middle and right diagrams represent the cells infected by UV irradiated-virus and PDZbased PDI virus, which do not express ZsGreen protein. FITC rate indicates the green fluorescent emission from ZsGreen. Data are ± means S.E.M. (*n* = 2).

#### *3.7. Observation of ZsGreen Expression by Confocal Microscopy*

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 11 of 15

*3.6. Green Fluorescent Measurement by Flow Cytometry* 

able to express the ZsGreen (Figure 5C).

Forty-eight h after cell incubation with pseudovirus (with no treatment), the ZsGreen expression was observed using confocal microscopy. Figure 6A shows images of the field in spectral mode (Figure 6A—panel (a)), and in channel mode merged with a wide field transmission image (Figure 6A—panel (c)). The two spectral contributions for both ZsGreen emission and the cell autofluorescence can be separated by taking two regions of interest (ROI) in panel (a) (green and red circles), as depicted as two graphs in Figure 6A—panel (b). In Figure 6A—panel (c) demonstrates that the emission detected between 492 and 532 nm (assigned the bright-blue false color) mainly signals the expression of the ZsGreen protein while the cellular autofluorescence can be differentiated by taking the emission (orange false color) in the spectral range from 585 to 695 nm. *3.7. Observation of ZsGreen Expression by Confocal Microscopy*  Forty-eight h after cell incubation with pseudovirus (with no treatment), the ZsGreen expression was observed using confocal microscopy. Figure 6A shows images of the field in spectral mode (Figure 6A—panel (a)), and in channel mode merged with a wide field transmission image (Figure 6A—panel (c)). The two spectral contributions for both ZsGreen emission and the cell autofluorescence can be separated by taking two regions of interest (ROI) in panel (a) (green and red circles), as depicted as two graphs in Figure 6A panel (b). In Figure 6A—panel (c) demonstrates that the emission detected between 492 and 532 nm (assigned the bright-blue false color) mainly signals the expression of the ZsGreen protein while the cellular autofluorescence can be differentiated by taking the emission (orange false color) in the spectral range from 585 to 695 nm.

Furthermore, the cells were infected with viruses, which were treated with either 36 s of UV-C or PDZ (10 μg/mL) of PDI, to measure the expression of the ZsGreen protein. The flow cytometry results showed that 46.3% of the virus-infected cells were emitting green fluorescence, while the cells treated with UV-C or the PDI-treated viruses were not

**Figure 6.** (**A**) Confocal microscopy images showing ZsGreen expression in 293T-ACE2 cells at 48 h after incubation with Spike-pseudotyped lentiviral particles with the ZsGreen backbone. The images are represented in spectral mode (panel **a**) and channel mode merged with a wide field transmission image (panel **c**). In panel **b**, the green and red curves represent the regions of interest (ROI) of ZsGreen emission and the cell autofluorescence, respectively; (**B**) The positive control cells incubated with pseudovirus without treatment showed strong green fluorescent emission, indicating the expression of ZsGreen in comparison to negative control cells. The ZsGreen emission was **Figure 6.** (**A**) Confocal microscopy images showing ZsGreen expression in 293T-ACE2 cells at 48 h after incubation with Spike-pseudotyped lentiviral particles with the ZsGreen backbone. The images are represented in spectral mode (panel (**a**)) and channel mode merged with a wide field transmission image (panel (**c**)). In panel (**b**), the green and red curves represent the regions of interest (ROI) of ZsGreen emission and the cell autofluorescence, respectively; (**B**) The positive control cells incubated with pseudovirus without treatment showed strong green fluorescent emission, indicating the expression of ZsGreen in comparison to negative control cells. The ZsGreen emission was detected between 492 and 532 nm (assigned the bright-blue false color), while the cellular autofluorescence can be differentiated by taking the emission (orange false color) in the spectral range from 585 to 695 nm; (**C**) The results of viruses with 36 s UV-C irradiation (360 mJ/cm<sup>2</sup> ) did not show green fluorescent emission, while the cells with 1 s UV-C irradiation (10 mJ/cm<sup>2</sup> ) were still showing somewhat ZsGreen expression; (**D**) The viruses were treated with Photoditazine (PDZ) (10 µg/mL) and irradiated in a time-dependent manner of 0 (so-called dark), 1 and 20 min. The cells infected by PDI virus after 20 min do not express ZsGreen protein. (**A**,**B**) scale bar: 20 µm.

To study the expression of ZsGreen protein by confocal microscopy, the cells were infected with pseudovirus treated with UV-C or PDI. The positive control cells incubated with pseudovirus without treatment showed strong green fluorescent emission indicating the expression of ZsGreen in comparison to negative control cells (Figure 6B). The results of viruses with 36 s UV-C irradiation (360 mJ/cm<sup>2</sup> ) did not show green fluorescent emission (Figure 6C), while the cells with 1 s UV-C irradiation (10 mJ/cm<sup>2</sup> ) were still showing slight ZsGreen expression. The results were in agreement with our luciferase assay (Figure 3A), confirming that the 1 s UV-C irradiation is not sufficient to completely inactivate the viruses.

For the PDI study, the viruses were incubated with 10 µg/mL PDZ, and irradiated for 1 or 20 min, which equals the light doses of 1.8 and 36 J/cm<sup>2</sup> , respectively (Figure 6D). The dark control groups were submitted to the same procedure, except for light exposure. No green fluorescent emission was observed in the cells after PDI with 20 min irradiation. In contrast, the dark controls showed fluorescent emission of ZsGreen. Neither PDZ irradiated samples nor dark samples showed toxicity on the cell confluency, while an increase of autofluorescence was observable, compared to the negative control cells. These observations confirm our results of luciferase assay (Figure 3B), and are in agreement with our previous studies on PDZ-based PDI, as described elsewhere [15].

In sum, two distinct strategies (UV-C irradiation and PDZ-based PDI) were applied for the inactivation of SC2 pseudovirus produced using HIV-based lentiviral system which specifically infect ACE2-expressing cells. This specificity was demonstrated using luciferase assay compared to the control negative cells and the control naked viruses, which agreed with previous reports [21,23,33,34]. The viral inactivation could be the consequence of either viral protein damage, which affects the cell internalization, or viral genome damage affecting the viral load. Unlike the SC2 RNA virus with viral reproduction independent of the host genome [7,8,30,35,36], this pseudotyped model enabled us to study not only the RNA viral load, but also the DNA integration, as well as the presence or absence of a spike on the viral particle. Several reports demonstrating the results of viral inactivation assays have a high degree of concordance with a clinical isolate of SC2 [33,34]; however, the results cannot be used for the inactivation of the actual SC2 virus unless tested.

#### **4. Conclusions**

Considering the advantages of pseudovirus over the actual SC2 virus, which was discussed above, we followed a simple protocol for calculating the RNA and DNA load and compared the qPCR data with luciferase assay results. Hence, we studied the viral inactivation by UV-C and PDI in dose and time-dependent manners via biochemical characterizations and quantitative PCR on four levels; virion damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDI treatments could destroy ssRNA and the spike protein of the virus in different ratios; however, the virus was still capable of binding and entering into the ACE-2 expressing 293T cells. UV-C irradiation disinfected the virus mainly through viral genome damage, with no apparent effects on the viral size and virus–cell binding ability. On the other side, PDZ-based PDI mostly destroyed the spike and viral membrane. Ignoring the type of viral destruction (ssRNA or spike), the cells infected by the photo-inactivated virus could not express the luciferase reporter gene. Our findings emphasize the advantages of PDI over UV-C viral inactivation. ROSmediated damages on the viral envelope may generate debris or the fragments which could stimulate host immune defense. Moreover, viral PDI has affordability compared to other therapeutics like monoclonal antibodies (e.g., Ronapreve), which can be important factors for preventative use at home [37]. Other advantages of PDI include high repeatability without viral resistance or UV-signature mutations, with fast removal of the virus in a very short time.

The other advantage of this model is comparing the viral particles in two forms of enveloped and non-enveloped (naked) viruses, as a matter of importance for side-by-side comparison. Therefore, comparing two viruses with similar genomes but different in their protein envelope enables us to study the effect of each inactivation strategy on damaging the RNA genome in the presence and absence of a spike. Besides, this pseudotyped model can be used for other radiation-based strategies for understanding their mechanism of viral inactivation, with no need to work in BSL-3.

**Author Contributions:** F.E.G.G., M.M. and M.S. conceptualized and wrote the main manuscript text and prepared figures; F.E.G.G., M.M., R.S.D. and M.S. designed and performed the biochemical and microscopy experiments; F.F.P.J. designed and performed the DLS assay; R.S.D., R.S.F., E.F.d.C., M.S. and J.G. designed and performed the viral assays; L.F., L.Z. and G.C.-M. reviewed and edited the manuscript; M.S., R.S.D. and F.E.G.G. supervised the study. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors acknowledge the support provided by: FAPESP (Sao Paulo Research Foundation)–grant numbers: 2017/10910-5 (M. Sadraeian-Pós-Doutorado-Fluxo Contínuo), 2013/07276-1 (CEPOF–CEPID Program), 2019/14526-0 and 2020/05146-7 (G. Cabral-Miranda, JP FAPESP), and CAPES Project (COMBA TE-COVID1673158P) Program (PHYSICS 33002045002P9). The COVID-19 reagents were supported by BEI Resources Repository of ATCC and the NIH.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data available are reported in the article.

**Acknowledgments:** We thank BEI Resources Repository of ATCC and the NIH for supporting the reagent related to COVID-19. We thank Seth Pincus, M.D. for his support in the Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT.

**Conflicts of Interest:** Authors declare no competing interests. Graphical figures were created with BioRender software ((https://biorender.com/ (accessed on 15 July 2021)).

#### **Abbreviations**


#### **References**


## *Article* **Multiple Light-Activated Photodynamic Therapy of Tetraphenylethylene Derivative with AIE Characteristics for Hepatocellular Carcinoma via Dual-Organelles Targeting**

**Chuxing Chai 1,†, Tao Zhou 2,†, Jianfang Zhu 3,†, Yong Tang <sup>1</sup> , Jun Xiong <sup>1</sup> , Xiaobo Min <sup>1</sup> , Qi Qin <sup>1</sup> , Min Li 1,\* , Na Zhao 4,5,6 and Chidan Wan 1,\***


**Abstract:** Photodynamic therapy (PDT) has emerged as a promising locoregional therapy of hepatocellular carcinoma (HCC). The utilization of luminogens with aggregation-induced emission (AIE) characteristics provides a new opportunity to design functional photosensitizers (PS). PSs targeting the critical organelles that are susceptible to reactive oxygen species damage is a promising strategy to enhance the effectiveness of PDT. In this paper, a new PS, 1-[2-hydroxyethyl]-4-[4-(1,2,2 triphenylvinyl)styryl]pyridinium bromide (TPE-Py-OH) of tetraphenylethylene derivative with AIE feature was designed and synthesized for PDT. The TPE-Py-OH can not only simultaneously target lipid droplets and mitochondria, but also stay in cells for a long period (more than 7 days). Taking advantage of the long retention ability of TPE-Py-OH in tumor, the PDT effect of TPE-Py-OH can be activated through multiple irradiations after one injection, which provides a specific multiple light-activated PDT effect. We believe that this AIE-active PS will be promising for the tracking and photodynamic ablation of HCC with sustained effectiveness.

**Keywords:** photodynamic therapy; aggregation-induced emission; organelles targeting; hepatocellular carcinoma

## **1. Introduction**

Locoregional ablation therapy was recommended as an effective treatment for patients with primary hepatocellular carcinoma (HCC) and liver metastases [1,2]. The mechanism of ablation is to cause the destruction of local tumor by physical or chemical damage. Thermal ablation, including radiofrequency ablation and microwave ablation, has been applied as a safe, low-cost, effective alternative of surgery in small and multifocal liver tumor [3]. The ablation procedure often relies on the placement of the needle electrode into target tumor under the guidance of imaging technology which requires additional competence and fails to provide real-time self-monitoring. Multiple treatment sessions and repetitive puncture operation may be required due to difficult tumor conditions and

**Citation:** Chai, C.; Zhou, T.; Zhu, J.; Tang, Y.; Xiong, J.; Min, X.; Qin, Q.; Li, M.; Zhao, N.; Wan, C. Multiple Light-Activated Photodynamic Therapy of Tetraphenylethylene Derivative with AIE Characteristics for Hepatocellular Carcinoma via Dual-Organelles Targeting. *Pharmaceutics* **2022**, *14*, 459. https://doi.org/10.3390/ pharmaceutics14020459

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis, Gerhard Litscher and Patrick J. Sinko

Received: 8 December 2021 Accepted: 17 January 2022 Published: 21 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

limitation of imaging technology, which may cause additional suffering of patients [4]. Alternatively, photodynamic therapy (PDT) is a potential tumor-ablative locoregional therapy [5–7]. Fluorescent molecules with photodynamic effect can provide precise and safe ablation of tumor at the cellular level while enabling dynamic monitoring of tumor lesion, which has great promise in building cancer-targeted theragnostic platforms [8,9]. PDT can induce both cancer cell death and immune response against the tumor [10,11]. Additionally, intraoperative fluorescence image-guided surgery combined with PDT can eliminate residual tumors and reduce the recurrence of cancer [12–15]. The use of PDT has gained much attention in the treatment of HCC, and has been approved as a feasible approach for HCC treatment in preclinical studies [6,16].

The three crucial elements of PDT are photosensitizer (PS), light irradiation, and oxygen. PS is the core element of PDT. The procedure of PDT involves administration of PSs which accumulate in the tumor tissue, followed by local illumination with specific wavelength to active PSs [17]. After exposure to the light, the PSs can transfer energy from light to molecular oxygen, contribute to their change of energy states, to generate reactive oxygen species (ROS), which then induce apoptosis, necrosis, and autophagy in treated cells and cause cell death [18,19]. However, traditional PSs—such as porphyrin, phthalocyanine, and analogs—are prone to aggregate in the treated cells or aqueous solution, which will lead to a dramatic reduction in ROS production and suppression in fluorescence emission [20,21].

In 2001, Tang's group discovered a novel fluorogen with the opposite characteristics, namely aggregation-induced emission (AIE) [22], which is nonemissive in a molecularly dissolved state, but is highly emissive in their aggregate/solid state due to the mechanism of restriction of intramolecular motions/rotations [23]. Meanwhile, as organic molecules, AIE luminogens (AIEgens) were highly editable. The different additional groups and molecular structures can provide different optical, physical, and chemical properties to AIEgens, which enables broad biological medicine application [24–27]. A series of AIEgens have been developed for translational applications in sensing, imaging, and theranostics with excellent performance than conventional fluorescent probes [28,29]. Recently, some AIEgens were designed to exhibit excellent photosensitization and ROS generation ability, which broadens their potential applications in PDT of cancer. The unique features of AIEgens provide new opportunities for the facile design of PS with special function, high accuracy, and efficacy for image-guided PDT [30–33].

A critical limitation of PDT is that the half-life of ROS (<40 ns) is usually very short so that it can only act on the production site (<20 nm) [34], which means simple transportation PSs into tumor tissues or cancer cells are not efficient enough to achieve expected outcomes [34,35]. Designing PSs to target the critical organelles that are susceptible to ROS damage is a promising strategy to enhance the effectiveness of PDT. The mitochondrion is a promising target for PSs owing to its crucial role in ROS generation, oxidation-reduction status modulation, and cell apoptosis mediation. Mitochondria in tumor cells are sensitive to ROS generation induced by PDT, which ultimately induces tumor cell apoptosis by disrupting the balance of mitochondrial ROS [36]. Recently, several AIEgens have been designed for targeting mitochondria and have exhibited remarkable photostability, high brightness, and excellent PDT effect [37–40]. However, multiple doses and repeated injections of PSs are always required for better outcomes in PDT procedures, which may lead to superfluous side effects and additional toxicity to patients [36]. The destruction of tumor vessels of PDT may also reduce the accumulation of PSs in tumor tissue and influence the efficiency of second PDT. Lipid droplets (LDs), also known as adiposomes or lipid bodies, are intracellular lipid-rich organelles for the long-term storage of lipids [41]. The synthesis of lipid droplets is at a lower level in normal cells, whereas tumor cells actively synthesize lipid droplets owing to their high-metabolism state [42,43]. Due to the suitable metabolic activity and stability, as well as their interactions with other organelles, especially mitochondrial, LDs can be valuable targets for PDT [44,45]. Therefore, AIEgens that could dual-target mitochondria and LDs may provide long-term photodynamic effect and better outcomes in PDT treatment procedure after administration of single-dose PSs.

In this work, for developing a new PS with multiple light-activated photodynamic effect and organelle-targeting abilities, the 1-[2-hydroxyethyl]-4-[4-(1,2,2-triphenylvinyl) styryl]pyridinium bromide (TPE-Py-OH) was designed and synthesized. TPE-Py-OH possesses a typical AIE character and exhibits strong fluorescence emission in the aggregated state. It could simultaneously target LDs and mitochondria in living cells. Due to its lipophilic feature, it can aggregate and store in LDs in a relatively stable state for long-term intracellular retention. Meanwhile, TPE-Py-OH also has efficient ROS generation ability and PDT effect both in vitro and in vivo. All these features suggest its superior performance in long-term tracking of living cancer cells and organelles-targeted PDT effect of cancer cells. Benefiting from the strong fluorescence, good photostability, and efficient ROS generation ability of TPE-Py-OH, the long-term cancer cell tracking and multiple light-activated PDT effect of HCC for more than 7 days were achieved after one PS administration. This AIE-active PS exhibit sustained effectiveness in vitro and in vivo, which suggests that the PS can hopefully be applied in long-term intracellular organelle imaging and multiple light-activated photodynamic ablation of HCC.

## **2. Materials and Methods**

### *2.1. Materials and Instruments*

4-Methylpyridine, 2-bromoethanol, piperidine, anhydrous acetonitrile, and ethanol were purchased from Sigma-Aldrich and used without further purification unless specified otherwise. 4-(1,2,2-Triphenylvinyl)benzaldehyde was synthesized according to the literature method [46,47]. Other chemicals and solvents were all purchased from Aldrich and used as received without further purification. Distilled water was used throughout the experiments. HepG2 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle medium, fetal bovine serum (FBS), BODIPYTM 493/503 Dye, and JC-1 Dye were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell Counting Kit-8 (CCK8) was purchased from Dojindo Molecular Technologies, Inc. (Mashikimachi, Japan). Reactive oxygen species (ROS) assay kit, calcein AM/PI double stain kit, Annexin V-FITC/PI double stain kit and MitoTracker Red FM was purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Qcbio Science & Technologies (Shanghai, China). Tissue culture flask, 96-well plates, and 24-well plates were purchased from Corning Incorporated (Corning, NY, USA). Confocal dishes were purchased from Biosharp Life Science (Hefei, China).

<sup>1</sup>H and <sup>13</sup>C NMR spectra were measured on a Bruker ARX 400 NMR spectrometer (Bruker, Tucson, AZ, USA) using DMSO-*d<sup>6</sup>* as solvents. High-resolution mass spectra (HRMS) were recorded on a Finnigan MAT TSQ 7000 Mass Spectrometer System (Finnigan MAT, San Jose, CA, USA) operating in a MALDI-TOF mode. UV–vis absorption spectra were recorded on a Biochrom UV visible spectrometer (Biochrom, Berlin, Germany). Photoluminescence (PL) spectra were recorded on a PerkinElmer spectrofluorometer LS 55 (PerkinElmer, Singapore, Singapore). The cells were imaged under LSM7 DUO confocal laser scanning microscope (CLSM; Carl Zeiss Microscopy, Jena, Germany) and fluorescence microscope (IX71, Olympus, Tokyo, Japan). The absorbance was measured using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.2. Synthesis of 1-(2-Hydroxyethyl)-4-methylpyridinium Bromide (Compound 1)*

An acetonitrile (20 mL) solution of 4-methylpyridine (0.93 g, 10.00 mmol) had 2 bromoethanol (2.5 g, 20.00 mmol) added to it. The mixture was stirred at reflux temperature (82 ◦C) under nitrogen for 24 h and then cooled to room temperature. Then, the reaction mixture was precipitated in ethyl acetate (EA) (200 mL). The solid residue was filtrated and washed with excess EA. The filtrate was collected and dried in a vacuum oven. Compound 1 was a light brown powder in 86% yield. <sup>1</sup>H NMR (400 MHz, DMSO-*d6*, δ): 8.91 (d, 2H, *J* = 6.8 Hz), 7.99 (d, 2H, *J* = 6.4 Hz), 4.64 (t, 2H, *J* = 15.6 Hz), 3.81 (t, 2H, *J* = 15.6 Hz), 2.60 (s, 3H). <sup>13</sup>C NMR (100 MHz, DMSO-*d6*) δ: 158.5, 143.9, 127.7, 61.9, 59.7, 21.2. HRMS (LDI-TOF) m/z calcd for [C8H12NO]<sup>+</sup> , 138.0919 [M-Br]+; found, 138.0920 [M-Br]+.

#### *2.3. 1-[2-Hydroxyethyl]-4-[4-(1,2,2-triphenylvinyl)styryl]pyridinium Bromide (TPE-Py-OH)*

A solution of 1 (0.5 g, 2 mmol) and 4-(1,2,2-triphenylvinyl)benzaldehyde (1.01 g, 2.8 mmol) was refluxed at temperature (78 ◦C) under nitrogen in dry ethanol (20 mL) catalyzed by three drops of piperidine. After cooling to ambient temperature, the solvent was evaporated under reduced pressure. The residue was purified by a silica gel column chromatography (50 mesh) using dichloromethane/methanol mixture (5:1 *v*/*v*) as eluent to obtain TPE-Py-OH as a yellow solid in 64% yield (0.72 g, 1.29 mmol). <sup>1</sup>H NMR (400 MHz, DMSO-*d6*) δ: 8.82 (d, 2H, *J* = 6.8 Hz), 8.16 (d, 2H, *J* = 6.8 Hz), 7.88 (d, 1H, *J* = 16.0 Hz), 7.50 (d, 2H, *J* = 8.4 Hz), 7.42 (d, 1H, *J* = 16.4 Hz), 7.16–6.94 (m, 17H), 4.51 (t, 2H, *J* = 10.0 Hz), 3.82 (m, 2H), 3.5(brs, 1H). <sup>13</sup>C NMR (100 MHz, DMSO-*d6*) δ: 152.70, 145.35, 144.50, 142.79, 142.71, 142.56, 141.40, 140.07, 139.72, 133.10, 131.25, 130.53, 130.51, 130.43, 127.79, 127.68, 127.51, 126.69, 126.62, 123.22, 123.02, 61.92, 59.83. HRMS (LDI-TOF) *m*/*z* calcd for [C15H30NO]<sup>+</sup> , 480.2327 [M-Br]+; found, 480.2317 [M-Br]+.

#### *2.4. Cellular Uptake and Subcellular Distribution*

HepG2 human HCC cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 ◦C in a 5% CO2, 90% relative humidity incubator. For cell imaging, the HepG2 cells were seeded in slide chambers at a density of 10<sup>5</sup> and cultured overnight for adhesion. Then, cells were incubated with TPE-Py-OH at various concentrations (2, 5, and 10 µM) for 30 min and continued to incubate with fresh medium for 12 h. Nuclei were labeled with DAPI (1 µg/mL) overnight. After rinsing with PBS, the cells were imaged under a confocal laser scanning microscope (CLSM, Nikon Corporation, Tokyo, Japan). To further explore the subcellular distribution of TPE-Py-OH, 2 <sup>×</sup> <sup>10</sup><sup>5</sup> HepG2 cells were plated in the 35-mm confocal dish and cultured overnight, then incubated with 5 µM TPE-Py-OH for 30 min. After rising with PBS, the cells were then stained with 50 nM MitoTracker Red FM, or continuously incubated with fresh culture medium and then stained with 1 µg/mL BODIPY for 30 min, respectively. After three washes, the cell images were captured under CLSM, and the data was analyzed by NIS-Elements Imaging Software (Nikon Corporation, Tokyo, Japan) and Image-J (National Institutes of Health freeware, Bethesda, MD, USA). For TPE-Py-OH: λex = 405 nm and band-pass filter λ = 550–600 nm. For DAPI: λex = 405 nm and band-pass filter λ = 425–475 nm. For MitoTracker Red: λex = 581 nm and band-pass filter λ = 600–650 nm. For BODIPY 493/503: λex = 488 nm and band-pass filter λ = 500–550 nm.

#### *2.5. Cytotoxicity Studies In Vitro*

Cell viability after incubation with various concentrations of TPE-Py-OH was evaluated by CCK8 assays. Briefly, the cells at a density of 1 <sup>×</sup> <sup>10</sup><sup>4</sup> were seeded into a 96-well plate overnight and then incubated with a series of concentrations of TPE-Py-OH (0, 1, 3, 5, 7, 10, and 15 µM). After pre-treatment, all the cells were incubated with 100 µL fresh medium including with 10 µL CCK8 solution per well and then continued to incubate for 4 h. The absorbance of the solution at 450 nm was measured using a multimode plate reader (Perkin Elmer Pte. Ltd., Singapore, Singapore).

For cytotoxicity effect induced by PDT, the cells were incubated with various concentrations of TPE-Py-OH for 30 min and continued to incubate with fresh medium for 12 h and then exposed to blue laser irradiation with different duration (450 nm, 30 mW/cm<sup>2</sup> , 1.8 J/cm2–18 J/cm<sup>2</sup> ). The cells were as negative controls without both laser irradiation and TPE-Py-OH treatment. Sequentially, the cells continued to incubate for 24 h and the cell viability was measured according to the above description.

#### *2.6. Intracellular ROS Detection*

Intracellular ROS production was measured by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe according to the manufacturer's instructions. After incubation in the presence or absence of TPE-Py-OH for 30 min, the cells were loaded with 10 µM DCFH-DA at 37 ◦C for 30 min in dark. The cells were replaced with fresh medium and then exposed to 450 nm blue laser irradiation at the power density of 30 mW/cm<sup>2</sup> , then immediately imaged under a fluorescence microscope (IX71, Olympus, Tokyo, Japan). For DCFH-DA: λex = 488 nm and band-pass filter λ = 500–550 nm.

#### *2.7. Calcein-AM and Propidium Iodine (PI) Staining Assay*

The live and dead cells were identified using a calcein/PI double stain kit according to the protocol. After treatment, the cells were incubated with 2 µM calcein-AM and 5 µM PI for 30 min at 37 ◦C. The fluorescence images were captured under a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

#### *2.8. Flow Cytometry Analysis*

The apoptotic and necrotic cells were detected by Annexin V-FITC/PI dual staining using flow cytometry. The cells were seeded into a 24-well plate overnight and then incubated with TPE-Py-OH for 30 min. After exposure to laser irradiation (450 nm, 30 mW/cm<sup>2</sup> , 4 min, or 8 min), the cells were further incubated for 24 h, then collected and resuspended in 100 µL of binding buffer. According to the manufacturer's instructions, the cells were incubated with 5 µL Annexin V-FITC and 10 µL PI for 15 min in the dark at room temperature and then immediately measured by flow cytometry (BD Falcon, San Jose, CA, USA).

#### *2.9. Mitochondrial Membrane Potential (*∆*ψm) Measurement*

The change of mitochondrial membrane potential (∆ψm) was measured by JC-1 Dye according to the manufacturer's instructions. Briefly, the cells were incubated with 2 µg/mL of JC-1 for 20 min at 37 ◦C after different concentrations of TPE-Py-OH administration and different laser irradiation times, washed twice with PBS, and then imaged under CLSM. For JC-1 (monomer): λex = 488 nm and band-pass filter λ = 500–530 nm; (J-aggregate): λex = 585 nm and band-pass filter λ = 590 nm.

#### *2.10. Long-Term Cell Tracking and In Vitro PDT*

The cells were first stained with 10 µM TPE-Py-OH for 12 h and then rinsed with PBS to remove the extracellular molecules. The image was captured as a reference. The cells were incubated continuously and one-third of them were sub-cultured into another dish for continual tracking every 2 days. Other cells were seeded into the 35-mm confocal dish. After attachment for 24 h, the fluorescence of TPE-Py-OH in cells was imaged by CLSM, the ROS level in cells was performed according to the above description. The step was repeated every 2 days until non-fluorescence in cells.

To confirm the long-term localization of intracellular TPE-Py-OH, the HepG2 cells were incubated with 10 µM TPE-Py-OH for 12 h and then cultured with fresh DMEM medium, the daughter cells were sequentially co-stained with 1 µg/mL BODIPY for 30 min on days 1, 3, 5, and 7 and their fluorescence images were captured under a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

For long-term PDT in vitro, the HepG2 cells were incubated with 10 µM TPE-Py-OH for 12 h and replaced with fresh medium for following experiments. On days 1, 3, 5, and 7 after TPE-Py-OH treatment, the cells were seeded into 35-mm confocal dishes. After attachment, the cells were exposed to blue laser irradiation with different duration (450 nm, 30 mW/cm<sup>2</sup> , 18–45 J/cm<sup>2</sup> ) with half of the confocal dish covered by foil paper to avoid light irradiation. Then the calcein/PI double stain was used to detect the effect of PDT, and the JC-1 mitochondrial membrane potential probe was used for mitochondrial membrane

potential measurement. The fluorescence images were captured under a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

#### *2.11. Animal Model*

Male BALB/c mice (4 weeks old, 20 g) were purchased from HFK Bioscience Co, Ltd. (Beijing, China). All animal experiments were approved by the Ethics Committee of the Union Hospital of Huazhong University of Science and Technology and conducted in accordance with the guidelines of the Department of Laboratory Animals of Tongji Medical College. H22 cells (5 <sup>×</sup> <sup>10</sup><sup>5</sup> ) were subcutaneous injection into the right axilla of each mouse to establish xenograft liver tumor model, and the tumor volume reached 60 mm<sup>3</sup> after 5 days.

### *2.12. In Vivo Multiple Light-Activated PDT*

Tumor-bearing mice were randomized into 5 groups of 8 animals per group: (Group 1: TPE-Py-OH 3 IR) 50 ug/100 uL TPE-Py-OH injected intratumorally, and then underwent laser irradiation (450 nm, 100 mw/cm<sup>2</sup> , 10 min; 60 J/cm<sup>2</sup> ) on days 1, 3, and 5 after injection; (Group 2: TPE-Py-OH 1 IR) 50 ug/100 uL TPE-Py-OH injected intratumorally, and then underwent laser irradiation (450nm, 100 mw/cm<sup>2</sup> , 10min; 60 J/cm<sup>2</sup> ) on the first day after injection; (Group 3: TPE-Py-OH non IR) 50 ug/100 uL TPE-Py-OH injected intratumorally without irradiation; (Group 4: 3 IR only) 100 µL of PBS injected intratumorally, and then underwent laser irradiation (450 nm, 100 mw/cm<sup>2</sup> , 10 min; 60 J/cm<sup>2</sup> ) on days 1, 3, and 5 after injection; (Group 5: NC) negative control.

Seven days after injection, 3 mice of every group were sacrificed. Tumors were collected and kept in 4% formalin followed by embedded in paraffin. Slices cut from the paraffin sections were stained by hematoxylin and eosin (H&E) and TUNEL before being scanned with a fluorescence microscope (IX71, Olympus, Tokyo, Japan). The tumor size was measured by a caliper every 2 days and calculated as the volume = (tumor length) × (tumor width)<sup>2</sup> <sup>×</sup> 0.5.

#### **3. Results and Discussions**

#### *3.1. Synthesis and Characterization*

TPE-Py-OH was synthesized according to the synthetic route shown in Figure 1A. Compound 1 was obtained by quaternization of 4-methylpyridine with 2-bromoethanol. The product was purified by precipitation of the reaction mixture in excess EA. The product was obtained by filtrating and washing with EA, which was suitable to be carried forward. 4- (1,2,2-Triphenylvinyl)benzaldehyde (TPE-CHO) was synthesized according to the reported method [46,47]. Finally, compound 1 and TPE-CHO were refluxed in ethanol to give TPE-Py-OH in reasonable yield by aldol condensation. Unexpectedly, TPE-Py-OH can be purified with silica column by using high polarity solvent mixture (DCM: MeOH = 5:1) as eluent. Compound 1 and TPE-Py-OH were fully characterized by HRMS, and <sup>1</sup>H and <sup>13</sup>C NMR spectroscopies, and gave satisfactory analysis results corresponding to their chemical structures (Figures S1–S4). Due to its ionic character, TPE-Py-OH has good solubility in most of organic solvents like THF, DCM, and DMSO, but it is not soluble in water.

#### *3.2. Aggregation and Micellization of TPE-Py-OH*

Figure S5 shows the UV spectrum of TPE-Py-OH (10 µM) in DMSO solutions. The maximum absorption peak of TPE-Py-OH is located at 395 nm. For convenient bio-application, 405 nm was utilized as excitation wavelength for PL measurement. Photoexcitation of its DMSO solution (100 µM) induces a red emission at 650 nm (Figure 1B,C), giving a large Stokes shift of 9932 cm−<sup>1</sup> due to its extended conjugation as well as the intramolecular charge transfer (ICT) effect from the electron-donating TPE moiety to the electronaccepting pyridinium unit [47]. Due to the positively charged characteristics and pendant hydroxy group, TPE-Py-OH is amphiphilic and shows reversible aggregation behaviors in DMSO/H2O mixture (Figure S6A). Normally, solutions with a concentration of 10−5–10−<sup>6</sup>

M are used in PL measurements. A highly concentrated solution (100 µM) of TPE-Py-OH is employed for the analysis because TPE-Py-OH only forms aggregates or micelles at higher concentrations. According to previous studies, the aromatic rings on AIEgens have larger twisted angles in the aggregated state than in the solution state [23]. Thus, the AIEgens generally show blue-shifted in the aggregated state compared to that in the solution state. As shown in Figure 1B, TPE-Py-OH emits strongly in aqueous solution at 565 nm due to the activation of the AIE effect by micelles formation. The emission becomes weaker and shifts to 650 nm upon addition of a small amount of DMSO, presumably owing to the disintegration of the micelles, which releases free molecules with a more planar conformation to the solution [48]. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 7 of 19

**Figure 1.** (**A**) Synthesis of molecule 2 (TPE-Py-OH). (**B**) PL spectra of TPE-Py-OH in DMSO/H2O mixtures with different water fractions (fw). Concentration: 100 µM; excitation wavelength: 405 nm. (**C**) Plot of PL intensity versus the composition of the DMSO/H2O mixtures of TPE-Py-OH. **Figure 1.** (**A**) Synthesis of molecule 2 (TPE-Py-OH). (**B**) PL spectra of TPE-Py-OH in DMSO/H2O mixtures with different water fractions (fw). Concentration: 100 µM; excitation wavelength: 405 nm. (**C**) Plot of PL intensity versus the composition of the DMSO/H2O mixtures of TPE-Py-OH.

*3.2. Aggregation and Micellization of TPE-Py-OH* Figure S5 shows the UV spectrum of TPE-Py-OH (10 µM) in DMSO solutions. The maximum absorption peak of TPE-Py-OH is located at 395 nm. For convenient bio-application, 405 nm was utilized as excitation wavelength for PL measurement. Photoexcitation of its DMSO solution (100 µM) induces a red emission at 650 nm (Figure 1B,C), giving a large Stokes shift of 9932 cm−<sup>1</sup> due to its extended conjugation as well as the intramolecular charge transfer (ICT) effect from the electron-donating TPE moiety to the electron-accepting pyridinium unit [47]. Due to the positively charged characteristics and pendant hydroxy group, TPE-Py-OH is amphiphilic and shows reversible aggregation behaviors in To study the micellization behaviors of TPE-Py-OH, the PL of its solutions at different concentrations were investigated by taking advantage of its AIE property. The PL intensity of TPE-Py-OH increases with increasing the solution concentration with stable emission spectra (Figure S6B). The plot of relative PL intensity (*I*/*I*0) against the logarithm solution concentration generates two lines, the intersection of which determines the critical micelle concentration (CMC) and is found to be 0.3 mM in H2O/DMSO mixtures (*v*/*v* 90%). TPE-Py-OH is molecularly dispersed in solutions with concentrations of below 0.3 mM and displayed weak emission. When the concentration of TPE-Py-OH was more than or equal to 0.3 mM, the FL intensity was remarkably increased due to the AIE feature.

#### DMSO/H2O mixture (Figure S6A). Normally, solutions with a concentration of 10−5–10−6 M are used in PL measurements. A highly concentrated solution (100 µM) of TPE-Py-OH *3.3. Dual-Organelles Targeting and Cell Imaging of TPE-Py-OH*

is employed for the analysis because TPE-Py-OH only forms aggregates or micelles at higher concentrations. According to previous studies, the aromatic rings on AIEgens have larger twisted angles in the aggregated state than in the solution state [23]. Thus, the AIEgens generally show blue-shifted in the aggregated state compared to that in the solution state. As shown in Figure 1B, TPE-Py-OH emits strongly in aqueous solution at 565 nm due to the activation of the AIE effect by micelles formation. The emission becomes We next studied the cellular uptake of TPE-Py-OH in HepG2 cells. The cells were treated with TPE-Py-OH and then observed by CLSM upon excitation at 405 nm with a collection of fluorescent signals above 585 nm. As shown in Figure S7, the bright yellow fluorescence could be detected from the cytoplasm after incubation with TPE-Py-OH for 30 min, which demonstrated that TPE-Py-OH could be internalized with high efficiency by HepG2 cells. Meanwhile, the fluorescence structure in cells was mainly in funicular shape

weaker and shifts to 650 nm upon addition of a small amount of DMSO, presumably owing to the disintegration of the micelles, which releases free molecules with a more planar

To study the micellization behaviors of TPE-Py-OH, the PL of its solutions at different concentrations were investigated by taking advantage of its AIE property. The PL intensity of TPE-Py-OH increases with increasing the solution concentration with stable emission spectra (Figure S6B). The plot of relative PL intensity (*I*/*I*0) against the logarithm solution concentration generates two lines, the intersection of which determines the critical micelle concentration (CMC) and is found to be 0.3 mM in H2O/DMSO mixtures (*v*/*v* 90%). TPE-Py-OH is molecularly dispersed in solutions with concentrations of below 0.3

when TPE-Py-OH entered into cells in short time and then fluorescence in punctate shape gradually enhanced, which demonstrated the aggregation of free TPE-Py-OH molecules in cytoplasm as the incubation time prolonged. This phenomenon indicated that the organelles targeting of TPE-Py-OH maybe occur in live cells.

To explore the subcellular location of TPE-Py-OH in living cells, HepG2 cells were costained with TPE-Py-OH and commercial fluorescent probes of mitochondria (MitoTracker Red) and LDs (BODIPY 493/503), respectively. As shown in Figure 2A, a large amount of yellow fluorescence from TPE-Py-OH merged with red fluorescence from MitoTracker Red in cells. However, some extra bright yellow fluorescence signals with punctate shape did not overlay the punctate red mitochondrial fluorescence. Sequentially, the co-localization experiment with BODIPY showed that the yellow punctate shape fluorescence from TPE-Py-OH and green fluorescence from BODIPY were co-localized well in live cells (Figure 2B). The Pearson's correlation was further quantitatively determined, which is high as 0.839 (Figure 2C). The intensity profile for the region of interest line across the cells also varied in close synchrony (Figure 2D). All the results indicated that TPE-Py-OH has a dual-targeting ability of mitochondria and LDs with high selectivity due to their structures of membrane phospholipid. Furthermore, it seems that the more dissociative TPE-Py-OH in cytoplasm could be finally inclined to LDs, and then stored in these organelles and kept stable in cells. Additionally, the fluorescent intensity of TPE-Py-OH in cells was dependent on its concentration in culture medium, which demonstrated that stronger fluorescence was captured in cells incubated with higher concentration (Figure 2E).

#### *3.4. In Vitro Anti-Cancer Efficacy of TPE-Py-OH*

Biocompatibility is a vital parameter of the PS for cancer therapy in vivo, and thus the biocompatibility of TPE-Py-OH on HepG2 cells was assessed via CCK8 assay before the in vitro PDT experiments. The cytotoxicity of TPE-Py-OH with different concentrations upon incubation with HepG2 cells in dark condition was first evaluated. As shown in Figure 3A, no significant change was observed in cell viability at 24 h after 30 min incubation without light irradiation. Even after 12 h incubation with 15 µM TPE-Py-OH, the cell viability was more than 80%, which indicated minimal cytotoxicity of TPE-Py-OH to cells and excellent biocompatibility. Then the PDT anti-cancer efficiency of TPE-Py-OH against HepG2 cells was further assessed. As shown in Figure 3B,C, the cell viability was significantly decreased in the presence of 450 nm blue laser irradiation. Meanwhile, the amount of cell death was associated with the light duration and the incubation concentration of TPE-Py-OH. The process of ROS generation and mitochondrial-reliant photodynamic killing of TPE-Py-OH was illustrated in Figure 3D. The intracellular ROS level after PDT treatment was measured via ROS indicator 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), which is highly fluorescent in the presence of ROS in living cells. A JC-1 dye was applied to monitor the change of mitochondrial membrane potential (∆ψm). The decline of relative intensity of red to green fluorescence of JC-1 dye could indicate the depolarization of mitochondria, which is commonly used as an indicator of mitochondrial damage [49]. Additionally, after incubation with TPE-Py-OH followed by light irradiation, the appearance change of HepG2 cells was observed. As the concentration of TPE-Py-OH and irradiation time increased, HepG2 cells gradually shrank and turned round, indicating early apoptosis. After staining with DCFH-DA, green fluorescence was negligible without light irradiation or TPE-Py-OH. In contrast, cells incubated with TPE-Py-OH showed bright green fluorescence with light irradiation, which indicated significantly increased ROS production. The results of JC-1 imaging showed that TPE-Py-OH could efficiently promote the decline of ∆ψm under light irradiation and the decline of ∆ψm was concentration- and irradiation time-dependent. To further explore the PDT effect of TPE-Py-OH, HepG2 cells were stained with calcein-AM (green fluorescence) and PI (red fluorescence) to label live and dead cells (Figure 3E). The increased red fluorescence in TPE-Py-OH plus light irradiation group indicated good photodynamic cell death effect. An Annexin V-FITC/PI apoptosis detection kit was used to further investigate the cell death modality induced by PDT treatment

(Figure 3E). An increased number of both necrotic cells and apoptotic cells were observed with an elevation of the TPE-Py-OH concentration and irradiation time. All of the above results indicated that TPE-Py-OH could induce cell death through ROS generation and mitochondrial damage in the presence of light irradiation, and the dominant cell death is closely related to the light duration and incubation concentration. *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 9 of 19

**Figure 2.** Fluorescence imaging of TPE-Py-OH in HepG2 cells. (**A**) Co-staining of 5 μM TPE-Py-OH (λex = 405 nm) and 50 nM MitoTracker Red (λex = 581 nm) for 30 min. (**B**) Co-staining of cells incubated with 5 μM TPE-Py-OH for 30 min, followed by continuous incubation in fresh medium for 12 h and then staining with 1 μg/L BODIPY (λex = 488 nm) for 30 min. (**C**,**D**) Quantitation analysis of the cells co-stained with TPE-Py-OH and BODIPY. (**E**) Images of TPE-Py-OH in HepG2 cells incubated at various concentrations for 30 min. *3.4. In Vitro Anti-Cancer Efficacy of TPE-Py-OH* **Figure 2.** Fluorescence imaging of TPE-Py-OH in HepG2 cells. (**A**) Co-staining of 5 µM TPE-Py-OH (λex = 405 nm) and 50 nM MitoTracker Red (λex = 581 nm) for 30 min. (**B**) Co-staining of cells incubated with 5 µM TPE-Py-OH for 30 min, followed by continuous incubation in fresh medium for 12 h and then staining with 1 µg/L BODIPY (λex = 488 nm) for 30 min. (**C**,**D**) Quantitation analysis of the cells co-stained with TPE-Py-OH and BODIPY. (**E**) Images of TPE-Py-OH in HepG2 cells incubated at various concentrations for 30 min.

Biocompatibility is a vital parameter of the PS for cancer therapy in vivo, and thus the biocompatibility of TPE-Py-OH on HepG2 cells was assessed via CCK8 assay before the in vitro PDT experiments. The cytotoxicity of TPE-Py-OH with different concentrations upon incubation with HepG2 cells in dark condition was first evaluated. As shown in Figure 3A, no significant change was observed in cell viability at 24 h after 30 min incubation without light irradiation. Even after 12 h incubation with 15 µM TPE-Py-OH, the cell viability was more than 80%, which indicated minimal cytotoxicity of TPE-Py-OH to

against HepG2 cells was further assessed. As shown in Figure 3B,C, the cell viability was

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 11 of 19

**Figure 3.** In vitro ROS generation and anti-tumor efficacy of TPE-Py-OH. (**A**) The viability of HepG2

cells pretreated with different concentrations of TPE-Py-OH for different times without light irradiation. (**B**) The viability of HepG2 cells pretreated with TPE-Py-OH, followed by different irradiation times (450 nm, 30 mW/cm<sup>2</sup> ). (**C**) The viability of HepG2 cells pretreated with different concentrations of TPE-Py-OH, followed by 4 min or 8 min irradiation (450 nm, 30 mW/cm<sup>2</sup> ; 7.2 J/cm<sup>2</sup> or 14.4 J/cm<sup>2</sup> ). (**D**) Detection cell morphology, ROS generation, and mitochondrial membrane potential (∆Ψm) after different treatment of HepG2 cells. For DCFH-DA: λex = 488 nm and band-pass filter λ = 500–550 nm. For JC-1 (monomer): λex = 488 nm and band-pass filter λ = 500–530 nm; (J-aggregate): λex = 585 nm and band-pass filter λ = 590 nm. (**E**) Fluorescence images stained with calcein-AM/PI (For calcein-AM λex = 490 nm, For PI λex = 545 nm) and flow cytometry stain with Annexin V-FITC/PI after different treatment of HepG2 cells. For flow cytometry: Q1: Necrotic cells; Q2: Late apoptotic cells; Q3: Early apoptotic cells; Q4: Normal cells.

### *3.5. In Vitro Long-Term Tracking and PDT of TPE-Py-OH*

To investigate the long-term retention of TPE-Py-OH in living cells, the HepG2 cells stained with TPE-Py-OH were continuously cultured and their fluorescent signal was monitored under CLSM. As shown in Figure 4A, the yellow fluorescence from TPE-Py-OH was recorded after 12 h of incubation as a reference. Sequentially, the fluorescence signal was captured every 2 days, and the intracellular yellow fluorescence still existed even on the seventh day after incubation with TPE-Py-OH, which demonstrated the long-term intracellular retention ability of TPE-Py-OH. Although the fluorescence intensity decreased as cell divided due to the reduction of the number of the AIEgen in each cell, the residual PSs could still be activated by light irradiation and induce ROS generation, which was confirmed by the DCFH-DA staining. Since previous result showed intracellular storage of TPE-Py-OH, we then explored the location of long-term retention of TPE-Py-OH in live HepG2 cells. Although TPE-Py-OH decreased in single cell as cell division, the yellow fluorescence was still co-localized with BODIPY, indicating that they could keep staying in LDs with high binding characteristics in Figure 4B. To verify long-term photodynamic effect of the remaining TPE-Py-OH in cells, the PDT efficiency was investigated using calcein-AM/PI dual staining. As shown in Figure 4C, the region of light irradiation (right part of the dotted line) showed increased red fluorescence of PI stain even on the seventh day after treatment with TPE-Py-OH in contrast with the region without light irradiation (left part of the dotted line). In the region of light irradiation, cell morphologic changes of the early stage of apoptosis were also observed in the bright field under optical microscope. Although the efficiency of PDT was decreased with the passage of time, the obvious photodynamic killing effect could last for at least 1 week after primary TPE-Py-OH treated with prolonged light irradiation. It seems that TPE-Py-OH can combine with and store in intracellular LDs with stable fluorescence and photodynamic tumorkilling effect. Previous results also showed that TPE-Py-OH could induce mitochondrial damage and dysfunction due to its ability to target mitochondria. Since the TPE-Py-OH could aggregate in LDs after long-term incubation, we further explored the change of damage mechanism. Interestingly, JC-1 staining demonstrated that TPE-Py-OH could still induce dramatically mitochondrial damage in spite of tending to accumulate into LDs in Figure 4D. Singlet oxygen species produced by PDT can oxidize intracellular LDs to form lipid oxide or lipid peroxide [50]. The intracellular lipid peroxidation could initiate cellular ferroptosis, which can induce mitochondrial morphology disruption and depolarization in this process [51]. This may be the mitochondrial damage mechanism of TPE-Py-OH stored in LDs. Hence, the TPE-Py-OH could store in LDs in a relatively stable state and maintain its ROS generation ability for more than 7 days, which allowed multiple-light activated PDT effect and mitochondrial damage.

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 13 of 19

**Figure 4.** Long-term tracking and PDT effect of HepG2 cells stained with 10 μM TPE-Py-OH for 12 h and continuously cultured for different time. (**A**) Fluorescent images of TPE-Py-OH and intracellular ROS generation after light irradiation (450 nm, 30 mW/cm<sup>2</sup> , 10 min). (**B**) Co-staining of HepG2 cells stained with TPE-Py-OH and continuously cultured for different time, followed by staining with 1 μg/mL BODIPY for 30 min. (**C**) Fluorescence images stained with calcein-AM/PI of HepG2 **Figure 4.** Long-term tracking and PDT effect of HepG2 cells stained with 10 µM TPE-Py-OH for 12 h and continuously cultured for different time. (**A**) Fluorescent images of TPE-Py-OH and intracellular ROS generation after light irradiation (450 nm, 30 mW/cm<sup>2</sup> , 10 min). (**B**) Co-staining of HepG2 cells stained with TPE-Py-OH and continuously cultured for different time, followed by staining with 1 µg/mL BODIPY for 30 min. (**C**) Fluorescence images stained with calcein-AM/PI of HepG2 cells cultured for different time. left part of the dotted line: without light irradiation, right part of the dotted line: with light irradiation. (**D**) Fluorescence images stained with JC-1 of HepG2 cells cultured for different time with irradiation.

#### *3.6. In Vivo Multiple Light-Activated PDT Effect of TPE-Py-OH*

To further evaluate the potential of PDT performance of TPE-Py-OH in vivo, we carried out a mice H22 tumor model. The tumor model was established by subcutaneous injection of 5 <sup>×</sup> <sup>10</sup><sup>5</sup> H22 cells in BALB/c mice. After the tumors had become 60 mm<sup>3</sup> in size, TPE-Py-OH were injected intratumorally. In vivo distribution of TPE-Py-OH in mice over time was monitored by whole animal fluorescence. As shown in Figure S8A, local fluorescence still existed even at 12th day after TPE-Py-OH administration. The compression splice of tumor tissue shows bright yellow fluorescence in tumor cells (Figure S8B). Based on the long-term retention of TPE-Py-OH in tumor foci after administration, we hypothesized that the remaining TPE-Py-OH could kill the tumor cells by repeated irradiation after one administration via PDT effect. Subsequently, to explore the multiple light-activated PDT performance of TPE-Py-OH, H22 tumor-bearing mice were randomized into five groups: (1) TPE-Py-OH with three irradiations; (2) TPE-Py-OH with one irradiation; (3) TPE-Py-OH without irradiation; (4) PBS with irradiation; (5) negative control. TPE-Py-OH was intratumorally injected and further irradiated with laser (450 nm, 100 mW/cm<sup>2</sup> , 10 min) and the course of treatment is shown in Figure 5A. As shown in Figure 5B, the mice injected TPE-Py-OH with laser irradiation three times exhibited larger scale local necrosis of tumor and better tumor inhibition than the group treated with irradiation one time. TPE-Py-OH without irradiation group, PBS with irradiation group and negative control group were found to exhibit negligible tumor necrosis and inhibition. The tumor tissues were harvested after the mice were sacrificed on the seventh Day. Hemotoxylin/eosin (H&E) staining of tumor tissue showed a majority of dead cells in TPE-Py-OH with the three-time irradiation group and relative mild tumor cells death in TPE-Py-OH with the one-time irradiation group (Figure 5C). A high apoptosis rate was also observed by TUNEL stain, which demonstrated the multiple light-activated PDT effect of TPE-Py-OH after once injection for more than 1 week. This result was consistent with previous in vitro experiments. Tumor growth in the different groups was monitored by recording the tumor volume every 2 days for a period of 10 days. The tumor volume of TPE-Py-OH with three-time irradiation group was obviously smaller than other groups (Figure 5D). Although TPE-Py-OH with the one-irradiation group showed mild tumor inhibition after treatment, the tumor volume significantly increased after treatment ended. Mice survival was monitored, as shown in Figure 5E. Mice treated with TPE-Py-OH and irradiation three times showed better survival than other groups. All these results demonstrate that TPE-Py-OH could accumulate in the tumor region for a long time and provide multiple light-activated PDT effect for a long time.

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 15 of 19

**Figure 5.** In vivo multiple light-activated PDT effect of TPE-Py-OH in H22 tumor-bearded mice. (**A**) Scheme of in vivo PDT experiment. (**B**) Representative photographs of mice with treatments at different days, the irradiation was performed with 450 nm laser (100 mW/cm<sup>2</sup> ) for 10 min. (**C**) H&E stained and TUNEL stained tumor tissue slices after 7 days of treatments. (**D**) Tumor growth curves of mice with the various treatments. (**E**) Survival of mice with the various treatments.

#### **4. Conclusions**

In summary, we have developed a dual-organelles-targeting AIEgen based on tetraphenylethylene derivative (TPE-Py-OH). TPE-Py-OH can target both mitochondria and LDs, exhibits excellent ability of long-term retention, has stable fluorescence emission and multiple light-activated PDT effect in cancer cells. By storing in intracellular LDs, the fluorescence of TPE-Py-OH in cells can be maintained for a long time, and the photodynamic tumor-killing effect could still be activated by light irradiation at 7 days after a single treatment with TPE-Py-OH in vitro and in vivo. We believe that this AIEgen will be promising for the long-term tracking and multiple light-activated photodynamic ablation of HCC with sustained effectiveness. Although the tumor-targeting ability and the approach of drug administration is less than perfect, the hydroxy group (-OH) of this AIEgen provides excellent molecular modifiability and carrier combinability. Further modification of molecular structure and combination with nanocarrier could improve the cancer selectivity and fluorescence properties of TPE-Py-OH. Based on molecular characteristics of TPE-Py-OH, new PSs with improved function and a better administration approach can be designed to achieve multiple light-activated PDT.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10.3 390/pharmaceutics14020459/s1, Figure S1: HRMS spectra of molecule 1; Figure S2: HRMS spectra of TPE-Py-OH; Figure S3: <sup>1</sup>H NMR spectra of (A) TPE-Py-OH and (B) molecular 1 in DMSO-*d*<sup>6</sup> ; Figure S4: <sup>13</sup>C NMR spectra of (A) TPE-Py-OH and (B) molecular 1 in DMSO-*d*<sup>6</sup> ; Figure S5: UV spectra of TPE-Py-OH at the concentration of 10 µM in DMSO; Figure S6: (A) PL spectra of TPE-Py-OH in H2O/DMSO mixtures (*v*/*v* 90%) with different concentrations. Excitation wavelength: 405 nm. (B) Plot of PL intensity versus the concentrations of TPE-Py-OH. Inset: Photograph of TPE-Py-OH with various concentrations taken under 365 nm UV irradiation; Figure S7: The dynamic monitoring of the distribution of TPE-Py-OH in living cells. The HepG2 cells were incubated with 5 µM TPE-Py-OH for 30 min and then replaced with fresh medium, Figure S8: (A) The distribution of TPE-Py-OH in vivo after intratumoral injection over time. (B) Imaging of compression slice of H22 tumor on day 12 after TPE-Py-OH administration.

**Author Contributions:** Conceptualization, M.L. and C.W.; Methodology, M.L., C.C., T.Z. and J.Z.; TPE-Py-OH synthesis and characterization, N.Z.; In vitro and in vivo studies, C.C., T.Z., J.Z. and X.M.; Writing—original draft preparation, C.C.; Writing—review and editing, M.L., Q.Q., J.X. and Y.T.; Supervision, C.W., M.L. and J.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the National Natural Science Foundation of China (81672919, 81874208, 82172754, 81771005, 81771718).

**Institutional Review Board Statement:** The animal procedures were conducted at the guidelines of the Department of Laboratory Animals of Tongji Medical College and approved by Institutional animal care and use Committee of Huazhong University of Science and Technology (approval number 2526, date: 12 November 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** The authors would like to thank Yilong Chen for his help in the synthesis of TPE-Py-OH. Furthermore, we are grateful to Shunchang Zhou for his help during the animal experiment.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Insight into the Web of Stress Responses Triggered at Gene Expression Level by Porphyrin-PDT in HT29 Human Colon Carcinoma Cells**

**Maria Dobre <sup>1</sup> , Rica Boscencu <sup>2</sup> , Ionela Victoria Neagoe <sup>1</sup> , Mihaela Surcel <sup>1</sup> , Elena Milanesi <sup>1</sup> and Gina Manda 1,\***


**Abstract:** Photodynamic therapy (PDT), a highly targeted therapy with acceptable side effects, has emerged as a promising therapeutic option in oncologic pathology. One of the issues that needs to be addressed is related to the complex network of cellular responses developed by tumor cells in response to PDT. In this context, this study aims to characterize in vitro the stressors and the corresponding cellular responses triggered by PDT in the human colon carcinoma HT29 cell line, using a new asymmetric porphyrin derivative (P2.2) as a photosensitizer. Besides investigating the ability of P2.2-PDT to reduce the number of viable tumor cells at various P2.2 concentrations and fluences of the activating light, we assessed, using qRT-PCR, the expression levels of 84 genes critically involved in the stress response of PDT-treated cells. Results showed a fluence-dependent decrease of viable tumor cells at 24 h post-PDT, with few cells that seem to escape from PDT. We highlighted following P2.2-PDT the concomitant activation of particular cellular responses to oxidative stress, hypoxia, DNA damage and unfolded protein responses and inflammation. A web of inter-connected stressors was induced by P2.2-PDT, which underlies cell death but also elicits protective mechanisms that may delay tumor cell death or even defend these cells against the deleterious effects of PDT.

**Keywords:** photodynamic therapy; colon carcinoma cells; stress responses

## **1. Introduction**

Photodynamic therapy (PDT) has lately emerged as a promising targeted therapy for solid tumors. As reviewed by Agostinis et al. [1], PDT consists of the administration of a biocompatible photosensitizer (PS) that is inactive in "dark" conditions and is more or less selectively accumulated by tumor cells. Local activation of PS using visible light in a PS-specific wavelength spectrum triggers a strong singlet oxygen burst that induces locally important oxidative damages and therefore destabilizes the tumor niche. Because PSs should not exhibit "dark" cytotoxicity, and the activating light is highly targeted to the diseased tissue, PDT has minimal effects on healthy tissues. PDT has fewer side-effects than conventional anticancer therapies like radio- and chemotherapy and is sufficiently safe for repeated therapy sessions. Moreover, PDT does not induce immune suppression and can even boost the antitumor immune response which will complement the therapeutic action [2]. Major technical issues raised by PDT, recently summarized [3], are related to PS properties which, besides having to be non-cytotoxic in "dark" conditions, should have convenient amphiphilic properties to load cells and minimal aggregation in physiologic fluids and be activated with light of sufficiently high wavelength to penetrate tissues. Additionally, technical limitations in PDT are mostly related to the devices through which light can be guided to deep-seated tumors for activating PDT locally. An interesting issue

**Citation:** Dobre, M.; Boscencu, R.; Neagoe, I.V.; Surcel, M.; Milanesi, E.; Manda, G. Insight into the Web of Stress Responses Triggered at Gene Expression Level by Porphyrin-PDT in HT29 Human Colon Carcinoma Cells. *Pharmaceutics* **2021**, *13*, 1032. https://doi.org/10.3390/ pharmaceutics13071032

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 27 May 2021 Accepted: 4 July 2021 Published: 7 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for further improving PDT is the characterization of the complex mechanisms underlying its therapeutic effects and the potential therapy-induced resistance [4], both aspects being highly dependent on the tumor type, the structure and properties of the PS and on the PDT settings in terms of light wavelength and fluence. Particular aspects characterizing PDT-induced responses in tumor cells have been already documented, connecting the PDT-induced oxidative stress, cell death, survival mechanisms and danger-associated molecular patterns (DAMPs) production and immunity [5–7], but a detailed picture of the intra-network connections is still missing. interesting issue for further improving PDT is the characterization of the complex mechanisms underlying its therapeutic effects and the potential therapy-induced resistance [4], both aspects being highly dependent on the tumor type, the structure and properties of the PS and on the PDT settings in terms of light wavelength and fluence. Particular aspects characterizing PDT-induced responses in tumor cells have been already documented, connecting the PDT-induced oxidative stress, cell death, survival mechanisms and danger-associated molecular patterns (DAMPs) production and immunity [5–7], but a detailed picture of the intra-network connections is still missing.

tissues. Additionally, technical limitations in PDT are mostly related to the devices through which light can be guided to deep-seated tumors for activating PDT locally. An

*Pharmaceutics* **2021**, *13*, x 2 of 25

For characterizing at the molecular level the response of tumor cells to PDT, we investigated by qRT-PCR the gene expression pattern in HT29 tumor cells subjected to PDT, addressing 84 genes that are critically involved in cellular responses to stress, aiming to describe the web of stressors elicited by a porphyrin-PDT regimen. As a porphyrinic photosensitizer we used an unsymmetrical meso-tetrasubstituted phenyl porphyrin (4-acetoxy-3-methoxyphenyl)porphyrin, P2.2) that we have previously designed and characterized (Figure 1). As described in [8], P2.2 has remarkable amphyphylic properties, has good solubility in biologically friendly media and long-term stability in polyethylen glycol 200 (PEG 200) and is able to generate PDT-acceptable singlet oxygen yields when activated with light in the spectral domain 600–650 nm. P2.2 was shown to accumulate well into tumor cells following a dose-dependent linear relationship, was significantly less uptaken by blood cells, exhibited good fluorescence for imagistic detection and did not exert in "dark" conditions important in vitro cytotoxicity on cells specific for the tumor niche (tumor colon carcinoma cells and tumorigenic fibroblasts) or on blood cells (peripheral blood mononuclear cells). For characterizing at the molecular level the response of tumor cells to PDT, we investigated by qRT-PCR the gene expression pattern in HT29 tumor cells subjected to PDT, addressing 84 genes that are critically involved in cellular responses to stress, aiming to describe the web of stressors elicited by a porphyrin-PDT regimen. As a porphyrinic photosensitizer we used an unsymmetrical meso-tetrasubstituted phenyl porphyrin (4 acetoxy-3-methoxyphenyl)porphyrin, P2.2) that we have previously designed and characterized (Figure 1). As described in [8], P2.2 has remarkable amphyphylic properties, has good solubility in biologically friendly media and long-term stability in polyethylen glycol 200 (PEG 200) and is able to generate PDT-acceptable singlet oxygen yields when activated with light in the spectral domain 600–650 nm. P2.2 was shown to accumulate well into tumor cells following a dose-dependent linearrelationship, was significantly less uptaken by blood cells, exhibited good fluorescence for imagistic detection and did not exert in "dark" conditions important in vitro cytotoxicity on cells specific for the tumor niche (tumor colon carcinoma cells and tumorigenic fibroblasts) or on blood cells (peripheral blood mononuclear cells).

**Figure 1.** Chemical structure of 5-(4-hydroxy-3-methoxyphenyl)-10, 15, 20-tris-(4-acetoxy-3 methoxyphenyl) porphyrin (P2.2). **Figure 1.** Chemical structure of 5-(4-hydroxy-3-methoxyphenyl)-10, 15, 20-tris-(4-acetoxy-3 methoxyphenyl) porphyrin (P2.2).

Results showed that the new P2.2 photosensitizer produced in vitro PDT in a concentration- and fluence-dependent manner. The gene expression study highlighted that P2.2-PDT generated a particular molecular fingerprint of oxidative stress, hypoxia signaling, DNA damage, endoplasmic reticulum (ER) stress and unfolded protein response (UPR), along with inflammation. The web of inter-connected stress responses elicited by P2.2-PDT in HT29 tumor cells might sustain or limit the therapeutic effect of PDT. The identified genes could represent valuable molecular targets for co-therapies Results showed that the new P2.2 photosensitizer produced in vitro PDT in a concentrationand fluence-dependent manner. The gene expression study highlighted that P2.2-PDT generated a particular molecular fingerprint of oxidative stress, hypoxia signaling, DNA damage, endoplasmic reticulum (ER) stress and unfolded protein response (UPR), along with inflammation. The web of inter-connected stress responses elicited by P2.2-PDT in HT29 tumor cells might sustain or limit the therapeutic effect of PDT. The identified genes could represent valuable molecular targets for co-therapies aimed at reinforcing PDT.

#### aimed at reinforcing PDT. **2. Results**

In vitro PDT was performed on the human colon carcinoma cell line HT29 using the new porphyrinic photosensitizer P2.2 (Figure 1) and activating laser light of 635 nm.

The fluorescent P2.2 photosensitizer was incorporated into HT29 tumor cells following a linear concentration-dependent relationship in the range (2.5–40) µM (Figure 2a). The difference between fluorescence values in samples loaded with consecutive P2.2 concentra-

tions was statistically significant (*p* < 0.01). In the investigated concentration range, P2.2 did not exert "dark" cytotoxicity, according to the lactate dehydrogenase (LDH) release data (not shown). Additionally, the distribution of fluorescence values within a P2.2-treated sample and the corresponding SD value of the distribution (measure of the fluorescence values spread around the mean value in a defined cellular population) showed that cells had incorporated variable amounts of P2.2 (Figure 2b). This may affect the strength of PDT, depending on the cellular P2.2 load. 2a). The difference between fluorescence values in samples loaded with consecutive P2.2 concentrations was statistically significant (*p* < 0.01). In the investigated concentration range, P2.2 did not exert "dark" cytotoxicity, according to the lactate dehydrogenase (LDH) release data (not shown). Additionally, the distribution of fluorescence values within a P2.2-treated sample and the corresponding SD value of the distribution (measure of the fluorescence values spread around the mean value in a defined cellular population) showed that cells had incorporated variable amounts of P2.2 (Figure 2b). This may affect the strength of PDT, depending on the cellular P2.2 load.

In vitro PDT was performed on the human colon carcinoma cell line HT29 using the

The fluorescent P2.2 photosensitizer was incorporated into HT29 tumor cells following a linear concentration-dependent relationship in the range (2.5–40) µM (Figure

new porphyrinic photosensitizer P2.2 (Figure 1) and activating laser light of 635 nm.

*Pharmaceutics* **2021**, *13*, x 3 of 25

**2. Results**

(**a**) Mean cellular fluorescence. (**b**) Fluorescence distribution in P2.2-loaded cells.

**Figure 2.** The uptake of the fluorescent P2.2 photosensitizer by HT29 tumor cells. Cells were incubated for 24 h with 2.5– 40 µM P2.2. Intracellular P2.2 was evaluated by flow cytometry, based on the red P2.2 fluorescence. Fluorescence data, expressed in arbitrary units, and their median values in each sample were obtained with the BD FACSDiva software (Becton Dickinson). (**a**) Median cellular fluorescence. Data are presented as the mean value of the median fluorescence of each sample of the triplicate (mean ± SEM). Comparison between samples treated with consecutive P2.2 concentrations was performed using Student's *t*-test considering unequal variances: \*\* *p* < 0.01, \*\*\* *p* < 0.001. (**b**) Fluorescence distribution in samples treated with various P2.2 concentrations. The represented SD values, obtained with the BD FACSDiva 6.1 software, are a measure of the fluorescence values spread around the mean value in a defined cellular population. **Figure 2.** The uptake of the fluorescent P2.2 photosensitizer by HT29 tumor cells. Cells were incubated for 24 h with 2.5–40 µM P2.2. Intracellular P2.2 was evaluated by flow cytometry, based on the red P2.2 fluorescence. Fluorescence data, expressed in arbitrary units, and their median values in each sample were obtained with the BD FACSDiva software (Becton Dickinson). (**a**) Median cellular fluorescence. Data are presented as the mean value of the median fluorescence of each sample of the triplicate (mean ± SEM). Comparison between samples treated with consecutive P2.2 concentrations was performed using Student's *t*-test considering unequal variances: \*\* *p* < 0.01, \*\*\* *p* < 0.001. (**b**) Fluorescence distribution in samples treated with various P2.2 concentrations. The represented SD values, obtained with the BD FACSDiva 6.1 software, are a measure of the fluorescence values spread around the mean value in a defined cellular population.

> The viability of tumor cells following exposure to P2.2-PDT was investigated as a preparative step for the gene expression analysis in PDT-treated samples as compared to non-treated controls. The viability of tumor cells following exposure to P2.2-PDT was investigated as a preparative step for the gene expression analysis in PDT-treated samples as compared to non-treated controls.

#### *2.1. PDT-Induced Changes of Cell Viability 2.1. PDT-Induced Changes of Cell Viability*

#### 2.1.1. PDT-Induced Decrease of Viable Tumor Cells 2.1.1. PDT-Induced Decrease of Viable Tumor Cells

The dependence of the in vitro PDT outcome (viability of HT29 tumor cells) on P2.2 concentration (2.5–40 µM) was investigated by MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]) reduction at 24 h post-PDT (10 J/cm<sup>2</sup> , 50 mW/cm<sup>2</sup> ). As shown in Figure 3, MTS reduction had a linear decrease in the P2.2 concentration range 2.5–10 µM. In the case of higher P2.2 concentrations (20–40 µM), MTS reduction almost dropped to zero, indicating that only few metabolically active cells remained at 24 h after PDT with high P2.2 concentrations. The dependence of the in vitro PDT outcome (viability of HT29 tumor cells) on P2.2 concentration (2.5–40 µM) was investigated by MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]) reduction at 24 h post-PDT (10 J/cm<sup>2</sup> , 50 mW/cm<sup>2</sup> ). As shown in Figure 3, MTS reduction had a linear decrease in the P2.2 concentration range 2.5–10µM. In the case of higher P2.2 concentrations (20–40 µM), MTS reduction almost dropped to zero, indicating that only few metabolically active cells remained at 24 h after PDT with high P2.2 concentrations.

An IC50 value of 8.07 ± 1.69 µM (mean ± SD) was computed using the Quest Graph™ IC50 Calculator (https://www.aatbio.com/tools/ic50-calculator, accessed on 27 June 2021). A concentration of 10 µM P2.2 was further chosen for performing PDT at various light fluences (5–25 J/cm<sup>2</sup> ), for further differential investigation of both live and dead cells regarding gene expression.

The number of metabolically active tumor cells in culture dramatically decreased at 24 h post-PDT in comparison with non-treated control cells, as shown by the significant fluence-dependent decrease of MTS reduction (PDT effect < 0.3) at all the tested light fluences (10 J/cm<sup>2</sup> , 15 J/cm<sup>2</sup> and 25 J/cm<sup>2</sup> , delivered at 50 mW/cm<sup>2</sup> ) (Figure 4). A subunit PDT effect (≤0.5) following a fluence-dependent curve was also registered at 72 h (Figure 4). A stronger PDT regimen (25 J/cm<sup>2</sup> ) rendered almost all tumor cells metabolically inactive

in the first 24 h after PDT and this effect was maintained until 72 h. Meanwhile, PDT exerted a lower effect on MTS reduction at 72 h than at 24 h post-PDT (*p* < 0.001) at the fluences of 10 J/cm<sup>2</sup> and 15 J/cm<sup>2</sup> , indicating that the PDT effect was attenuated over time. *Pharmaceutics* **2021**, *13*, x 4 of 25

*Pharmaceutics* **2021**, *13*, x 4 of 25

**Figure 3.** The dependence on P2.2 concentration of MTS reduction by HT29 tumor cells at 24 h post-PDT. Results are presented as PDT effect (mean ± SEM) in triplicate samples. PDT effect was calculated as OD in PDT-treated samples divided by the mean value of OD in control samples. Comparison between samples was performed using Student's *t*-test: paired two sample for means: \* *p* < 0.05, \*\* *p* < 0.01. **Figure 3.** The dependence on P2.2 concentration of MTS reduction by HT29 tumor cells at 24 h post-PDT. Results are presented as PDT effect (mean ± SEM) in triplicate samples. PDT effect was calculated as OD in PDT-treated samples divided by the mean value of OD in control samples. Comparison between samples was performed using Student's *t*-test: paired two sample for means: \* *p* < 0.05, \*\* *p* < 0.01. PDT effect (≤0.5) following a fluence-dependent curve was also registered at 72 h (Figure 4). A stronger PDT regimen (25 J/cm<sup>2</sup> ) rendered almost all tumor cells metabolically inactive in the first 24 h after PDT and this effect was maintained until 72 h. Meanwhile, PDT exerted a lower effect on MTS reduction at 72 h than at 24 h post-PDT (*p* < 0.001) at the fluences of 10 J/cm<sup>2</sup> and 15 J/cm<sup>2</sup> , indicating that the PDT effect was attenuated over time.

**Figure 4.** The dependence of MTS reduction by PDT-treated HT29 tumor cells on the fluence of the activation light delivered at a fluence rate of 50 mW/cm<sup>2</sup> . Results are presented as PDT effect (mean ± SEM) in triplicate samples. PDT effect was calculated as OD in PDT-treated samples divided by the mean value of OD in control samples. Comparison between samples was performed using Student's *t*-test considering unequal variances: comparison between PDT regimens at a specific time-point: \* *p* < 0.05, \*\*\* *p* < 0.001; comparison between samples at 24 h and 72 h: ### *p* < 0.001.

This observation was sustained by microscopic investigations that showed the presence of adhered cells at 24 h and 72 h post-PDT, most probably viable cells, even in the case of the stronger PDT regimen of 25 J/cm<sup>2</sup> (Figure 5A,B). While the number of adhered cells decreased over time, a few cells that appeared to be less affected by PDT continued to slowly proliferate and formed cell islets at 120 h post-PDT (Figure 5C).

bar).

**Figure 4.** The dependence of MTS reduction by PDT-treated HT29 tumor cells on the fluence of the

± SEM) in triplicate samples. PDT effect was calculated as OD in PDT-treated samples divided by the mean value of OD in control samples. Comparison between samples was performed using Student's *t*-test considering unequal variances: comparison between PDT regimens at a specific time-point: \* *p* < 0.05, \*\*\* *p* < 0.001; comparison between samples at 24 h and 72 h: ### *p* < 0.001.

This observation was sustained by microscopic investigations that showed the presence of adhered cells at 24 h and 72 h post-PDT, most probably viable cells, even in the case of the stronger PDT regimen of 25 J/cm<sup>2</sup> (Figure 5A,B). While the number of adhered cells decreased over time, a few cells that appeared to be less affected by PDT continued to slowly proliferate and formed cell islets at 120 h post-PDT (Figure 5C).

. Results are presented as PDT effect (mean

activation light delivered at a fluence rate of 50 mW/cm<sup>2</sup>

**Figure 5.** Images of HT29 tumor cells at various time points after PDT performed with various light fluences (10–25 J/cm<sup>2</sup> ), which were delivered with the fluence rate of 50 mW/cm<sup>2</sup> . Cells were visualized by bright-field microscopy at 24 h (**A**), 72 h (**B**) and 120 h (**C**) post-PDT (100 µm scale **Figure 5.** Images of HT29 tumor cells at various time points after PDT performed with various light fluences (10–25 J/cm<sup>2</sup> ), which were delivered with the fluence rate of 50 mW/cm<sup>2</sup> . Cells were visualized by bright-field microscopy at 24 h (**A**), 72 h (**B**) and 120 h (**C**) post-PDT (100 µm scale bar).

The observation that some tumor cells might have escaped from the deleterious action of PDT was also evidenced when cell proliferation was assessed at 72 h post-PDT by flow cytometry with CFDA-SE. Most of the cells exposed to 10 J/cm<sup>2</sup> or 15 J/cm<sup>2</sup> PDT were found at 72 h in lower-order daughter generations as compared to control cells, indicating that their overall proliferation was slower (Figure 6). Concurrently, a low cell percentage was found in higher-order daughter generations at 72 h post-PDT, suggesting that they were not harmed by PDT and actively proliferated at higher rates than control cells. These few cells seem to have gained a proliferation advantage. The observation that some tumor cells might have escaped from the deleterious action of PDT was also evidenced when cell proliferation was assessed at 72 h post-PDT by flow cytometry with CFDA-SE. Most of the cells exposed to 10 J/cm<sup>2</sup> or 15 J/cm<sup>2</sup> PDT were found at 72 h in lower-order daughter generations as compared to control cells, indicating that their overall proliferation was slower (Figure 6). Concurrently, a low cell percentage was found in higher-order daughter generations at 72 h post-PDT, suggesting that they were not harmed by PDT and actively proliferated at higher rates than control cells. These few cells seem to have gained a proliferation advantage. *Pharmaceutics* **2021**, *13*, x 6 of 25

**Figure 6.** Demonstrative data on the proliferation of HT29 tumor cells subjected to PDT (fluences: 10 J/cm<sup>2</sup> and 15 J/cm<sup>2</sup> ; fluence rate: 50 mW/cm<sup>2</sup> ) vs. non-treated control cells at 72 h post-PDT. Results are presented as a percentage of cells in various consecutive daughter generations in comparison with the parent population distanced by X previous generations. Data were obtained by flow cytometry with CFDA-SE and were processed using the ModFit LT software. **Figure 6.** Demonstrative data on the proliferation of HT29 tumor cells subjected to PDT (fluences: 10 J/cm<sup>2</sup> and 15 J/cm<sup>2</sup> ; fluence rate: 50 mW/cm<sup>2</sup> ) vs. non-treated control cells at 72 h post-PDT. Results are presented as a percentage of cells in various consecutive daughter generations in comparison with the parent population distanced by X previous generations. Data were obtained by flow cytometry with CFDA-SE and were processed using the ModFit LT software.

LDH release, indicating alteration of the plasma membrane (Figure 7). The PDT-induced increase of LDH release was negatively correlated with the decrease of MTS reduction (Pearson r = 0.986, *p* = 0.106), following a linear regression equation (y = −0.09X + 0.64). Results indicated that, at least partly, the decrease of metabolically active tumor cells in PDT-treated samples was due to membrane alterations generally occurring in necrotic

The PDT effect on LDH release was lower at 72 h compared to 24 h for all the investigated light fluences and became fluence-independent in time (Figure 7). Results suggest that PDT induced persistent plasma membrane alterations in the time frame of

**\*\*\***

**Figure 7.** The dependence of LDH release by PDT-treated HT29 tumor cells on the fluence of the

**)**

post-PDT. Results are presented as PDT effect (mean ± SEM) in triplicate samples. PDT effect was calculated as OD in samples subjected to PDT divided by the mean value of OD in control samples.

. Analysis was performed at 24 h and 72 h

**\***

**\*\***

**24 h 72 h**

0 5 10 15 20 25 30

**Fluence (J/cm<sup>2</sup>**

**# ###** y = 0.1303x + 3.3461

and necroptototic cells [9,10], which allow significant LDH release.

2.1.2. PDT-Induced Alteration of Membrane Integrity

24–72 h post-PDT, especially in the first 24 h.

R² = 0.9979

**PDT effect**

activating light delivered at a fluence rate of 50 mW/cm<sup>2</sup>

#### 2.1.2. PDT-Induced Alteration of Membrane Integrity The significant decrease of the number of metabolically active tumor cells registered

2.1.2. PDT-Induced Alteration of Membrane Integrity

; fluence rate: 50 mW/cm<sup>2</sup>

cytometry with CFDA-SE and were processed using the ModFit LT software.

10 J/cm<sup>2</sup> and 15 J/cm<sup>2</sup>

*Pharmaceutics* **2021**, *13*, x 6 of 25

The significant decrease of the number of metabolically active tumor cells registered at 24 h post-PDT (Figure 4) was associated with a fluence-dependent linear increase of LDH release, indicating alteration of the plasma membrane (Figure 7). The PDT-induced increase of LDH release was negatively correlated with the decrease of MTS reduction (Pearson r = 0.986, *p* = 0.106), following a linear regression equation (y = −0.09X + 0.64). Results indicated that, at least partly, the decrease of metabolically active tumor cells in PDT-treated samples was due to membrane alterations generally occurring in necrotic and necroptototic cells [9,10], which allow significant LDH release. at 24 h post-PDT (Figure 4) was associated with a fluence-dependent linear increase of LDH release, indicating alteration of the plasma membrane (Figure 7). The PDT-induced increase of LDH release was negatively correlated with the decrease of MTS reduction (Pearson r = 0.986, *p* = 0.106), following a linear regression equation (y = −0.09X + 0.64). Results indicated that, at least partly, the decrease of metabolically active tumor cells in PDT-treated samples was due to membrane alterations generally occurring in necrotic and necroptototic cells [9,10], which allow significant LDH release.

**Figure 6.** Demonstrative data on the proliferation of HT29 tumor cells subjected to PDT (fluences:

are presented as a percentage of cells in various consecutive daughter generations in comparison with the parent population distanced by X previous generations. Data were obtained by flow

) vs. non-treated control cells at 72 h post-PDT. Results

The PDT effect on LDH release was lower at 72 h compared to 24 h for all the investigated light fluences and became fluence-independent in time (Figure 7). Results suggest that PDT induced persistent plasma membrane alterations in the time frame of 24–72 h post-PDT, especially in the first 24 h. The PDT effect on LDH release was lower at 72 h compared to 24 h for all the investigated light fluences and became fluence-independent in time (Figure 7). Results suggest that PDT induced persistent plasma membrane alterations in the time frame of 24–72 h post-PDT, especially in the first 24 h.

**Figure 7.** The dependence of LDH release by PDT-treated HT29 tumor cells on the fluence of the activating light delivered at a fluence rate of 50 mW/cm<sup>2</sup> . Analysis was performed at 24 h and 72 h post-PDT. Results are presented as PDT effect (mean ± SEM) in triplicate samples. PDT effect was calculated as OD in samples subjected to PDT divided by the mean value of OD in control samples. **Figure 7.** The dependence of LDH release by PDT-treated HT29 tumor cells on the fluence of the activating light delivered at a fluence rate of 50 mW/cm<sup>2</sup> . Analysis was performed at 24 h and 72 h post-PDT. Results are presented as PDT effect (mean ± SEM) in triplicate samples. PDT effect was calculated as OD in samples subjected to PDT divided by the mean value of OD in control samples. Comparison between samples was performed using Student's *t*-test considering unequal variances: comparison between PDT regimens at a specific time-point: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001; comparison between samples at 24 h and 72 h: # *p* < 0.05, ### *p* < 0.001.

At 24 h post-PDT, when the percentage of metabolically active cells decreased drastically below 50% in a fluence-dependent manner (Figure 4), many cells were found detached and rounded up, as seen in Figure 5A. For clarifying cell death, apoptosis and necrosis were investigated by flow cytometry with annexin V-propidium iodide. An important increase of the percentage of apoptotic cells, more specifically late apoptotic cells, accompanied by a smaller increase of necrotic cells was registered in PDT-treated vs. untreated cultures at 24 h post-PDT (Figure 8).

**Figure 8.** The percentage of apoptotic and necrotic HT29 cells in samples exposed to PDT (10 J/cm<sup>2</sup> and 25 J/cm<sup>2</sup> , 50 mW/cm<sup>2</sup> ) and in non-treated controls. Apoptosis and necrosis were assessed by flow cytometry using FITC-annexin V and propidium iodide at 24 h post-PDT. Results are presented as percentage (mean ± SEM) of cells in triplicate samples, regarding early, late and total apoptosis, as well as necrosis. Comparison between treated and non-treated samples was performed using Student's *t*-test considering unequal variances. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. **Figure 8.** The percentage of apoptotic and necrotic HT29 cells in samples exposed to PDT (10 J/cm<sup>2</sup> and 25 J/cm<sup>2</sup> , 50 mW/cm<sup>2</sup> ) and in non-treated controls. Apoptosis and necrosis were assessed by flow cytometry using FITC-annexin V and propidium iodide at 24 h post-PDT. Results are presented as percentage (mean ± SEM) of cells in triplicate samples, regarding early, late and total apoptosis, as well as necrosis. Comparison between treated and non-treated samples was performed using Student's *t*-test considering unequal variances. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

Comparison between samples was performed using Student's *t*-test considering unequal variances: comparison between PDT regimens at a specific time-point: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001;

At 24 h post-PDT, when the percentage of metabolically active cells decreased drastically below 50% in a fluence-dependent manner (Figure 4), many cells were found detached and rounded up, as seen in Figure 5A. For clarifying cell death, apoptosis and necrosis were investigated by flow cytometry with annexin V-propidium iodide. An important increase of the percentage of apoptotic cells, more specifically late apoptotic cells, accompanied by a smaller increase of necrotic cells was registered in PDT-treated

comparison between samples at 24 h and 72 h: # *p* < 0.05, ### *p* < 0.001.

vs. untreated cultures at 24 h post-PDT (Figure 8).

We further investigated whether P2.2-PDT is also efficient in milder PDT conditions with lower light fluences (5–10 J/cm<sup>2</sup> ) delivered at a lower fluence rate of 10 mW/cm<sup>2</sup> . A linear fluence-dependent increase of PDT effect on LDH release was registered at 24 h post-PDT, with significant differences between untreated controls and samples exposed to the higher investigated light fluences of 7.5 J/cm<sup>2</sup> and 10 J/cm<sup>2</sup> (Figure 9). Results emphasized once again that important damages at the level of plasma membrane, resulting in LDH release, were induced by PDT within 24 h post-treatment. Moreover, it appears that LDH release is not influenced by the fluence rate, as demonstrated by similar PDT effects on LDH release at 10 mW/cm<sup>2</sup> and 50 mW/cm<sup>2</sup> for a light fluence of 10 J/cm<sup>2</sup> (PDT effect at 10 mW/cm<sup>2</sup> was 4.2 ± 0.7 and at 50 mW/cm<sup>2</sup> it was 4.6 ± 0.6). We further investigated whether P2.2-PDT is also efficient in milder PDT conditions with lower light fluences (5–10 J/cm<sup>2</sup> ) delivered at a lower fluence rate of 10 mW/cm<sup>2</sup> . A linear fluence-dependent increase of PDT effect on LDH release was registered at 24 h post-PDT, with significant differences between untreated controls and samples exposed to the higher investigated light fluences of 7.5 J/cm<sup>2</sup> and 10 J/cm<sup>2</sup> (Figure 9). Results emphasized once again that important damages at the level of plasma membrane, resulting in LDH release, were induced by PDT within 24 h post-treatment. Moreover, it appears that LDH release is not influenced by the fluence rate, as demonstrated by similar PDT effects on LDH release at 10 mW/cm<sup>2</sup> and 50 mW/cm<sup>2</sup> for a light fluence of 10 J/cm<sup>2</sup> (PDT effect at 10 mW/cm<sup>2</sup> was 4.2 <sup>±</sup> 0.7 and at 50 mW/cm<sup>2</sup> it was 4.6 ± 0.6). *Pharmaceutics* **2021**, *13*, x 8 of 25

**Figure 9.** The dependence of LDH release by HT29 tumor cells exposed to milder PDT conditions on the fluence of the activating light (5–7.5–10 J/cm<sup>2</sup> ) that was delivered at a lower fluence rate of 10 mW/cm<sup>2</sup> . Results are presented as PDT effect (mean ± SEM) in triplicate samples. PDT effect was calculated as OD in samples subjected to PDT divided by the mean OD value in controls. Comparison between samples was performed using Student's *t*-test considering unequal variances: \* *p* < 0.05. **Figure 9.** The dependence of LDH release by HT29 tumor cells exposed to milder PDT conditions on the fluence of the activating light (5–7.5–10 J/cm<sup>2</sup> ) that was delivered at a lower fluence rate of 10 mW/cm<sup>2</sup> . Results are presented as PDT effect (mean ± SEM) in triplicate samples. PDT effect was calculated as OD in samples subjected to PDT divided by the mean OD value in controls. Comparison between samples was performed using Student's *t*-test considering unequal variances: \* *p* < 0.05.

Altogether, results showed that, at least from the point of view of LDH release, which provides information on plasma membrane integrity, P2.2-PDT was efficacious on tumor Altogether, results showed that, at least from the point of view of LDH release, which provides information on plasma membrane integrity, P2.2-PDT was efficacious on tumor

We have shown above that in approximately 24 h, more than 50% of HT29 tumor cells were affected by P2.2-PDT, despite the fact that an acute oxidative burst is known to be generated instantaneously in the course of PDT [11]. Possibly, tumor cells develop rescue mechanisms that delay cell death and may even protect some cells against the

Therefore, we investigated by qRT-PCR the expression profile of 84 genes (Table 1) that are critically involved in cellular responses to stressors such as oxidative stress, hypoxia, osmotic stress, genotoxic stress, unfolded protein response and inflammation, as well as in cell death by apoptosis, necrosis and autophagy. Briefly, cells exposed in vitro

was evaluated separately in adhered and detached cells as potentially living and

**Table 1.** The list of analyzed genes, contained in the RT² Profiler™ PCR Array Human Stress and

ADM, ARNT, BNIP3L, CA9, EPO, HMOX1, LDHA, MMP9, SERPINE1 (PAI-1), SLC2A1, VEGFA

CASP1 (ICE), FAS, MCL1, TNFRSF10A (TRAIL-R), TNFRSF10B (DR5), TNFRSF1A (TNFR1).

FAS, GRB2, PARP1 (ADPRT1), PVR, RIPK1, TNFRSF10A (TRAIL-R), TNFRSF1A (TNFR1),

FTH1, GCLC, GCLM, GSR, GSTP1, HMOX1, NQO1, PRDX1, SQSTM1, TXN, TXNRD1

AKR1B1, AQP1, AQP2, AQP4, CFTR, EDN1, HSPA4L (OSP94), NFAT5, SLC5A3

) were harvested 24 h post-PDT. The gene expression pattern

, with the highest damaging effects

HT29 cells in the light fluence domain of 7.5–25 J/cm<sup>2</sup>

.

*2.2. PDT-Induced Gene Expression Changes* 

deleterious action of PDT (Figure 5C).

apoptotic/necrotic cells, respectively.

, 50 mW/cm<sup>2</sup>

Toxicity PathwayFinder (Qiagen, PAHS-003Z).

AutophagyATG12, ATG5, ATG7, BECN1, FAS, ULK1

registered at 25 J/cm<sup>2</sup>

to PDT (10 J/cm<sup>2</sup>

**Oxidative Stress**

**Osmotic Stress**

**Cell Death** *Apoptosis:*

*Necrosis:*

TXNL4B

**DNA Damage and Repair**

**Hypoxia Signaling**

HT29 cells in the light fluence domain of 7.5–25 J/cm<sup>2</sup> , with the highest damaging effects registered at 25 J/cm<sup>2</sup> .

#### *2.2. PDT-Induced Gene Expression Changes*

We have shown above that in approximately 24 h, more than 50% of HT29 tumor cells were affected by P2.2-PDT, despite the fact that an acute oxidative burst is known to be generated instantaneously in the course of PDT [11]. Possibly, tumor cells develop rescue mechanisms that delay cell death and may even protect some cells against the deleterious action of PDT (Figure 5C).

Therefore, we investigated by qRT-PCR the expression profile of 84 genes (Table 1) that are critically involved in cellular responses to stressors such as oxidative stress, hypoxia, osmotic stress, genotoxic stress, unfolded protein response and inflammation, as well as in cell death by apoptosis, necrosis and autophagy. Briefly, cells exposed in vitro to PDT (10 J/cm<sup>2</sup> , 50 mW/cm<sup>2</sup> ) were harvested 24 h post-PDT. The gene expression pattern was evaluated separately in adhered and detached cells as potentially living and apoptotic/necrotic cells, respectively.

**Table 1.** The list of analyzed genes, contained in the RT<sup>2</sup> Profiler™ PCR Array Human Stress and Toxicity PathwayFinder (Qiagen, PAHS-003Z).


According to the data presented in Table 2, 43 genes were found differentially expressed in P2.2-treated cells compared to controls (1.5 < FC < −1.5, *p* < 0.05). Among these genes, 38 were upregulated and 5 were downregulated either in adhered or detached cells. Selected genes were classified according to their known association with various types of stress (oxidative stress, hypoxia, genotoxic and proteotoxic stress, along with inflammation), some of them being part of several signaling networks.

**Table 2.** Genes with statistically significant expression changes (1.5 < FR < −1.5, *p* < 0.05) in PDTtreated cells (10 J/cm<sup>2</sup> , 50 mW/cm<sup>2</sup> ), either adhered or detached, as compared to untreated controls. Gene expression levels were assessed at 24 h post-PDT. Genes were classified according to the type of stress in which they are involved. Some genes are common to several types of stress. ns = not significant.


#### **b. Hypoxia signaling**

**PDT-treated vs. non-treated HT29 tumor cells at 24 h post-PDT**


**c. Cell death**

**PDT-treated vs. non-treated HT29 tumor cells at 24 h post-PDT**


**Table 2.** *Cont.*



**f. Inflammation**


*BID* 1.75 ns 3.96 <0.05 *HSPA4 (HSP70)* 2.96 ns 2.95 <0.05 *HSPA5 (GRP78)* −2.27 <0.01 −2.42 <0.001 *DNAJC3* −1.59 <0.05 −4.07 <0.001 *HSP90B1* −1.77 ns −3.59 <0.001 *CALR* −1.89 <0.001 ns ns

#### 2.2.1. Oxidative Stress

A strong molecular fingerprint of oxidative stress was evidenced in PDT-treated samples by the upregulation of 11 redox genes involved in antioxidant defense (Table 2). The activation of antioxidant mechanisms demonstrated on the one hand that cells were indeed subjected to an oxidative challenge during PDT, as expected considering the PDTgenerated burst of singlet oxygen [3,12]. On the other hand, gene expression data pointed out the upregulation of several protective redox genes that aim to counteract or delay cell death induced by the oxidative challenge generated by PDT. The identified antioxidant genes are involved in iron metabolism (*HMOX1*, *FTH1*) [13], in glutathione (*GCLC*, *GCLM*, *GSR*, *GSTP1*) [14] or thioredoxin (*TXN*, *TXNRD1*) [15] metabolism, as well as in other cytoprotective pathways (*PRDX1* [16], *NQO1* [17,18] and *SQSTM1* [19,20]). Some of these upregulated genes were common to adhered and detached cells, while other genes were preferentially upregulated either in detached or adhered cells (Figure 10a). Thus, *PRDX1* (*p* < 0.001), *NQO1* (*p* < 0.05), *GSTP1* (*p* < 0.05) and *TXN* (*p* < 0.05) were distinctively expressed in detached cells, while *SQSTM1* (*p* < 0.05) had a higher transcript level in detached cells as compared to adhered cells. Alongside this, *TXNRD1* (*p* < 0.05) was exclusively overexpressed in adhered cells. Altogether, results indicated that a robust antioxidant response is generated by PDT, which triggers antioxidant defense mechanisms that appear to be insufficient to counteract the deleterious action of PDT, resulting in the significant decrease of viable tumor cells (Figures 4, 7 and 8). Surprisingly, the protective antioxidant response was stronger in detached cells than in adhered cells (Table 2). This observation might be explained by a higher photosensitizer load in some cells (Figure 3), that possibly led to a stronger singlet oxygen burst triggered by PDT and, consequently, to robust activation of antioxidant mechanisms. *Pharmaceutics* **2021**, *13*, x 11 of 25 upregulated genes were common to adhered and detached cells, while other genes were preferentially upregulated either in detached or adhered cells (Figure 10a). Thus, *PRDX1* (*p* < 0.001), *NQO1* (*p* < 0.05), *GSTP1* (*p* < 0.05) and *TXN* (*p* < 0.05) were distinctively expressed in detached cells, while *SQSTM1* (*p* < 0.05) had a higher transcript level in detached cells as compared to adhered cells. Alongside this, *TXNRD1* (*p* < 0.05) was exclusively overexpressed in adhered cells. Altogether, results indicated that a robust antioxidant response is generated by PDT, which triggers antioxidant defense mechanisms that appear to be insufficient to counteract the deleterious action of PDT, resulting in the significant decrease of viable tumor cells (Figures 4, 7 and 8). Surprisingly, the protective antioxidant response was stronger in detached cells than in adhered cells (Table 2). This observation might be explained by a higher photosensitizer load in some cells (Figure 3), that possibly led to a stronger singlet oxygen burst triggered by PDT and, consequently, to robust activation of antioxidant mechanisms.

**Figure 10.** *Cont*.

was performed with a paired samples *t*-test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001).

cells that were exposed to P2.2-PDT (10 J/cm<sup>2</sup>

**Figure 10.** Common and distinctive gene expression patterns in adhered and attached HT29 tumor

with modified expression in PDT-treated cells as compared to untreated cells (1.5 < FR < −1.5, *p* < 0.05) are represented. These genes are involved in (**a**) oxidative stress, (**b**) Hypoxia signaling, (**c**) cell death, (**d**) DNA damage, (**e**) unfolded protein response and (**f**) inflammation. All the reported genes were upregulated, excepting those marked with downwards arrows. Genes placed at the borders of the two circles are expressed both in adhered and detached cells, but have higher expression in one of the two cell types. This comparison between gene expression levels in adhered and detached cells

, 50 mW/cm<sup>2</sup>

) at 24 h post-PDT. Some selected genes

(**a**) (**b**)

upregulated genes were common to adhered and detached cells, while other genes were preferentially upregulated either in detached or adhered cells (Figure 10a). Thus, *PRDX1* (*p* < 0.001), *NQO1* (*p* < 0.05), *GSTP1* (*p* < 0.05) and *TXN* (*p* < 0.05) were distinctively expressed in detached cells, while *SQSTM1* (*p* < 0.05) had a higher transcript level in detached cells as compared to adhered cells. Alongside this, *TXNRD1* (*p* < 0.05) was exclusively overexpressed in adhered cells. Altogether, results indicated that a robust antioxidant response is generated by PDT, which triggers antioxidant defense mechanisms that appear to be insufficient to counteract the deleterious action of PDT, resulting in the significant decrease of viable tumor cells (Figures 4, 7 and 8). Surprisingly, the protective antioxidant response was stronger in detached cells than in adhered cells (Table 2). This observation might be explained by a higher photosensitizer load in some cells (Figure 3), that possibly led to a stronger singlet oxygen burst triggered by PDT and,

consequently, to robust activation of antioxidant mechanisms.

**Figure 10.** Common and distinctive gene expression patterns in adhered and attached HT29 tumor cells that were exposed to P2.2-PDT (10 J/cm<sup>2</sup> , 50 mW/cm<sup>2</sup> ) at 24 h post-PDT. Some selected genes with modified expression in PDT-treated cells as compared to untreated cells (1.5 < FR < −1.5, *p* < 0.05) are represented. These genes are involved in (**a**) oxidative stress, (**b**) Hypoxia signaling, (**c**) cell death, (**d**) DNA damage, (**e**) unfolded protein response and (**f**) inflammation. All the reported genes were upregulated, excepting those marked with downwards arrows. Genes placed at the borders of the two circles are expressed both in adhered and detached cells, but have higher expression in one of the two cell types. This comparison between gene expression levels in adhered and detached cells was performed with a paired samples *t*-test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). **Figure 10.** Common and distinctive gene expression patterns in adhered and attached HT29 tumor cells that were exposed to P2.2-PDT (10 J/cm<sup>2</sup> , 50 mW/cm<sup>2</sup> ) at 24 h post-PDT. Some selected genes with modified expression in PDT-treated cells as compared to untreated cells (1.5 < FR < −1.5, *p* < 0.05) are represented. These genes are involved in (**a**) oxidative stress, (**b**) Hypoxia signaling, (**c**) cell death, (**d**) DNA damage, (**e**) unfolded protein response and (**f**) inflammation. All the reported genes were upregulated, excepting those marked with downwards arrows. Genes placed at the borders of the two circles are expressed both in adhered and detached cells, but have higher expression in one of the two cell types. This comparison between gene expression levels in adhered and detached cells was performed with a paired samples *t*-test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001).

#### 2.2.2. Hypoxia Signaling

Gene expression data indicated that P2.2-PDT induced the activation of hypoxia signaling, as evidenced by the upregulation of six pathway-relevant genes (Table 2), namely *SER-PINE1* [21], *ADM* [22], *ARNT* [23], *VEGFA* [24] and *BNIP3L* [25]. Additionally, *HMOX1* [26], which is at the crossroad of oxidative stress, hypoxia and inflammation [27], was also found highly overexpressed. As shown in Figure 10b, *HMOX1*, *SERPINE1* and *VEGFA* were upregulated both in adhered and detached cells, *ARNT* was upregulated only in detached cells (*p* < 0.01), while the transcript levels of *BNIP3L* and *ADM* were increased in detached vs. adhered cells (*p* < 0.05).

#### 2.2.3. Cell Death

As previously explained, P2.2-PDT was shown to induce significant apoptotic and necrotic cell death within 24 h post-treatment (Figure 8). We also found that several cell death-related genes were upregulated in PDT-exposed cells (Table 2), complementing the functional and phenotypic data on cell death following PDT (Figures 7 and 8). The identified genes are involved in apoptosis (*TNFRSF10B* [28], *BID* [29], *BNIP3L* [25] and *BBC3* [30]), necrosis (*RIPK1* [31]) and autophagy (*ATG12* [32], *SQSTM1* [33]). As shown in Figure 10c, *TNFRSF10B* was found overexpressed both in adhered and detached cells. Meanwhile, *BID*, *ATG12*, *SQSTM1* and *BNIP3L* were preferentially expressed in detached cells (*p* < 0.05) and *RIPK1* (*p* < 0.05) in adhered cells, albeit not exclusively. This gene expression pattern at 24 h post-PDT suggests that distinctive death mechanisms might be activated in detached and adhered cells. Of note, death genes were shown to be overexpressed in adhered cells (presumably living cells), indicating that they were in fact committed to death that may occur sometimes after the first 24 h post-PDT.

In the context of cell death, we identified several genes that were found to be upregulated by P2.2-PDT, which are related to cellular responses to genotoxic or proteotoxic stress, as will be presented below.

#### 2.2.4. DNA Damage

PDT-induced DNA damage was indirectly evidenced by the upregulation of eight genes involved in DNA damage repair mechanisms, such as cell cycle arrest (*CDKN1A* [34], *GADD45A*/*G* [35], *CHEK2* [36] and *HUS1* [37], or other DNA damage responses (*DDIT3* [38], *DDB2* [39] and *XPC* [40] (Table 2). As shown in Figure 10d, the *CDKN1A* and *GADD45A*

genes involved in cell cycle arrest were found overexpressed in both adhered and detached cells. This finding suggests that the observed decrease of the number of metabolically active cells in PDT-treated samples (Figure 4) might be due not only to cell death (Figures 7 and 8, Table 2), but also to a proliferation inhibition (Figure 6). *DDIT3*, *GADD45G* and *DDB2* had higher transcript levels in detached vs. adhered cells (*p* < 0.05), probably due to increased DNA damage in detached cells, while *HUS1* was moderately overexpressed only in adhered cells (FC = 1.62, *p* < 0.05).

#### 2.2.5. Unfolded Protein Response

P2.2-PDT was shown to induce proteotoxic stress, probably due to the PDT-inflicted oxidative damage of proteins. Proof for ER stress and the consequent UPR was provided by significant expression changes of nine pathway-specific genes, out of which five genes were upregulated and four genes were downregulated (Table 2). Thus, *DDIT3*, which is induced by ER stress [41], along with *BBC3* and *BID*, which link the ER stress response to the mitochondrial apoptosis pathway [42], were concurrently found overexpressed. Some other genes encoding protective ER chaperones (*HSP90AA1* and *HSPA4*) were also found upregulated. They may target misfolded proteins for degradation and even shut down UPR when the stress subsides [43]. Meanwhile, some other chaperone-encoding genes, such as *HSP90AA1* [44], *HSPA5* [45] and *HSP90B1* [46], were found to be downregulated in PDTtreated cells, along with *CALR* [47] and *DNAJC3* [48]. Accordingly, important mechanisms in UPR might be suppressed in cells exposed to PDT, potentially leading in time to cell death. As shown in Figure 10e, while *HSP90AA1* was markedly overexpressed both in adhered and detached cells, *HSPA5* and *DNAJC3* were found concurrently downregulated in both types of cells. Meanwhile, the genes connecting ER stress and apoptosis (*DDIT3* and *BID*) were preferentially overexpressed in detached cells (*p* < 0.05), which also exhibited downregulation of the protective *HSP90B1* gene (Figure 10e). Surprisingly, *CALR* was downregulated by PDT mostly in adherent cells, thus making them more susceptible to ER stress and to its deleterious consequences. In turn, *CALR* downregulation may have beneficial effects in tumors, considering that this can induce inhibition of cell growth, invasion and cell cycle progression [49].

#### 2.2.6. Inflammation

A pro-inflammatory cytokine response was shown to be elicited by PDT in HT29 tumor cells, as seen from the significant overexpression of the pro-inflammatory *CXCL8* and *IL1B* genes in both adhered and detached cells (Table 2, Figure 10f). Surprisingly, *IL1A*, encoding for a danger signal that senses genotoxic stress and is released by cells with damaged plasma membrane [50], was found distinctively downregulated in detached cells (*p* < 0.05) as shown in Table 2 and Figure 10f. Possibly, *IL1A* transcription was inhibited after the activation of the apoptotic machinery, cells having already sensed the death-inducing signals.

#### **3. Discussion**

The study brought first experimental in vitro proof on the photosensitizing ability of the new asymmetric porphyrinic compound P2.2 [8] for efficient PDT in the human colon carcinoma HT29 cell line. Following P2.2-PDT, we showed that tumor cells massively died by necrosis and apoptosis within 24 h, especially at high activating light fluences of 25 J/cm<sup>2</sup> , as demonstrated using functional and phenotypic tests.

Gene expression data highlighted the molecular mechanisms underlying cell death and survival following a milder P2.2-PDT regimen (10 J/cm<sup>2</sup> ). Thus, the observed increased apoptosis of PDT treated-cells might be partly mediated by the Death receptor 5 (DR5), also known as TRAIL receptor 2 (TRAILR2) or tumor necrosis factor receptor superfamily member 10B (TNFRSF10B), which triggers the extrinsic apoptotic cascade [51]. DR5 has been found upregulated in various types of tumors, including colorectal carcinomas, and can be exploited for selective apoptotic killing of cancer cells through caspase 8 [52]. Recent

studies have shown that PDT extensively sensitizes refractory colon tumors to death signals delivered by long-acting TRAIL [53], possibly by increasing the levels of TRAIL receptors on tumor cells. Furthermore, the increased levels of the *BID* transcripts evidenced by us may sustain the connection of death receptor signaling to the mitochondrial apoptotic machinery [54]. The activation of the mitochondrial apoptotic pathway in PDT-treated cells is also demonstrated by the increased transcript levels of the *BBC3* gene which is under TP53 transcriptional control but can be independently upregulated also by ER stress [55]. A complementary death mechanism, which is highly relevant for tumors and for PDT [56], was evidenced by the overexpression of the *BNIP3L* gene, which is associated with TP53-dependent apoptosis under hypoxia [25].

A particular death pathway bridging apoptosis and necrosis (necroptosis or programmed necrosis) might occur in PDT, as evidenced by *RIPK1* overexpression [57,58]. It is known that necroptosis and necrosis can initiate inflammatory reactions, albeit through distinctive mechanisms, and this may defend or sustain tumor progression, depending on the context [57]. Finally, we have found that autophagy might also be activated by P2.2-PDT, as shown by *ATG12* and *SQSTM1* upregulation, indicating that damaged cells attempt to tolerate the photo-damage or are partly driven to autophagic cell death [59].

We have evidenced that PDT-triggered genotoxic stress may drive tumor cells towards death. In turn, PDT-treated cells responded by upregulating genes involved in cell cycle arrest and in other DNA damage responses (DDR) as defense mechanisms against DNA injury [60]. PDT-induced DNA damage, accompanied by the failure of DNA repair mechanisms, can result in the death of a high percentage of the treated cells [61], as also shown by our cell viability data. Most interesting, among the overexpressed genes involved in DDR, we identified genes with dual roles, which can facilitate DNA repair or induce apoptosis, depending on the context. For instance, the multifunctional DDB2 protein (damaged DNA binding protein 2) is known to contribute to DNA repair through the induction of nucleotide excision repair [62], but can also drive damaged cells towards apoptosis [63,64], for instance by downregulating *CDKN1A* expression [65]. Moreover, it has been shown that DDB2 can regulate the transcription of the antioxidant *SOD2* gene, hence regulating superoxide levels and consequently the redox balance [66].

Molecular analysis of cell death revealed that PDT triggered a complex web of stressors that may lead either to cell death or to resistance of treated tumor cells, as detailed below. We have emphasized that, in time, the few cells surviving PDT continued to grow slowly, probably accounting for disease relapse sometimes after PDT. Therefore, unraveling the rescue mechanisms triggered by PDT may be of highest importance for designing new adjuvant therapeutic strategies aimed at sustaining PDT.

P2.2-PDT induced a strong oxidative burst that was indirectly evidenced in this study through the upregulation of several potent antioxidant genes. Surprisingly, a robust antioxidant response was generated by PDT not only in detached cells (apoptotic cells) but also in adhered cells (presumably living cells), and this protective response was even stronger in detached cells. This observation might be explained by a potentially higher photosensitizer load in some cells, probably resulting in stronger PDT-induced singlet oxygen burst and extensive activation of antioxidant mechanisms. Nonetheless, the elicited antioxidant response was not sufficiently protective to completely counteract the observed PDT-driven decrease of viable cells number. In turn, the remaining viable cells may get shielded against oxidative damage and become resistant to future PDT sessions as well as to other anticancer therapies that rely on oxidative stress for cytotoxicity [3].

The hypoxia signature detected in this study in PDT-treated cells sustains the assumption that the acute oxidative burst triggered by PDT can transiently reduce local oxygen availability due to its utilization for singlet oxygen generation [3]. PDT-induced hypoxia adds to the intrinsic hypoxia of large tumors [67]. Through a feedforward loop, acute hypoxia can induce within minutes a superoxide burst that intensifies the oxidative stress triggered by PDT through the generated singlet oxygen [68]. It has been also shown that reactive oxygen species (ROS) can directly stabilize and activate the transcriptional

activity of the hypoxia-inducible factor-1 alpha (HIF1α), which regulates the expression of many hypoxia-responsive genes [69]. Unfortunately, hypoxia signaling is generally associated with increased angiogenesis and epidermal to mesenchymal transition (EMT), both processes favoring tumor progression [70]. Therefore, besides being cytotoxic against most of the treated tumor cells, the PDT regimen investigated by us appears also to support the survival of residual cancer cells that escaped from the therapeutic hit through hypoxia signaling.

The accumulation of oxidatively damaged proteins resulting from the severe oxidative burst triggered by PDT may account for the ER stress signature and the consequent UPR detected by us at transcriptional level in PDT-treated cells [71]. Alternatively, we cannot rule out that ER stress might be induced directly by PDT if the photosensitizer localizes, at least partly, in ER, and a massive amount of ROS is produced locally [72]. Considering the high reactivity of photo-generated ROS, autophagy was shown by us to be initiated to remove damaged organelles [73], if cells were not driven to apoptosis by DDIT3 in conjunction with BID and BBC3 [59,74,75]. Additionally, we evidenced the upregulation of the heat shock protein-encoding genes *HSP90AA1* and *HSPA4*, which may limit the PDT-induced proteotoxic stress. In turn, other protective genes were found downregulated (*HSPA5*, *DNAJC3*, *HSP90B1* and *CALR*), indicating that important cytoprotective mechanisms might have been suppressed by the investigated PDT regimen.

As will be detailed below, several transcription factors regulate the expression of the genes found significantly modified in the present study.

All the investigated redox genes are known targets of the transcription factor NRF2 which controls at the transcriptional level more than 250 cytoprotective genes, most of them having an antioxidant role [76]. It has been shown that singlet oxygen, the peculiar form of ROS generated during PDT, can activate the NRF2 system directly or via derived oxidized products, therefore inducing antioxidant shielding against the PDT-inflicted injuries. Moreover, NRF2 links oxidative conditions with DDR through the p21 protein which is encoded by *CDKN1A* and is mainly involved in cell cycle arrest [34]. NRF2 can induce the transcription of the *CDKN1A* gene which is also under the transcriptional control of TP53 [77]. Through a feed forward loop, enhanced levels of p21 compete with NRF2 for binding to its KEAP1 repressor, hence inducing the activation of the NRF2 pathway [78]. Altogether, NRF2 silencing would be a promising co-therapy for increasing PDT efficacy [79] but, for the moment, there are no NRF2 inhibitors approved for therapeutic use, due to their numerous off-target effects [80].

Most of the proteins encoded by the genes identified in this study are directly or indirectly connected to the redox-sensitive tumor suppressor TP53 (Figure 11). TP53 is one of the most important genome guardians that protects cells against various insults, including oxidative stress, by driving damaged cells towards death or by activating rescue mechanisms [81–83]. Interestingly, it has been shown that TP53 might play a significant role in the death of cells subjected to porphyrin-PDT, through direct interaction with the drug itself, leading to induction of TP53-dependent cell death both in the dark and upon PDT [81]. Nevertheless, our data also point towards a TP53-mediated activation of cytoprotective mechanisms that might render treated cells resistant to future PDT sessions. It is worth mentioning that many genes that are known to be under TP53 control can be also upregulated by other transcription factors, depending on the stressors to which cells were exposed. Therefore, the involvement of TP53 has to be carefully analyzed in PDT for finding the proper therapy regimen that drives tumor cells towards death.

The PDT-mediated upregulation of some gene targets of the redox- and hypoxiasensitive transcription factor HIF1α, such as *VEGFA*, give evidence that a hypoxia response is indeed triggered by PDT. HIF1α may elicit protective mechanisms that not only defend tumor cells against PDT, but may further drive tumor progression by sustaining angiogenesis and EMT [84,85].

**Figure 11.** Functional associations between the tumor suppressor TP53 and the proteins encoded by genes with modified expression in PDT-treated HT29 tumor cells, as compared to non-treated cells (Table 2). The network was built using the online tool "STRING: functional protein association **Figure 11.** Functional associations between the tumor suppressor TP53 and the proteins encoded by genes with modified expression in PDT-treated HT29 tumor cells, as compared to non-treated cells (Table 2). The network was built using the online tool "STRING: functional protein association network". Only connections deriving from experiments and databases, with a high confidence of 0.7, are represented.

genes were found downregulated (*HSPA5*, *DNAJC3*, *HSP90B1* and *CALR*), indicating that important cytoprotective mechanisms might have been suppressed by the investigated

genes found significantly modified in the present study.

therapeutic use, due to their numerous off-target effects [80].

As will be detailed below, several transcription factors regulate the expression of the

All the investigated redox genes are known targets of the transcription factor NRF2 which controls at the transcriptional level more than 250 cytoprotective genes, most of them having an antioxidant role [76]. It has been shown that singlet oxygen, the peculiar form of ROS generated during PDT, can activate the NRF2 system directly or via derived oxidized products, therefore inducing antioxidant shielding against the PDT-inflicted injuries. Moreover, NRF2 links oxidative conditions with DDR through the p21 protein which is encoded by *CDKN1A* and is mainly involved in cell cycle arrest [34]. NRF2 can induce the transcription of the *CDKN1A* gene which is also under the transcriptional control of TP53 [77]. Through a feed forward loop, enhanced levels of p21 compete with NRF2 for binding to its KEAP1 repressor, hence inducing the activation of the NRF2 pathway [78]. Altogether, NRF2 silencing would be a promising co-therapy for increasing PDT efficacy [79] but, for the moment, there are no NRF2 inhibitors approved for

Most of the proteins encoded by the genes identified in this study are directly or indirectly connected to the redox-sensitive tumor suppressor TP53 (Figure 11). TP53 is one of the most important genome guardians that protects cells against various insults, including oxidative stress, by driving damaged cells towards death or by activating rescue mechanisms [81–83]. Interestingly, it has been shown that TP53 might play a significant role in the death of cells subjected to porphyrin-PDT, through direct interaction with the drug itself, leading to induction of TP53-dependent cell death both in the dark and upon PDT [81]. Nevertheless, our data also point towards a TP53-mediated activation of cytoprotective mechanisms that might render treated cells resistant to future PDT sessions. It is worth mentioning that many genes that are known to be under TP53 control can be also upregulated by other transcription factors, depending on the stressors to which cells were exposed. Therefore, the involvement of TP53 has to be carefully analyzed in PDT for finding the proper therapy regimen that drives tumor cells towards death.

PDT regimen.

A transcription factor revealed by our results to be transcriptionally upregulated by P2.2-PDT is DDIT3 (CHOP). This stress-induced transcription factor has been shown to integrate the signals from multiple stressors such as DNA damage and ER stress. It mediates a vast array of cellular responses, from UPR to stress-induced cell death, either per se or through interaction with other transcription factors [38]. As evidenced in this study, *DDIT3* overexpression was paralleled by the upregulation of the *TNFRS10B* gene that encodes the death receptor DR5. This might be a functional association between cell death and ER stress, considering that *TNFRS10B* transcription is under the control of DDIT3, in addition to TP53 and NFκB [86,87]. Moreover, it has been shown that DR5 along with other TRAIL receptors serve as stress-associated molecular patterns (SAMPs) to promote ER stress-induced inflammation [88]. Besides the involvement of DDIT3 in apoptotic and autophagic cell death [75], it has been demonstrated that this transcription factor can activate inflammatory responses that generally sustain tumor progression [89]. This can explain, at least partly, some of our results regarding the PDT-induced inflammatory response in HT29 colon carcinoma cells. For instance, we detected in PDT-treated cells a markedly enhanced transcription of the *CXCL8* gene encoding the IL-8 chemokine and this might be mediated by DDIT3 [90]. IL-8 triggers enhanced recruitment of neutrophils in the tumor niche [91], their cytotoxic activity increasing PDT efficacy against tumor cells [92], hence limiting disease progression [93,94]. In turn, increased levels of IL-8 were shown to boost colorectal liver metastasis, hence worsening disease outcome [95]. We do not rule out that enhanced *CXCL8* expression could derive also from NFκB-mediated gene transcription under the PDT pressure [96].

Altogether, molecular data highlighted that the investigated PDT regimen triggered increased transcription of critical genes that underlies the therapeutic effect, but some of the investigated genes may confer a survival advantage to tumor cells. The differential analysis of the gene expression pattern in adhered and detached cells could not provide definite evidence on the particular genes that determine the difference between tumor cells killed by PDT and the surviving ones at 24 h post-PDT. Most of the selected genes were common to adhered and detached cells, indicating that adhered cells might be in fact committed to delayed death. Only *TXNRD1* and *RIPK1* were upregulated specifically in adhered cells, being involved in antioxidant protection and cell death, respectively. The antioxidant genes *NQO1*, *GSTP1*, *PRDX1* and *NQO1* were upregulated specifically in detached cells, indicating that antioxidant mechanisms were not fully capable of protecting tumor cells against the PDT-inflicted oxidative injuries.

#### **4. Materials and Methods**

#### *4.1. Photosensitizer*

The unsymmetrical porphyrin 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Figure 1), abbreviated as P2.2, was obtained according to the method previously described by us [8]. The method is based on the interaction between 4-hydroxy-3-methoxybenzaldehyde, pyrrol and 4-acetoxy-3-methoxybenzaldehyde (3:1 ratio) for approximately 3 h in an acid environment, under strict temperature control (t = 125 ◦C). P2.2 purification was obtained by column chromatography using Al2O<sup>3</sup> 90 (Merck, 63–200 µm and 70–230 µm mesh) as stationary phase and dichloromethane/diethyl ether (30:1 *v*/*v*) as eluent. The synthesis yield was 7% and the purity was 100%, as previously shown by NMR analysis. The fluorescence emission and singlet oxygen formation quantum yields, lifetimes and the methods used for quantification are described in [8] and [97]. P2.2 has a Q band with a peak at 628 nm and was therefore activated for PDT using a 635 nm laser in the Modulight ML6600 equipment (Modullight, Tampere, Finland). The obtained P2.2 was dissolved at 10 mM concentration in PEG 200, a biocompatible solvent, and was stored at room temperature in the dark until use.

#### *4.2. Cells*

The human colon adenocarcinoma cell line HT29 purchased from the American Tissue and Cell Collection (ATCC® HTB-38™, Manassas, VA, USA) was used. Cells were grown in DMEM-F12 culture medium with GlutaMAX (Gibco), supplemented with 10% fetal bovine serum (FBS, Sigma, Saint Louis, MO, USA), that will be hereinafter referred to as complete culture medium. Twice per week cells were detached with 0.05%/0.02% (*w*/*v*) Trypsin-EDTA (Biochrom). After trypsin inactivation with two volumes of complete culture medium, cells were washed by centrifugation (1200 rpm, 5 min, 4 ◦C), were suspended in complete culture medium and were counted by optical microscopy in a Burker–Turk counting chamber, using Trypan blue as dead cells stain. Only cell cultures with a viability >95% were used for experiments. For multiplication, cells were seeded in 25 cm<sup>2</sup> cell culture flasks (40,000 live cells/cm<sup>2</sup> ) and were cultivated at 37 ◦C in 5% CO<sup>2</sup> atmosphere.

#### *4.3. Loading of Cells with P2.2*

HT29 cells (0.5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells) were seeded in 35 mm Petri dishes in 2 mL complete culture medium, in triplicates for each experimental condition. Cells were cultivated for 24 h in 5% CO<sup>2</sup> atmosphere at 37 ◦C for allowing their adherence. Thereafter, the complete culture medium was discarded and was replaced with 2 mL DMEM-F12 culture medium with GlutaMAX, supplemented with 2% FBS and 10 µM P2.2 (referred as P2.2-loading culture medium). Cells were cultivated for another 24 h to allow P2.2 loading. All the procedures with P2.2 and P2.2-loaded cells were performed in "dark" conditions (no direct light falling on the samples) for avoiding uncontrolled activation of the photosensitizer.

#### *4.4. P2.2 Uptake in HT29 Cells*

For measuring P2.2 uptake into HT29 cells, 0.1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells were seeded in triplicates in 24 well plates and were cultivated for 24 h in 0.5 mL complete culture medium for allowing their adherence. The culture medium was then discarded and was replaced with P2.2 loading culture medium (see Section 4.3). At 24 h after P2.2 addition to cell cultures, cells were detached with Trypsin-EDTA (see Section 4.2), were washed by centrifugation and were finally suspended in Live Cell Imaging Solution (Thermo Fisher Scientific, Waltham, MA, USA). The uptake control samples were not incubated with P2.2 and were processed

exactly as P2.2-treated cells. All the procedures with P2.2-loaded cells were performed in "dark" conditions. P2.2 incorporation into HT29 cells was measured based on P2.2 red fluorescence by flow cytometry on a BD FACSCanto II cytometer with BD FACSDiva 6.1 software (Becton Dickinson, Franklin Lakes, NJ, USA). Data from a minimum of 10,000 events were acquired. Fluorescence was expressed in arbitrary units. Fluorescence data were processed in each sample as median fluorescence value or fluorescence distribution, using the above-mentioned software.

## *4.5. In Vitro PDT*

After loading of cells with P2.2 in 35 mm Petri dishes (see Section 4.3), the P2.2 loading culture medium was discarded and cells were gently washed with complete culture medium at room temperature. Two mL Hank's balanced salt solution supplemented with 2% FBS (PDT culture medium) was added and cells were subjected to in vitro PDT. PDT was performed in test samples at room temperature, using a Modulight ML6600 instrument (Modulight, Tampere, Finland) equipped with a 635 nm laser, illumination chamber for 35 mm Petri dishes and software control of temperature and PDT parameters (light fluence, fluence rate, power and time). The PDT parameters applied in various experimental settings were: 5, 7.5, 10, 15 and 25 J/cm<sup>2</sup> . Light was delivered at a fluence rate of 50 mW/cm<sup>2</sup> . A comparison between 10 and 50 mW/cm<sup>2</sup> fluence rate was performed for a fluence of 10 J/cm<sup>2</sup> . Control samples loaded with P2.2 were prepared by applying the same procedure as described above, were not subjected to PDT and were kept at room temperature during the time when test samples were exposed to PDT. Immediately after PDT, the PDT culture medium was removed and was replaced with 2 mL complete culture medium. Both test samples and controls were further cultivated in 5% CO<sup>2</sup> atmosphere at 37 ◦C for performing various post-PDT investigations.

#### *4.6. Post-PDT Investigations*

4.6.1. Preparation of Samples for Post-PDT Investigations


#### 4.6.2. MTS Reduction

MTS reduction was used for evaluating the relative number of metabolically active cells in PDT-treated and non-treated samples. The method therefore provides information on cell viability and proliferation [8].

Detached and adhered cells harvested at 24 h post-PDT (see Section 4.6.1) were mixed for each sample. Cells in non-treated samples were counted and the volume containing 30,000 cells was calculated. This volume of cell suspension was harvested from all samples, both PDT-treated and non-treated, was adjusted to 100 µL and the resulting samples were analyzed for MTS reduction at 24 h post-PDT. MTS reduction was tested in the cell cultures prepared for 72 h investigations, as follows. A total of 50 µL of supernatant was collected from the cell cultures (see Section 4.6.1) following centrifugation of the 96 well plates at 150 g for 5 min and was used for assessing LDH release (see Section 4.6.3). Then, 50 µL of fresh complete culture medium was added to cell cultures for assessing MTS reduction.

MTS reduction was measured using the colorimetric CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) from Promega (Madison, WI, USA), according to the manufacturer's procedure. Briefly, 20 µL of the kit's reagent was added to each well and samples were cultivated 2 h at 37 ◦C for allowing MTS reduction by metabolically active cells. Finally, the optical density at 490 measured against a 620 nm wavelength reference was measured in each sample using a Sunrise Tecan microplate reader equiped with universal reader control and Magellan data analysis software (Tecan, Männedorf, Switzerland). Data were processed as corrected optical density (OD) obtained by subtrating the mean optical density of the background samples from the the optical density of test samples. Data were processed as PDT effect calculated by dividing the OD of each PDTtreated sample to the mean value of the OD of the corresponding control samples (not treated by PDT).

#### 4.6.3. LDH Release

LDH release was used for evaluating membrane integrity of PDT-treated and non-treated cells [8]. The method provides information on cell death through necrosis/necroptosis [9].

Cell-free supernatants obtained immediately after PDT and at 24 h or 72 h post-PDT were tested for LDH release.

LDH release was measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega). The colorimetric test was performed according to the manufacturer's procedure. Briefly, 50 µL of cell-free supernatant and 50 µL of LDH substrate were incubated in the dark at room temperature for 30 min. The reaction was interrupted by the addition of 50 µL stop solution. Finally, the optical density at 490 nm was measured in each sample using a Sunrise Tecan microplate reader equipped with universal reader control and Magellan data analysis software (Tecan). Data were processed as corrected optical density (OD) obtained by subtracting the mean optical density of background samples from the optical density of test samples. Data were processed as PDT effect calculated by dividing the OD of each PDT-treated sample to the mean OD value of control samples (not treated by PDT).

#### 4.6.4. Apoptosis and Necrosis Evaluation by Flow Cytometry

The percentage of apoptotic and necrotic cells in P2.2-treated samples and in untreated controls was evaluated at 24 h post-PDT by flow cytometry using the Annexin A5 Apoptosis Detection Kit (BioLegend, San Diego, CA, USA). The test was performed according to the procedure indicated by the manufacturer.

Briefly, detached and adhered cells harvested at 24 h post-PDT (see Section 4.6.1) were mixed for each samples. Cells were counted and approximately 300,000 cells from each sample were harvested. Cells were washed once with phosphate-buffered saline (PBS), were suspended in 100 µL annexin V binding buffer and were labelled with 5 µL FITC Annexin V and 10 µL of propidium iodide solution. Cell suspensions were incubated for 15 min at room temperature, in the dark. The reaction was stopped by the addition of 400 µL annexin V binding buffer. Samples were analyzed within 30 min by flow cytometry on a BD FACSCanto II cytometer with BD FACSDiva 6.1 software (Becton Dickinson). Data from a minimum of 5000 events were acquired. For PE-FITC the compensation was set at 31, while for FITC-PE it was set at 5. Flow cytometry data were presented as percentage of

early apoptotic cells (annexin V+/PI−), late apoptotic cells (annexin V+/PI<sup>+</sup> ) and necrotic cells (annexin V−/PI<sup>+</sup> ). The total pecentage of apoptotic cells was calculated as sum of early and late apoptotic cells percentages.

#### 4.6.5. Cell Proliferation Evaluation

Cell proliferation was assessed by flow cytometry with CFDA-SE (Vybrant® CFDA SE Cell Tracer Kit, Invitrogen, Waltham, MA, USA). According to the information provided by the supplier, CFDA-SE passively diffuses into cells. It is colorless and nonfluorescent until its acetate groups are cleaved by intracellular esterases to yield highly fluorescent, amine-reactive carboxy-fluorescein succinimidyl ester. The succinimidyl ester group reacts with intracellular amines, forming fluorescent conjugates that are well-retained within cells. The dye–protein adducts that form in labeled cells are retained by the cells throughout development, meiosis and in vivo tracing. The label is inherited by daughter cells after cell division and is not transferred to adjacent cells in a population, therefore providing reliable information on cell proliferation.

Adhered cells harvested at 24 h post-PDT (see Section 4.6.1) were counted. From each sample 300,000 cells were collected and were washed twice in cold PBS (1200 rpm, 5 min, 4 ◦C). Sedimented cells were incubated for 15 min with 5 µM CFDA-SE in 300 µL pre-warmed PBS (37 ◦C). Cells were washed once in PBS by centrifugation. Sedimented cells were suspended in 500 µL fresh complete culture medium and were incubated for another 30 min at 37 ◦C to ensure stable loading of cells. The suspension was washed by centrifugation and the cell pellet was suspended in 300 µL complete culture medium. Then, 100 µL of cell suspension from each sample was placed in 24 well plates and the culture volume was adjusted to 1 mL. Cell cultures were incubated for another 48 h at 37 ◦C in 5% CO<sup>2</sup> atmosphere and were analyzed at 72 h post-PDT. Before investigation, cells were detached with Tripsin-EDTA solution (see Section 4.2) and were washed twice in ice-cold PBS. The intracellular fluorescence of CFDA-SE was measured by flow cytometry using a BD FACSCalibur flow cytometer and CellQuest Pro software (Becton Dickinson, Franklin Lakes, NJ, USA). CFDA-SE fluorescence was activated with a 482 nm laser, while emmission was registered in the FL-1 channel for fluorescein isothiocyanate (FITC). The data from a minimum of 50,000 events were acquired. Flow cytometry data were further processed using the ModFit LT software (Verity Sotware House, Topsham, ME, USA), which provides the distribution of proliferative cells in consequtive daughter generations.

#### 4.6.6. Microscopic Monitoring of Cell Cultures

Cell cultures were monitored in time, up to 120 h post-PDT, by optical microscopy using an EVOS XL Core Cell Imaging System equiped with image acquisition software (ThermoFisher Scientific). Of note is that separate cell cultures were prepared for imaging, equivalent to those described at 4.5, considering that exposure of P2.2-loaded cells to visible light might trigger an artificial activation of PDT and might compromise the results of the post-PDT tests. Due to the same reason, even the samples dedicated to imaging were only rarely subjected to microscopic evaluation (1 time/day, with only short exposure to light).

#### 4.6.7. Gene Expression

Cell cultures in 35 mm Petri dishes were prepared and processed as described in Sections 4.3–4.5.

At 24 h post-PDT, cell supernatants containing detached cells were collected and centrifuged and cellular sediments were finally collected in 1 mL RiboZol reagent (VWR, Radnor, PA, USA). Attached cells were gently washed with warm PBS (37 ◦C) and 1 mL RiboZol reagent was then added for RNA extraction. Processed samples in RiboZol reagent were stored at −80 ◦C until use.

Total RNA isolation from cells preserved in RiboZol reagent (VWR Life science) was performed according to the manufacturer's instructions. RNA concentration was quantified using the Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Both

the 260/280 nm and 260/230 nm ratios were above 1.8, indicating a high RNA quality. cDNA synthesis was performed using 600 ng of total RNA with the RT<sup>2</sup> First Strand Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The expression of 84 genes involved in stress and toxicity was assessed using the RT<sup>2</sup> Profiler™ PCR Array Human Stress and Toxicity PathwayFinder (PAHS-003Z) from Qiagen Table 1), using the SYBR Green chemistry on ABI7500 Fast PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The expression level of each gene was normalized on the geometric mean values of two housekeeping genes (ACTB and HPRT1), which were selected according to RefFinder algorithm [98] after the analysis of five candidate reference genes (ACTB, B2M, GAPDH, HPRT1 and RPLP0) both in PDT-treated samples and non-treated controls. Gene expression data were analyzed with the RT<sup>2</sup> Profiler PCR Array software package (Qiagen). Gene expression levels were calculated as 2−∆CT values. Fold change (FC) in gene expression was calculated as the 2−∆CT mean values in patients divided by 2−∆CT mean values in controls. Results are presented as fold regulation (FR) as follows: when the FC value was above 1, FR was equal to FC and results were reported as fold upregulation; when the FC value was less than 1, FR was expressed as the negative inverse of FC and results were reported as fold downregulation.

#### *4.7. Data Processing*

Whenever possible data were presented as mean value ± standard error of the mean (SEM) for triplicate samples. The IC50 value of P2.2 was computed using the online Quest Graph™ IC50 Calculator (https://www.aatbio.com/tools/ic50-calculator, accessed on 27 June 2021) and was expressed as mean ± standard deviation (SD). PDT effect was calculated as a parameter value in PDT-treated samples divided by the mean parameter value in non-treated controls. Supraunit values of PDT effect indicate activation, while subunit values indicate an inhibition. Comparison of the phenotypic and functional data from cells exposed to PDT and from non-treated controls was performed in Excel using the Student's t-test (unequal variances or paired two samples for mean, depending on the case). Comparison of gene expression data between PDT-treated samples and controls was performed with the Student's t-test (equal variances) using the RT<sup>2</sup> Profiler PCR Array software package (Qiagen, Hilden, Germany). Comparison between adhered and attached cells regarding gene expression (FR values) was performed in Excel using the Student's t-test (paired two samples for mean). Differences were considered significant for *p* values < 0.05. The Pearson test was used for evaluating correlations between parameters, considered as significant for r values > 0.6 and *p* < 0.05.

#### **5. Conclusions**

Using an integrative systems biology approach on the expression of stress genes in HT29 human colon carcinoma cells subjected to porphyrin-PDT, the study highlighted the network of molecular mechanisms underlying therapy-induced cell death and other cellular responses to the complex web of stressors triggered concomitantly by P2.2-PDT, encompassing oxidative stress, hypoxia, DNA damage, ER stress and UPR, along with inflammation. The transcription factors potentially responsible for the observed gene expression profile (NRF2, HIF1α, p53, DDIT3 and, possibly, NFκB) were highlighted. Particular cytoprotective mechanisms and upregulated pathway-specific genes were found, which represent promising therapeutic targets for improving PDT efficacy. Nevertheless, this study does not provide information on the impact of the observed gene expression changes at protein and functional level, an aspect that should be addressed in future studies. The identified expression profile of stress genes triggered by the particular P2.2- PDT regimen used in the present study might be further extended to other porphyrinic photosensitizers and PDT regimens.

**Author Contributions:** M.D. original draft preparation, investigations-gene expression, methodology; R.B. conceptualization-photosensitizers; investigations-P2.2 synthesis, methodology; I.V.N. investigations-cell cultures, in vitro PDT, viability tests; M.S. investigations-flow cytometry, methodology; E.M. methodology, formal analysis, draft revision; G.M. conceptualization, draft preparation and revision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Romanian Ministry of Research, Innovation and Digitization, grant number PCCDI 64/2018. Publication charges were funded by the Romanian Ministry of Research, Innovation and Digitization, grant PN 19.29.02.02 (Nucleu Programme). EM is recipient of a post-doctoral contract of the European Regional Development Fund, Competitiveness Operational Program 2014–2020, through the grant P\_37\_732/2016 REDBRAIN.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are presented in the paper, and raw data are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Article* **Improvement of Pulmonary Photodynamic Therapy: Nebulisation of Curcumin-Loaded Tetraether Liposomes**

**Jennifer Lehmann <sup>1</sup> , Michael R. Agel <sup>1</sup> , Konrad H. Engelhardt <sup>1</sup> , Shashank R. Pinnapireddy <sup>1</sup> , Sabine Agel <sup>2</sup> , Lili Duse <sup>1</sup> , Eduard Preis <sup>1</sup> , Matthias Wojcik <sup>1</sup> and Udo Bakowsky 1,\***


**Abstract:** Lung cancer is one of the most common causes for a high number of cancer related mortalities worldwide. Therefore, it is important to improve the therapy by finding new targets and developing convenient therapies. One of these novel non-invasive strategies is the combination of pulmonary delivered tetraether liposomes and photodynamic therapy. In this study, liposomal model formulations containing the photosensitiser curcumin were nebulised via two different technologies, vibrating-mesh nebulisation and air-jet nebulisation, and compared with each other. Particle size and ζ-potential of the liposomes were investigated using dynamic light scattering and laser Doppler anemometry, respectively. Furthermore, atomic force microscopy and transmission electron microscopy were used to determine the morphological characteristics. Using a twin glass impinger, suitable aerodynamic properties were observed, with the fine particle fraction of the aerosols being ≤62.7 ± 1.6%. In vitro irradiation experiments on lung carcinoma cells (A549) revealed an excellent cytotoxic response of the nebulised liposomes in which the stabilisation of the lipid bilayer was the determining factor. Internalisation of nebulised curcumin-loaded liposomes was visualised utilising confocal laser scanning microscopy. Based on these results, the pulmonary application of curcuminloaded tetraether liposomes can be considered as a promising approach for the photodynamic therapy against lung cancer.

**Keywords:** tetraether liposomes; nebulisation; liposomal stability; photodynamic therapy; curcumin; A549 cells

#### **1. Introduction**

Cancer remains as one of the major challenges in healthcare worldwide and is currently responsible for the majority of global deaths [1]. In recent years, lung cancer was the most common type of cancer and with 18% of the total cancer deaths, it was the leading cause of cancer death in both sexes combined [2]. According to the type of lung cancer and individual risk assessment, standard therapeutic strategies involve chemotherapy, radiotherapy, a combination of both or less frequently, treatment with monoclonal antibodies [3,4]. One of the basic problems in cancer therapy is the high rate of side effects due to undesired tissue destruction and a loss of anatomical and physiological integrity [5]. Therefore, scientists aim to improve the current treatment guidelines as well as to develop treatments with novel mechanisms and targets.

One of these promising mechanisms is the application of light, which itself has a long tradition in the history of medicine, in combination with a photoactive drug collectively termed photodynamic therapy (PDT). The basic principle of PDT is a combination of

**Citation:** Lehmann, J.; Agel, M.R.; Engelhardt, K.H.; Pinnapireddy, S.R.; Agel, S.; Duse, L.; Preis, E.; Wojcik, M.; Bakowsky, U. Improvement of Pulmonary Photodynamic Therapy: Nebulisation of Curcumin-Loaded Tetraether Liposomes. *Pharmaceutics* **2021**, *13*, 1243. https://doi.org/ 10.3390/pharmaceutics13081243

Academic Editor: Armin Mooranian

Received: 5 July 2021 Accepted: 5 August 2021 Published: 12 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

three otherwise non-toxic components, a photosensitiser, light and oxygen [6,7]. The photosensitiser is a molecule that can be excited by light, mostly due to a conjugated π-electron system. Upon excitation with light of a specific wavelength, the photosensitiser generates reactive oxygen species (ROS) in the presence of oxygen, thereby exhibiting a cytotoxic effect towards surrounding cancer cells [8,9]. The emerged oxygen species has a half-life of only 0.01–0.04 µs and a restricted site of action of 0.01–0.02 µm [8,10]. Due to this, an effect occurs only within a very small range to the irradiation site [11]. Additionally, PDT is able to damage tumour-associated vasculature, resulting in an anaemic infarction of the tumour and can activate the innate immune response against cancer cells. The three mechanisms of PDT can act synergistically and influence each other [12,13]. Other advantages of the PDT are a minor impact towards healthy tissues, its effectiveness against chemo-resistant cancer cell types and being combinable with other therapies such as conventional chemotherapy, microbial adjuvants or cytokines for a T-cell therapy [12,14,15].

There are many different photosensitisers that have been tested in studies, but only very few have reached the stage of advanced human clinical trials or approval for clinical use [14,16]. One such photosensitiser with a huge therapeutic potential is curcumin [17–19]. It is a polyphenol extracted from the rhizomes of turmeric (*Curcuma longa*), naturally occurring in south and southeast Asia [20]. Curcumin has an orange-yellow colour due to its phenylogous π-electron system, and is the most active ingredient of turmeric making approximately 0.5–3% of its weight [14]. Since several decades, it has been an object to pharmaceutical research due to its anti-inflammatory, anti-oxidative, anti-microbial and anti-carcinogenic effects [21–23]. However, curcumin is prone to an extensive first-pass metabolism after oral administration, undergoes a fast metabolic reduction and is subject to biliary excretion [24]. Besides, due to the highly lipophilic properties of this photosensitiser, with a solubility of merely 11 ng/mL in phosphate buffer saline (pH 5), it has a very limited bioavailability [24,25].

These limitations can be overcome by the liposomal encapsulation of curcumin. Within the lipid bilayer of the liposomes, the active ingredient is protected from the physiological degradation on the one hand [9], and the cellular uptake as well as bio-membrane permeability are enhanced on the other hand [9,26]. Altogether, bioavailability, biocompatibility and the circulation time can be improved through liposomal encapsulation, enhancing the overall effectiveness of the photosensitiser [27].

Nebulisers have turned into a trendsetting option in the treatment of a multitude of diseases due to increased patient compliance. For instance with the usage of glycerosomes and hyalurosomes, which are stable towards the process of nebulisation and proved to be good vehicles for a pulmonary application [28,29]. The nebulisation of liposomes can also lead to a considerable improvement in lung cancer disease management.

The major focus of this study was to investigate a non-invasive convenient approach for the treatment of lung cancer by combining nebulised liposomes and PDT. For this purpose, behaviour and stability of three liposomal model formulations were investigated before and after the process of nebulisation and their in vitro efficacy was assessed using A549 lung adenocarcinoma cells. The liposomal formulations had different degrees of membrane stabilisation, amongst others through the usage of bipolar tetraether lipids extracted from the biomass of the archaea *Sulfolobus acidocaldarius*. This plays an important role in membrane integrity and thus the ability to successfully deliver the active ingredient. Curcumin was chosen as it is a commonly used photosensitizer with a well-known impact [14,17,30]. Additionally, the suitability of two different nebulisation techniques, vibrating-mesh and air-jet, was tested and compared for the nebulisation of liposomes in small amounts.

#### **2. Materials and Methods**

#### *2.1. Materials*

Curcumin (≥80%), cholesterol (≥99%), HEPES (≥99.5%), ethanol (absolute, ≥99.8%), D-(+)-trehalose dehydrate (≥99%) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). The lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE) were a gift from Lipoid GmbH (Ludwigshafen am Rhein, Germany). Fractions containing the polar tetraether lipids caldarchaeol (GDGT, approx. 10% of the extract) and calditoglycerocaldarchaeol (GDNT, approximately 90% of the extract) were extracted from the biomass of *Sulfolobus acidocaldarius* (Transmit GmbH, Gießen, Germany) [31]. The ends of GDNT contain phosphatidylmyoinositol and β-glucose respectively while GDGT contains phosphatidylmyoinositol and β-D-galactosyl-D-glucose on either ends respectively [32]. Ultrapure water from PURELAB® flex 4 (ELGA LabWater, High Wycombe, UK) was used for all experiments in this study. All solvents used were of analytical or HPLC grade.

#### *2.2. Cell Culture*

A549 adenocarcinomic human alveolar basal epithelial cells were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). The cells were cultured in DMEM (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 10% foetal bovine serum (Capricorn Scientific GmbH). The cells were cultivated at 5% CO<sup>2</sup> and 37 ◦C under humid conditions. They were grown as monolayers and passaged upon reaching 80% confluency.

### *2.3. Irradiation Device*

For photodynamic activation, a low power prototype LED device consisting of an array of light emitting diodes designed to fit multiwall plates was used. The device was custom manufactured by Lumundus GmbH (Eisenach, Germany), equipped with two different arrays of LEDs capable of emitting light at different wavelengths. Activation took place at 457 nm, the actual fluence used in this study was calculated based on the amperage and irradiation time [26].

#### *2.4. Preparation of Liposomes*

Liposomes were prepared via thin-film-hydration method using stock solutions of curcumin in ethanol and lipids dissolved in chloroform:methanol (2:1 (*v*/*v*)). The ingredients were mixed in a 10 mL round bottom flask with a curcumin to lipid ratio of 1:10. To gain a thin film, the mixture was evaporated at 40 ◦C using a Laborota 4000 rotary evaporator (Heidolph Instruments, Schwabach, Germany) fitted with a vacuum pump (KNF Neuberger GmbH, Freiburg im Breisgau, Germany). Afterwards, the film was rehydrated with HEPES (20 mM, pH 7.4) to gain a final concentration of 1 mg/mL. The liposomal formulations were sonicated in a bath sonicator (Elamsonic P30H, Elma Schmidbauer GmbH, Singen, Germany) above the phase transition temperature (Tc) for 30 min.

To homogenise the vesicles, all liposomes were extruded with an Avanti Mini Extruder (Avanti Polar Lipids, INC., Alabaster, AL, USA) using polycarbonate membranes with a pore size of 200 nm. This step was carried out above the Tc. The obtained liposomes were stored at 4 ◦C.

In total three formulations were compared with each other in this study (Table 1).

**Table 1.** Lipid compositions of the liposomal formulations. The lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), tetraether lipids (TEL), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol (Chol) were used.


#### *2.5. Lyophilisation*

To improve long-term storage stability, the liposomes were lyophilised using an Alpha 1-4 LSC freeze-dryer (Martin Christ, Osterrode am Harz, Germany). In this process, D-(+)-trehalose dihydrate was used to protect the liposomes from collapsing or aggregating during lyophilisation.

Briefly, the liposomal formulations were prepared as stated in Section 2.4 and rehydrated in filtrated HEPES containing 4 mg/mL D-(+)-trehalose dihydrate. A total of 500 µL of the freshly produced liposomes in round bottom flasks were rapidly frozen by shaking in liquid nitrogen to increase the surface and lyophilised with the following parameters: primary drying for 24 h at +15 ◦C shelf temperature and a partial vacuum of 0.120 mbar, followed by secondary drying for 24 h at +25 ◦C shelf temperature and 0.100 mbar. The lyophilised residual underwent visual examination. Prior to use and nebulisation with PARI VELOX®, the freeze-dried liposomes were redispersed in ultrapure water.

#### *2.6. Nebulisation, Aerosol Output and Emitted Volume*

For the nebulisation of previously produced liposomes, two nebulisers representing two different techniques of nebulisation were used and compared: PARI VELOX® (PARI GmbH, Starnberg, Germany), a metal-based vibrating-mesh nebuliser with a resonance frequency of 160 kHz and PARI BOY® SX utilising compressed air with an operating pressure of approximately 1.6 bar and a nozzle size of 0.48 mm. Briefly, 2 mL of each liposomal formulation were pipetted in the sample reservoir and collected again after nebulisation directly into 5 mL tubes (Sarstedt AG & Co. KG, Nümbrecht, Germany). The nebulisation time and volume of nebulised liposomes were measured for each formulation to calculate the aerosol output rate (L/min) and emitted volume (%). All formulations were nebulised at a total lipid concentration of 1 mg/mL.

#### *2.7. Dynamic Light Scattering and Laser Doppler Anemometry*

Particle size (hydrodynamic diameter applying intensity mode) and PdI (polydispersity index) of all liposomal formulations were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Kassel, Germany). All measurements were carried at a wavelength of 633 nm (HeNe laser) and 20.5 ◦C. For this purpose, a clear disposable folded capillary cell (DTS1060, Malvern Instruments, Kassel, Germany) was utilised. The instrument adjusted attenuation and measurement position of the laser automatically; the detection angle was 173◦ . Immediately before measurement, 900 µL liposomes were diluted with 100 µL HEPES (20 mM, pH 7.4), thus at the ratio of 1:10. All samples were measured three times with the instrument performing 3 runs with 15 subruns each, all sub-runs lasting 10 s.

Laser Doppler anemometry (LDA) was used to determine the ζ-potential with the Zetasizer Nano ZS. The liposomes were diluted as described above. All samples were measured three times and the instrument performed automatically 15–100 sub-runs. Values from three independent samples were considered.

#### *2.8. Encapsulation Efficiency and Loading Capacity*

To extract curcumin from the liposomes, the method of Duse et al. was applied [14,26]. Briefly, 300 µL of each formulation were centrifuged for 90 min at 2000× *g* (Eppendorf 5418 Centrifuge, Eppendorf AG, Hamburg, Germany) following further centrifugation steps to remove all non-dissolved lipids. The pellet was dissolved in 200 µL ethanol and 200 µL HEPES (20 mM, pH 7.4), 200 µL of the supernatant was mixed with 200 µL ethanol. Subsequently, curcumin in all samples was quantified spectrophotometrically at a wavelength of 425 nm using Multiskan GO plate reader (Thermo Fischer Scientific GmbH, Dreieich, Germany). The required calibration curve was generated with predetermined concentrations of curcumin in the same solvent composition and unloaded liposomes were

used as blank. Calculations of the encapsulation efficiency (EE) and loading capacity (LC) were then carried out with the following equations:

$$\text{EE}[\%] = \frac{\text{Current}\_{\text{encapusted}}}{\text{Current}\_{\text{total}}} \times 100\tag{1}$$

$$\text{LC}[\%] = \frac{\text{Current}\_{\text{encapulated}}}{\text{Ingradients}\_{\text{total}}} \times 100\tag{2}$$

#### *2.9. Atomic Force Microscopy*

The morphological characteristics of the liposomal formulations were studied with atomic force microscopy (AFM) using a NanoWizard® 3 (JPK Instruments AG, Berlin, Germany) with silicon cantilevers (HQ:NSC14/AL BS, Mikromasch Europe, Wetzlar, Germany). The measurements were carried out using intermittent contact mode at scan rates between 0.5 to 1 Hz to avoid damaging the lipid membrane [33]. A resonance frequency of 120 kHz and the force constant 5 N/m was used. Different formulations of nebulised liposomes were diluted 1:10 with ultrapure water, 10 µL of the mixture was placed onto glass slides and left to dry at room temperature. To gain the final images, the amplitude signal of the cantilever in the trace direction and the height signal in retrace direction were used and the images were processed using JPKSPM data processing software (JPK Instruments AG).

In addition, AFM images were analysed for size confirmation. For this purpose, the average liposomal size was determined by analysing a representative number of height images using ImageJ software (v1.52a, National Institutes of Health, Bethesda, MD, USA) [33,34].

#### *2.10. Transmission Electron Microscopy*

As a second method to examine the liposomal morphology, transmission electron microscopy (TEM) was used. The sample preparation was carried out on 400 mesh copper grids, coated with 1.2% Formvar and carbon (Plano GmbH, Wetzlar, Germany). Briefly, the liposomes were diluted to 1:10 ratio with HEPES (20 mM, pH 7.4) and 10 µL were placed on a grid. After 5 min of incubation, the liquid was withdrawn by suction with a Whatman 4 filter paper (Whatman plc, Maidstone, UK). Then the grids were placed for 5 min on 20 µL 2% uranyl acetate, which was used as contrast agent for the negative staining. The liquid was withdrawn by suction again and the grids were placed on 20 µL H2O as a washing step. After the remaining liquid was carefully removed, the grids were left to dry. To obtain the images, samples were examined using a Leo 912 AB TEM (Carl Zeiss Microscopy GmbH, Jena, Germany) with different magnifications and an accelerating voltage of 100 kV.

#### *2.11. Aerodynamic Properties*

To define the aerodynamic properties, a determination of the fine particle fraction (FPF) was carried out according to monograph 2.9.18 with device A (twin glass impinger) mentioned in the European Pharmacopoeia 9.5. Briefly, 7 mL and 30 mL of water:acetonitrile (1:1 (*v*/*v*)) were added to the top and bottom chamber, respectively. The device was assembled and connected to an Erweka vacuum pump VP 1000 (Erweka GmbH, Heusenstamm, Germany). Using a flow meter DFM 2000 (Copley Scientific, Nottingham, UK), the flow rate was adjusted to 60 ± 5 L/min. The liposomal formulations were pipetted into the sample reservoir of a PARI VELOX® nebuliser, which was linked to the glass twin impinger (Hannes & König GmbH, Heusenstamm, Germany) via mouthpiece. The vacuum pump was turned on 10 s before starting the nebulisation. After 60 s, the nebulisation process was stopped, and the vacuum pump was turned off 5 s afterwards. The amount of curcumin in

each chamber was quantified spectrophotometrically. FPF was then calculated according to the following equation:

$$\text{FFF}[\%] = \frac{\text{Current in bottom chamber}}{\text{Current in total}} \times 100\tag{3}$$

## *2.12. Mucous Membrane Compatibility*

The determination of mucosal tolerance for all liposomal formulations was performed by HET-CAM assay [35]. The chorio-allantoic membrane (CAM) is an extraembryonic membrane that results from the fusion of the shell membrane (chorion) with the allantoic cavity (rectal protrusion of the embryo) in a fertilized chicken egg [36]. For preparation, the fertilized chicken eggs had to be thoroughly cleaned with ethanol 70% (*v*/*v*) after delivery and incubated at 37.8 ◦C and 60% relative humidity in a Thermo de Luxe 250 incubator (Hemel Brutgeräte GmbH). Eggs were continuously turned 12 times per day until egg development day (EDD) 3. On EDD 3, a round incision (ø 28 mm) was added to the blunt side of the eggs with the aid of an EggPunch pneumatic egg opener (Schuett-Biotec GmbH). An opening was then created by carefully removing the shell as well as the shell membrane with curved Dumostar Style 7 forceps (Manufactures D'Outils Dumont SA). For protection, the opened eggs subsequently had to be covered with a sterile Petri dish and incubated in a vertical position in the incubator.

On EDD 9300 µL of nebulised liposomes were applied to the CAM and observed during 5 min under a Stemi 2000-C stereomicroscope (Carl Zeiss AG) at 13-fold magnification. The study was performed with 6 eggs per sample. HEPES (20 mM, pH 7.4) served as a negative control, sodium dodecyl sulfate (SDS) 1% (m/m) and 0.1 N sodium hydroxide were used as positive control. The determination was based on stereomicroscopic images taken with a Moticam 5 MP digital camera (Motic Deutschland GmbH) and an irritation score (*IS*), which included an assessment of haemolysis (*H*), vessel lysis (*L*), and blood coagulation (*C*) [37]. Events were photographed at the time of their occurrence and the irritation score was calculated as follows according to the literature [38]:

$$IS = \frac{(301 - H[s])}{300} \times 5 + \frac{(301 - L[s])}{300} \times 7 + \frac{(301 - C[s])}{300} \times 9 \tag{4}$$

#### *2.13. In Vitro Cytotoxicity*

A549 cells were seeded onto 96-well plates (NUNC, Thermo Fischer Scientific GmbH, Dreieich, Germany) with a seeding density of 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/0.35 cm<sup>2</sup> (per well) and the plates were incubated for 24 h. For the in vitro cytotoxicity assay, the cells were treated with various concentrations of all liposomal formulations using a serial dilution starting with 100 µM of curcumin. After 4 h of incubation, liposomal formulations were aspirated and replaced with fresh medium. The cells were irradiated at 457 nm using an LED device (Lumundus GmbH) with a fluence of 6.61 J cm−<sup>2</sup> . After 24 h, the cells were incubated with MTT reagent (0.2 mg/mL) for 4 h and DMSO was then used to dissolve the resulting formazan crystals. The absorbance was determined at a wavelength of 570 nm using FLUOstar® Optima (BMG Labtech, Ortenberg, Germany). Untreated cells (blank) and an unirradiated microtiter plate (dark plate) were used as controls. The viability of blank cells was considered as 100%.

#### *2.14. Cellular Uptake Studies*

Liposomal formulations can be absorbed into the cells in different ways and thus develop their effect. For a better understanding of these effects, the uptake pathways were determined. This was done by inhibiting two major pathways respectively, determining the cell survival rate and comparing it to that of untreated cells. Chlorpromazine served as an inhibitor of clathrin-mediated endocytosis, and filipin III inhibited caveolae-mediated endocytosis. As previously described, A549 cells were seeded at a density of 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well in 96-well plates and incubated for 24 h. Subsequently, these had to be washed

twice with PBS buffer containing Ca2+/Mg2+ to ensure removal of serum residues that could interfere with the effect of the inhibitors. Then, preincubation of chlorpromazine and filipin III (5 µg/mL each) for 30 min took place, followed by incubation with liposomes (100 µM each) for 4 h. Afterwards, the liposomes were replaced with fresh medium and irradiation of the cells took place at λ = 457 nm and with a fluence of 6.61 J/cm<sup>2</sup> . After further incubation of the cells for 24 h, a determination of cell viability was performed by in vitro cytotoxicity assay as described in Section 2.13. Untreated and treated but unirradiated cells as well as cells treated with inhibitor only were used as controls.

#### *2.15. Confocal Laser Scanning Microscopy (CLSM)*

A549 cells were seeded onto 15 mm coverslips inside 12-well plates (NUNC, Thermo Fischer Scientific GmbH, Dreieich, Germany) with a seeding density of 9 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/3.5 cm<sup>2</sup> (per well). The cells were treated with liposomal formulations containing 100 µM of curcumin and incubated for 4 h. After irradiation (6.61 J cm−<sup>2</sup> at 457 nm), the cells were washed twice with PBS buffer containing Ca2+ and Mg2+ (pH 7.4) and fixed with 4% formaldehyde solution. Afterwards, the cells were washed twice with PBS buffer and the cell nuclei were counterstained with 0.1 µg/mL 40 ,6-diamidino-2-phenylindole (DAPI). The cells were then washed with PBS buffer and the coverslips were mounted onto slides and sealed using FluorSave™ (Calbiochem Corp., La Jolla, CA, USA). The cells were examined using an LSM700 confocal laser-scanning microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). All micrographs were recorded at a similar adjustment.

#### *2.16. Cellular Migration*

The so called "in vitro scratch assay" was performed to determine the growth inhibitory effect of PDT with nebulised liposomes on A549 cells. For this assay, 9 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well were also seeded in 12-well plates and cell growth in the wells was observed until a confluent monolayer was formed (approximately after 48 h). After 4 h of incubation with nebulised liposomes of 100 µM concentration and a subsequent exchange with fresh DMEM medium, it was possible to scratch through the wells with a pipette tip and thus create a gap in the cell monolayer.

#### *2.17. Statistical Analysis*

All experiments were carried out in triplicates and the values are presented as mean ± standard deviation unless otherwise stated. The statistical significance of measured values was determined by using a two-tailed *t*-test and the probability values of *p* < 0.05 were considered statistically significant.

#### **3. Results and Discussion**

#### *3.1. Suitability of Nebulisers*

In this study, two nebulisers of different technology were compared to identify the device best suited for the nebulisation of liposomes. PARI VELOX® represents vibratingmesh nebulisation and PARI BOY® SX air-jet nebulisation. Vibrating-mesh nebulisers extrude liquid through a perforated mesh to generate aerosols, whereas air-jet nebulisers use compressed air to press the liquid through a nozzle of defined size (Figure 1a,b) [39,40].

Generally, PARI VELOX® showed a slightly slower output rate compared to PARI BOY® SX, but a much higher emitted dose. On the vibrating-mesh nebuliser, ultrapure water had a low output rate of 0.27 ± 0.04 mL/min due to a lower concentration of electrolytes [41]. However, the liposomal formulations rehydrated in 20 mM HEPES (pH 7.4) showed output rates of 0.48 ± 0.07 mL/min (DT), 0.50 ± 0.01 mL/min (DC) and 0.53 ± 0.03 mL/min (DD). According to the literature, the average output rate of PARI VELOX® for normal saline (0.9% NaCl) is 0.76 <sup>±</sup> 0.18 mL/min [42]. Air-jet nebulisation showed an output rate of 0.29 ± 0.03 mL/min for ultrapure water, 0.65 ± 0.05 mL/min for DT, 0.63 ± 0.03 mL/min for DC and 0.69 ± 0.01 mL/min for DD. The emitted dose of vibrating-mesh nebulisation was 93.5 ± 3.3% (*v*/*v*) for ultrapure water, 94.0 ± 1.5% (*v*/*v*) for

DT, 95.5 ± 1.3% (*v*/*v*) for DC and 94.7 ± 1.0% (*v*/*v*) for DD. Air-jet nebulisation revealed an emitted dose of 18.5 ± 2.3% (*v*/*v*) for ultrapure water and 15.3 ± 0.8% (*v*/*v*) (DT), 17.3 ± 1.3% (*v*/*v*) (DC), 14.8 ± 1.5% (*v*/*v*) (DD) for the liposomal formulations. Looking at the liposomal formulations, a slight difference is visible. For both nebulisers, DC had the highest emitted dose. However, the results of all formulations regarding output rate and emitted dose were in the same range for both nebulisers, which was probably because all liposomes were rehydrated with HEPES (20 mM, pH 7.4). The output rate of ultrapure water was much lower and, according to literature, the one of 0.9% NaCl much higher. Thus, an increased conductivity leads to a higher output rate. This confirms previous findings, that the concentration of electrolytes is a significant parameter influencing output rate due to a more efficient liquid breakdown during atomisation [43]. More fluidic liposomes also seem to promote a higher output rate, which can be seen in the case of DD with both nebulisers. It could be explained by smaller and easier separable liposomes which may decrease the liquid resistance. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 8 of 24 mesh nebulisation and PARI BOY® SX air-jet nebulisation. Vibrating-mesh nebulisers extrude liquid through a perforated mesh to generate aerosols, whereas air-jet nebulisers use compressed air to press the liquid through a nozzle of defined size (Figure 1a,b) [39,40].

**Figure 1.** Operating mode of both techniques: (**a**) vibrating-mesh nebulization, (**b**) air-jet nebulisa-**Figure 1.** Operating mode of both techniques: (**a**) vibrating-mesh nebulization, (**b**) air-jet nebulisation.

Generally, PARI VELOX® showed a slightly slower output rate compared to PARI BOY® SX, but a much higher emitted dose. On the vibrating-mesh nebuliser, ultrapure water had a low output rate of 0.27 ± 0.04 mL/min due to a lower concentration of electrolytes [41]. However, the liposomal formulations rehydrated in 20 mM HEPES (pH 7.4) showed output rates of 0.48 ± 0.07 mL/min (DT), 0.50 ± 0.01 mL/min (DC) and 0.53 ± 0.03 mL/min (DD). According to the literature, the average output rate of PARI VELOX® for normal saline (0.9% NaCl) is 0.76 ± 0.18 mL/min [42]. Air-jet nebulisation showed an output rate of 0.29 ± 0.03 mL/min for ultrapure water, 0.65 ± 0.05 mL/min for DT, 0.63 ± 0.03 mL/min for DC and 0.69 ± 0.01 mL/min for DD. The emitted dose of vibrating-mesh nebulisation was 93.5 ± 3.3% (*v/v*) for ultrapure water, 94.0 ± 1.5% (*v/v*) for DT, 95.5 ± 1.3% (*v/v*) for DC and 94.7 ± 1.0% (*v/v*) for DD. Air-jet nebulisation revealed an emitted dose of 18.5 ± 2.3% (*v/v*) for ultrapure water and 15.3 ± 0.8% (*v/v*) (DT), 17.3 ± 1.3% (*v/v*) (DC), 14.8 ± 1.5% (*v/v*) (DD) for the liposomal formulations. Looking at the liposomal formulations, In the recent literature the two nebulisation techniques are described to function with nanoscaled drug carrier systems [44,45] and are suitable for therapies with small volumes since both require a minimum sample volume of only 2 mL. They provide comparable lung deposition profiles and show reportedly no damage of precursor liposomes [39,45]. A comparison of the measured size values for both nebulisation techniques of all formulations (Table 2) revealed no significant difference (*p* > 0.05) between PARI VELOX® and PARI BOY® SX. PdI and ζ-potential likewise showed no major differences for both nebulisation techniques among all formulations, leading to the assumption that both nebulisers are capable of emitting intact liposomes, suitable for deep lung deposition. However, vibratingmesh nebulisation seemed more suitable than air-jet nebulisation due to a very high emitted dose and a much better handling with similar output rates. Additionally, PARI VELOX® combines other advantages such as being portable, quiet and cost-effective, making the therapy patient-friendly as well. It was therefore used for all further experiments in this study.

#### a slight difference is visible. For both nebulisers, DC had the highest emitted dose. However, the results of all formulations regarding output rate and emitted dose were in the *3.2. Physicochemical Properties of the Liposomal Formulations*

tion.

liquid resistance.

same range for both nebulisers, which was probably because all liposomes were rehydrated with HEPES (20 mM, pH 7.4). The output rate of ultrapure water was much lower and, according to literature, the one of 0.9% NaCl much higher. Thus, an increased conductivity leads to a higher output rate. This confirms previous findings, that the concentration of electrolytes is a significant parameter influencing output rate due to a more efficient liquid breakdown during atomisation [43]. More fluidic liposomes also seem to promote a higher output rate, which can be seen in the case of DD with both nebulisers. It could be explained by smaller and easier separable liposomes which may decrease the One focus in the course of drug nebulisation was the application of liposomes [39,46]. State of the art in pulmonary drug delivery for the treatment of lung cancer is the nebulisation of different nanocarriers such as nanoparticles, nanoemulsions and liposomes [47–50]. To achieve a more localised drug delivery, different modifications and targets were tested so far [47,51]. The liposomes tested for the treatment of lung cancer in this study have a great advantage, as the used lipids are biocompatible, biodegradable and present in clinical trials or even approved for clinical use [52]. In the field of pulmonary delivery, several liposomal formulations have been tested in animal studies and clinical trials as well [53].

In the recent literature the two nebulisation techniques are described to function with nanoscaled drug carrier systems [44,45] and are suitable for therapies with small volumes since both require a minimum sample volume of only 2 mL. They provide comparable lung deposition profiles and show reportedly no damage of precursor liposomes [39,45]. A comparison of the measured size values for both nebulisation techniques of all formu-

PARI BOY® SX. PdI and ζ-potential likewise showed no major differences for both nebulisation techniques among all formulations, leading to the assumption that both nebulisers are capable of emitting intact liposomes, suitable for deep lung deposition. However, vibrating-mesh nebulisation seemed more suitable than air-jet nebulisation due to a very high emitted dose and a much better handling with similar output rates. Additionally, PARI VELOX® combines other advantages such as being portable, quiet and cost-effective, **Table 2.** DLS analysis of DT (DPPC:TEL), DC (DPPC:Chol) and DD (DPPC:DOPE). Curcumin-loaded liposomes nebulised via vibrating-mesh (PARI VELOX®) and air-jet nebulisation (PARI BOY® SX) were compared with each other, respectively. Unnebulised curcumin-loaded liposomes were used as control. Values of particle size represent the distribution by intensity. Independent formulations were used to measure the triplicates and all results were expressed as mean ± standard deviation.


<sup>1</sup> Polydispersity index.

As there are many suitable lipids for the preparation of liposomes, the present study aimed to compare different representative liposomal compositions. DT (DPPC:TEL 90:10) is the composition with the highest degree of membrane stabilisation as the tetraether lipids are crossing the entire lipid bilayer. This is due to the ring structure of these lipids, originated from four ether bonds (Figure 2b), implementing a tight packing of the lipid bilayer and a rigid behaviour of these liposomes. In addition, TEL reduces liposomal sensitivity towards oxidation [26]. DC (DPPC:Chol 70:30) is a standard medium rigid composition using cholesterol for membrane stabilisation, which is incorporated in the lipid bilayer. The composition DD (DPPC:DOPE 75:25) is more fluidic than the others, with no stabilisation of the membrane (Figure 2a). Upon rehydration of the lipid films, curcumin is packed inside the liposomal bilayer of all formulations having hydrogen bonds to the lipid acyl chains. This encapsulation protects curcumin from degradation and greatly increases its solubility in aqueous media [26,27].

Particle size, PdI and ζ-potential of unnebulised and nebulised liposomal formulations were compared (Table 2), as stable delivery systems are key requirements for a successful application. Looking closer at the results, a few differences between the formulations were noticed. Prior to nebulisation, DT liposomes were slightly larger in diameter than the other formulations due to TEL crossing the lipid bilayer. Particle size and PdI were in the same range before and after nebulisation for this formulation, making it inured to nebulisation. This may be explained by increased packing and stronger hydrogen bonds within the lipid bilayer, reducing the effects of nebulisation on these liposomes [54]. DT also had a more negative ζ-potential compared to the other formulations, which can be explained by the ether bonds of TEL. DC liposomes were smaller than DT, probably due to the smaller cholesterol molecule. Likewise, particle size and PdI were in the same range after nebulisation, revealing them stable to nebulisation as well. Cholesterol can decrease the bilayer fluidity by reducing the movement of the lipid hydrocarbon chains and increase the packing of lipid head groups, which can lead to a smaller particle size [40]. In contrast, DD was a more fluidic formulation due to DOPE, which can destabilise the membrane as it may stimulate a transition from a lamellar to a hexagonal phase [55,56]. This fluidic behaviour could cause the increase in size and PdI after nebulisation, which can be seen in Table 2. Therefore, DD seemed less stable to forces during the process of nebulisation. Liposomal compositions and the total lipid concentration can influence the nebulisation, but conversely, the different nebulisation techniques can have an impact on size, uniformity and integrity of liposomes [57]. It can be seen in this study, that

nebulisation with compressed air leads to a lower uniformity of all liposomal formulations. This could be explained by higher shearing forces while pushing the liquid through the nozzle. Both techniques also have a slight impact on liposome sizes and ζ-potentials. The negative ζ-potential of all formulations could be related to the entrapment of curcumin, the different extent seems depending on the liposomal formulation [26]. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 10 of 24

**Figure 2.** (**a**) Model compositions of liposomes: DT with tetraether lipids providing the highest degree of membrane stabilisation, DC as medium stabilised standard composition with cholesterol and the fluidic composition of DD with no stabilisation of the membrane. (**b**) Structures of the tetraether lipids used in this study. (**1**) GDGT, glycerol dialkylglycerol tetraether and (**2**) GDNT, glycerol dialkylnonitol tetraether with R1: inositol, R2: β-D-glucopyranose and R3: β-D-galactosyl-β-Dglucopyranose. **Figure 2.** (**a**) Model compositions of liposomes: DT with tetraether lipids providing the highest degree of membrane stabilisation, DC as medium stabilised standard composition with cholesterol and the fluidic composition of DD with no stabilisation of the membrane. (**b**) Structures of the tetraether lipids used in this study. (**1**) GDGT, glycerol dialkylglycerol tetraether and (**2**) GDNT, glycerol dialkylnonitol tetraether with R1: inositol, R2: β-D-glucopyranose and R3: β-D-galactosylβ-D-glucopyranose.

Particle size, PdI and ζ-potential of unnebulised and nebulised liposomal formulations were compared (Table 2), as stable delivery systems are key requirements for a successful application. Looking closer at the results, a few differences between the formulations were noticed. Prior to nebulisation, DT liposomes were slightly larger in diameter than the other formulations due to TEL crossing the lipid bilayer. Particle size and PdI were in the same range before and after nebulisation for this formulation, making it inured to nebulisation. This may be explained by increased packing and stronger hydrogen bonds within the lipid bilayer, reducing the effects of nebulisation on these liposomes [54]. DT also had a more negative ζ-potential compared to the other formulations, which can be explained by the ether bonds of TEL. DC liposomes were smaller than DT, probably due to the smaller cholesterol molecule. Likewise, particle size and PdI were in the same To increase the storage stability, the three liposomal formulations were lyophilised directly after preparation, rehydrated with ultrapure water and nebulised using PARI VELOX®. It was observed that the ζ-potential of these formulations was more negative, which could be explained by a leakage and attachment of curcumin to the outer surface of the liposomes. This observation is confirmed by results of the encapsulation efficiency (Table 3). A change in the ζ-potential can affect the mucoadhesive properties of the liposomes as they are based on the interaction between charged molecules [58]. Size and PdI of all formulations were also increased. Source of this increase could be the cryoprotectant D-(+)-trehalose dehydrate itself, due to its penetration into the liposomes as it leads to a stabilisation of liposomes from inside and outside [59]. According to literature, lyophilising

range after nebulisation, revealing them stable to nebulisation as well. Cholesterol can

and increase the packing of lipid head groups, which can lead to a smaller particle size [40]. In contrast, DD was a more fluidic formulation due to DOPE, which can destabilise the membrane as it may stimulate a transition from a lamellar to a hexagonal phase [55,56]. without cryoprotectant leads usually to intense aggregation or fusion [59,60]. Possible causes for the good protective properties of D-(+)-trehalose dehydrate can be the high glass transition temperature and the low tendency to crystallisation [59]. However, lyophilisation prior to nebulisation seemed to have a negative influence on all three formulations.

**Table 3.** Encapsulation efficiency (EE) and loading capacity (LC) of DT (DPPC:TEL), DC (DPPC:Chol) and DD (DPPC:DOPE) liposomes. The results in this table were calculated according to Equations (1) and (2) in Section 2.8 for liposomes containing a curcumin concentration of 0.1 mg per 1 mg lipids. Unnebulised curcumin-loaded liposomes were used as control.


Altogether, the membrane stabilised formulations DT and DC appeared stable to nebulisation whereas DD as fluidic formulation did not seem eligible.

Generally, liposomes are very convenient as vehicles for all kinds of drugs due to their relatively high encapsulation efficiency [9,26,46]. The lipophilic drug curcumin was used in this study with 100 µg curcumin per 1 mL liposomes. Typically, curcumin is non-uniformly distributed in the lipid bilayer and entirely located inside the hydrophobic interior, which is important for a high drug loading capacity [27]. Previous research indicated that EE and LC are dependent on the composition of lipid acyl chains forming the liposomal bilayer. Lipids containing shorter acyl chains and hence, a lower Tc, are mostly able to incorporate a larger amount of hydrophobic substances such as curcumin due to altered van der Waals linkages [60]. Additionally, unilamellar vesicles, as they emerge after extrusion through polycarbonate membranes, are considered to have better drug incorporation into the bilayer than multilamellar vesicles [46]. These findings stand in accordance with the results of this study, as Table 3 showed relatively high encapsulation efficiencies for all un-nebulised liposomes. Looking closer at the three formulations, a difference in behaviour was visible. Upon addition of TEL, a high encapsulation efficiency to the extent of 93.9% was achieved. This could be due to the fact that TEL, by crossing the lipid bilayer with its acyl chains containing cyclopentane structures, provided a tight membrane packing through more van der Waals linkages. Resultant in a very good capability of encapsulating hydrophobic substances [61,62]. Both, EE and LC seemed quite stable after the process of nebulisation, which speaks again for the high degree of membrane stability as it makes the liposomes less permeable by preserving membrane integrity. Stabilisation through the bipolar structure of TEL also provides thermal stability [63] and better long-term storage stability rates [64]. Besides, TEL can positively affect the intermembrane exchange of the active ingredient and thereby enhance the delivery [64]. The linkages of Cholesterol inside the lipid bilayer of DC similarly promote the encapsulation of curcumin [60], but with 88.4% (Table 3) to a lower extent. DD, which contains the fluidic lipid DOPE, showed the lowest encapsulation efficiency of 85.1%. Compared to the un-nebulised liposomes, EE values of nebulised curcumin-loaded liposomes were decreased. The slight effect of DT can be explained by leftover curcumin attached to the outer surface of the liposomes, remaining even after extrusion [46]. The residual amount of encapsulated curcumin in DC was also relatively high, as the moderate stabilisation with Cholesterol also leads to decreased leakage rates, whereas for DD a much stronger effect was visible probably due to a disintegration of the fluidic liposomes followed by a loss of curcumin. These

findings stand in accordance with the ones of DLS analysis as the changes in size, PdI and ζ-potential were correspondent to the different behaviour of the formulations. This is also the case for lyophilised and nebulised curcumin-liposomes, which had the lowest EE and LC. Again, the cryoprotectant D-(+)-trehalose dehydrate could be the cause, as it can replace curcumin inside the liposomal bilayer [58].

With respect to EE and LC of liposomes, another aspect that must be taken under consideration is the state of entrapped curcumin. Hydrophilic drugs encapsulated in the aqueous core have a high tendency to aggregate which can compromise their therapeutic impact, whereas lipophilic drugs such as curcumin have a much lower tendency to aggregate within the bilayer, probably also due to the presence of van der Waals linkages with the lipid acyl chains building a barrier [60].

Again, the membrane stabilisation of DT and DC seemed to be an advantage. The EE and LC of both formulations were high, and the values of DT were quite stable after nebulisation. However, DD, with no membrane stabilisation, exhibited an unstable behaviour since the EE decreased constantly during lyophilisation and nebulisation. The lack of membrane stabilisation revealed a higher loss of entrapped curcumin making DD not suitable for nebulisation after all.

#### *3.3. AFM Visualisation*

AFM images revealed spherically shaped vesicles for all formulations after nebulisation (Figure 3). Smooth surfaces of the vesicles indicated complete incorporation of curcumin. Images of DD (Figure 3e,f), the most fluidic formulation, exhibited several small spherically shaped vesicles, presumably due to a disintegration through the vibrating mesh during the process of nebulisation. This disintegration could be explained by the fluidic behaviour and positive charge, that both lipids have in this formulation. Conductivity greatly affects the process of nebulisation [65], in this case the charge probably led to an interaction with the surface of the metal-based mesh promoting further disintegration. Another explanation can be the high Tc, originated by long hydrocarbon chains. Below this temperature, liposomes act more like solids than as fluids, increasing the probability of decomposition [60]. This stands in accordance with the results of EE and LC, as DD revealed to be not suitable for nebulisation. In contrast, the highly stabilised formulation DT (Figure 3a,b) and the medium stabilised formulation DC (Figure 3c,d) showed constant behaviour in terms of their size. Although the effectiveness of inhalable therapies depends on the aerodynamic properties of the nebulised vehicles, the output of intact liposomes from the nebuliser, which was achieved in this study, is a fundamental requirement for the applicability of the therapy [66]. Liposomes of all formulations appeared slightly bigger in the AFM images. On the one hand, probably due to spreading on the glass slides while they were left to dry as a part of the sample preparation, on the other hand, due to different measuring conditions compared to DLS. DLS results were obtained in aqueous conditions whereas AFM measurements took place under dry conditions. However, size analysis using the height images (Figure 3) stood in agreement with the results obtained with DLS.

#### *3.4. TEM Visualisation*

Transmission electron microscopy enabled a precise examination of morphological differences between the liposomal formulations after nebulisation with PARI VELOX®. Through the method of negative staining with uranyl acetate it was possible to image the liposomes in their original environment, which was HEPES (20 mM, pH 7.4) in this study. Likewise seen in the particular AFM images, spherically shaped vesicles were visible for the three formulations. The most rigid formulation DT appeared as well-defined vesicles, which were slightly dented (Figure 4a,b). This was in accordance with AFM measurements. The liposomal diameter determined from TEM images corresponded to the ones obtained via DLS and AFM. These findings indicated the stability of tetraether liposomes during the process of nebulisation. The appearance of the membrane stabilised DC also matched the one in AFM images, as the vesicles were equal round and with nearly uniform sizes

(Figure 4d). Additionally, TEM visualisation revealed the lipid bilayer of DC liposomes in the magnified image (Figure 4c) and hence their intactness. Likewise, the vesicle diameter determined from TEM images corresponded to previous ones indicating DC's stability towards nebulisation. DD, as the most fluidic formulation in this study, showed different behaviour. A few vesicles were identified as intact liposomes due to the visible lipid bilayer (Figure 4e), but the majority was found to be spread on the grid (Figure 4f). For this reason, the diameter determined from TEM images appeared larger compared to the ones obtained by DLS, excluded the intact vesicles. Looking closer at Figure 4f, tiny black spots were visible, matching the results of CLSM for DD. A possible explanation for these spots could be the agglomeration of curcumin after rupture and leakage of the fluidic liposomes during nebulisation. These results indicated the infeasibility of the fluidic formulation DD for nebulisation and were in accordance with the results of AFM, CLSM, DLS and measurements of the encapsulation efficiency. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 13 of 24 constant behaviour in terms of their size. Although the effectiveness of inhalable therapies depends on the aerodynamic properties of the nebulised vehicles, the output of intact liposomes from the nebuliser, which was achieved in this study, is a fundamental require-

#### *3.5. Aerodynamic Properties* ment for the applicability of the therapy [66]. Liposomes of all formulations appeared slightly bigger in the AFM images. On the one hand, probably due to spreading on the

Aerosols are the products generated from drug solutions or dispersions during the process of nebulisation and their aerodynamic properties determine the location and intensity of the effect [57]. Measurements of the FPF according to monograph 2.9.18 of the 10th Edition of the European Pharmacopoeia were used to specify the aerodynamic characteristics after nebulisation with PARI VELOX®. glass slides while they were left to dry as a part of the sample preparation, on the other hand, due to different measuring conditions compared to DLS. DLS results were obtained in aqueous conditions whereas AFM measurements took place under dry conditions. However, size analysis using the height images (Figure 3) stood in agreement with the results obtained with DLS.

**Figure 3.** Atomic force microscopy (AFM) visualisation of DT, DC and DD after nebulisation with PARI VELOX® , containing a curcumin concentration of 100 µM. Images (**a**,**c**,**e**) are amplitude signals and (**b**,**d**,**f**) are height signals. Liposomes in height images together with their cross-sectional profiles were used for a size evaluation along the shown lines and a morphological characterisation. The stated sizes are mean values ± standard deviation gathered by analysing representative height images using ImageJ software. Scale bars represent 200 nm. **Figure 3.** Atomic force microscopy (AFM) visualisation of DT, DC and DD after nebulisation with PARI VELOX®, containing a curcumin concentration of 100 µM. Images (**a**,**c**,**e**) are amplitude signals and (**b**,**d**,**f**) are height signals. Liposomes in height images together with their cross-sectional profiles were used for a size evaluation along the shown lines and a morphological characterisation. The stated sizes are mean values ± standard deviation gathered by analysing representative height images using ImageJ software. Scale bars represent 200 nm.

Transmission electron microscopy enabled a precise examination of morphological differences between the liposomal formulations after nebulisation with PARI VELOX®

Through the method of negative staining with uranyl acetate it was possible to image the

the three formulations. The most rigid formulation DT appeared as well-defined vesicles, which were slightly dented (Figure 4a,b). This was in accordance with AFM measurements. The liposomal diameter determined from TEM images corresponded to the ones obtained via DLS and AFM. These findings indicated the stability of tetraether liposomes during the process of nebulisation. The appearance of the membrane stabilised DC also matched the one in AFM images, as the vesicles were equal round and with nearly uniform sizes (Figure 4d). Additionally, TEM visualisation revealed the lipid bilayer of DC

.

*3.4. TEM Visualisation*

AFM, CLSM, DLS and measurements of the encapsulation efficiency.

liposomes in the magnified image (Figure 4c) and hence their intactness. Likewise, the vesicle diameter determined from TEM images corresponded to previous ones indicating DC's stability towards nebulisation. DD, as the most fluidic formulation in this study, showed different behaviour. A few vesicles were identified as intact liposomes due to the visible lipid bilayer (Figure 4e), but the majority was found to be spread on the grid (Figure 4f). For this reason, the diameter determined from TEM images appeared larger compared to the ones obtained by DLS, excluded the intact vesicles. Looking closer at Figure 4f, tiny black spots were visible, matching the results of CLSM for DD. A possible explanation for these spots could be the agglomeration of curcumin after rupture and leakage of the fluidic liposomes during nebulisation. These results indicated the infeasibility of the fluidic formulation DD for nebulisation and were in accordance with the results of

**Figure 4.** Transmission electron microscope (TEM) images of the liposomal formulations DT, DC and DD after nebulisation, containing a curcumin concentration of 100 µM. The samples were negatively stained with 2% uranyl acetate. Scale bars represent 100 nm for the images (**a**,**c**,**e**) and 1 µm for the images (**b**,**d**,**f**)**. Figure 4.** Transmission electron microscope (TEM) images of the liposomal formulations DT, DC and DD after nebulisation, containing a curcumin concentration of 100 µM. The samples were negatively stained with 2% uranyl acetate. Scale bars represent 100 nm for the images (**a**,**c**,**e**) and 1 µm for the images (**b**,**d**,**f**).

Suitable droplet size distribution, velocity and trajectory of an aerosol optimises the desired pulmonary deposition. Hence, it is important to know which parameters have an impact on these properties and which have none. Higher conductivity of the precursor fluid leads to a more efficient liquid breakdown [65]. Therefore, 20 mM HEPES pH 7.4 seems appropriate for the rehydration of lipid films as it facilitates a good output rate during nebulisation. However, the precise differences between the HEPES buffered liposomal formulations and ultrapure water regarding the output rate are shown in Section 3.1. Surface tension and reservoir volume do not have a relevant impact on aerodynamic properties, as described in the literature [65]. In addition, the vibrating mesh is moving too fast to give surface tension a chance to improve the liquid breakdown [65]. Since nanocarriers are mostly used to encapsulate small amounts of highly potent active ingredients, they are produced in small volumes. To ensure a successful therapy, it has to be certain that these small volumes get nebulised in a reliable manner, which again speaks for the suitability of vibrating-mesh nebulisers in this context, as the hydrostatic pressure on the nebuliser mesh does not affect the aerosol diameter [65]. Previous studies revealed that aerosol droplet sizes have to be between 1 and 6 µm for an optimal effect. Droplets larger than 6 µm may

deposit in the mouth or throat, droplets smaller than 1 µm can be exhaled, both cases lead to ineffectiveness [67,68]. According to literature, with PARI VELOX® up to 75% of the produced aerosol droplets are smaller than 6 µm and the average volumetric-median-diameter of the aerosol droplets is 3.6 to 4.4 µm [42]. The FPF measured in this study was defined by monograph 2.9.18 of the European Pharmacopoeia as droplets smaller than 6.4 µm and was stated as percentage ± standard deviation of active ingredient from the total nebulised amount of the latter. The liposomal formulation DC showed the best result with an FPF of 62.7 ± 1.6%, DT showed 59.5 ± 2.4% and DD 58.2 ± 1.4%. For the previously lyophilised liposomes, the FPF was 57.3 ± 3.8% for DT, 55.9 ± 6.2% for DC and 58.0 ± 5.4% for DD.

Overall, the FPF of all three nebulised formulations was relatively high and in the same range. This indicates that the composition and stability of liposomes does not affect the liquid breakdown and that 20 mM HEPES pH 7.4 provides a sufficient conductivity for nebulisation. Among the previously lyophilised liposomes, the stabilisation of DT seemed to be a slight advantage.

#### *3.6. Mucous Membrane Compatibility*

The mucous membrane compatibility of the nebulised curcumin-loaded liposomes was tested on the CAM and resulted in an irritation index of 0 for all three formulations. This means that none of the liposomal formulations caused a negative reaction, consequently mucosal intolerance. Therefore, the images were taken at the beginning as well as at the endpoint of the test (5 min) and show no changes (Figure 5). The positive control shows the occurrence of bleeding (Figure 5j). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 16 of 24

**Figure 5.** HET-CAM assay for mucous membrane compatibility of nebulised curcumin-loaded liposomes (**a**–**f**). HEPES 20 mM pH 7.4 served as negative control (**g**,**i**); 0.1 N NaOH as positive control (**h**,**j**)**.** Scale bars represent 1000 nm. **Figure 5.** HET-CAM assay for mucous membrane compatibility of nebulised curcumin-loaded liposomes (**a**–**f**). HEPES 20 mM pH 7.4 served as negative control (**g**,**i**); 0.1 N NaOH as positive control (**h**,**j**)**.** Scale bars represent 1000 nm.

To determine the photodynamic effect of the nebulised curcumin-liposomes, MTT

Qualitative evidence of the internalisation of nebulised curcumin-loaded liposomes into A549 cells was given via visualisation by confocal laser scanning microscopy. After

acceptable range for all formulations [14,69]. The cell viabilities after treatment with irradiated nebulised liposomes were 10.99 ± 3.04% for DT, 12.76 ± 0.58% for DC and 24.90 ± 2.70% for DD (Figure 6a). All formulations showed a significant difference (*p* < 0.05) between dark control and irradiated nebulised samples, as well as between dark control and irradiated un-nebulised samples, respectively. This indicates an excellent cytotoxic efficacy and an improved nebulisation suitability of liposomal formulations containing tetraether lipids due to the strong adhesion forces within their lipid bilayer [62]. Besides, the ability of TEL to positively affect the intermembrane exchange of the active ingredient, thus enhancing the delivery [64], was also confirmed by these results. The difference between dark control and irradiated lyophilised and nebulised curcumin-liposomes was also significant (*p* < 0.05) for DT and DC, but not for DD. A decreasing cytotoxic effect from DT to DD was visible. Again, these results validate the improved stability of DT and DC liposomes towards nebulisation and the general eligibility of nebulised liposomes for pulmonary PDT. In addition, the results confirm the fact that curcumin is a suitable pho-

*3.7. In Vitro Cytotoxicity, Cellular Uptake and Migration*

tosensitiser for a PDT of tumours [70,71].

#### *3.7. In Vitro Cytotoxicity, Cellular Uptake and Migration*

To determine the photodynamic effect of the nebulised curcumin-liposomes, MTT cytotoxicity studies were made. They revealed low dark toxicities of 88.32 ± 4.14% cell viability for DT, 85.03 ± 3.30% for DC and 85.73 ± 1.26% for DD, which was within the acceptable range for all formulations [14,69]. The cell viabilities after treatment with irradiated nebulised liposomes were 10.99 ± 3.04% for DT, 12.76 ± 0.58% for DC and 24.90 ± 2.70% for DD (Figure 6a). All formulations showed a significant difference (*p* < 0.05) between dark control and irradiated nebulised samples, as well as between dark control and irradiated un-nebulised samples, respectively. This indicates an excellent cytotoxic efficacy and an improved nebulisation suitability of liposomal formulations containing tetraether lipids due to the strong adhesion forces within their lipid bilayer [62]. Besides, the ability of TEL to positively affect the intermembrane exchange of the active ingredient, thus enhancing the delivery [64], was also confirmed by these results. The difference between dark control and irradiated lyophilised and nebulised curcumin-liposomes was also significant (*p* < 0.05) for DT and DC, but not for DD. A decreasing cytotoxic effect from DT to DD was visible. Again, these results validate the improved stability of DT and DC liposomes towards nebulisation and the general eligibility of nebulised liposomes for pulmonary PDT. In addition, the results confirm the fact that curcumin is a suitable photosensitiser for a PDT of tumours [70,71].

Qualitative evidence of the internalisation of nebulised curcumin-loaded liposomes into A549 cells was given via visualisation by confocal laser scanning microscopy. After incubation with nebulised formulations containing 100 µM curcumin, a considerable accumulation of the photosensitiser was detected within the cells, which is evident from the z-stack images. Furthermore, for DT and DC a homogenous distribution of curcumin inside the cells was clearly visible (Figure 6b–f). Curcumin from the formulation DD appeared as crystal-like agglomerates (Figure 6b,g,h), probably due to its instability towards nebulisation resulting in a leakage of curcumin. AFM micrographs of DD (Figure 3e,f), recorded after nebulisation of the curcumin-loaded liposomes, showed small liposomal fragments which confirms the thesis of fluidic liposomes being unstable towards nebulisation. Results of the encapsulation efficiency confirm this thesis as well, as the value dropped from 85.1% to 57.5% (Table 3) for DD. Curcumin, originally encapsulated in the lipid bilayer of DD liposomes, was liberated and hence, given the opportunity to aggregate. Similar behaviour of ultra-deformable liposomes regarding fragmentation, decreasing encapsulation efficiency and aggregation of active ingredient occurred in the study of Elhissi et al. [40]. Although no difference in the cell appearance was observable in these micrographs, the assumption that the irradiated nebulised curcumin-loaded liposomes were the source of the cytotoxic effect (Figure 6a), seems very likely and is consistent with the literature [26].

One of the aims of encapsulating a drug in nanocarriers such as liposomes is to transport it to the site of action, in this study adenocarcinoma cells of the lung. For this purpose, the active ingredient must be taken up into the cells as a final step, which can take place by various mechanisms. The most important mechanism is endocytosis, which can play a significant role especially for hydrophobic drugs [72]. This active process can occur without external stimuli or can be induced by ligands [73]. Initially, an insertion is formed in the cell membrane for this purpose, in which extracellular substances or particles are trapped and which is subsequently internalized by constriction. Different endocytosis pathways can be distinguished; of relevance in this study were clathrinmediated endocytosis on the one hand, and calveolae-mediated endocytosis on the other. Macropinocytosis as well as non-clathrin-non-calveolae-mediated endocytosis can also occur, but were not considered in this work.

**Figure 6.** (**a**) MTT in vitro cytotoxicity assay of DT, DC and DD containing a curcumin concentration up to 100 µM. The figure shows a comparison of dark control, irradiated (IR) unnebulised, IR nebulised (PARI VELOX® ) and IR lyophilised & nebulised curcumin-loaded liposomes. Irradiation always took place at 457 nm with a fluence of 6.61 J cm<sup>−</sup><sup>2</sup> ; (**b**) CLSM micrographs of A549 cells with nebulised curcumin-loaded liposomes containing a curcumin concentration of 100 µM. Dark and irradiated (457 nm, 6.61 J cm<sup>−</sup><sup>2</sup> ) samples were compared for the formulations: DT, (**c**,**d**); DC, (**e**,**f**); and DD, (**g**,**h**). Untreated cells were used for the blank micrographs (**a**,**b**). The cell nuclei were counterstained with 0.1 µg/mL DAPI and fixed with a 4% formaldehyde solution for all micrographs. Scale bars represent 20 µM. **Figure 6.** (**a**) MTT in vitro cytotoxicity assay of DT, DC and DD containing a curcumin concentration up to 100 µM. The figure shows a comparison of dark control, irradiated (IR) unnebulised, IR nebulised (PARI VELOX®) and IR lyophilised & nebulised curcumin-loaded liposomes. Irradiation always took place at 457 nm with a fluence of 6.61 J cm−<sup>2</sup> ; (**b**) CLSM micrographs of A549 cells with nebulised curcumin-loaded liposomes containing a curcumin concentration of 100 µM. Dark and irradiated (457 nm, 6.61 J cm−<sup>2</sup> ) samples were compared for the formulations: DT, (**c**,**d**); DC, (**e**,**f**); and DD, (**g**,**h**). Untreated cells were used for the blank micrographs (**a**,**b**). The cell nuclei were counterstained with 0.1 µg/mL DAPI and fixed with a 4% formaldehyde solution for all micrographs. Scale bars represent 20 µM.

One of the aims of encapsulating a drug in nanocarriers such as liposomes is to transport it to the site of action, in this study adenocarcinoma cells of the lung. For this purpose, the active ingredient must be taken up into the cells as a final step, which can take place by various mechanisms. The most important mechanism is endocytosis, which To identify the preferred endocytosis pathway of the nebulised curcumin liposomes, appropriate controls were first used to ensure that the inhibitors alone did not reduce cell viability. It can be seen that they have little effect on cell viability (Figure 7). Chlorpromazine

served as an inhibitor of clathrin-mediated endocytosis, and filipin III inhibited caveolaemediated endocytosis. When liposomes were applied after preincubation with filipin III, DT and DC after irradiation showed that the effects without inhibition were comparable to the effects after inhibition of caveolae-mediated endocytosis. This implies that liposome uptake into cells was possible in both cases and suggests that this endocytosis pathway is not the preferred one. The crossmatch after preincubation with chlorpromazine confirms this, as cell viability is significantly increased. Blockade of clathrin-mediated endocytosis inhibited liposome uptake to a large extent. These results agree with previous studies, according to which liposomes with a diameter of up to 200 nm are mainly taken up via the formation of so-called "clathrin-coated pits" and subsequent endocytosis [74]. In contrast, the DD formulation also appears to be taken up via caveolae-mediated to a lesser extent, with clathrin-mediated endocytosis still being preferred (Figure 7). Again, this could be related to the altered size distribution of these fluid liposomes. zine served as an inhibitor of clathrin-mediated endocytosis, and filipin III inhibited caveolae-mediated endocytosis. When liposomes were applied after preincubation with filipin III, DT and DC after irradiation showed that the effects without inhibition were comparable to the effects after inhibition of caveolae-mediated endocytosis. This implies that liposome uptake into cells was possible in both cases and suggests that this endocytosis pathway is not the preferred one. The crossmatch after preincubation with chlorpromazine confirms this, as cell viability is significantly increased. Blockade of clathrin-mediated endocytosis inhibited liposome uptake to a large extent. These results agree with previous studies, according to which liposomes with a diameter of up to 200 nm are mainly taken up via the formation of so-called "clathrin-coated pits" and subsequent endocytosis [74]. In contrast, the DD formulation also appears to be taken up via caveolae-mediated to a lesser extent, with clathrin-mediated endocytosis still being preferred (Figure 7). Again, this could be related to the altered size distribution of these fluid liposomes.

can play a significant role especially for hydrophobic drugs [72]. This active process can occur without external stimuli or can be induced by ligands [73]. Initially, an insertion is formed in the cell membrane for this purpose, in which extracellular substances or particles are trapped and which is subsequently internalized by constriction. Different endocytosis pathways can be distinguished; of relevance in this study were clathrin-mediated endocytosis on the one hand, and calveolae-mediated endocytosis on the other. Macropinocytosis as well as non-clathrin-non-calveolae-mediated endocytosis can also

To identify the preferred endocytosis pathway of the nebulised curcumin liposomes, appropriate controls were first used to ensure that the inhibitors alone did not reduce cell viability. It can be seen that they have little effect on cell viability (Figure 7). Chlorproma-

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 19 of 24

occur, but were not considered in this work.

**Figure 7.** Determination of the endocytotic uptake mechanism of nebulised curcumin liposomes after irradiation with a fluence of 6.61 J cm−2 at λ = 457 nm. The endocytosis pathways were either inhibited by filipin III or chlorpromazine or were not inhibited. The incubation time of liposomes was 4 h, and untreated cells represented the 100% value of cell viability. **Figure 7.** Determination of the endocytotic uptake mechanism of nebulised curcumin liposomes after irradiation with a fluence of 6.61 J cm−<sup>2</sup> at λ = 457 nm. The endocytosis pathways were either inhibited by filipin III or chlorpromazine or were not inhibited. The incubation time of liposomes was 4 h, and untreated cells represented the 100% value of cell viability.

In addition, the influence of PDT with nebulised liposomes on the migration behaviour of A549 cells was determined in this study. This was done by means of an "in vitro In addition, the influence of PDT with nebulised liposomes on the migration behaviour of A549 cells was determined in this study. This was done by means of an "in vitro scratch assay", a method based on the observation of an artificially created scratch in the confluent cell monolayer. The cells at the edge of this gap move towards the open area, which happens with the aim of closing the gap as quickly as possible until new cell-cell contacts have been created [75].

The results of this assay show that the natural migration of untreated cells closed the gap in the cell monolayer within 48 h (Figure 8a,e,i,n,r,v). In comparison, it can be seen that the gap in the cells treated with PDT does not close within this time period. The effects on the cell migration behaviour seems to be more distinct for DT and DC, in agreement with the previous results. From the time of irradiation onwards, especially in the case of DT, many cells can be seen to have detached from the bottom of the well (Figure 8f). This can be recognised by the round shape and a bright glow of the cells and is usually a sign of their death. After treatment with the formulation DD, there is still a slight migration or growth

of the cells (Figure 8h,m,q,u,y). An explanation for the good migration inhibition of all three formulations could be the fact that curcumin can, in addition to the ROS generation during PDT, inhibit matrix metalloproteinases 2 and 9 in A549 cells, which play a known role in migration, invasion and angiogenesis of these cells [76]. growth of the cells (Figure 8h,m,q,u,y). An explanation for the good migration inhibition of all three formulations could be the fact that curcumin can, in addition to the ROS generation during PDT, inhibit matrix metalloproteinases 2 and 9 in A549 cells, which play a known role in migration, invasion and angiogenesis of these cells [76].

scratch assay", a method based on the observation of an artificially created scratch in the confluent cell monolayer. The cells at the edge of this gap move towards the open area, which happens with the aim of closing the gap as quickly as possible until new cell-cell

The results of this assay show that the natural migration of untreated cells closed the gap in the cell monolayer within 48 h (Figure 8a,e,i,n,r,v). In comparison, it can be seen that the gap in the cells treated with PDT does not close within this time period. The effects on the cell migration behaviour seems to be more distinct for DT and DC, in agreement with the previous results. From the time of irradiation onwards, especially in the case of DT, many cells can be seen to have detached from the bottom of the well (Figure 8f). This can be recognised by the round shape and a bright glow of the cells and is usually a sign of their death. After treatment with the formulation DD, there is still a slight migration or

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 20 of 24

contacts have been created [75].

**Figure 8.** Microscopic images of the A549 cells during 48 h of the "in vitro scratch assay". The images a, e, i, n, r and v show the natural migration of untreated cells (blank) compared to cell migration after PDT treatment with nebulised curcumin liposomes (b,e,d,f,g,h,k,l,m,o,p,q,s,t,u,w,x,y). **Figure 8.** Microscopic images of the A549 cells during 48 h of the "in vitro scratch assay". The images a, e, i, n, r and v show the natural migration of untreated cells (blank) compared to cell migration after PDT treatment with nebulised curcumin liposomes (b,e,d,f,g,h,k,l,m,o,p,q,s,t,u,w,x,y).

#### **4. Conclusions**

Summing up, curcumin was taken up via clathrin-coated pits and visualised inside the cells. The good efficacy of nebulised curcumin liposomes with membrane stabilisation on cell viability and migration confirms their eligibility to improve pulmonary PDT.

**Author Contributions:** Conceptualization, J.L.; methodology, J.L., L.D., K.H.E., S.A. and S.R.P.; software, M.R.A.; validation, J.L.; formal analysis, J.L.; investigation, J.L.; resources, U.B.; data curation, J.L.; writing—original draft preparation, J.L.; writing—review and editing, J.L. and M.R.A.; visualization, J.L.; supervision, E.P., M.W. and U.B.; project administration, U.B.; funding acquisition, U.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to express very special thanks to Nikola Schubert for her outstanding support, motivation and technical assistance. Gratitude goes also to Elias Baghdan and Eva Maria Mohr for their support and technical assistance. The authors would also like to thank Lipoid GmbH for providing some of the lipids used in this study and PARI GmbH for kindly providing PARI BOY® SX and PARI VELOX®.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Novel Pyropheophorbide Phosphatydic Acids Photosensitizer Combined EGFR siRNA Gene Therapy for Head and Neck Cancer Treatment**

**Chia-Hsien Yeh <sup>1</sup> , Juan Chen <sup>2</sup> , Gang Zheng 2,3, Leaf Huang 4,5 and Yih-Chih Hsu 1,5,6,\***


**Abstract:** This study combined two novel nanomedicines, a novel LCP Pyro PA photodynamic therapy (PDT) and LCP EGFR siRNA gene therapy, to treat head and neck cancer. A novel photosensitizer, pyropheophorbide phosphatydic acids (Pyro PA), was first modified into Lipid-Calcium phosphate nanoparticles named LCP Pyro PA NPs, and targeted with aminoethylanisamide as a novel PDT photosensitizer. EGFR siRNA was encapsulated into LCP NPs to silence EGFR expression. Measured sizes of LCP EGFR siRNA NPs and LCP Pyro-PA NPs were 34.9 ± 3.0 and 20 nm respectively, and their zeta potentials were 51.8 ± 1.8 and 52.0 ± 7.6 mV respectively. In vitro studies showed that EGFR siRNA was effectively knocked down after photodynamic therapy (PDT) with significant inhibition of cancer growth. SCC4 or SAS xenografted nude mice were used to verify therapeutic efficacy. The LCP Control siRNA+PDT group of SCC4 and SAS showed significantly reduced tumor volume compared to the phosphate buffered saline (PBS) group. In the LCP-EGFR siRNA+LCP Pyro PA without light group and LCP EGFR siRNA + PBS with light group, SCC4 and SAS tumor volumes were reduced by ~140% and ~150%, respectively, compared to the PBS group. The LCP EGFR siRNA+PDT group of SCC4 and SAS tumor volumes were reduced by ~205% and ~220%, respectively, compared to the PBS group. Combined therapy showed significant tumor volume reduction compared to PBS, control siRNA, or PDT alone. QPCR results showed EGFR expression was significantly reduced after treatment with EGFR siRNA with PDT in SCC4 and SAS compared to control siRNA or PDT alone. Western blot results confirmed decreased EGFR protein expression in the combined therapy group. No toxic results were found in serum biomarkers. No inflammatory factors were found in heart, liver and kidney tissues. Results suggest that the novel LCP Pyro PA mediated PDT combined with LCP siEGFR NPs could be developed in clinical modalities for treating human head and neck cancer in the future.

**Keywords:** targeted delivery; head and neck cancer; nanoparticles; photodynamic therapy

## **1. Introduction**

Head and neck cancers develop mainly in the human oral cavity [1], larynx, hypopharynx and sinonasal areas. Ninety-five percent of head and neck cancer is caused by squamous cell carcinoma (SCC) [2]. Major risk factors include tobacco, betel nut chewing, alcohol drinking and HPV infections, which are the four major causes for carcinogenesis [2]. Clinical practices to treat human head and neck cancers include surgery, chemotherapy

**Citation:** Yeh, C.-H.; Chen, J.; Zheng, G.; Huang, L.; Hsu, Y.-C. Novel Pyropheophorbide Phosphatydic Acids Photosensitizer Combined EGFR siRNA Gene Therapy for Head and Neck Cancer Treatment. *Pharmaceutics* **2021**, *13*, 1435. https://doi.org/ 10.3390/pharmaceutics13091435

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 20 July 2021 Accepted: 26 August 2021 Published: 9 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and radiotherapy. Another new modality is photodynamic therapy (PDT), a noninvasive alternative for oral cancer therapy with proven low cumulative side effects after repeated therapies, including little to no observed scarring in the oral cavity [3,4]. PDT uses a specific photosensitizer activated by a specific light wavelength that selectively kills cancerous cells [5]. Photodynamic reactions generate reactive oxygen species that can kill cancer cells. In this study, we are the first to develop a novel Lipid-Calcium phosphate– pyropheophorbide phosphatydic acids nanoparticles (LCP-Pyro PA NPs) technology. LCP Pyro PA NPs consist of LCP encapsulated with a novel photosensitizer, Pyro PA, with targeting ligand aminoethylanisamide (AEAA) on the outer-left layer of LCP NPs. Previous studies have found evidence of EGFR overexpression in human head and neck SCC, and oral cancers have been reported to demonstrate evidence of mRNA expression for EGFR [6,7]. Up-regulation of EGFR by squamous epithelial cells from 24 HNSCC patients with head and neck squamous cell carcinoma (HNSCC) suggests that therapies targeting these genes may be effective in the prevention of head and neck cancer [8]. We delivered small interfering RNA (siRNA) to target genes with specific siRNA to tumor cells without distinct adverse effects. The concept has been proven with transfected HIF1a siRNA [9] and VEGF siRNA [10] and induces in vitro cancer cell apoptosis and in vivo tumor destruction. Therefore, we delivered EGFR siRNA to head and neck tumor cells using lipid-based LCP NPs with AEAA targeting ligands formulated on the outer layer portion of the nanoparticles to target the over-expressed sigma receptors [11,12] on the HNSCC cell surface. The goal of this study was to investigate the therapeutic outcome of integrating a novel targeting photosensitizer, Pyro PA (LCP Pyro PA NPs mediated PDT), with LCP NPs with EGFR siRNA gene therapy and targeting AEAA ligands loaded to treat HNSCC in xenograft animal models (Scheme 1). reactive oxygen species that can kill cancer cells. In this study, we are the first to develop a novel lipid–calcium phosphate–pyropheophorbide phosphatydic acids nanoparticles (LCP-Pyro PA NPs) technology. LCP Pyro PA NPs consist of LCP encapsulated with a novel photosensitizer, Pyro PA, with targeting ligand aminoethylanisamide (AEAA) on the outer-left layer of LCP NPs. Previous studies have found evidence of EGFR overexpression in human head and neck SCC, and oral cancers have been reported to demonstrate evidence of mRNA expression for EGFR [6,7]. Up-regulation of EGFR by squamous epithelial cells from 24 HNSCC patients with head and neck squamous cell carcinoma (HNSCC) suggests that therapies targeting these genes may be effective in the prevention of head and neck cancer [8]. We delivered small interfering RNA (siRNA) to target genes with specific siRNA to tumor cells without distinct adverse effects. The concept has been proven with transfected HIF1a siRNA [9] and VEGF siRNA [10] and induces in vitro cancer cell apoptosis and in vivo tumor destruction. Therefore, we delivered EGFR siRNA to head and neck tumor cells using lipid-based LCP NPs with AEAA targeting ligands formulated on the outer layer portion of the nanoparticles to target the over-expressed sigma receptors [11,12] on the HNSCC cell surface. The goal of this study was to investigate the therapeutic outcome of integrating a novel targeting photosensitizer, Pyro PA (LCP Pyro PA NPs mediated PDT), with LCP NPs with EGFR siRNA gene therapy and targeting AEAA ligands loaded to treat HNSCC in xenograft animal models (Scheme 1).

chewing, alcohol drinking and HPV infections, which are the four major causes for carcinogenesis [2]. Clinical practices to treat human head and neck cancers include surgery, chemotherapy and radiotherapy. Another new modality is photodynamic therapy (PDT), a noninvasive alternative for oral cancer therapy with proven low cumulative side effects after repeated therapies, including little to no observed scarring in the oral cavity [3,4]. PDT uses a specific photosensitizer activated by a specific light wavelength that selectively kills cancerous cells [5]. Photodynamic reactions generate

*Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 2 of 18

**Scheme 1.** Schematic illustration of LCP EGFR siRNA and LCP Pyro PA to xenograft models. We used SCC4 and SAS xenografts of Balb/c nude mice to verify the efficacy of the combination therapy. Combined therapy showed greater tumor growth inhibition for both SCC4 and SAS. **Scheme 1.** Schematic illustration of LCP EGFR siRNA and LCP Pyro PA to xenograft models. We used SCC4 and SAS xenografts of Balb/c nude mice to verify the efficacy of the combination therapy. Combined therapy showed greater tumor growth inhibition for both SCC4 and SAS.

#### **2. Materials and Methods**

#### *2.1. EGFR siRNA (siEGFR)*

The EGFR siRNA (siEGFR) with sense strand 50 -AAG UGC UGG AUG AUA GAC GCA dTdT-30 was designed using the provided Genome webserver (National Center for Biotechnology Information, Bethesda, MD, USA) [10]. The control siRNA (siControl) was the siGENOME non-targeting siRNA with sense strand 50 -AUGUAUUGGCCUGUAUUAG dTdT-3<sup>0</sup> and was purchased from Thermo Scientific (Waltham, MA, USA). LipofectamineTM RNAiMAX was purchased from Invitrogen (Carlsbad, CA, USA). Dioleoylphosphatydic acid (DOPA), 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP), cholesterol and 1,2-distearoryl-*sn*-glycero-3-phosphoethanolamine-*N*-[methoxy(polyethyleneglycol-2000)] ammonium salt (DSPE-PEG2000) were all purchased from Avanti Polar Lipids (Alabaster, AL, USA). DSPE-PEG-AEAA was chemically synthesized in the Hsu lab [12]. Other chemicals used in the study were purchased from Sigma-Aldrich (St. Louis, MO, USA).

#### *2.2. Novel Synthesis Method of Pyro PA Photosensitizer*

Pyropheophorbide-phosphatydic acid (Pyro PA) was synthesized and provided by Professor Zheng (Toronto, ON, Canada). Forty mg of pyropheophrobide-phospholipid (Pyro-lipid) was suspended in 3 mL of sodium acetate buffer (40 mM sodium acetate, 80 mM CaCl2, pH 5.0) and sonicated for 5 min for efficient dispersion, after which 3 mL of diethyl ether and 1000 U of phospholipase D obtained from *S. chromofuscus* were added. The mixture was light sensitive and therefore was protected from light and mixed aggressively at 33 ◦C. The reaction was completed in 5 h to yield Pyro PA, evidenced by HPLC-MS analysis. The HPLC profiles of the Pyro-lipid and Pyro PA and their corresponding UV spectra and mass spectra were further examined and verified to ensure the success of the synthesis.

#### *2.3. Novel Formulation of LCP Pyro PA NP*

First, phosphatydic acid was conjugated into Pyro PA for encapsulating into the lipid calcium phosphate nanoparticles. Then, 100 µL of 0.5 M CaCl<sup>2</sup> was dispersed in an 8 mL oil phase (cyclohexane/Igepal CO-250 [71/29, *v*/*v*]) to form a homogenous water-in-oil well-dispersed microemulsion. The phosphate micro-emulsion was prepared by dispersing 100 µL of 100 mM Na2HPO<sup>4</sup> in a separate 8-mL oil phase (cyclohexane/Igepal CO-250 [71/29, *v*/*v*]). Concentrations of 17.3 mM DOPA and 17.3 mM Pyro PA were dissolved in chloroform and added to the phosphate micro-emulsion solution. Then, the two microemulsions were mixed for 30 min, and ethanol (16 mL) was added to the combined solution. The well-mixed solution was then centrifuged to remove unnecessary ingredients. After three repetitions of the ethanol wash, the CaP core pellets were collected as white precipitates. The final LCP NPs were prepared in a separate step: 50 µL of LCP CaP cores were mixed with 20 µL of 20 mM DOTAP, 20 mM cholesterol, 20 mM DSPE-PEG-AEAA, and 20 mM DSPE-PEG-2000. Finally, residual lipids were evaporated. LCP NPs were formed when rehydrated in 50 µL of 20% glucose solution for tail vein injection into mice. Aminoethylanisamide (AEAA) was modified on the outer leaflet of LCP NPs to target the sigma receptors that are overexpressed on HNSCC cells [13].

#### *2.4. Formulation of LCP Nanoparticles*

LCP NPs were formulated as described with slight modifications [13–16]. Sigma receptors are highly expressed on the surface of SCC4 or SAS cell lines, and AEAA is the target moiety against sigma receptors on the surface of LCP NPs [12]. In an oil-phase solution (20 mL) comprising cyclohexane and Igepal CO-250 at 71/29 (*v*/*v*) ratio, a homogenous water-in-oil microemulsion solution was formed. Subsequently, 300 µL of 2.5 M CaCl<sup>2</sup> with 60 µg of siRNA was dispersed into the microemulsion system. A phosphate microemulsion was formulated by adding 300 µL of 12.5 mM Na2HPO<sup>4</sup> in a 20 mL oil phase bottle. One hundred microliters of 20 mM DOPA dissolved in chloroform solvent was transferred to the phosphate solution. After mixing the two microemulsions homogenously for 20 min, ethanol (40 mL) was added to the final solution and then centrifuged to spin down the LCP NPs. It was crucial to conduct the ethanol wash procedure 3–4 times, after which the CaP core pellets were collected. The final LCP NPs were prepared in a separate step; 50 µL of LCP CaP cores was mixed with 20 µL of 20 mM DOTAP, 20 mM cholesterol, 20 mM DSPE-PEG-AEAA, and 20 mM DSPE-PEG-2000. Finally, residual lipids were evaporated. LCP NPs were formed when rehydrated in 50 µL of 20% glucose solution for tail vein injection into mice.

#### *2.5. Characterization of LCP Nanoparticles*

Transmission electron microscope images of LCP were obtained using the Bio-TEM Hitachi HT7700 (Hitachi, Ibaraki, Japan). LCP NP size and zeta potential were determined using the Malvern Zetasizer Nano series (Westborough, MA, USA).

#### *2.6. Human SCC Cell Cultures*

The SCC4 cell line was acquired from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and the SAS cell line was a gift from Prof. Chun-Ji Liu, Yang Ming University. Both SCC4 and SAS cells were cultured using DMEM/F-12 medium (Invitrogen, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, NY, USA) and incubated at 37 ◦C with 5% CO2. SCC4 and SAS cells were removed from culture plates using 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA) when cancer cells reached 80% confluence on plate.

#### *2.7. In Vitro siRNA Transfection Study*

In a six-well plate, 3 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per well of SCC4 or SAS cells were cultured on each well and incubated for 24 h. Transfection was conducted with 25 nM siRNA concentration in Opti-MEM Reduced medium (Invitrogen, Grand Island, NY, USA) using LipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA) based on manual. SCC4 or SAS cells were then placed into an incubator at 37 ◦C for 4 h in the Opti-MEM Reduced medium. The medium was then changed to a growth medium containing 10% FBS for 48 h, after which cells were lysated.

#### *2.8. SCC4 and SAS Xenograft Model Establishment*

Eight week old male BALB/cAnN.Cg-Foxn1 nude mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan. To establish SCC4 or SAS xenograft mice models, 5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells of SCC4 or SAS in DMEM/F12 media were combined gently with matrigel (Corning, Bedford, MA, USA) and infused subcutaneously at the right side of the lower flank of each mouse. SCC4 and SAS xenograft mice were randomly divided into five groups with five mice in each group: (1) PBS; (2) LCP siControl NPs+PDT; (3) LCP Pyro PA NPs without light+LCP siEGFR; (4) LCP siEGFR NPs+PBS+light; and (5) LCP siEGFR NPs+PDT. The five treatments were given intravenously via tail vein infusion. The dosage of LCP siControl NPs and LCP siEGFR NPs was 0.36 mg/kg. The dosage of LCP Pyro PA NPs was 0.78 mg/kg, prepared by solubilizing 0.3 mg Pyro PA in 200 µL PBS for 25 g of weight and given via tail vein. After 55 min, red light at 663 ± 9 nm wavelength with 320 mW/cm<sup>2</sup> power density delivered 100 J/cm<sup>2</sup> energy dosage for 11 min. Tumor size reached 200 <sup>±</sup> 5% mm<sup>3</sup> (190~210 mm<sup>3</sup> ) and was sufficient for treatment. The formula used to calculate tumor volume was V = (L × W × H)/2, where V stands for tumor volume, L stands for length, W stands for width perpendicular to length, and H stands for height of the tumor. Tumor size was determined daily using calipers and all mice were sacrificed on the 13th day for further analysis. Excised tumors and organs were divided and fixed in formalin for H&E and IHC stainings. This animal study was approved on August 6, 2015 (case number #103030) and carried out under strict guidelines based on the recommendations of the Guide for the Care and Use produced by the Institutional Animal Care and Use Committee of Chung Yuan Christian University, Chungli, Taoyuan, Taiwan.

#### *2.9. Western Blot Experiments*

Cells were harvested or tumor specimens removed and homogenized using a lysis buffer (PRO-PREPTMTM protein extraction solution, Intron Biotechnology Inc., Seoul, Korea). These samples were assayed. Identical amounts of protein determined using BCA protein assays (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA) were assayed for these specimens. Lysated cells or tissues were first heated to denatured soluble proteins in the sample buffer at 100 ◦C for 5 min. Then, these prepared specimens were loaded in individual wells of 5%/12% Bis-Tris acrylamide gels (stacking/separating gel) with protein markers. SDS-PAGE electrophoresis was conducted at a constant voltage (150 V) at 25 ◦C. Proteins on the SDS-PAGE gels were transferred to a PVDF membrane (Millipore Corporation, Billerica, MA, USA) electrophoretically at a constant voltage for 2 h at 4 ◦C. After the protein transfer step, the PVDF membranes were blocked with a blocking buffer (BlockPROTMTM, Visual Protein Biotechnology Corporation, Taipei, Taiwan), then each fraction of the membranes was separated and incubated with rabbit primary antibodies against EGFR (GTX121919; GeneTex, Taipei, Taiwan) (1:1000 dilution) and sigma receptor (SIGMAR1 GTX115389; GeneTex, Taipei, Taiwan) (1:1000 dilution), followed by peroxidaseconjugated goat anti-rabbit IgG (GTX213110 GeneTex, Taipei, Taiwan) (1:10,000 dilution), and then developed in ELC (enhanced chemiluminescence) substrate (PerkinElmer, Inc., Boston, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH GTX100118; GeneTex, Taipei, Taiwan) (1:1000 dilution) was used as the internal control.

#### *2.10. Quantitation of EGFR Gene Knock-Down Using Real Time-PCR*

First, RNA was extracted from the tumor tissues. Tumor specimens were divided into small pieces and several pieces of 0.05 g were randomly chosen from each experimental group. Tumor specimens were homogenized for extraction of total tissue RNA using RNAzol® RT solution (Molecular Research Center Inc., Cincinnati, OH, USA). The obtained cDNAs were synthesized using RevertAid cDNA synthesis kits (Fermentas, Thermo Scientific Inc., Waltham, MA, USA). FastStart Universal Master Probe (Roche Applied Science, Mannheim, Germany) was used for quantitative real-time PCR. PCR were operated using a regular cycling schedule: 95 ◦C for 10 min, 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min on a real-time PCR system (AB7300, Applied Biosystems, Foster City, CA, USA). The forward primer sequences for human EGFR were 50 -TTCCTCCCAGTGCCTGAA-30and the reverse primer sequences for human EGFR were 50 -GGGTTCAGAGGCTGATTGTG-30 . The forward primer sequences for human GAPDH were 50 -AGCCACATCGCTCAGACAC-30 and the reverse primer sequences for human EGFR were 50 -AGCCACATCGCTCAGACAC-30 . The forward and reverse primer pairs were produced by Roche (Roche Applied Science, Mannheim, Germany). Calculated results were based on the relative times of threshold schedules in which the calculated EGFR concentration data were normalized against GAPDH concentration as internal control.

### *2.11. H&E Staining and Immunohistochemistry (IHC)*

Harvested organs and tumor tissues were embedded in paraffin and divided into several connective sections followed by standard H&E staining protocol. Tissue sections of paraffin-embedded SCC4 and SAS tumors were deparaffinized and rehydrated to retrieve epitopes on the antigens for IHC. Tissue slides were treated with either monoclonal or polyclonal antibodies, such as rabbit polyclonal anti-CD31 (1:100, ab28364, Abcam, Cambridge, MA, USA), rabbit monoclonal anti-Ki-67 (1:200, ab16667, Abcam, Cambridge, MA, USA), and rabbit polyclonal anti-EGFR (1:100, GTX121919; GeneTex, Taipei, Taiwan). Sections of tumor tissues were then incubated with HRP-conjugated anti-rabbit antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min. Slide observation was conducted using a DAB detection kit (Pierce, Rockland, IL, USA). All specimens were examined using an Olympus light microscope (BX53F model, Olympus, Tokyo, Japan). Intensities of IHC stains were further quantified for comparison at 40× magnification

(10 images per group) using ImageJ software on 21 July 2016 (version 1.8, National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/).

#### *2.12. TUNEL Assays*

SCC4 and SAS tumor paraffin-embedded tissue sections were deparaffinized, rehydrated and pretreated for protease. TUNEL assays was executed using in situ Cell Death Detection Kits, POD (Roche, Mannheim, Germany) following the manufacturer's guidelines. Specimens were examined using an Olympus BX53F light microscope (Olympus, Tokyo, Japan). Positive TUNEL cell images were recorded and quantified at 40× magnification using ImageJ software (National Institutes of Health, http://imagej.nih.gov/ij/).

#### *2.13. In Vivo Toxicity Assays*

In vivo toxicity assays followed the same protocol as treatment for tail vein infusion of four treated groups including LCP NPs of PBS, siControl, siEGFR, and Pyro PA. Three mice were used in each group. The 7- to 8-week-old C57BL/6JNarl mice were tail vein injected with PBS, LCP siControl NPs, and LCP siEGFR NPs on days 0, 1 and 2, with injections repeated on days 7, 8 and 9, and a 4-day interval between injections (of three experiment groups). The LCP Pyro PA NPs group was also tail vein injected into mice on days 3 and 10. Mice were anesthetized and cardiac punctured to collect blood for toxicity assays and sacrificed on the 14th day. To avoid hemolysis of mouse blood, blood was carefully transferred to a 1.5 mL vial in a slow mode, and blood was set to clot at 25 ◦C for 20 min prior to centrifugation to separate serum from the supernatant portion (Hermle Z233 MK-2, Hermle, Wehingen, Germany). The collected serum was analyzed for concentrations of liver biomarkers (AST and ALT), kidney biomarkers (CREA and BUN), calcium, and phosphorus. Concentrations of toll-like receptor 3 (TLR3) were also examined using TLR3 ELISA kits (MyBioSource, Inc., San Diego, CA, USA) to ensure no immune activation. The above assays used mouse IL-6 ELISA kits (RayBiotech, Norcross, GA, USA), mouse IL-12 p40/70 ELISA kits (RayBiotech, Norcross, GA, USA), mouse IFN gamma ELISA kits (RayBiotech, Norcross, GA, USA) and TLR3 ELISA kits (MyBioSource Inc., San Diego, CA, USA), respectively.

## *2.14. Statistical Analysis*

All data were presented as mean ± standard deviation. Statistical significance was analyzed using one-way ANOVAs with Tukey's test using SigmaPlot® (Version 12.5, Systat Software, Inc., San Jose, CA, USA).

#### **3. Results**

#### *3.1. Results of Novel Synthesis Method of Pyropheophrobide-Phosphatydic Acid (Pyro Pa) from Pyropheophrobide-Phospholipid (Pyro-Lipid)*

The innovative synthesis of pyropheophrobide-phosphatydic acid (Pyro Pa) from pyropheophrobide-phospholipid (Pyro-lipid) used phospholipase D (from *S. chromofuscus*) as described in the Section 2 (Figure 1a). The reaction was completed to gain Pyro PA, as evidenced by HPLC-MS analysis (Figure 1b,c). The figures show HPLC profiles of the Pyro-lipid (Figure 1b) and Pyro PA (Figure 1c) and their corresponding UV spectra and mass spectra. The compound absorption spectrum had no significant changes during the reaction. The identified mass signals for Pyro-lipid were: *m*/*z* calculated for C57H82N5O9P [M]<sup>+</sup> 1012.3, found [M]<sup>+</sup> 1013.2; [M]2+ 507.2; for Pyro PA: *m*/*z* calculated for C52H70N4O9P [M]<sup>+</sup> 927.1, found 928.1.

*m*/*z* calculated for C57H82N5O9P [M]+ 1012.3, found [M]+ 1013.2; [M]2+ 507.2; for Pyro PA:

*m*/*z* calculated for C52H70N4O9P [M]+ 927.1, found 928.1.

**Figure 1.** Novel synthesis method of Pyro PA. (**a**) 40mg of pyropheophrobide-phospholipid (Pyro-lipid) was suspended in 3mL of sodium acetate buffer (40 mM sodium acetate, 80 mM CaCl2, pH 5.0) and sonicated for 5 min for efficient dispersion, after which 3 mL of diethyl ether and 1000 unit of phospholipase D obtained from *S. chromofuscus* were added. The mixture was shaded against light and mixed extensively at 33 °C. (**b**,**c**) The reaction was completed in 5 h to gain pyropheophrobide-phosphatydic acid (Pyro PA) evidenced by HPLC-MS analysis. The HPLC profiles of the Pyro-lipid and Pyro PA and their corresponding UV spectra and mass spectra were examined and verified to ensure success. **Figure 1.** Novel synthesis method of Pyro PA. (**a**) 40 mg of pyropheophrobide-phospholipid (Pyro-lipid) was suspended in 3 mL of sodium acetate buffer (40 mM sodium acetate, 80 mM CaCl<sup>2</sup> , pH 5.0) and sonicated for 5 min for efficient dispersion, after which 3 mL of diethyl ether and 1000 unit of phospholipase D obtained from *S. chromofuscus* were added. The mixture was shaded against light and mixed extensively at 33 ◦C. (**b**,**c**) The reaction was completed in 5 h to gain pyropheophrobide-phosphatydic acid (Pyro PA) evidenced by HPLC-MS analysis. The HPLC profiles of the Pyro-lipid and Pyro PA and their corresponding UV spectra and mass spectra were examined and verified to ensure success.

*3.2. Characterization of Lipid-Calcium-Phosphate Pyropheophrobide Phosphatydic Acid Nanoparticles (LCP Pyro Pa NPs) or Lipid-Calcium-Phosphate EGFR siRNA Nanoparticles (LCP EGFR siRNA NPs) EGFR siRNA NPs)*  3.2.1. Preparation of Lipid-Calcium-Phosphate Pyropheophrobide Phosphatydic Acid

*3.2. Characterization of Lipid-Calcium-Phosphate Pyropheophrobide Phosphatydic Acid* 

*Nanoparticles (LCP Pyro Pa NPs) or Lipid-Calcium-Phosphate EGFR siRNA Nanoparticles (LCP* 

*Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 8 of 18

3.2.1. Preparation of Lipid-Calcium-Phosphate Pyropheophrobide Phosphatydic Acid Nanoparticles (LCP-Pyro Pa NPs) Nanoparticles (LCP-Pyro Pa NPs) LCP-Pyro PA NPs were formulated with two different types of phospholipids to

LCP-Pyro PA NPs were formulated with two different types of phospholipids to form double-layer liposomal nanoparticles that were composed of pH-sensitive calcium phosphate cores stabilized by DOPA and coated with DOTAP. The outer part of the lipid layer was modified with polyethylene glycol (PEG) chains and aminoethylanisamide (AEAA). AEAA is the targeting ligand for sigma receptors on cancer cell surfaces (Figure 2a). TEM photomicrographs indicated that nanoparticles were 15 to 20 nm in size. The zeta potential determined for LCP Pyro Pa NPs was 52.0 ± 7.6 mV with AEAA and 3.5 ± 0.6 mV without AEAA (Figure 2). form double-layer liposomal nanoparticles that were composed of pH-sensitive calcium phosphate cores stabilized by DOPA and coated with DOTAP. The outer part of the lipid layer was modified with polyethylene glycol (PEG) chains and aminoethylanisamide (AEAA). AEAA is the targeting ligand for sigma receptors on cancer cell surfaces (Figure 2a). TEM photomicrographs indicated that nanoparticles were 15 to 20 nm in size. The zeta potential determined for LCP Pyro Pa NPs was 52.0 ± 7.6 mV with AEAA and 3.5 ± 0.6 mV without AEAA (Figure 2).

**Figure 2***.* Novel synthesis method of LCP Pyro-Pa nanoparticles. LCP Pyro-Pa NP was composed of a biodegradable calcium phosphate core stabilized using DOPA and DOTAP. The outer layer of lipid was modified with polyethylene glycol (PEG) chains and aminoethylanisamide. **Figure 2.** Novel synthesis method of LCP Pyro-Pa nanoparticles. LCP Pyro-Pa NP was composed of a biodegradable calcium phosphate core stabilized using DOPA and DOTAP. The outer layer of lipid was modified with polyethylene glycol (PEG) chains and aminoethylanisamide.

3.2.2. Preparation of Lipid-Calcium Phosphate EGFR siRNA Nanoparticles (LCP EGFR siRNA NPs)

Refer to the similar descriptions shown above for LCP-Pyro PA NPs (Figure 3a). As TEM was conducted in dehydrated conditions, it showed nanoparticle sizes ranging from 25 to 30 nm. The LCP NP sizes detected by dynamic light scattering technology were larger than the TEM-measured NP sizes, from 31 to 37 nm, because of the hydrodynamic diameter. The zeta potential for LCP siEGFR NPs was 50.1 ± 1.8 mV with AEAA and −6.1 ± 0.4 mV without AEAA. Data were presented as mean ± SD (in triplicate) (Figure 3b–d).

siRNA NPs)

triplicate) (Figure 3b–d).

**Figure 3.** Characteristic Properties of LCP siEGFR NPs. (**a**) Demonstration of LCP siEGFR NPs. LCP Pyro PA NPs were formulated with two different types of phospholipids to form double-layer liposomal nanoparticles that were composed of pH sensitive calcium phosphate cores stabilized by DOPA and coated with DOTAP. The outer part of the lipid layer was modified with polyethylene glycol (PEG) chains and aminoethylanisamide (AEAA). AEAA is the targeting ligands for the sigma receptors on cancer cell surfaces (**b**–**d**). Data presented as mean ± SD (in triplicate). **Figure 3.** Characteristic Properties of LCP siEGFR NPs. (**a**) Demonstration of LCP siEGFR NPs. LCP Pyro PA NPs were formulated with two different types of phospholipids to form double-layer liposomal nanoparticles that were composed of pH sensitive calcium phosphate cores stabilized by DOPA and coated with DOTAP. The outer part of the lipid layer was modified with polyethylene glycol (PEG) chains and aminoethylanisamide (AEAA). AEAA is the targeting ligands for the sigma receptors on cancer cell surfaces (**b**–**d**). Data presented as mean ± SD (in triplicate).

#### *3.3. In Vitro Sigma Receptor and EGFR Protein Expression 3.3. In Vitro Sigma Receptor and EGFR Protein Expression*

Our past studies have reported that sigma receptors are expressed on the surface of HNSCC in vitro [9,10]. We examined the expression of sigma receptors and EGFR proteins at one day after photodynamic therapy. Either SCC4 or SAS cells were then treated with 0 ~ 0.25µg /mL Pyro Pa NPs µg/mL irradiated with 663 ± 9 nm at 10 J/cm2 described as the PDT group in the in vitro study. Subsequently, they were incubated in the medium for 24 h. Cell viability was decreased due to the EGFR siRNA knockdown effect and follow-up PDT treatment in SCC4 (Figure 4a) and SAS (Figure 4b) cell lines. Either SCC4 or SAS cells were then transfected with 12.5 nM self-designed EGFR siRNA. After two days' incubation, EGFR protein amount was examined using Western blots. An EGFR silencing effect was observed for SCC4 and SAS cells treated with EGFR siRNA+PDT as compared to the groups treated with phosphate buffered saline (PBS)+PDT or Control siRNA+PDT. This indicates that the novel EGFR siRNA sequence could knockdown EGFR expression (Figure 4c,d). Our past studies have reported that sigma receptors are expressed on the surface of HNSCC in vitro [9,10]. We examined the expression of sigma receptors and EGFR proteins at one day after photodynamic therapy. Either SCC4 or SAS cells were then treated with 0~0.25 <sup>µ</sup>g /mL Pyro Pa NPs <sup>µ</sup>g/mL irradiated with 663 <sup>±</sup> 9 nm at 10 J/cm<sup>2</sup> described as the PDT group in the in vitro study. Subsequently, they were incubated in the medium for 24 h. Cell viability was decreased due to the EGFR siRNA knockdown effect and follow-up PDT treatment in SCC4 (Figure 4a) and SAS (Figure 4b) cell lines. Either SCC4 or SAS cells were then transfected with 12.5 nM self-designed EGFR siRNA. After two days' incubation, EGFR protein amount was examined using Western blots. An EGFR silencing effect was observed for SCC4 and SAS cells treated with EGFR siRNA+PDT as compared to the groups treated with phosphate buffered saline (PBS)+PDT or Control siRNA+PDT. This indicates that the novel EGFR siRNA sequence could knockdown EGFR expression (Figure 4c,d).

3.2.2. Preparation of Lipid–Calcium Phosphate EGFR siRNA Nanoparticles (LCP EGFR

Refer to the similar descriptions shown above for LCP-Pyro PA NPs (Figure 3a). As TEM was conducted in dehydrated conditions, it showed nanoparticle sizes ranging from 25 to 30 nm. The LCP NP sizes detected by dynamic light scattering technology were larger than the TEM-measured NP sizes, from 31 to 37 nm, because of the hydrodynamic diameter. The zeta potential for LCP siEGFR NPs was 50.1 ± 1.8 mV with AEAA and −6.1 ± 0.4 mV without AEAA. Data were presented as mean ± SD (in

#### *3.4. In Vivo Treatment Efficacy of Combination Therapy*

Two HNSCC xenograft models, SCC4 and SAS models of 204~209 mm<sup>3</sup> tumor volume, were then randomly distributed to five study subgroups: (1) PBS; (2) PDT (LCP siControl+PDT); +LCP Control siRNA, (3) LCP-Pyro PA+LCP EGFR siRNA, (4) PBS+light+LCP EGFR siRNA and (5) PDT (LCP-Pyro PA+light)+LCP EGFR siRNA. The complete therapeutic protocol was conducted for 13 days. All mice received two treatments on days 0, 1, or 2 and 7, 8, or 9. Either photosensitizer or LCP NPs containing siControl or siEGFR were injected into mice via the tail vein. On days 3 and 10, the mice received either PBS or LCP Pyro PA NPs (2 mg/kg) and were given 663 ± 9 nm red light to reach 100 joules after a 55 min metabolic period. For further analysis, all mice were humanely anaesthetized to death on the 13th day to collect major organs and tumors.

**Figure 4.** In vitro study of combined therapy. Combined therapy of EGFR siRNA or Control siRNA and photodynamic therapy (PDT) with different concentrations of Photosan photosensitizers in SCC4 (**a**) or SAS (**b**) cell lines. (**c**,**d**) Western blots of EGFR protein level in siRNA treated SCC4 and SAS cells. Columns, mean (*n* = 3); \* stands for *p* < 0.05 compared to PBS group. \*\* stands for *p* < 0.01 compared to PBS group. **Figure 4.** In vitro study of combined therapy. Combined therapy of EGFR siRNA or Control siRNA and photodynamic therapy (PDT) with different concentrations of Photosan photosensitizers in SCC4 (**a**) or SAS (**b**) cell lines. (**c**,**d**) Western blots of EGFR protein level in siRNA treated SCC4 and SAS cells. Columns, mean (*n* = 3); \* stands for *p* < 0.05 compared to PBS group. \*\* stands for *p* < 0.01 compared to PBS group.

*3.4. In Vivo Treatment Efficacy of Combination Therapy*  Two HNSCC xenograft models, SCC4 and SAS models of 204~209 mm3 tumor volume, were then randomly distributed to five study subgroups: (1) PBS; (2) PDT (LCP siControl+PDT); +LCP Control siRNA, (3) LCP-Pyro PA+LCP EGFR siRNA, (4) PBS+light+LCP EGFR siRNA and (5) PDT (LCP-Pyro PA+light)+LCP EGFR siRNA. The complete therapeutic protocol was conducted for 13 days. All mice received two treatments on days 0, 1, or 2 and 7, 8, or 9. Either photosensitizer or LCP NPs containing siControl or siEGFR were injected into mice via the tail vein. On days 3 and 10, the mice received either PBS or LCP Pyro PA NPs (2 mg/kg) and were given 663 ± 9nm red light to reach 100 joules after a 55 min metabolic period. For further analysis, all mice were humanely anaesthetized to death on the 13th day to collect major organs and tumors. The tumors of the PBS group increased in size by up to 213% and 270% after 13 days The tumors of the PBS group increased in size by up to 213% and 270% after 13 days in the SCC4 and SAS xenograft models, respectively (Figure 5a,b). On day 13, the LCP siControl+PDT group showed decreased tumor size after the PDT treatments on days 3 and 10 with ~100% and ~145% inhibition of tumor volume in the SCC4 and SAS xenograft models, respectively. Both SCC4 and SAS models demonstrated significant differences (*p* < 0.01) when compared with PBS. On day 13, LCP siEGFR+PS and LCP siEGFR+PBS+light groups showed ~130% and ~175% inhibition of tumor volume in the SCC4 and SAS xenograft models, respectively. Both SCC4 and SAS groups showed tumor volume inhibition resulting from LCP siEGFR in the absence of a complete PDT treatment. The combined group, LCP Pyro PA NPs–mediated PDT, showed significant tumor volume inhibition after LCP siEGFR silencing as compared to the other four groups (*p* < 0.01); combined therapy demonstrated significant tumor volume decrease of ~200% and ~220% in the SCC4 and SAS models, respectively, when compared with untreated PBS groups, with significant differences for both models (*p* < 0.01).

in the SCC4 and SAS xenograft models, respectively (Figure 5a,b). On day 13, the LCP siControl+PDT group showed decreased tumor size after the PDT treatments on days 3 and 10 with ~100% and ~145% inhibition of tumor volume in the SCC4 and SAS xenograft models, respectively. Both SCC4 and SAS models demonstrated significant differences (*p* < 0.01) when compared with PBS. On day 13, LCP siEGFR+PS and LCP siEGFR+PBS+light groups showed ~130% and ~175% inhibition of tumor volume in the

treatment. The combined group, LCP Pyro PA NPs–mediated PDT, showed significant tumor volume inhibition after LCP siEGFR silencing as compared to the other four

and the three other treated groups.

groups (*p* < 0.01); combined therapy demonstrated significant tumor volume decrease of ~200% and ~220% in the SCC4 and SAS models, respectively, when compared with

Quantitative RT-PCR and Western blot tests were conducted to examine SCC4 and SAS tumor tissues obtained on the 13th day. Protein expression of EGFR for LCP siEGFR NPs groups decreased significantly (*p* < 0.01). All data were analyzed and normalized with GAPDH densities when compared with PBS and LCP siControl+PDT groups for SCC4 and SAS xenograft models (Figures 5c). EGFR expression was consistent with assayed siEGFR mRNA expression for SCC4 or SAS xenograft animal models (Figure 5d) and significant differences (*p* < 0.01) were found in LCP siEGFR NPs compared with PBS

untreated PBS groups, with significant differences for both models (*p* < 0.01).

**Figure 5.** In vivo study of combined therapy. Tumor curves of SCC4 (**a**) and SAS (**b**) xenograft models. The experiments were conducted approximately 3–5 weeks post cell inoculation once the tumor volume reached approximately 200 mm3. △ indicated IV infusion of LCP with either Control siRNA or EGFR siRNA or PBS; ▲ indicated IV infusion of LCP Pyro PA with or without light. Tumor images of collected SCC4 (**a**) and SAS (**b**) xenograft tumors on day 13. (**c**) Quantified relative EGFR phosphorylated-EGFR (tyr1173) phosphorylated-EGFR (tyr1092) phosphorylated-EGFR (tyr1045), Her2, AKT, phosphorylated-AKT, cleaved caspase-3 and GAPDH protein expression band intensity in SCC4 and SAS cell line. (**d**) QPCR analysis of EGFR protein expression after treatment for SCC4 and SAS xenograft model. Quantified relative EGFR protein expression normalized against GAPDH. GAPDH protein was applied as the internal normalized protein marker with their respective relative EGFR protein expression. Columns, mean (*n* = 3); \*, *p* < 0.05; \*\*, *p* < 0.01. **Figure 5.** In vivo study of combined therapy. Tumor curves of SCC4 (**a**) and SAS (**b**) xenograft models. The experiments were conducted approximately 3–5 weeks post cell inoculation once the tumor volume reached approximately 200 mm<sup>3</sup> . 4 indicated IV infusion of LCP with either Control siRNA or EGFR siRNA or PBS; N indicated IV infusion of LCP Pyro PA with or without light. Tumor images of collected SCC4 (**a**) and SAS (**b**) xenograft tumors on day 13. (**c**) Quantified relative EGFR phosphorylated-EGFR (tyr1173) phosphorylated-EGFR (tyr1092) phosphorylated-EGFR (tyr1045), Her2, AKT, phosphorylated-AKT, cleaved caspase-3 and GAPDH protein expression band intensity in SCC4 and SAS cell line. (**d**) QPCR analysis of EGFR protein expression after treatment for SCC4 and SAS xenograft model. Quantified relative EGFR protein expression normalized against GAPDH. GAPDH protein was applied as the internal normalized protein marker with their respective relative EGFR protein expression. Columns, mean (*n* = 3); \*, *p* < 0.05; \*\*, *p* < 0.01.

> Quantitative RT-PCR and Western blot tests were conducted to examine SCC4 and SAS tumor tissues obtained on the 13th day. Protein expression of EGFR for LCP siEGFR NPs groups decreased significantly (*p* < 0.01). All data were analyzed and normalized with GAPDH densities when compared with PBS and LCP siControl+PDT groups for SCC4 and SAS xenograft models (Figure 5c). EGFR expression was consistent with assayed siEGFR mRNA expression for SCC4 or SAS xenograft animal models (Figure 5d) and significant differences (*p* < 0.01) were found in LCP siEGFR NPs compared with PBS and the three other treated groups.

#### *3.5. Combination Therapy Does Inhibit HNSCC Tumor Growth Efficiently*

Hematoxylin and eosin (H&E) staining assays for SCC4 or SAS tumors tissues obtained from xenografted mice after treatments were conducted to observe tumor, liver and kidney damage. No cell damage was found in livers or kidneys (Figure 6a,b).

*3.5. Combination Therapy Does Inhibit HNSCC Tumor Growth Efficiently* 

Hematoxylin and eosin (H&E) staining assays for SCC4 or SAS tumors tissues obtained from xenografted mice after treatments were conducted to observe tumor, liver and kidney damage. No cell damage was found in livers or kidneys (Figure 6a,b).

The following assays were conducted on SCC4 or SAS tumors for the EGFR marker:

**Figure 6.** Hematoxylin and eosin staining of SCC4 and SAS Tumors. Hematoxylin and eosin histopathological stains of liver and kidney organs and SCC4 (**a**) and SAS tumors (**b**) of xenograft mice. Original magnification 40×. Scale bar stands for 100 µm. **Figure 6.** Hematoxylin and eosin staining of SCC4 and SAS Tumors. Hematoxylin and eosin histopathological stains of liver and kidney organs and SCC4 (**a**) and SAS tumors (**b**) of xenograft mice. Original magnification 40×. Scale bar stands for 100 µm.

Ki67 marker was used for cell proliferation staining; CD31 marker was used for microvessel formation; cleaved caspase-3 was used to mark tumor cell apoptosis and TUNEL was used to stain DNA fragmentation. All of these assays were evaluated and quantified using immunohistochemistry staining (IHC) (Figure 7b–f,h–l). EGFR expression was observed at high levels with tumors of the PBS and LCP siControl+PDT groups, suggesting it was highly expressed in SCC4 and SAS cells. Both SCC4 and SAS tumors showed higher EGFR protein expression levels in the PBS and LCP siControl+PDT groups than in the three siEGFR-loaded LCP NPs groups. These three groups treated with LCP siEGFR nanoparticles showed lower EGFR protein expression (Figure 7b,h), which suggests that EGFR genes were silenced. This EGFR gene knockdown effect could lead to tumor cell apoptosis, DNA fragmentation, and reduced microvessel blood vessel formation, especially with the LCP siEGFR NPs+PDT combined therapy. For the LCP Pyro PA NPs–mediated PDT, LCP siEGFR, and both combined treatments groups, there were increased caspase-3 levels, representing apoptotic cells. Significant differences in the expression of TUNEL positive cells and cleaved caspase-3 between all treatment groups compared to the PBS group (*p* < 0.01, *p* < 0.05) were found in both SCC4 and SAS xenograft models (Figure 7c,d,i,j). The combined LCP siEGFR+PDT group showed significantly drastic cell destruction effects that led to 55% and 40% cell apoptosis detected via TUNEL assay (Figure 7c,i) and 35% and 50% cleaved caspase-3 (Figure 7d,j) expression in SCC4 and SAS models, respectively. Moreover, the LCP siEGFR+LCP-Pyro PA–mediated PDT showed a significant difference from LCP siControl+PDT, indicating that the LCP siEGFR NPs enhanced the apoptotic effect on cancer cells when combined with LCP Pyro PA–mediated PDT in SCC4 and SAS xenograft models. The quantified positive field of Ki67 cells indicated a stronger The following assays were conducted on SCC4 or SAS tumors for the EGFR marker: Ki67 marker was used for cell proliferation staining; CD31 marker was used for microvessel formation; cleaved caspase-3 was used to mark tumor cell apoptosis and TUNEL was used to stain DNA fragmentation. All of these assays were evaluated and quantified using immunohistochemistry staining (IHC) (Figure 7b–f,h–l). EGFR expression was observed at high levels with tumors of the PBS and LCP siControl+PDT groups, suggesting it was highly expressed in SCC4 and SAS cells. Both SCC4 and SAS tumors showed higher EGFR protein expression levels in the PBS and LCP siControl+PDT groups than in the three siEGFR-loaded LCP NPs groups. These three groups treated with LCP siEGFR nanoparticles showed lower EGFR protein expression (Figure 7b,h), which suggests that EGFR genes were silenced. This EGFR gene knockdown effect could lead to tumor cell apoptosis, DNA fragmentation, and reduced microvessel blood vessel formation, especially with the LCP siEGFR NPs+PDT combined therapy. For the LCP Pyro PA NPs–mediated PDT, LCP siEGFR, and both combined treatments groups, there were increased caspase-3 levels, representing apoptotic cells. Significant differences in the expression of TUNEL positive cells and cleaved caspase-3 between all treatment groups compared to the PBS group (*p* < 0.01, *p* < 0.05) were found in both SCC4 and SAS xenograft models (Figure 7c,d,i,j). The combined LCP siEGFR+PDT group showed significantly drastic cell destruction effects that led to 55% and 40% cell apoptosis detected via TUNEL assay (Figure 7c,i) and 35% and 50% cleaved caspase-3 (Figure 7d,j) expression in SCC4 and SAS models, respectively. Moreover, the LCP siEGFR+LCP-Pyro PA–mediated PDT showed a significant difference from LCP siControl+PDT, indicating that the LCP siEGFR NPs enhanced the apoptotic effect on cancer cells when combined with LCP Pyro PA–mediated PDT in SCC4 and SAS xenograft models. The quantified positive field of Ki67 cells indicated a stronger inhibition effect in the LCP siEGFR+PDT group compared to PBS, LCP siControl+PDT, LCP siEGFR+LCP Pyro PA without light, and LCP siEGFR+PBS+light groups (*p* < 0.01) for SCC4 (Figure 7e) and SAS (Figure 7k). This showed that the combination of LCP siEGFR NPs and LCP Pyro PA– mediated PDT enhanced the inhibition effect of the cell proliferation activity significantly. LCP siControl+PDT, LCP siEGFR+LCP-Pyro PA without light, and LCP siEGFR+PBS+light groups demonstrated significant inhibition (*p* < 0.01) in Ki67 amounts compared to PBS group for SCC4 and SAS xenograft models, revealing that LCP-Pyro PA–mediated PDT alone or siEGFR-loaded LCP NPs alone contributed significantly decreasing tumor cell proliferation. The quantified CD31 microvessel intensities of SCC4 and SAS tumors (Figure 7f,l) showed a dramatic decrease in groups treated with LCP siEGFR NPs for SCC4 and SAS models compared with PBS group (*p* < 0.01 for both). These groups exhibited

significant differences compared with the LCP siControl+PDT (*p* < 0.01) group, suggesting that LCP siEGFR NPs showed a positive impact on the inhibition of microvasculature formation of the HNSCC tumors. in groups treated with LCP siEGFR NPs for SCC4 and SAS models compared with PBS group (*p* < 0.01 for both). These groups exhibited significant differences compared with the LCP siControl+PDT (*p* < 0.01) group, suggesting that LCP siEGFR NPs showed a positive impact on the inhibition of microvasculature formation of the HNSCC tumors.

inhibition effect in the LCP siEGFR+PDT group compared to PBS, LCP siControl+PDT, LCP siEGFR+LCP Pyro PA without light, and LCP siEGFR+PBS+light groups (*p* < 0.01) for SCC4 (Figure 7e) and SAS (Figure 7k). This showed that the combination of LCP siEGFR NPs and LCP Pyro PA–mediated PDT enhanced the inhibition effect of the cell proliferation activity significantly. LCP siControl+PDT, LCP siEGFR+LCP-Pyro PA without light, and LCP siEGFR+PBS+light groups demonstrated significant inhibition (*p* < 0.01) in Ki67 amounts compared to PBS group for SCC4 and SAS xenograft models, revealing that LCP-Pyro PA–mediated PDT alone or siEGFR-loaded LCP NPs alone contributed significantly decreasing tumor cell proliferation. The quantified CD31 microvessel intensities of SCC4 and SAS tumors (Figure 7f,l) showed a dramatic decrease

*Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 13 of 18

**Figure 7.** Immunohistochemistry of SCC4 and SAS Tumors; SCC4 tumor cell proliferation and apoptotic cells. Phosphoylated-EGFR (**a**), TUNEL, cleaved caspase-3, Ki-67 and CD31 assays for SCC4 tumor tissue sections. Original magnification x40. Scale bar stands for 100µm. Quantitative analysis for Phosphoylated-EGFR (1092) (**b**), TUNEL (**c**), Cleaved Caspase-3 (**d**), Ki-67 (**e**) and CD31 (**f**). SCC4 tumor cell proliferation and apoptotic cells. Phosphoylated-EGFR (**g**), TUNEL, cleaved caspase-3, Ki-67 and CD31 assays for SAS tumor tissue sections. Original magnification x40. Scale bar stands for 100 µm. Quantitative analysis for phosphoylated-EGFR (1092) (**h**), TUNEL (**i**), cleaved caspase-3 (**j**), Ki-67 (**k**) and CD31 (**l**). Columns, mean (7 images); \* *p* < 0.05; \*\* *p* < 0.01. *3.6. Toxicity and Inflammatory Cytokine Studies In Vivo*  The siEGFR-loaded LCP NPs, siControl-loaded LCP NPs and LCP-Pyro PA NPs were investigated for liver and kidney toxicity. C57BL/6 mice were used for the studies. **Figure 7.** Immunohistochemistry of SCC4 and SAS Tumors; SCC4 tumor cell proliferation and apoptotic cells. Phosphoylated-EGFR (**a**), TUNEL, cleaved caspase-3, Ki-67 and CD31 assays for SCC4 tumor tissue sections. Original magnification 40×. Scale bar stands for 100 µm. Quantitative analysis for Phosphoylated-EGFR (1092) (**b**), TUNEL (**c**), Cleaved Caspase-3 (**d**), Ki-67 (**e**) and CD31 (**f**). SCC4 tumor cell proliferation and apoptotic cells. Phosphoylated-EGFR (**g**), TUNEL, cleaved caspase-3, Ki-67 and CD31 assays for SAS tumor tissue sections. Original magnification 40×. Scale bar stands for 100 µm. Quantitative analysis for phosphoylated-EGFR (1092) (**h**), TUNEL (**i**), cleaved caspase-3 (**j**), Ki-67 (**k**) and CD31 (**l**). Columns, mean (7 images); \* *p* < 0.05; \*\* *p* < 0.01.

Twelve mice were distributed into four groups that were given three IV infusions of PBS, LCP Control siRNA NPs, LCP EGFR siRNA NPs and LCP-Pyro PA NPs for three days,

assays on the fourth day. For liver function, there was no significant difference in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total-bilirubin (T-BIL) (*p* > 0.05) for LCP siControl, LCP siEGFR, and LCP Pyro PA groups compared to the PBS group. This suggests no liver function damage. Similarly in the kidney index, the creatinine (CREA), blood urea nitrogen (BUN), uric acid (UA), phosphorus (P), and calcium (CA) amounts indicated no significant effect (*p* > 0.05) (Figure 8a). No abnormal effect of LCP NPs on kidney function was noted, even though their cores are made of calcium phosphate. In Figure 8b–e, no significant difference was observed on the levels of interleukin 6 (IL-6), interleukin 12 (IL-12 P40/P70), interferon gamma (INF-γ), and Toll-like receptor 3 (TLR3) in triplicate (*p* > 0.05) for all groups (PBS, LCP siControl, LCP siEGFR, and LCP Pyro Pa). This further indicates no toxicity resulting from significant activation by LCP NPs of IL-6, IL-12, INF-γand TLR3 in mouse serum (*p* > 0.05). These in

#### *3.6. Toxicity and Inflammatory Cytokine Studies In Vivo*

The siEGFR-loaded LCP NPs, siControl-loaded LCP NPs and LCP-Pyro PA NPs were investigated for liver and kidney toxicity. C57BL/6 mice were used for the studies. Twelve mice were distributed into four groups that were given three IV infusions of PBS, LCP Control siRNA NPs, LCP EGFR siRNA NPs and LCP-Pyro PA NPs for three days, one injection per day. Mice were sacrificed by cardiac puncture to collect the blood for assays on the fourth day. For liver function, there was no significant difference in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total-bilirubin (T-BIL) (*p* > 0.05) for LCP siControl, LCP siEGFR, and LCP Pyro PA groups compared to the PBS group. This suggests no liver function damage. Similarly in the kidney index, the creatinine (CREA), blood urea nitrogen (BUN), uric acid (UA), phosphorus (P), and calcium (CA) amounts indicated no significant effect (*p* > 0.05) (Figure 8a). No abnormal effect of LCP NPs on kidney function was noted, even though their cores are made of calcium phosphate. In Figure 8b–e, no significant difference was observed on the levels of interleukin 6 (IL-6), interleukin 12 (IL-12 P40/P70), interferon gamma (INF-γ), and Toll-like receptor 3 (TLR3) in triplicate (*p* > 0.05) for all groups (PBS, LCP siControl, LCP siEGFR, and LCP Pyro Pa). This further indicates no toxicity resulting from significant activation by LCP NPs of IL-6, IL-12, INF-γand TLR3 in mouse serum (*p* > 0.05). These in vivo toxicity assays suggest that both novel LCP Pyro PA NPs and LCP siRNA NPs are safe based on SCC4 and SAS animal model studies (Figure 8b–e). *Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 15 of 18 vivo toxicity assays suggest that both novel LCP Pyro PA NPs and LCP siRNA NPs are safe based on SCC4 and SAS animal model studies (Figure 8b–e).

**Figure 8.** In vivo toxicity and inflammatory response. (**a**) The serum levels of C57BL/6 mice for liver Figure 6. (**b**), interleukin-12 p40/70 (**c**), interferon gamma (**d**), Toll-like receptor 3 (**e**). Data presented as mean ± SD (*n* = 3), \* *p* > 0.05 compared to PBS (control) group. **Figure 8.** In vivo toxicity and inflammatory response. (**a**) The serum levels of C57BL/6 mice for liver Figure 6. Mouse Interleukin 6 (IL-6) (**b**), interleukin-12 p40/70 (**c**), interferon gamma (**d**), Toll-like receptor 3 (**e**). Data presented as mean ± SD (*n* = 3), \* *p* > 0.05 compared to PBS (control) group.

and neck cancer patients. Clinical practices for head and neck cancer therapy include surgery, chemotherapy and radiotherapy. PDT is a noninvasive modality for cancer therapy with minimum side effects [3,4]. In this study, we were the first to design and formulate novel liposomal Pyro PA nanoparticles with targeted ligand AEAA for photodynamic therapy. The combined treatment of targeted LCP Pyro PA with targeted LCP siRNA NPs in vitro and in vivo showed positive preclinical outcomes. Significantly, this is the first design and application of the novel EGFR siRNA sequence in LCP EGFR siRNA targeting EGFR to human HNSCC. The design mechanism of drug release of the LCP NPs was based on cores of calcium phosphate LCP nanoparticles. LCP NPs are pH-sensitive nanoparticles that release drugs locally and transiently. LCP NPs are quickly taken up into endosomes and release encapsulated Pyro PA or encapsulated EGFR siRNA

**4. Discussion** 

### **4. Discussion**

Combined therapy has become the mainstream present-day strategy for treating head and neck cancer patients. Clinical practices for head and neck cancer therapy include surgery, chemotherapy and radiotherapy. PDT is a noninvasive modality for cancer therapy with minimum side effects [3,4]. In this study, we were the first to design and formulate novel liposomal Pyro PA nanoparticles with targeted ligand AEAA for photodynamic therapy. The combined treatment of targeted LCP Pyro PA with targeted LCP siRNA NPs in vitro and in vivo showed positive preclinical outcomes. Significantly, this is the first design and application of the novel EGFR siRNA sequence in LCP EGFR siRNA targeting EGFR to human HNSCC. The design mechanism of drug release of the LCP NPs was based on cores of calcium phosphate LCP nanoparticles. LCP NPs are pHsensitive nanoparticles that release drugs locally and transiently. LCP NPs are quickly taken up into endosomes and release encapsulated Pyro PA or encapsulated EGFR siRNA into cell cytoplasmic compartments [11,14]. The asymmetric bilayer structure of LCP NPs was made of anionic lipids (DOPA), cationic lipids (DOTAP) and cholesterol, and LCP NPs were PEGylated to avoid uptake by the immune system and to extend the circulation period in order to enhance the endocytosis of LCP NPs to cancer cells [11,14]. The second leaflet of LCP NPs was PEGylated and also modified with AEAA target ligands, which target the over-expressed sigma receptors in several kinds of cancer cells, including HNSCC cells [9]. LCP Pyro PA NPs and EGFR siRNA NPs were dispersed evenly with similar particle sizes on TEM photomicrographs (Figures 2 and 3). The particle sizes of targeted EGFR siRNA NPs and targeted LCP Pyro PA NPs averaged 34.9 ± 3.0 nm and 15–20 nm, with zeta potentials of 50.1 ± 1.8 mV and 52.0 ± 7.6 mV, respectively. Cabral et al. compared a range of nanoparticle sizes including 30, 50, 70 and 100 nm for accumulation and effectiveness in several poorly and highly penetrable tumor models. Results suggest that 30~50 nm nanomedicines could be the optimal nanoparticle size to penetrate poorly permeable tumors for achieving a better therapeutic effect [16]. Perrault et al. reported that nanoparticle sizes larger than 100 nm exhibited limited permeation into tumors but could be feasible for antiangiogenic therapy; however, the therapeutic effect on malignant tumors was limited due to the larger nanoparticle sizes [17]. The nanoparticle sizes obtained for both LCP Pyro PA NPs and LCP siEGFR NPs were within the suggested 30~50 nm range to enter into tumor cells via endocytosis [16,17]. From our toxicity studies, the CaP cores were shown to be biodegradable and the double lipid bilayer structure of the nanoparticles was relatively functional with regard to safety, with no observed liver and kidney damage caused by LPC NPs (Figure 8). Edmonds et al. reported that EGFR signaling pathway inhibition leads to enhanced PDT cell toxicity with cell apoptosis upregulation. This suggests that targeting EGFR pathways could be a promising solution for PDT clinical trials for patients with cancer metastasis [18]. Protocols of treatment sequence were investigated in vitro to obtain the best feasible order of therapy, which involved conducting LCP siEGFR NPs gene therapy first, followed by PDT (data not shown). We proved that our novel EGFR siRNA sequence did silence EGFR expression. Combined treatment was implemented to apply siEGFR by IV infusion of LCP siRNA NPs for one injection/day for three days, then conducting LCP Pyro PA NPs–mediated PDT on the fourth day. The therapeutic outcome of PDT was demonstrated in the siControl+PDT group. LCP Pyro PA–mediated PDT enhanced inhibition of tumor proliferation and increased tumor apoptosis. In the incomplete PDT treatment groups (LCP siEGFR+LCP Pyro PA without light and LCP siEGFR+PBS+light), the anti-tumorigenic effects of the LCP siEGFR NPs were observed as LCP siEGFR was effectively delivered to cancer cells resulting in lower levels of EGFR mRNA and protein expression. Significant reduction in tumor volume and cell proliferation (Ki67) and upregulation in cell apoptosis (TUNEL and cleaved caspase-3) were observed. LCP siEGFR gene therapy functioned effectively in vivo. Overall, the combination treatment of siEGFR-loaded LCP NPs and LCP Pyro PA mediated PDT demonstrated the greatest inhibition of tumor volume while maintaining inhibition effect. For combined therapy, mRNA and protein expression of EGFR were

significantly inhibited, and consistent results in EGFR immunohistochemical stains were documented. The inhibition of EGFR protein expression was consistent with a decrease in micro-vessel density (CD31 biomarker), which may have been caused by the decrease in Ki67 protein tumor proliferation and enhanced tumor apoptosis (cleaved caspase-3 protein and TUNEL data). In a previous study, liposomal porphysomes were previously formed using pyropheophorbide, with a nanoparticle size of 100nm. Porphysomes have large, tunable extinction coefficients and are effective agents for either photothermal or photoacoustic application [17,18]. In the past, porphysomes were intended for use as a photosensitizer for tumors irradiated under PDT conditions. However, porphysomes did not induce PDT–mediated effects on tissue damage, indicating that porphysomes were ineffective for PDT [18]. Cho et al. investigated a nanocomplex containing dextran sulfate and poly-L-arginine-based polyelectrolyte to deliver EGFR siRNA in a HNSCC xenografted mouse model. The size of this polyelectrolyte nanocomplex was less than 200 nm with a positive zeta potential surface charge. Results showed an enhanced EGFR siRNA uptake efficiency in EGFR gene silencing–tumor cells as well as tumor growth inhibition [19]. However, Cabral et al. reported that nanoparticle size is crucial to the therapeutic success of drug delivery to tumor cells [16]. Nanoparticle sizes less than 50 nm can pass through poorly permeable tumors. These findings suggest that LCP NPs are a better nanodelivery system at <50 nm sizes, promoting effective drug delivery and payloads released to targeted cancer cells. In this study, LCP Pyro PA NPs and LCP siEGFR NPs were both within 20–50 nm in size. A PDT effect was observed for the in vivo LCPcontrol siRNA+PDT group compared to the PBS group (*p* < 0.01). Most importantly, LCP Pyro PA NPs enhanced EGFR siRNA gene therapy, resulting in a significant decrease in tumor volume (*p* < 0.01) compared with the four other experiment groups (Figure 5).

#### **5. Conclusions**

In summary, self-designed siEGFR loaded into LCP NPs and combined with novel LCP Pyro PA NPs–mediated PDT promises to improve the treatment effectiveness in HNSCC. This novel nanomedicine combined targeted LCP Pyro PA mediated PDT and gene therapy in order to effectively silence the EGFR oncogene marker.

**Author Contributions:** All authors discussed the results and contributed to the final manuscript. Conceptualization, L.H. and Y.-C.H.; Methodology, L.H., C.-H.Y., G.Z., J.C. and Y.-C.H.; Writing—original draft preparation, Pyro PA and Pyro lipid synthesis, G.Z. and J.C.; LPC synthesis, characterization, in vitro and in vivo SAS studies, western blots, histology, qPRC, C.-H.Y.; Writing—review & editing, L.H. and Y.-C.H.; Supervision, Y.-C.H.; Funding acquisition, Y.-C.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work conducted in Hsu lab at Departement of Bioscience Technology, Chung Yuan Christian University was supported by Ministry of Science and Technology grant 104-2221-E-033-018- MY3, 108-2119-M-033-001, 109-2221-E-033-008 and 110-2823-8-033-001, Taiwan. The work conducted in Huang lab was supported by NIH grants CA149363, CA151652 and CA149387. The work conducted in Zheng lab was supported by CIHR Foundation Grant #154326, Canada Research Chair Programs and Princess Margaret Cancer Foundation.

**Institutional Review Board Statement:** These studies were approved (approval number 104011) and carried out in strict accordance with the recommendations in the Guide for the Care and Use produced by the Institutional Animal Care and Use Committee of Chung Yuan Christian University, Chungli, Taoyuan, Taiwan.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Thalia Gray and Mary Lin for reviewing, language editing assistance and inputs on manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Article* **Topical Photodynamic Therapy with Different Forms of 5-Aminolevulinic Acid in the Treatment of Actinic Keratosis**

**Joanna Bartosi ´nska 1,\* , Paulina Szczepanik-Kułak <sup>2</sup> , Dorota Raczkiewicz <sup>3</sup> , Marta Niewiedzioł <sup>2</sup> , Agnieszka Gerkowicz <sup>2</sup> , Dorota Kowalczuk <sup>4</sup> , Mirosław Kwa´sny <sup>5</sup> and Dorota Krasowska <sup>2</sup>**


**Abstract:** Photodynamic therapy (PDT) is safe and effective in the treatment of patients with actinic keratosis (AK). The aim of the study was to assess the efficacy, tolerability and cosmetic outcome of topical PDT in the treatment of AKs with three forms of photosensitizers: 5-Aminolevulinic acid hydrochloride (ALA-HCl), 5-Aminolevulinate methyl ester hydrochloride (MAL-HCl) and 5-Aminolevulinate phosphate (ALA-P). The formulations were applied onto selected scalp/face areas. Fluorescence was assessed with a FotoFinder Dermoscope 800 attachment. Skin areas were irradiated with Red Beam Pro+, Model APRO (MedLight GmbH, Herford, Germany). Applied treatments were assessed during the PDT as well as 7 days and 12 weeks after its completion. Ninety-four percent of patients rated obtained cosmetic effect excellent. The efficacy of applied PSs did not differ significantly. However, pain intensity during the PDT procedure was significantly lower in the area treated with ALA-P (5.8 on average) in comparison to the areas treated with ALA-HCl or MAL-HCl (7.0 on average on 0–10 scale). Obtained results show that ALA-P may undergo more selective accumulation than ALA-HCl and MAL-HCl. Our promising results suggest that PDT with the use of ALA-P in AK treatment may be an advantageous alternative to the already used ALA-HCl and MAL-HCl.

**Keywords:** photodynamic therapy; actinic keratosis; photosensitizer; 5-aminolevulinic acid

#### **1. Introduction**

Melanoma and non-melanoma skin cancer are the most common types of skin malignancies in the Caucasian population. Their incidence rates continue to rise, which is a matter of great concern to the patient and a substantial economic burden for the health care system [1,2]. Squamous cell carcinoma (SCC) deserves particular attention because of its tendency to metastasize. The risk of metastasis in invasive SCC is estimated to be about 4% and up to 2–3 times higher in immunosuppressed patients. Therefore, in addition to prevention, identification and therapy of early SCC is vital to avoid neoplastic progression [3].

One of premalignant conditions, with a malignant transformation rate ranging from 0.025% to 16%, is actinic keratosis (AK), an epidermal keratinocytic disorder induced by chronic exposure to ultraviolet (UV) light. A precise prediction of the risk of AK evolution into invasive SCC is infeasible [4]. However, it could be stated that AK plays an important

**Citation:** Bartosi ´nska, J.;

Szczepanik-Kułak, P.; Raczkiewicz, D.; Niewiedzioł, M.; Gerkowicz, A.; Kowalczuk, D.; Kwa´sny, M.; Krasowska, D. Topical Photodynamic Therapy with Different Forms of 5-Aminolevulinic Acid in the Treatment of Actinic Keratosis. *Pharmaceutics* **2022**, *14*, 346. https://doi.org/10.3390/ pharmaceutics14020346

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 20 December 2021 Accepted: 29 January 2022 Published: 1 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

role in the development of SCC. According to Criscione et al. [5], who studied a highrisk population to estimate the risk of progression of AK to SCC, approximately 65% of all primary squamous cell cancers developed from previously diagnosed AK. Moreover, subclinical and early AK lesions are also capable of direct transformation into invasive malignant disease [6]. All this information is sufficient to conclude that each detected AK lesion needs to undergo appropriate therapy, which is especially relevant to patients with multiple AK lesions in whom the risk of progression to SCC is higher [4,7].

Two separate treatment modalities are distinguished for AK, i.e., lesion-directed therapy and field-directed therapy [4]. The main focus of the latter includes treating subclinical lesions, reducing AK recurrence rates, and potentially lowering the risk of developing SCC. Application of the field-directed therapy is based on the theory of "field cancerization", according to which the skin surrounding the lesions, due to chronic exposure to UV radiation, has the potential to transform to malignancy [8].

Photodynamic therapy (PDT) with the use of 5-aminolevulinic acid hydrochloride (ALA-HCl) as a photosensitizer (PS) and irradiation with blue light for the treatment of AK was approved by the FDA in the year 2000. In 2016, the FDA approved the use of ALA-HCl in combination with red light [8]. At present, due to the high treatment efficacy and a good cosmetic outcome, PDT is a routine, first-line treatment for AK. This type of therapy causes local and selective destruction of the neoplastic skin lesions without damage to the healthy tissue. PDT is a hardly invasive method, is generally well tolerated, is repeatable [9] and is applied either directly onto the lesion or onto the entire field of cancerization [4]. (Figure 1).

**Figure 1.** The methods of lesion- and field-directed therapies in actinic keratosis.

PDT has been shown to eliminate AK lesions and prevent their recurrence [8,10]. The action of PDT is based on selective photooxidation of the lesional tissue with the simultaneous involvement of three indispensable components, i.e., a PS, oxygen, and light of appropriate wavelength. The used PS accumulates in the dysplastic cells as well as in all the cells with high proliferation rates, where it is enzymatically metabolized via the heme pathway into the active endogenous photosensitizer protoporphyrin IX (PpIX). When the PS-treated skin is exposed to a light source that spans the absorption spectrum of PpIX (400–730 nm), the compound becomes photoactivated and triggers a photochemical reaction that generates cytotoxic singlet oxygen and free radicals, resulting in subsequent tissue loss [11,12]. The course of PDT and its efficacy are substantially dependent on PpIX production, distribution and depth of its penetration into the skin [13].

ALA-HCl and methyl aminolevulinate (MAL) are the most widely investigated PSs for the treatment of AK lesions. ALA-HCl and MAL are commercially available as Levulan® and Metvix®, respectively [1]. There are reports of some disadvantages associated with these two PSs, especially pain experienced by patients during PDT, which seems to be the main adverse effect and a discouraging factor [14]. Therefore, new substances with more versatile properties are in the pipeline. Unlike ALA-HCl and MAL, the use of 5-aminolevulinic acid phosphate (ALA-P) has not yet been investigated as a PS in PDT of AKs.

The aim of the study was to assess the efficacy, tolerability and cosmetic outcome of topical PDT in the treatment of AK lesions with the use of three different forms of 5-ALA, i.e., 5-Aminolevulinic acid hydrochloride (ALA-HCl), 5-Aminolevulinate methyl ester hydrochloride (MAL-HCl) and 5-Aminolevulinic acid phosphate (ALA-P).

#### **2. Materials and Methods**

### *2.1. Study Group*

Twenty-two Caucasian adults (of II and III skin phototypes), within the age range from 60 to 84 years, with multiple mild to severe AK lesions (Grade I-III according to Olsen) localized on the face and/or scalp were included in the study. The patients received PDT treatment at the outpatient clinic of the Department of Dermatology at the Medical University of Lublin, Poland between April 2018 and July 2021.

Inclusion criteria were as follows: multiple AK lesions Grade I-III according to Olsen and willingness to receive therapy and to participate in follow-up visits. Exclusion criteria were: pregnancy, epilepsy, history of photodermatosis, taking photosensitizing medication, and receiving any AK topical therapies at least three months prior to the beginning of the study. Written informed consent was obtained from all the patients before enrollment in the study. The study was approved by the local ethic committee (KE-0254/286/2019).

#### *2.2. Study Formulations*

In the study, 5-Aminolevulinic acid hydrochloride (ALA-HCl; 5-amino-4-oxopentanoic acid hydrochloride; C5H10ClNO3; [HOOC–CH2–CH2–CO–CH2–NH<sup>3</sup> + ]Cl−); 5-Aminolevulinic acid phosphate (ALA-P; pentanoic acid, 5-amino-4-oxo-, phosphate (1:1); C5H12NO7P; [HOOC–CH2–CH2–CO–CH2–NH<sup>3</sup> + ]H2PO<sup>4</sup> −); 5-Aminolevulinate methyl ester hydrochloride (MAL-HCl; methyl 5-amino-4-oxopentanoate hydrochloride; C6H12ClNO3; H3COOC– CH2–CH2–CO–CH2–NH<sup>3</sup> + ]Cl−), obtained from Arisun chempharm Co., Ltd., Xi'an, China, were used PSs. The purity of ALA-HCl was 99.5% and of ALA-P was 99.2%, whereas MAL-HCl (purity of 97.0%) had to be further purified (recrystallization from methyl alcohol in the acidic environment) until its purity reached 99.5%. The purity of the used substances was confirmed with the standards by high-performance liquid chromatography (HPLC). The lipophilicity (LogP value) for the substances to be examined were calculated using the computer programs based on different calculation methods: ACD/LogP (Advanced Chemistry Development Inc., Toronto, ON, Canada; http://www.acdlabs.com (accessed on 17 January 2022)) ALogPs (VCCLAB, Virtual Computational Chemistry Laboratory, German Research Center for Environmental Health, Neuherberg, Munich, Germany; http://www.vcclab.org (accessed on 17 January 2022)) and miLogP (Molinspiration Software; http://www.molinspiration.com/cgi-bin/properties (accessed on 17 January 2022).

In order to obtain the study formulations, each of the three substances was added to the creamy LIPOBAZA. Since each formulation was to contain 10% of pure 5-ALA, the following concentrations of the study formulations were used: ALA-HCl—12.7%, MAL-HCl—12.5%, ALA-P—17.5%. The degree of homogenization was controlled with the use of an optical microscope.

#### *2.3. Treatment Protocol*

### 2.3.1. Application of Study Formulations

In order to avoid the patients' subjective pain assessment, after the scales and crusts were gently removed from a selected face or scalp field designated for the treatment, in each patient, the skin field to be treated was divided into three roughly equal areas onto which a 1 mm-thick layer of the ALA-HCl, MAL-HCl or ALA-P formulation was applied. Caution was taken to avoid overlapping and mixing of the used formulations. Finally, an occlusive plastic dressing and aluminum foil were placed over the treated skin surface. (Figure 2).

(Figure 2)

(Figure 2)

In order to avoid the patients' subjective pain assessment, after the scales and crusts were gently removed from a selected face or scalp field designated for the treatment, in each patient, the skin field to be treated was divided into three roughly equal areas onto which a 1 mm‐thick layer of the ALA‐HCl, MAL‐HCl or ALA‐P formulation was applied. Caution was taken to avoid overlapping and mixing of the used formulations. Finally, an occlusive plastic dressing and aluminum foil were placed over the treated skin surface.

In order to avoid the patients' subjective pain assessment, after the scales and crusts were gently removed from a selected face or scalp field designated for the treatment, in each patient, the skin field to be treated was divided into three roughly equal areas onto which a 1 mm-thick layer of the ALA-HCl, MAL-HCl or ALA-P formulation was applied. Caution was taken to avoid overlapping and mixing of the used formulations. Finally, an occlusive plastic dressing and aluminum foil were placed over the treated skin surface.

**Figure 2.** Application of study formulations in PDT of AK lesions. **Figure 2.** Application of study formulations in PDT of AK lesions. After a 3 h incubation period, the applied dressing and the remains of the study

*Pharmaceutics* **2022**, *14*, 346 4 of 16

After a 3 h incubation period, the applied dressing and the remains of the study formulations were removed with 0.9% saline solution swabs. After a 3 h incubation period, the applied dressing and the remains of the study formulations were removed with 0.9% saline solution swabs. formulations were removed with 0.9% saline solution swabs. 2.3.2. Assessment of Skin Fluorescence Following Application of Study Formulations

#### 2.3.2. Assessment of Skin Fluorescence Following Application of Study Formulations 2.3.2. Assessment of Skin Fluorescence Following Application of Study Formulations Qualitative absorption of the PSs contained in each of the study formulations was

Qualitative absorption of the PSs contained in each of the study formulations was assessed with the use of a special attachment of FotoFinderDermoscope 800 with white and violet LED diodes emitting LED light. The opaque special material of the FotoFinder FD lens was used. (Figure 3a) The fluorescence coming from PpIX formed after application of each PS was rated as high, medium and low, as well as diffuse, confluent Qualitative absorption of the PSs contained in each of the study formulations was assessed with the use of a special attachment of FotoFinderDermoscope 800 with white and violet LED diodes emitting LED light. The opaque special material of the FotoFinder FD lens was used. (Figure 3a) The fluorescence coming from PpIX formed after application of each PS was rated as high, medium and low, as well as diffuse, confluent and blotchy. assessed with the use of a special attachment of FotoFinderDermoscope 800 with white and violet LED diodes emitting LED light. The opaque special material of the FotoFinder FD lens was used. (Figure 3a) The fluorescence coming from PpIX formed after application of each PS was rated as high, medium and low, as well as diffuse, confluent and blotchy.

(**a**) (**b**) **Figure 3.** The use of FotoFinderDermoscope 800 to assess the fluorescence (**a**); the use of MedLight GmbH red light 630 ± 5 nm (**b**). **Figure 3.** The use of FotoFinderDermoscope 800 to assess the fluorescence (**a**); the use of MedLight GmbH red light 630 ± 5 nm (**b**).

**Figure 3.** The use of FotoFinderDermoscope 800 to assess the fluorescence (**a**); the use of MedLight

#### GmbH red light 630 ± 5 nm (**b**). 2.3.3. Irradiation with Red Light 630 ± 5 nm 2.3.3. Irradiation with Red Light 630 ± 5 nm

2.3.3. Irradiation with Red Light 630 ± 5 nm The field-directed PDT was performed on the selected areas of the face and/or scalp. The study participants were instructed to wear protective glasses during irradiation. The field-directed PDT was performed on the selected areas of the face and/or scalp. The study participants were instructed to wear protective glasses during irradiation.

The field‐directed PDT was performed on the selected areas of the face and/or scalp. The study participants were instructed to wear protective glasses during irradiation. Irradiation sessions were conducted with the use of Red Beam Pro+, Model APRO (MedLight GmbH, Herford, Germany), which insures optimal light penetration. (Figure 3b).

The Red Beam Pro+ lamp contains three movable units with 78 high power red lightemitting diodes, which provide red light operating at 630 ± 5 nm [9]. A total dose per session was 37 J/cm<sup>2</sup> , and the light power intensity was equal to 68 mW/cm<sup>2</sup> . The distance from the face and/or scalp was 10 cm. The patients were instructed to avoid sun exposure for 48 h following the treatment.

#### 2.3.4. PDT with Study Formulations Efficacy Assessment

Assessment of the percent of AK lesion clearance in each treated area was performed 12 weeks after PDT completion.

### 2.3.5. PDT with Study Formulations Cosmetic Outcome and Patient Satisfaction Assessment

Twelve weeks after the PDT completion, cosmetic outcome assessment was performed with the use of a four-grade scale (Table 1), and the study participants were asked to express their level of satisfaction with the PDT and willingness to repeat the therapy if the need be.

**Table 1.** Cosmetic outcome of photodynamic therapy assessment.


#### 2.3.6. PDT with Study Formulations Tolerability Assessment

All the study participants were assessed by the same dermatologists during the procedures, shortly after their completion, and 7 days after the PDT completion.

During the procedure and 7 days after, the study participants were asked to evaluate pain intensity in each treated area on a 10-point VAS scale, and their exact location was determined with a pointer.

Frequency and severity of occurrence of PDT side effects, i.e., erythema, edema, desquamation and crusting, were defined as: none, mild, moderate, or severe.

Erythema and edema were assessed shortly after and 7 days after PDT.

Exfoliation, crusting and pigmentation were assessed 7 days after PDT.

Photographs of the skin lesions were taken at every visit.

#### *2.4. Statistical Methods*

The data were analyzed using STATISTICA 13 software (Statsoft, Kraków, Poland). The mean and standard deviation were estimated for numerical variables, as well as for absolute numbers (n) and percentages (%) of the occurrence of items for categorical variables.

Wilcoxon's signed rank test for paired samples was used to compare severity of pain (10-point scale from 0 to 10) or clearance (in percentage) between every pair of two photosensitizers between the first and the second procedures.

The significance level was assumed to be 0.05 in all statistical tests.

#### **3. Results**

#### *3.1. Characteristics of the Study Group*

Characteristics of the AK patients and AK lesions are presented in Table 2.

**Table 2.** Characteristics of the studied actinic keratosis patients.


Since extensive AK lesions are more frequently observed in males, we selected 21 men (18 with AK lesions on the scalp and 3 on the face) as well as 1 woman presenting with facial AK lesions. men (18 with AK lesions on the scalp and 3 on the face) as well as 1 woman presenting with facial AK lesions. Each of the study subjects completed the first PDT procedure, and because of

Since extensive AK lesions are more frequently observed in males, we selected 21

I grade (thin)

Characteristics of the AK patients and AK lesions are presented in Table 2.

**Variable Category Parameter Estimate** Age years Min‐max 60‐84

<sup>n</sup> (%) <sup>21</sup> (95%)

<sup>n</sup> (%) <sup>4</sup> (18%)

9 (24%)

female 1 (5%)

scalp 18 (82%)

II grade (moderately thick) 18 (49%) III grade (thick) 10 (27%)

n (%)

Each of the study subjects completed the first PDT procedure, and because of extensive AK lesions, 16 of them qualified for a second PDT session. One patient refused to continue the treatment because of severe pain experienced during the first PDT procedure. extensive AK lesions, 16 of them qualified for a second PDT session. One patient refused to continue the treatment because of severe pain experienced during the first PDT procedure.

#### *3.2. Fluorescence Assessment Following Application of Study Formulations 3.2. Fluorescence Assessment Following Application of Study Formulations*

*Pharmaceutics* **2022**, *14*, 346 6 of 16

**Table 2.** Characteristics of the studied actinic keratosis patients.

Sex male

Localizations of lesions face

**3. Results**

*3.1. Characteristics of the Study Group*

Thickness grade, according to Olsen et al. [14]

Assessment of fluorescence intensity was performed for each of the study subjects before application of irradiation with red light 630 ± 5 nm. In the majority of patients (88% of them in the areas treated with ALA-HCl and 85% of them in the areas treated with MAL-HCl), the fluorescence intensity was high and confluent, while in the majority of patients (82% of them in the areas treated with ALA-P), the fluorescence intensity was lower and blotchy. (Figure 4). Assessment of fluorescence intensity was performed for each of the study subjects before application of irradiation with red light 630 ± 5 nm. In the majority of patients (88% of them in the areas treated with ALA‐HCl and 85% of them in the areas treated with MAL‐HCl), the fluorescence intensity was high and confluent, while in the majority of patients (82% of them in the areas treated with ALA‐P), the fluorescence intensity was lower and blotchy. (Figure 4).

**Figure 4.** Intensity and distribution of fluorescence after application of ALA-HCl, MAL-HCl, and ALA-P.

#### *3.3. PDT with Study Formulations Efficacy*

Clearance 12 weeks after the first and second PDT procedures performed with ALA-HCl, MAL-HCl and ALA-P is presented in Figure 5. The clearance of AK lesions 12 weeks after the first PDT procedure in 22 patients was 83.9 ± 7.7%, 88.2 ± 7.5% and 86.6 ± 7.6% on average for ALA-HCl, MAL-HCl and ALA-P, respectively, while 12 weeks after the second PDT procedure in 15 patients, it was 82.7 ± 5.6%, 86.3 ± 7.2% and 83.7 ± 6.9%, respectively. The efficacy of any of the applied PSs did not differ significantly in the overall treatment of AK lesions (*p* > 0.05) both after the first and second PDT procedure.

ALA‐P.

average).

*3.3. PDT with Study Formulations Efficacy*

overall treatment of AK lesions (*p* > 0.05) both after the first and second PDT procedure.

**Figure 4.** Intensity and distribution of fluorescence after application of ALA‐HCl, MAL‐HCl, and

Clearance 12 weeks after the first and second PDT procedures performed with ALA‐HCl, MAL‐HCl and ALA‐P is presented in Figure 5. The clearance of AK lesions 12 weeks after the first PDT procedure in 22 patients was 83.9 ± 7.7%, 88.2 ± 7.5% and 86.6 ± 7.6% on average for ALA‐HCl, MAL‐HCl and ALA‐P, respectively, while 12 weeks after

**Figure 5.** Clearance of actinic keratosis lesions 12 weeks after the first and second PDT procedures with the use of study formulations. Midpoint, mean; box, mean ± standard deviation; whiskers, min–max. Clearance did not significantly differ among three studied PSs as well as between the first and second procedures (*p* > 0.05). p stands for Wilcoxon's signed rank test for paired samples. **Figure 5.** Clearance of actinic keratosis lesions 12 weeks after the first and second PDT procedures with the use of study formulations. Midpoint, mean; box, mean ± standard deviation; whiskers, min–max. Clearance did not significantly differ among three studied PSs as well as between the first and second procedures (*p* > 0.05). *p* stands for Wilcoxon's signed rank test for paired samples.

#### *3.4. PDT with Study Formulations COSMETIC Outcome and Patient Satisfaction 3.4. PDT with Study Formulations COSMETIC Outcome and Patient Satisfaction*

The vast majority of the study participants evaluated the overall cosmetic effect as excellent (94%), and the remaining 6% of them described it as good. Since scarring, atrophy, or induration were not observed, none of the participants found the PDT cosmetic outcome to be fair or poor. Patient satisfaction with the treatment was high, except for one patient who did not want to repeat the procedure because of excruciating pain experienced during the first PDT procedure. The vast majority of the study participants evaluated the overall cosmetic effect as excellent (94%), and the remaining 6% of them described it as good. Since scarring, atrophy, or induration were not observed, none of the participants found the PDT cosmetic outcome to be fair or poor. Patient satisfaction with the treatment was high, except for one patient who did not want to repeat the procedure because of excruciating pain experienced during the first PDT procedure.

#### *3.5. PDT with Study Formulations Tolerability*

*3.5. PDT with Study Formulations Tolerability* During PDT, all the studied patients reported at least one adverse reaction. Pain was their most frequent complaint, and if it was unbearable, short breaks in irradiation were During PDT, all the studied patients reported at least one adverse reaction. Pain was their most frequent complaint, and if it was unbearable, short breaks in irradiation were taken, or a cool dressing was applied.

taken, or a cool dressing was applied. Pain intensity (on 10‐point scale from 0 to 10) during PDT and 7 days after the PDT completion was compared between the PSs grouped in pairs, in both the first and second Pain intensity (on 10-point scale from 0 to 10) during PDT and 7 days after the PDT completion was compared between the PSs grouped in pairs, in both the first and second procedures separately (Figure 6a).

procedures separately (Figure 6a). In the 22 studied subjects, pain intensity during the first PDT procedure was significantly lower in the area treated with ALA‐P (5.8 on average) in comparison to the areas treated with either ALA‐HCl or MAL‐HCl (7.0 on average in 0–10 scale). However, pain intensity 7 days after the first PDT procedure was significantly lower for MAL‐HCl In the 22 studied subjects, pain intensity during the first PDT procedure was significantly lower in the area treated with ALA-P (5.8 on average) in comparison to the areas treated with either ALA-HCl or MAL-HCl (7.0 on average in 0–10 scale). However, pain intensity 7 days after the first PDT procedure was significantly lower for MAL-HCl and ALA-P (1.4 and 1.3 on average, respectively) in comparison to ALA-HCl (1.8 on average).

and ALA‐P (1.4 and 1.3 on average, respectively) in comparison to ALA‐HCl (1.8 on In 15 patients, pain intensity during the second PDT procedure did not significantly differ between the three used PSs (5.3 on average for ALA‐HCl; 5.2 on average for In 15 patients, pain intensity during the second PDT procedure did not significantly differ between the three used PSs (5.3 on average for ALA-HCl; 5.2 on average for MAL-HCl; 4.7 on average for ALA-P). Pain intensity 7 days after the second PDT procedure was low and did not significantly differ between the three PSs (1.1 on average for ALA-HCl; 1.3 on average for MAL-HCl; 0.9 on average for ALA-P).

MAL‐HCl; 4.7 on average for ALA‐P). Pain intensity 7 days after the second PDT Pain intensity (on 10-point scale from 0 to 10) during PDT and 7 days after PDT was compared between the first and second procedures in 15 patients who underwent both procedures (Figure 6b).

> Pain intensity was significantly lower during the second PDT procedure than during the first PDT procedure for each PS (*p* = 0.001 for ALA-HCl and MAL-HCl, *p* = 0.023 for ALA-P).

**Figure 6.** Pain intensity during PDT and 7 days after PDT in the first procedure (*n* = 22 patients) and in the second procedure (*n* = 15 patients): compared between every pair of two photosensitizers (**a**); compared between the first procedure and the second procedure (*n* = 15 patients) (**b**). Pain intensity on 10‐point scale from 0 to 10. Midpoint, mean; box, mean ± standard deviation; whiskers, min–max. *p* stands for Wilcoxon's signed rank test for paired samples. **Figure 6.** Pain intensity during PDT and 7 days after PDT in the first procedure (*n* = 22 patients) and in the second procedure (*n* = 15 patients): compared between every pair of two photosensitizers (**a**); compared between the first procedure and the second procedure (*n* = 15 patients) (**b**). Pain intensity on 10-point scale from 0 to 10. Midpoint, mean; box, mean ± standard deviation; whiskers, min–max. *p* stands for Wilcoxon's signed rank test for paired samples.

procedure was low and did not significantly differ between the three PSs (1.1 on average

Pain intensity (on 10‐point scale from 0 to 10) during PDT and 7 days after PDT was compared between the first and second procedures in 15 patients who underwent both

Pain intensity was significantly lower during the second PDT procedure than during the first PDT procedure for each PS (*p* = 0.001 for ALA‐HCl and MAL‐HCl, *p* = 0.023 for

Pain intensity 7 days after the second procedure was significantly lower than 7 days after the first PDT procedure only in ALA‐HCl (*p* = 0.003), while it was not significant in

for ALA‐HCl; 1.3 on average for MAL‐HCl; 0.9 on average for ALA‐P).

procedures (Figure 6b).

MAL‐HCl (*p* = 0.686) or ALA‐P (*p* = 0.091).

ALA‐P).

It was noted that the pain experienced during treatment tapered off/subsided together with the time of exposure to the red LED light. In our study, similar local responses to the three investigated PSs were observed. It Pain intensity 7 days after the second procedure was significantly lower than 7 days after the first PDT procedure only in ALA-HCl (*p* = 0.003), while it was not significant in MAL-HCl (*p* = 0.686) or ALA-P (*p* = 0.091).

was also observed that in the area treated with the ALA‐P formulation, the intensity of erythema was mild in over half of the study participants (59%), while in the area where It was noted that the pain experienced during treatment tapered off/subsided together with the time of exposure to the red LED light.

In our study, similar local responses to the three investigated PSs were observed. It was also observed that in the area treated with the ALA-P formulation, the intensity of erythema was mild in over half of the study participants (59%), while in the area where ALA-HCl and MAL-HCl were applied, the erythema intensity was moderate in the vast majority of the studied patients (82% and 91%, respectively). Seven days after the first PDT procedure, the same differences were still observed for the three studied PSs. No differences in erythema intensity were observed between the first and second PDT procedures, both shortly after and 7 days after PDT (Figure 7a).

**Figure 7.** The severity of erythema (**a**), edema (**b**), desquamation (**c**), crusting (**d**), and pigmentation (**e**) in the first and second PDT procedures.

ALA‐HCl and MAL‐HCl were applied, the erythema intensity was moderate in the vast majority of the studied patients (82% and 91%, respectively). Seven days after the first PDT procedure, the same differences were still observed for the three studied PSs. No differences in erythema intensity were observed between the first and second PDT

Shortly after and 7 days after PDT completion, edema intensity was mild in all or almost all study subjects regardless of the applied PS during both first and second PDT

Seven days after PDT completion, desquamation was mild in all the areas treated with MAL‐HCl and ALA‐P and in all but one patient treated with ALA‐HCl, both in the

Seven days after PDT completion, in three‐quarters of the study participants, crusting intensity was moderate, regardless of the used formulation in the first PDT procedure, whereas in 80% of the patients, it was moderate in the area treated with ALA‐HCl, and in 60% of the patients, it was mild in the areas treated with MAL‐HCl and

Seven days after PDT completion, the lowest pigmentation intensity was observed in the areas treated with ALA‐P both in the first and second PDT procedures (Figure 7e).

procedures, both shortly after and 7 days after PDT (Figure 7a).

procedures (Figure 7b).

ALA‐P (Figure 7d).

first and second PDT procedures (Figure 7c).

Shortly after and 7 days after PDT completion, edema intensity was mild in all or almost all study subjects regardless of the applied PS during both first and second PDT procedures (Figure 7b).

Seven days after PDT completion, desquamation was mild in all the areas treated with MAL-HCl and ALA-P and in all but one patient treated with ALA-HCl, both in the first and second PDT procedures (Figure 7c).

Seven days after PDT completion, in three-quarters of the study participants, crusting intensity was moderate, regardless of the used formulation in the first PDT procedure, whereas in 80% of the patients, it was moderate in the area treated with ALA-HCl, and in 60% of the patients, it was mild in the areas treated with MAL-HCl and ALA-P (Figure 7d).

Seven days after PDT completion, the lowest pigmentation intensity was observed in the areas treated with ALA-P both in the first and second PDT procedures (Figure 7e).

Some examples of local adverse reactions observed in the studied patients are presented in Figure 8.

procedures.

**Figure 7.** The severity of erythema (**a**), edema (**b**), desquamation (**c**), crusting (**d**), and pigmentation (**e**) in the first and second PDT

presented in Figure 8.

Some examples of local adverse reactions observed in the studied patients are

**Figure 8.** Local adverse reactions observed in the studied patients. **Figure 8.** Local adverse reactions observed in the studied patients.

The comparisons of the skin areas in the studied patients before PDT and 12 weeks after procedure completion are shown in Figures 9 and S1 (Supplementary Materials). The comparisons of the skin areas in the studied patients before PDT and 12 weeks after procedure completion are shown in Figures 9 and S1 (Supplementary Materials).

**Figure 9.** Comparisons of the skin areas in the studied patients before PDT and 12 weeks after procedure completion. **Figure 9.** Comparisons of the skin areas in the studied patients before PDT and 12 weeks after procedure completion.

#### **4. Discussion 4. Discussion**

In actinic keratoses, which are common skin lesions that may progress to invasive SCC, PDT is invaluable because of its minimal invasiveness and high efficacy. This paper presents the first observational, uncontrolled study of the efficacy and tolerability of a novel photosensitizer known as ALA‐P and draws comparisons with two other commercially available PSs, i.e., ALA‐HCl and MAL‐HCl used in PDT of AK lesions. Aminolevulinic acid phosphate (C5H12NO7P), with a molecular weight of 229.13 g/mol, is a fairly new synthetic chemical compound also known as UNII‐FM8DCR39GH; Pentanoic acid, 5‐amino‐4‐oxo‐, phosphate (1:1); 868074‐65‐1; 5‐Aminolevulinic acid phosphate. Due to its properties, it has already been approved as a nutritional supplement in Japan and a few other Asian countries [15]. When administered by mouth, ALA‐P has been shown to reduce blood glucose levels [16]. Furthermore, Higashikawa et al. observed that ALA‐P diminished the severity of negative emotions in individuals continuously feeling physically fatigued [17]. All this made us presume that if orally used ALA‐P is safe, effective and well tolerated, it could also be applied onto the skin with no need for in vivo tests to treat AK lesions with the use of PDT. In actinic keratoses, which are common skin lesions that may progress to invasiveSCC, PDT is invaluable because of its minimal invasiveness and high efficacy. This paperpresents the first observational, uncontrolled study of the efficacy and tolerability of a novel photosensitizer known as ALA-P and draws comparisons with two other commercially available PSs, i.e., ALA-HCl and MAL-HCl used in PDT of AK lesions. Aminolevulinic acid phosphate (C5H12NO7P), with a molecular weight of 229.13 g/mol, is a fairly new synthetic chemical compound also known as UNII-FM8DCR39GH; Pentanoic acid, 5-amino-4-oxo-, phosphate (1:1); 868074-65-1; 5-Aminolevulinic acid phosphate. Due to its properties, it has already been approved as a nutritional supplement in Japan and a few other Asian countries [15]. When administered by mouth, ALA-P has been shown to reduce blood glucose levels [16]. Furthermore, Higashikawa et al. observed that ALA-P diminished the severity of negative emotions in individuals continuously feeling physically fatigued [17]. All this made us presume that if orally used ALA-P is safe, effective and well tolerated, it could also be applied onto the skin with no need for in vivo tests to treat AK lesions with the use of PDT.

Thus, the novelty of the method presented in this study consists of the innovative, topical use of ALA-P as a photosensitizer, whereas assessment of the efficacy, tolerability and cosmetic outcome of ALA-P application in comparison with two other photosensitizers, i.e., ALA-HCl and MAL, revealed slightly better tolerance of the PDT procedure with the use of ALA-P.

The use of ALA-HCl and MAL in PDT has proven to be highly effective [18,19]. Nevertheless, the number of papers directly comparing the clinical outcomes of PDTs with ALA-HCl and MAL in AK patients is limited [18]. Moloney et al. performed a randomized, double-blind, prospective study comparing the efficacy and adverse effects of MAL-PDT and ALA-PDT in the treatment of scalp AKs. Their results showed that both ALA-PDT and MAL-PDT caused a significant reduction in the number of AK foci, with no significant difference in efficacy [20]. Fu et al., in a recent meta-analysis investigating the combination of PDT with BF-200 ALA (a 5-aminolevulinic acid nanoemulsion of ALA) versus MAL, indicated that PDT with the former (i.e., BF-200 ALA) had a 9% better chance of complete clearance of AK lesions at 3 months of treatment and a 24% better chance of grade II-III AK lesion clearance in comparison to the results of PDT with the use of the latter (i.e., MAL) [21]. In our study, the clearance assessed 12 weeks after treatment completion demonstrated similarly high PDT efficacy (clearance above 80%), regardless of the applied Ps (*p* > 0.05).

PDT is generally considered to give good cosmetic results with high patient satisfaction. This seems to be of particular importance in the treatment of the lesions localized on the exposed parts of the body [22]. In our study, 94% of the patients found the overall cosmetic effect to be excellent, while the remaining 6% assessed it as good, regardless of the used formulation. A study by Ko et al. compared PDT performed with ALA-HCl as well as MAL in AK patients. The cosmetic outcome after the treatment with the former was rated by 90% of the patients as excellent, while PDT performed with the latter was assessed as excellent by 97% of the study participants, which is in agreement with our results. The excellent result was still observed 12 months after the completion of treatment [23]. Räsänen et al. [24] compared the cosmetic outcome of PDT with the use of either BF-200 ALA or MAL in 69 patients with numerous AK lesions. In their study, 12 months after treatment completion, the cosmetic outcome was excellent or good in >90% of the studied patients, regardless of the used PS.

In order to make sure that PDT is highly effective, the following components need to be present, i.e., proper light energy source, oxygen and a PS with desirable properties such as water solubility, lipophilicity, penetration capability and accumulation in the skin.

In order to demonstrate the accumulation of the investigated PSs in the treated skin area, photodynamic diagnostics with white and violet LED diodes emitting LED light may be used [25]. This type of diagnostic involves the use of PpIX fluorescence, thereby enabling not only determination of PS accumulation in the diseased tissue but also establishing the boundaries between healthy and diseased tissues [26] and the extent and nature of the neoplastic skin lesions, malignant and non-malignant [27].

The intensity of red fluorescence depends on the skin penetration by a selected PS and production of PpIX in the epidermis determined by its lipophilic character. The calculated LogP values for the tested photosensitizers indicate a higher lipophilicity of MAL-HCl (ACD/LogP = −0.57, ALogPs = −1.30, miLogP = −1.62) as the ALA methyl ester compared to ALA-HCl and ALA-P (for both substances: ACD/LogP = −0.93, ALogPs = −2.85, miLogP = −1.93). In our study, confluent, intensive fluorescence seen in the ALA-HCl and MAL-HCl-treated skin areas indicates a remarkably fine accumulation of ALA in the skin and a high amount of PpIX production. However, the lower and blotchy fluorescence we observed in the skin areas treated with ALA-P may be suggestive of a more selective accumulation of ALA-P in the AK lesions. Therefore, because of their lower kinetics of PpIX formation, ALA-P may facilitate obtaining a better contrast between the healthy and diseased tissue [28,29]. It seems that the observed differences in the distribution of ALA-P in relation to ALA-HCl and MAL-HCl may result from the presence of the H2PO4- ion. Available studies suggest that the H2PO4- ion mediates the proton transfer required for the

enolization of amino acids by acting simultaneously as both a general base and a general acid. The dihydro-phosphate ion may catalyze various reactions in proteins, including the racemization of amino acid residues. Therefore, it may contribute to better cellular membrane permeability [30], as racemization of the amino acids influences the functions of many intracellular, extracellular and membrane-bound proteins, and it is considered as a critical factor of protein conformation [31]. However, the observations made must be confirmed in further studies.

PDT is not free of local side effects such as pain, erythema, edema, desquamation, crusting, and pustules, which may occur both during the PDT procedure and in the next hours/days. Urticaria, contact dermatitis, erosive pustular dermatosis of the scalp, pigmentary lesions, scarring, and bullous pemphigoid are less frequently reported [32].

One major drawback of PDT is pain, which may be the reason for discontinuation of the treatment altogether and which may discourage the patient from undergoing future treatments [26]. A number of studies have compared the intensity of pain experienced after application of ALA-HCl or MAL. The results, however, are difficult to interpret due to the use of different formulations and study protocols [33]. Therefore, bearing in mind the intensive pain accompanying PDT, the PDT procedure should be divided into a few stages with time intervals. In our study, which consisted of 22 patients, 16 of them qualified for the second PDT procedure because of the extensiveness of their AK lesions. Although all 22 patients completed the first PDT procedure successfully, one of the 16 patients requiring the second PDT procedure refused further treatment because of unbearable pain. We observed that the intensity of pain on a 10-point VAS scale during and shortly after the first PDT procedure performed in the skin areas treated with either ALA-HCl or MAL-HCl was similar (7.0 on average), whereas it was slightly lower in the skin areas treated with ALA-P (5.8 on average). However, 7 days after the first PDT procedure, the intensity of pain was significantly lower in the areas treated with MAL-HCl or ALA-P (1.4 and 1.3 on average, respectively) than in the areas treated with ALA-HCl (1.8 on average). In most of the studies, patients reported more acute pain at the site of ALA-HCl application [20,34–39]. However, in the reports by Yazdanyar et al., who compared the pain response to ALA-HCl and MAL treatment of the scalp and forehead AK lesions, no significant differences in pain intensity between these two formulations were found either during or 30 min after the completion of treatment [40]. Ibotson et al. [41] indicated that it was still unclear which of the tested compounds, i.e., ALA-HCl or MAL, caused more severe pain during irradiation.

In our study, the intensity of pain during the second PDT procedure and 7 days after its completion did not significantly differ regardless of the used PS, and it was slightly lower than during the first PDT procedure (approximately 5 points on average and approximately 1 point on average, respectively). It appears that the pain experienced during the second PDT procedure is better tolerated because of its foreseeable nature.

A limitation of our study is the quality of images showing intensity and distribution of fluorescence in the skin areas after application of ALA-HCl, MAL-HCl, ALA-P. Future studies, e.g., using an animal model and a higher-resolution device are needed.

#### **5. Conclusions**

ALA-P, a new PS first tested in our study turned out to be similarly effective as ALA-HCl and MAL-HCl. We suggest that the use of ALA-P in PDT of AK lesions should be further investigated, because despite in a small number of our study subjects, the obtained results are promising enough to acknowledge the fact that this new ALA photosensitizer may be an advantageous alternative to the already used ALA-HCl and MAL since its application appears to be slightly less painful and better tolerated.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics14020346/s1, Figure S1: Comparisons of the skin areas in the studied patients before PDT and 12 weeks after procedure completion.

**Author Contributions:** Conceptualization, D.K. (Dorota Krasowska), M.K., and J.B.; methodology, D.K. (Dorota Krasowska), M.K., J.B., A.G. and M.N.; software, J.B., P.S.-K. and D.R.; formal analysis, D.R., J.B., and D.K. (Dorota Kowalczuk); investigation, J.B., P.S.-K., M.N. and A.G.; data curation, P.S.-K., J.B., M.N. and D.K. (Dorota Kowalczuk) and D.R.; writing—original draft preparation, J.B., P.S.-K., A.G. and D.R.; writing—review and editing, D.K. (Dorota Krasowska), M.K. and D.K. (Dorota Kowalczuk); visualization, J.B., P.S.-K. and D.R.; supervision, D.K. (Dorota Krasowska) and M.K.; project administration, D.K. (Dorota Krasowska); funding acquisition, D.K. (Dorota Krasowska) and M.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of Medical University of Lublin, Poland (KE-0254/286/2019).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data sharing not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Photodynamic Therapy Targeting Macrophages Using IRDye700DX-Liposomes Decreases Experimental Arthritis Development**

**Daphne N. Dorst 1,2,\* ,† , Marti Boss 1,†, Mark Rijpkema <sup>1</sup> , Birgitte Walgreen <sup>2</sup> , Monique M. A. Helsen <sup>2</sup> , Desirée L. Bos <sup>1</sup> , Louis van Bloois <sup>3</sup> , Gerrit Storm 3,4 , Maarten Brom <sup>1</sup> , Peter Laverman <sup>1</sup> , Peter M. van der Kraan <sup>2</sup> , Mijke Buitinga 5,6, Marije I. Koenders <sup>2</sup> and Martin Gotthardt <sup>1</sup>**

	- 7522 NL Enschede, The Netherlands

**Abstract:** Macrophages play a crucial role in the initiation and progression of rheumatoid arthritis (RA). Liposomes can be used to deliver therapeutics to macrophages by exploiting their phagocytic ability. However, since macrophages serve as the immune system's first responders, it is inadvisable to systemically deplete these cells. By loading the liposomes with the photosensitizer IRDye700DX, we have developed and tested a novel way to perform photodynamic therapy (PDT) on macrophages in inflamed joints. PEGylated liposomes were created using the film method and post-inserted with micelles containing IRDye700DX. For radiolabeling, a chelator was also incorporated. RAW 264.7 cells were incubated with liposomes with or without IRDye700DX and exposed to 689 nm light. Viability was determined using CellTiterGlo. Subsequently, biodistribution and PDT studies were performed on mice with collagen-induced arthritis (CIA). PDT using IRDye700DX-loaded liposomes efficiently induced cell death in vitro, whilst no cell death was observed using the control liposomes. Biodistribution of the two compounds in CIA mice was comparable with excellent correlation of the uptake with macroscopic and microscopic arthritis scores. Treatment with 700DXloaded liposomes significantly delayed arthritis development. Here we have shown the proofof-principle of performing PDT in arthritic joints using IRDye700DX-loaded liposomes, allowing locoregional treatment of arthritis.

**Keywords:** photodynamic therapy; liposomes; experimental arthritis

## **1. Introduction**

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting the synovial joints in 0.5–1% of the general population [1]. The disease is characterized by relapsing and remitting inflammation associated with progressive damage to the joints [2]. Treatment of RA consists of disease-modifying anti-rheumatic drugs (DMARDs). Despite the treatment

**Citation:** Dorst, D.N.; Boss, M.; Rijpkema, M.; Walgreen, B.; Helsen, M.M.A.; Bos, D.L.; van Bloois, L.; Storm, G.; Brom, M.; Laverman, P.; et al. Photodynamic Therapy Targeting Macrophages Using IRDye700DX-Liposomes Decreases Experimental Arthritis Development. *Pharmaceutics* **2021**, *13*, 1868. https://doi.org/10.3390/ pharmaceutics13111868

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 14 September 2021 Accepted: 1 November 2021 Published: 5 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

paradigm having changed, a considerable percentage of patients do not respond adequately to both biological and synthetic DMARD treatment [3].

In RA, the synovial lining, a thin layer of cells providing a barrier between the synovial fluid and the joint tissue, becomes hyperproliferative, infiltrated with immune cells, and forms a tumor-like pannus. A key cell already present in this resident synovial lining is the macrophage. Its phagocytic function is crucial for maintaining joint homeostasis [4]. In RA, the function of the macrophage changes as it adapts a pro-inflammatory phenotype. Through its secretion of pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin (IL) −1, −6, −10 and granulocyte-macrophage colony-stimulating factor, it actively contributes to chemotaxis and activation of infiltrating immune cells [5,6]. Furthermore, these cytokines stimulate resident synovial fibroblasts to adapt and propagate an activated and destructive phenotype [6]. Through their production of enzymes such as matrix metalloproteinases, macrophages also directly contribute to joint destruction [6,7]. Due to the crucial role of macrophages in the initiation and propagation of arthritis, they are an interesting therapeutic target. Liposomes can be used to target macrophages since they are preferentially phagocytosed by these cells [8–11]. Depleting macrophages with clodronate-loaded liposomes decreased arthritis in rodents [12–14] and patients [15]. However, systemic administration of clodronate liposomes also resulted in depletion of these cells from the spleen and liver [14]. This may have serious side effects, such as a higher susceptibility to infection and cancer, and is therefore not an advisable strategy.

Instead, the selective depletion of macrophages only in the affected synovial joint would be a preferred treatment method. Therefore, loading the liposomes with a drug that is constantly active, such as clodronate, and that will thus exert its function in all cells that phagocytose it, is not optimal. Instead, a preferable approach would be to load the liposomes used for macrophage targeting with an inert molecule that can selectively be activated at the sites of inflammation. To this end, photodynamic therapy (PDT) may be a promising therapeutic approach. In PDT, a light-sensitive molecule, a so-called photosensitizer (PS), is used. By exposing the region to be treated to light of a specific wavelength, the PS will produce reactive oxygen species (ROS), which are cytotoxic to the cells in its vicinity. Off-target effects are limited, since the photosensitizer only becomes active upon excitation by locally applied light of a PS-specific wave-length (690 nm for the IRDye700DX used in this study), thus also avoiding side effects by daylight exposure. IRDye700DX (Pubchem ID: 102004325) is a phthalocyanine dye with two siloxane chains to improve water solubility. A NHS ester can be attached to the photosensitizer, which facilitates conjugation to free amino groups on macromolecules. This has previously been used by our group to modulate arthritis when conjugated to a monoclonal antibody targeting fibroblast activation protein [16].

Here, we investigate if targeting the macrophages using liposomes loaded with the PS IRDye700DX (700DX-liposomes) can specifically kill macrophages in vitro as well as decrease arthritis severity in the collagen-induced arthritis (CIA) mouse model.

#### **2. Materials and Methods**

#### *2.1. Animals*

Male DBA/1JRj mice were purchased from Janvier-Elevage at 10–12 weeks of age. Mice were housed in conventional cages on a 12 h day–night cycle and fed standard chow ad libitum. The study protocol was approved by the Radboud University animal ethics committee (RU-DEC-2016-0076, CCD number AVD103002016786). All procedures were performed according to the Institute of Laboratory Animal Research guide for Laboratory Animals.

#### *2.2. Liposome Preparation and Characterization*

Liposomes were prepared based on the film method [17]. Briefly, dipalmitoyl phosphatidylcholine (DPPC) and PEG-(2000)-distearoyl phosphatidylethanolamine (PEG-(2000)- DSPE), obtained from Lipoid AG (Steinhausen, Switzerland); DSPE-DTPA, from Avanti Polar Lipids (Birmingham, UK); cholesterol, obtained from Sigma (St. Louis, MO, USA), were used. All other chemicals were of reagent grade. A mix of chloroform and methanol (10:1 *v/v*) containing DPPC, PEG-(2000)-DSPE, cholesterol and DSPE-DTPA was prepared in a molar ratio of 1.85:0.15:1:0.15. The organic phase was evaporated with a rotavapor (BUCHI Labortechnik AG, Flawil, Switzerland), followed by nitrogen flushing to remove residual organic solvent. Liposomes were dispersed in phosphate-buffered saline (PBS). The liposomes were sequentially extruded through two stacked polycarbonate filters with pore sizes of 600, 200, and 100 nm (Nuclepore, Pleaston, CA, USA) under nitrogen pressure, using a Lipex high pressure extruder (Lipex, Nortern Lipids, Vancouver, BC, Canada). The retrieved liposomes had a mean particle size of 120 nm with a PDI (polydispersity index) of 0.02.

700DX-liposomes were prepared by post-insertion of micelles as described previously [18,19]. Briefly, micelles were prepared by mixing DSPE-NH2-PEG-2000 and DSPE-PEG-2000 (both Avanti Polar Lipids, Birmingham, UK) in a 1:1 molar ratio to enable covalent coupling of IRDye-700DX NHS ester (LI-COR Biosciences, Lincoln, NE, USA) to the NH<sup>2</sup> terminus of the PEG conjugates. Subsequently, micelles were incubated with the liposomes at 60 ◦C to allow post-insertion of the PEG conjugates. Unbound IRDye700DX was removed from solution using using a Slide-A-Lyzer Dialysis Cassette (20.000 MWCO, Thermofisher, Waltham, MA, USA) in phosphate-buffered saline (PBS) with 0.5% *w/v* Chelex (Bio-Rad, Hercules, CA, USA).

Fluorescence and absorbance spectra of the liposomes with and without IRDye700DX, as well as those of the free PS, were compared using a TECAN infinite M200 Pro plate reader (PerkinElmer, Groningen, The Netherlands). For fluorescence emission spectrum measurements, an excitation wavelength of 650 nm was used. Results were normalized to the peak absorbance for comparison of the different compounds.

#### *2.3. In Vitro Photodynamic Therapy*

RAW 264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L D-glucose and Glutamax, supplemented with 10% fetal calf serum (FCS), 100 IU/mL penicillin G and 10mg/mL streptomycin. Cells were seeded into 24-well plates (Thermo Scientific, Waltham, MA, USA) (150,000 cells/well) and grown overnight (*n* = 3 wells/group). Medium was replaced by binding buffer (medium with 0.1% bovine serum albumin (*w/v*) (BSA)) with 4 µL/mL liposomes with or without IRDye-700DX (200 µg dye/mL liposomes). After incubation at 37 ◦C for 24 h, cells were washed with binding buffer. Subsequently, cells were irradiated with a NIR light-emitting diode (LED) [20] (peak emission wavelength 690 nm (±10 nm for minimum and maximum emission), forward voltage: 2.6 V, power output: 490 mW) using 126 individual LED bulbs to ensure homogenous illumination at different radiant exposures (between 0 and 50 J/cm<sup>2</sup> ) (LEDfactory, Leeuwarden, The Netherlands). All experiments were carried out in triplicate. The LED device was calibrated using a FieldMaxII-TO power meter with a PM2 sensor (thermopile head), resolution 1 mW (Coherent, Richmond, CA, USA).

Cell viability was measured 4 h after irradiation. During this time, the cells were kept at 37 ◦C and 5% CO2. To determine the viability, ATP content was measured using a CellTiter-Glo® luminescent assay (Promega Benelux, Leiden, The Netherlands) according to the instructions of the manufacturer. Luminescence was measured using a TECAN infinite M200 Pro plate reader (PerkinElmer, Groningen, The Netherlands). The ATP content as a measure of cell viability was expressed as a percentage, determined by comparing the luminescent signal with the signal from untreated cells, which were considered 100% viable.

#### *2.4. Collagen-Induced Arthritis*

CIA was induced in male DBA/1JRj mice as described previously [21]. Briefly, animals received two injections of 100 µg bovine collagen type II (Radboudumc in-house production, batch 03-04-08). The first dose was emulsified in complete Freund's adjuvant (FCA) and injected intradermally at the base of the tail. The second dose of FCA was administered in PBS intraperitoneally at day 21, with subsequent arthritis developed between days 21 and 25. Mice were scored 3 times a week for development of arthritis on each paw according to

a 2-point scale of swelling and redness, as described previously [22]. Mice with a score ≥0.5 in one of the hind paws were included in the study. A cumulative inflammation score ≥7 was considered a humane endpoint.

#### *2.5. Biodistribution*

Male DBA/1JRj mice (starting weight 24.3 ± 0.8 g (*n* = 10/group)) with CIA were randomly divided between the treatment groups upon development of overt arthritis (macroscopic score of inflammation > 0.5). Upon inclusion, the mice were injected intravenously with 5 µL liposomes (in 200 µL PBS) with or without 1 µg IRDye700DX and labeled with 0.6 MBq <sup>111</sup>In for biodistribution analysis. A subset of mice (*n* = 2 per group) were injected with 18 MBq <sup>111</sup>In for SPECT/CT imaging. After 24 h, the mice were sacrificed, and the relevant tissues were dissected and weighed. Tissue uptake of <sup>111</sup>In was determined using a γ counter (WIZARD, 2480 Automatic Gamma Counter, Perkin Elmer, Waltham, MA, USA). Results are depicted as percentage of the injected amount per gram tissue (%IA/g). Mice which received a SPECT/CT dose of <sup>111</sup>In were scanned for 60 min (4 × 15 min frames) using a 1-mm-diameter pinhole ultra-high sensitivity mouse collimator (U-SPECT/CT-II, MILabs, Houten, The Netherlands). SPECT scans were followed by CT scans (65 kV, 615 µA). The SPECT scans were reconstructed using software from MILabs using a 0.2 mm voxel size, 1 iteration and 16 subsets.

#### *2.6. Microscopic Scoring of Inflammation*

To assess the joint inflammation, microscopically ankle joints were formalin-fixed, decalcified using formic acid and paraffin-embedded. The joints were subsequently cut into 7 µm sections, hematoxylin- and eosin-stained, and scored for the presence of inflammation according to the SMASH recommendations [23].

#### *2.7. In Vivo Photodynamic Therapy*

Male DBA/1JRj mice (starting weight 23.4 ± 1.4 g, (*n* = 8/group)) with CIA were randomly divided between the treatment groups upon development of overt arthritis in one of the hind paws (arbitrary arthritis score of >0.5). Mice were injected intravenously with 5 µL liposomes with or without 1 µg IRDye700DX in 200 µL of PBS, and 24 h after injection, the hind legs of the animals were exposed to 8.8 J/cm<sup>2</sup> or 26.4 J/cm<sup>2</sup> 690 nm light. The mice receiving the control liposomes also received the high dose of light. Arthritis development was subsequently scored daily. Mice were sacrificed 5 days after treatment, and the front and hind paws were formalin-fixed, decalcified using formic acid and paraffin-embedded. Histological sections of 7 µm were stained using hematoxylin and eosin, or safranin O, and scored for inflammation and proteoglycan depletion (PG depletion), respectively.

#### *2.8. Statistical Analysis*

Results are presented as mean ± SD. Statistical significance was determined using GraphPad Prism software (Version 5.03; GraphPad Software, San Diego, CA, USA). The tests used were: two-way ANOVA with Bonferroni post-test for the in vitro PDT, biodistribution, and therapy experiment. In the latter, we also accounted for repeated measures. The correlation between arthritis and liposome uptake in inflamed paws was determined using linear regression. A *p*-value < 0.05 was considered significant.

#### **3. Results**

#### *3.1. Fluorescence and Absorption of IRDye700DX-Loaded Liposomes Is Similar to Free IRDye700DX*

The fluorescence emission spectrum and absorbance spectrum were measured for liposomes with and without IRDye700DX loading, and these were compared to the respective spectra for free IRDye700DX. Results were normalized to peak fluorescence and absorbance for the corresponding measurement. The absorbance spectrum of 700DX-liposomes was very similar to that of free IRDye700DX, with both showing peak absorbance at 690 nm

**3. Results**

*IRDye700DX*

(Figure S1A). The fluorescence emission spectra were also very similar, with only a very minor shift in peak emission observed (Figure S1B). *3.2. PDT Using 700DX‐Liposomes Induces Cell Death in a Light Dose Dependent Manner*

#### *3.2. PDT Using 700DX-Liposomes Induces Cell Death in a Light Dose Dependent Manner* Already at light doses as low as 10 J/cm2 radiant exposure of 690 nm light, in vitro cell viability of cells incubated with 700DX‐liposomes was approximately 50% compared

The fluorescence emission spectrum and absorbance spectrum were measured for liposomes with and without IRDye700DX loading, and these were compared to the respective spectra for free IRDye700DX. Results were normalized to peak fluorescence and absorbance for the corresponding measurement. The absorbance spectrum of 700DX‐ liposomes was very similar to that of free IRDye700DX, with both showing peak absorbance at 690 nm (Figure S1A). The fluorescence emission spectra were also very

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 13

*3.1. Fluorescence and Absorption of IRDye700DX‐Loaded Liposomes Is Similar to Free*

similar, with only a very minor shift in peak emission observed (Figure S1B).

Already at light doses as low as 10 J/cm<sup>2</sup> radiant exposure of 690 nm light, in vitro cell viability of cells incubated with 700DX-liposomes was approximately 50% compared to the untreated control (Figure 1). By increasing the light dose even higher, cell death could be generated until, at a light dose of 50 J/cm<sup>2</sup> , only 3% of cells remained. Radiant exposure after incubation with control liposomes without IRDye700DX did not cause cell death, nor did incubation with 700DX-liposomes without light exposure (both *p* > 0.5). to the untreated control (Figure 1). By increasing the light dose even higher, cell death could be generated until, at a light dose of 50 J/cm2, only 3% of cells remained. Radiant exposure after incubation with control liposomes without IRDye700DX did not cause cell death, nor did incubation with 700DX‐liposomes without light exposure (both *p* > 0.5).

**Figure 1.** PDT using 700DX‐liposomes in RAW 264.7 cells. Cell viability decreased in a light dose dependent manner after incubation with IRDye700DX‐liposomes. Cell viability was not affected in cells incubated with control liposomes for any of the tested light doses. Data were normalized to the **Figure 1.** PDT using 700DX-liposomes in RAW 264.7 cells. Cell viability decreased in a light dose dependent manner after incubation with IRDye700DX-liposomes. Cell viability was not affected in cells incubated with control liposomes for any of the tested light doses. Data were normalized to the luminescent values of cells not incubated with liposomes and not exposed to light, and are depicted as mean ±SD (*n* = 3). Two-way ANOVA with Bonferroni post-test, \*\*\* = *p* < 0.001.

#### as mean ±SD (*n* = 3). Two‐way ANOVA with Bonferroni post‐test, \*\*\* = *p* < 0.001. *3.3. Liposomal Accumulation in the Arthritic Joint Is Not Negatively Affected by Loading with IRDye700DX*

luminescent values of cells not incubated with liposomes and not exposed to light, and are depicted

*3.3. Liposomal Accumulation in the Arthritic Joint Is Not Negatively Affected by Loading with IRDye700DX* The biodistribution of 111‐indium‐radiolabeled 700DX‐liposomes in mice with CIA was comparable to that of the control liposomes (Figure 2). No significant difference in uptake was noted, with the exception of the liver, where accumulation of the liposomes with the photosensitizer was significantly increased (24.9 ± 4.9%IA/g versus 14.5 ± 3%IA/g for the liposomes with and without the PS, respectively (*p* < 0.01)). Tracer uptake by the inflamed regions correlated significantly for both constructs with the macroscopic score of arthritis both in the front paw (Figure 3a,b) and ankle joint (Figure 3c,d), as well as with the microscopic score of inflammation (Figure S2). SPECT/CT images that were acquired The biodistribution of 111-indium-radiolabeled 700DX-liposomes in mice with CIA was comparable to that of the control liposomes (Figure 2). No significant difference in uptake was noted, with the exception of the liver, where accumulation of the liposomes with the photosensitizer was significantly increased (24.9 ± 4.9%IA/g versus 14.5 ± 3%IA/g for the liposomes with and without the PS, respectively (*p* < 0.01)). Tracer uptake by the inflamed regions correlated significantly for both constructs with the macroscopic score of arthritis both in the front paw (Figure 3a,b) and ankle joint (Figure 3c,d), as well as with the microscopic score of inflammation (Figure S2). SPECT/CT images that were acquired reflect these results, with clear uptake of the tracer observed in the inflamed joint, and with higher macroscopic inflammation scores also showing more uptake in the scans (Figure 2b,c).

#### reflect these results, with clear uptake of the tracer observed in the inflamed joint, and *3.4. PDT Using 700DX-Loaded Liposomes Ameliorates Arthritis Progression*

with higher macroscopic inflammation scores also showing more uptake in the scans (Figure 2b,c). The therapeutic effect of PDT using 700DX-liposomes was investigated in male DBA/1JRj mice with CIA. The mice were injected intravenously with the 700DX-liposomes or PBS as a control, and the inflamed paws were exposed to the 690 nm light 24 h postinjection. The arthritis scores for both the 8.8 and 26.4 J/cm<sup>2</sup> PDT treated groups were significantly lower than those for the PBS control at days 1–3 for the 8.8 J/cm<sup>2</sup> and day 1 and 2 for the 26.4 J/cm<sup>2</sup> PDT treated group (Figure 4). This difference became especially clear when accounting for the arthritis development of these mice as a whole using the area under the curve (Figure S3). After this initial delay in arthritis development in the treated paws, no differences in arthritis scores were observed at the later timepoints. In line with this, no differences in histological scores of inflammation or cartilage damage were observed after sacrifice of the animals at day 5 post-therapy (Figure S4).

**Figure 2.** (**A**) Biodistribution of liposomes with or without IRDye700DX. (*n* = 10 mice/group). (**B**) SPECT images of mice after injection of 111In-labeled IRDye700DX-liposomes. (**C**) SPECT images of mice after injection of 111In-labeled control liposomes. Arthritis scores are depicted in white next to the respective joints. (\*\* = *p* < 0.01). **Figure 2.** (**A**) Biodistribution of liposomes with or without IRDye700DX (*n* = 10 mice/group); (**B**) SPECT images of mice after injection of 111In-labeled IRDye700DX-liposomes; (**C**) SPECT images of mice after injection of <sup>111</sup>In-labeled control liposomes. Arthritis scores are depicted in white next to the respective joints (\*\* = *p* < 0.01). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 7 of 12

**Figure 3.** Uptake of 700DX-loaded and control liposomes positively correlated with macroscopic

The therapeutic effect of PDT using 700DX-liposomes was investigated in male DBA/1JRj mice with CIA. The mice were injected intravenously with the 700DX-liposomes or PBS as a control, and the inflamed paws were exposed to the 690 nm light 24 h postinjection. The arthritis scores for both the 8.8 and 26.4 J/cm2 PDT treated groups were significantly lower than those for the PBS control at days 1–3 for the 8.8 J/cm2 and day 1 and 2 for the 26.4 J/cm2 PDT treated group (Figure 4). This difference became especially clear when accounting for the arthritis development of these mice as a whole using the area under the curve (Figure S3). After this initial delay in arthritis development in the treated paws, no differences in arthritis scores were observed at the later timepoints. In line with this, no differences in histological scores of inflammation or cartilage damage were ob-

arthritis score in the hind ankle (**A**,**B**) and front paw (**C**,**D**) of mice with CIA. (*n* = 10 mice).

*3.4. PDT Using 700DX-Loaded Liposomes Ameliorates Arthritis Progression* 

served after sacrifice of the animals at day 5 post-therapy (Figure S4).

**Figure 3.** *Conts*.

**Figure 3.** Uptake of 700DX-loaded and control liposomes positively correlated with macroscopic arthritis score in the hind ankle (**A**,**B**) and front paw (**C**,**D**) of mice with CIA. (*n* = 10 mice). **Figure 3.** Uptake of 700DX-loaded and control liposomes positively correlated with macroscopic arthritis score in the hind ankle (**A**,**B**) and front paw (**C**,**D**) of mice with CIA (*n* = 10 mice). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 8 of 12

**Figure 4.** Development of arthritis over time after 700DX-liposome PDT. Initial arthritis development was slower after tPDT. Two-way ANOVA with Bonferroni post-test, \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001 (*n* = 8 mice/group). **Figure 4.** Development of arthritis over time after 700DX-liposome PDT. Initial arthritis development was slower after tPDT. Two-way ANOVA with Bonferroni post-test, \* = *p* < 0.05, \*\* = *p* < 0.01, \*\*\* = *p* < 0.001, ns: not significant (*n* = 8 mice/group).

In this study, we demonstrated the feasibility of depleting macrophages using PSloaded liposomes for tPDT both in vitro and in vivo in the CIA model of arthritis. Using PS-loaded liposomes, the macrophage cell line RAW264.7 was able to be efficiently depleted in a light dose dependent manner. The construct showed targeting of the arthritic

in these mice. Finally, 700DX-liposome PDT was able to ameliorate arthritis development

Macrophages are of critical importance for the development of RA. Depletion of macrophages using clodronate-loaded liposomes has previously been shown to be able to decrease the development of arthritis in mice when administered prior to overt arthritis development [12,13]. Additionally, arthritic flares were prevented, and administering the liposomes in the chronic phase of CIA ameliorated the development of the disease [24]. A drawback of the systemic action of clodronate-loaded liposomes is the resulting depletion of macrophages not only in the arthritic joints, but in many tissues, which interferes with the macrophage's normal function of maintaining tissue homeostasis and preventing infection. By using a construct that is inert in the absence of light, we can deplete macro-

Using PDT to treat arthritis in animal models has successfully been employed by several groups. Since these previous studies all used PDT, in which the PS preferentially accumulates in the arthritic joint due to the increased permeability of the vasculature,

phages specifically in the arthritic joints, eliminating this problem.

in the initial phase after treatment.

**4. Discussion** 

#### **4. Discussion**

In this study, we demonstrated the feasibility of depleting macrophages using PSloaded liposomes for tPDT both in vitro and in vivo in the CIA model of arthritis. Using PS-loaded liposomes, the macrophage cell line RAW264.7 was able to be efficiently depleted in a light dose dependent manner. The construct showed targeting of the arthritic joints in the CIA model of arthritis, with uptake closely correlating with arthritis severity in these mice. Finally, 700DX-liposome PDT was able to ameliorate arthritis development in the initial phase after treatment.

Macrophages are of critical importance for the development of RA. Depletion of macrophages using clodronate-loaded liposomes has previously been shown to be able to decrease the development of arthritis in mice when administered prior to overt arthritis development [12,13]. Additionally, arthritic flares were prevented, and administering the liposomes in the chronic phase of CIA ameliorated the development of the disease [24]. A drawback of the systemic action of clodronate-loaded liposomes is the resulting depletion of macrophages not only in the arthritic joints, but in many tissues, which interferes with the macrophage's normal function of maintaining tissue homeostasis and preventing infection. By using a construct that is inert in the absence of light, we can deplete macrophages specifically in the arthritic joints, eliminating this problem.

Using PDT to treat arthritis in animal models has successfully been employed by several groups. Since these previous studies all used PDT, in which the PS preferentially accumulates in the arthritic joint due to the increased permeability of the vasculature, treatment was accompanied by significant side effects, because accumulation of the photosensitizer in neighboring skin and muscle tissue, which were inadvertently also exposed to light, could not be sufficiently prevented by untargeted approaches, and this resulted in necrosis in the surrounding musculature [25]. By using liposomes to deliver the photosensitizer preferentially to the macrophages, and thus increasing the uptake in the inflamed joints relative to neighboring tissues, these unwanted side-effects were successfully prevented in this study [9].

Current treatment for RA consists of systemic immunosuppression through the administration of biologic or synthetic DMARDS. The advantage of using 700DX-liposome PDT instead of, or complimentary to DMARDS is that the side effects of systemic immunosuppression, such as an increased risk of infectious diseases, can be avoided [26,27]. Furthermore, since 700DX-liposomes are systemically delivered, in contrast to other local adjuvant treatments such as chemical-, surgical- or radio-synovectomy, the inflamed joint is not damaged by the insertion of needles or other instruments.

An important hurdle to achieving clinical translation is the limitation of light penetration. Near infrared light has a penetration depth in tissue between 5 and 10 mm, which is suitable for the treatment of small joints, e.g., interphalangeal joints [28,29]. However, for larger joints, the light will not be able to penetrate deep enough to excite the PS in the whole joint. An alternative to reach the whole synovium could be to deliver the light directly into the joint using endoscopic or fibre-optic systems (reviewed in [28]).

In this study, loading of the liposomes with the photosensitizer IRDye700DX did not significantly change the biodistribution of the compound, with the exception of increased liver accumulation, where the compound is cleared by sinusoidal epithelial cells [30]. This effect has previously been described for other fluorophores and may be due to changes in the charge and lipophilicity of the compounds [31,32]. The time required for elimination of this construct was not determined in this experiment, since biodistribution analysis was only performed 24 h after injection. However, since the liver and spleen are not exposed to light, and the PS has no dark toxicity at the concentrations used in this model, this should not result in any off-target damage.

The limited and transient treatment effect observed in this study may have several explanations. First, using CIA as a model for experimental arthritis to study the effect of PDT using 700DX-loaded liposomes proved challenging in this study. Timing of the treatment and analysis of outcome measures were complicated by the highly variable disease course

and incidence, and, since animals were included when they reached inclusion criteria, inclusion was spread over multiple weeks. At inclusion, the cumulative arthritis scores of the animals were variable, and we cannot rule out that the accompanying variation of systemic pro-inflammatory molecules influenced our treatment efficacy. Despite this limitation, we were able to demonstrate an amelioration of arthritis development. The transient nature of our therapy may also indicate that the treatment should be further optimized to give the best possible effect. Further optimization of the therapy could be achieved by performing light dosimetry, as well as by focusing on the timing of administration of the liposomes and subsequent light exposure [28,33]. Combining this therapy with low dose immunosuppressant therapy may also improve the longevity of the PDT effect. This is an especially relevant option for clinical translation since it allows for local treatment of disease flares without increasing systemic immunosuppression.

#### **5. Conclusions**

In conclusion, we have shown the proof-of-principle of performing photodynamic therapy in arthritic joints using liposomes loaded with a photosensitizer, which allows for locoregional treatment of RA and thus avoids systemic side effects. To expand on these findings and to assess their therapeutic value with respect to future clinical application, studies into PDT dosimetry and treatment effects in other animal models, as well as RA patient material ex vivo, should be performed.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics13111868/s1, Figure S1: Absorbance and fluorescence spectra of liposome with and without IRDye700DX compared to free IRDye700DX. Both absorbance and fluorescence spectra are similar when comparing IRDye700DX loaded in liposomes or free PS (all in PBS). Fluorescence emission was measured after excitation at 650 nm, Figure S2: Uptake of liposomes with (**a**) and without (**b**) IRDye700DX correlates with histological scores of inflammation in the ankle joints of mice with CIA (*n* = 10 mice), Figure S3: Area under the curve for the development of arthritis over time after 700DX-loaded liposome PDT. The AUC was significantly lower in the treated groups compared to PBS. Two-way ANOVA with Bonferroni post-test, \* = *p* < 0.05 (*n* = 8 mice/group), Figure S4: Histological scores of ankle and knee joint inflammation, proteoglycan depletion and knee chondrocyte death and cartilage erosion. Results are depicted as mean with SD (*n* = 8 mice, (*n* = 7 for the 90 s exposed liposomes group)).

**Author Contributions:** Conceptualization, D.N.D., M.B. (Marti Boss), M.R., G.S., M.B. (Maarten Brom), P.L., P.M.v.d.K., M.I.K. and M.G.; methodology, D.N.D., M.B. (Marti Boss), B.W., M.M.A.H., G.S. and L.v.B.; validation, D.N.D., M.B. (Marti Boss), B.W., M.M.A.H. and L.v.B.; formal analysis, D.N.D., M.B. (Marti Boss); investigation, D.N.D., M.B. (Marti Boss), B.W., M.M.A.H., D.L.B. and L.v.B.; resources, D.N.D., M.B. (Marti Boss), M.R., G.S., M.B. (Maarten Brom), P.L., P.M.v.d.K., M.I.K. and M.G.; data curation, D.N.D., M.B. (Marti Boss), M.R., G.S., M.B. (Maarten Brom), P.L., P.M.v.d.K., M.I.K. and M.G.; writing—original draft preparation, D.N.D. and M.B. (Marti Boss); writing—review and editing, D.N.D., M.B. (Marti Boss), M.R., B.W., M.M.A.H., D.L.B., L.v.B., G.S., M.B. (Maarten Brom), P.L., P.M.v.d.K., M.B. (Mijke Buitinga), M.I.K. and M.G.; visualization, D.N.D. and M.B. (Marti Boss); supervision, P.M.v.d.K., M.B. (Mijke Buitinga), M.I.K. and M.G.; project administration, D.N.D. and M.B. (Marti Boss). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The Radboud University animal ethics committee approved the study protocol (RU-DEC-2016-0076, CCD number AVD103002016786). All procedures were performed according to the Institute of Laboratory Animal Research guide for Laboratory Animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data can be made available upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **Abbreviations**

%IA/g: percentage of the injected amount per gram tissue; CIA: collagen-induced arthritis; DMARDs: disease-modifying anti-rheumatic drugs; DPPC: dipalmitoyl phosphatidylcholine; FCA: complete Freund's adjuvant; IL-: Interleukin; PDT: photodynamic therapy; PEG-(2000)-DSPE: PEG-(2000) distearoyl phosphatidylethanolamine; PG depletion: proteoglycan depletion; PS: photosensitizer; RA: rheumatoid arthritis; ROS: reactive oxygen species; SD: standard deviation; SPECT/CT: single photon emission computer tomography/computer tomography; TNF- α: tumor necrosis factor-α.

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**Eidi Christensen 1,2,3,\* , Olav A. Foss 4,5 , Petter Quist-Paulsen 3,6, Ingrid Staur <sup>1</sup> , Frode Pettersen <sup>7</sup> , Toril Holien 2,3,6, Petras Juzenas <sup>2</sup> and Qian Peng 2,8**

	- Fudan University, Shanghai 200433, China
	- **\*** Correspondence: eidi.christensen@ntnu.no

**Abstract:** Extracorporeal photopheresis (ECP), an immunomodulatory therapy for the treatment of chronic graft-versus-host disease (cGvHD), exposes isolated white blood cells to photoactivatable 8-methoxypsoralen (8-MOP) and UVA light to induce the apoptosis of T-cells and, hence, to modulate immune responses. However, 8-MOP-ECP kills diseased and healthy cells with no selectivity and has limited efficacy in many cases. The use of 5-aminolevulinic acid (ALA) and light (ALA-based photodynamic therapy) may be an alternative, as ex vivo investigations show that ALA-ECP kills T-cells from cGvHD patients more selectively and efficiently than those treated with 8-MOP-ECP. The purpose of this phase I-(II) study was to evaluate the safety and tolerability of ALA-ECP in cGvHD patients. The study included 82 treatments in five patients. One patient was discharged due to the progression of the haematological disease. No significant persistent changes in vital signs or laboratory values were detected. In total, 62 adverse events were reported. Two events were severe, 17 were moderate, and 43 were mild symptoms. None of the adverse events evaluated by the internal safety review committee were considered to be likely related to the study medication. The results indicate that ALA-ECP is safe and is mainly tolerated well by cGvHD patients.

**Keywords:** 5-aminolevulinic acid; ALA-based photodynamic therapy; phototherapy; extracorporeal; photopheresis; chronic graft-versus-host disease

## **1. Introduction**

The modification of today's standard extracorporeal photopheresis (ECP) with the introduction of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) may improve treatment efficacy. Since the treatment of cutaneous T-cell lymphoma was approved more than 30 years ago, ECP has been used to treat patients with other T-cell-mediated disorders, including chronic graft-versus-host disease (cGvHD) [1–3]. Graft-versus-host disease (GvHD) represents a serious immunologically mediated complication of allogeneic hematopoietic cell transplantation [4]. Donor immune cells, mostly T-lymphocytes (T-cells), are activated and attack the host cells, misrecognising them as foreign. GvHD is classified

**Citation:** Christensen, E.; Foss, O.A.; Quist-Paulsen, P.; Staur, I.; Pettersen, F.; Holien, T.; Juzenas, P.; Peng, Q. Application of Photodynamic Therapy with 5-Aminolevulinic Acid to Extracorporeal Photopheresis in the Treatment of Patients with Chronic Graft-versus-Host Disease: A First-in-Human Study. *Pharmaceutics* **2021**, *13*, 1558. https://doi.org/ 10.3390/pharmaceutics13101558

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 22 July 2021 Accepted: 19 September 2021 Published: 26 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

as chronic based on histological criteria [5]. It can involve multiple organs, of which the skin is the most often affected, and can cause severe morbidity in patients [6]. ECP is widely used in those patients with an initial non or partial response to first-line treatment, including systemic corticosteroids [2].

ECP is a leukapheresis-based therapeutic procedure. The mechanism of its immunomodulatory effect is not fully understood but involves altered T-cell function and the modulation of dendritic cell maturation, with the modification of the cytokine profile and stimulation of regulatory T-cells [2,7,8]. During ECP, leucocytes separated from the whole blood (buffy coat) are exposed to the photosensitising agent 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) light before reinfusion back into the patient. The combination of 8-MOP-UVA irradiation leads to deoxyribonucleic acid (DNA) crosslinking and, thereby, to the apoptosis of the treated cells. However, a major disadvantage of 8-MOP is that it binds to the DNA of diseased and normal cells with no selectivity, thus inducing the apoptosis of both types of cells after UVA activation. In addition, the treatment is time-consuming, expensive, and often ineffective. This demands the use of an alternative photosensitiser that selectively targets diseased cells to improve the effectiveness of today's standard treatment.

The idea of using 5-aminolevulinic acid (ALA) as a photosensitiser precursor for ECP has emerged from decades of research and experience with photodiagnosis and PDT. ALA-PDT is well established for topical use in the treatment of patients with non-melanoma skin cancer, and the oral intake of ALA is approved for clinical use in the photodetection of glioma and for the treatment of Barrett's oesophagus [9–11]. ALA is a naturally occurring amino acid and a precursor of heme that is metabolised intracellularly to endogenously porphyrin photosensitisers, mainly protoporphyrin IX (PpIX) [12]. It provides a highly selective accumulation of PpIX in the proliferative or activated cells, including tumour cells, due to their higher uptake of ALA and the alteration of the activities of enzymes involved in heme biosynthesis. The photoactivation of ALA-induced PpIX in the presence of oxygen through the formation of reactive oxygen species leads to the apoptosis and necrosis of the targeted cells [13,14]. After illumination, PpIX is partly or completely photobleached (faded). Under experimental conditions, ALA-UVA is shown to kill T-cells in the blood samples of patients with cGvHD more selectively and efficiently than those treated with 8-MOP-UVA [15]; hence, the number and duration of ECP treatments with the use of ALA can be reduced.

The aim of this study was to evaluate the safety and tolerability of multiple treatments with ALA-ECP in patients with cGvHD. In addition, data are presented from assessments of selected organs, performance, and quality of life in patients during the study period.

#### **2. Materials and Methods**

### *2.1. Patients*

Patients with cGvHD who responded inadequately to 8-MOP-ECP after a minimum of 3 months of treatment at St. Olavs Hospital, Trondheim University Hospital, Norway, were considered for study inclusion. Inadequate response was defined as the progression of cutaneous cGvHD (>25% worsening of skin score from baseline or insufficient response with a <15% improvement in skin score compared with baseline or a ≤25% reduction in corticosteroid dose). Excluded were patients under 18 years of age; with body weight below 40 kg; and those with photosensitive comorbidities such as porphyria or known hypersensitivity to ALA or porphyrins, aphakia, pregnancy or breastfeeding, polyneuropathy, ongoing cardiac and pulmonary diseases, uncontrolled infection or fever, history of heparin-induced thrombocytopenia, an absolute neutrophil count < 1 <sup>×</sup> <sup>10</sup>9/L, platelet count < 20 <sup>×</sup> <sup>10</sup>9/L, aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, or an international normalised ratio (INR) value ≥ 3× upper limit of normal or significant ECG findings. Patients considered unlikely to comply with the study procedures were also excluded

#### *2.2. Design and Procedures*

This prospective, open, single-centre, phase I–(II) study was approved by the regional committee for medical research ethics (REK-Nord 2014/2316), the Norwegian Medicines Agency (National Regulatory Authority 14/16760-29) and was registered at www.clinicaltrials.gov (accessed on 20 September 2017) as NCT03109353. The study was performed in accordance with the Helsinki Declaration, monitored by the Clinical Research Unit Central Norway, Norwegian University of Science and Technology (NTNU), and patients provided written informed consent.

The CELLEX Photopheresis machine (Therakos, Mallinckrodt Pharmaceuticals, Raritan, NJ, USA) was used. This is a closed system in which leukapheresis, photoactivation, and the reinfusion of white blood cells are achieved sequentially. The patients were connected to the machine through single or double venous access to collect whole blood. Then, through centrifugation, the fraction of white cells (buffy coat) was separated and collected into a treatment bag (buffy bag), while the other blood components, mainly red cells and plasma, were returned to the patient. A commercially available ALA-hydrochloride powder for systemic oral use (Gliolan, photonamic GmbH & Co. KG, Pinneberg, Germany) was used at a dose of 0.168% w/v (10mM). The pharmacy at St. Olavs Hospital reconstituted ALA-hydrochloride in 0.9% sodium chloride (NaCl) physiological saline under sterile condition to a stock solution of 30 mg/mL. Since the amount of buffy coat collected varied on different days for the same patients and between patients, the volume of the ALA solution added to the buffy coat was adjusted to the volume collected on each treatment to reach a dose of 10 mM. After the ALA-solution was added, a 1-h incubation period in the dark was allowed for intracellular PpIX production before UVA light exposure of the cells. Although the most effective light wavelength for PpIX activation is at 400 nm, the use of the UVA light source in the Therakos photopheresis machine can also effectively kill ALA-incubated human T-cell lines [16].

Finally, the treated buffy coat was reinfused into the patient. One treatment-cycle represents two treatments: one treatment given on two consecutive days. Each patient could receive up to 20 ALA-ECP treatments corresponding to 10 treatment-cycles within a year. The duration between two treatment-cycles varied depending on each patient's symptoms and treatment response.

Representative buffy coat samples for the investigation of ALA-induced PpIX were taken after a 1-h ALA incubation at room temperature. The plasma samples for PpIX determination were taken immediately after the reinfusion of the ex vivo ALA-ECP-treated buffy coat and 24-h after treatment. Measurements of ALA-induced PpIX in the buffy coat and plasma samples were performed in an extraction solution [17,18]. The solvent consisted of 1% sodium dodecyl sulfate in 1 N perchloric acid (HClO4) and methanol (CH3OH) (1:1 vol/vol). A 100-µL sample was thoroughly mixed with 900 µL of solvent for approximately 1 min in a 1.5-mL tube (Eppendorf, Hamburg, Germany). The sample homogenates were then kept at −20 ◦C until analysis. Before measurements, the samples were centrifuged for 5 min at 1000 rpm, and the supernatants were carefully transferred to new tubes. The fluorescence of PpIX was measured using a luminescence spectrometer (LS50B, Perkin-Elmer, Norwalk, CT, USA). The PpIX fluorescence in the sample supernatants was measured in a standard 10-mm quartz cuvette placed in the standard holder of the luminescence spectrometer. The fluorescence excitation wavelength was 400 nm, corresponding to the Soret band of the PpIX absorption spectrum. The fluorescence intensity was recorded at 605 nm, corresponding to the PpIX emission peak in the solvent. The amount of PpIX was determined by comparing the PpIX fluorescence intensities with the standard curve made by adding known amounts of PpIX to the solvent. The sensitivity level of our method, that is, the lowest concentration of PpIX that could be detected, was approximately 3 nM in the extraction solution.

#### *2.3. Safety and Tolerability*

Safety and tolerability were regularly monitored through clinical and laboratory examinations and patient reports (Table 1). Safety was monitored through frequency, seriousness, and intensity of adverse events, 12 lead electrocardiogram (ECG) recordings, vital signs (blood pressure as seated, pulse at rest, and forehead temperature), physical examinations, and laboratory measurements. The schedules of major events within each treatment-cycle and controls are presented in Table 1. Assessments performed either at screening, if it was less than one week before ALA-ECP or performed just before the first treatment were used as reference (baseline). Any abnormality was considered for clinical significance and for the need for action, for example, repetition of the test to verify the result and/or closer followup. Laboratory parameters included haematology (haemoglobin, white blood cell (WBC) count, WBC differential count, eosinophil count, platelet count, mean cell volume, mean cell haemoglobin, and INR), and clinical chemistry (albumin, ALT, alkaline phosphatase, AST, bilirubin, blood urea, cholesterol, C-reactive protein (CRP), calcium cation, creatinine, erythrocyte sedimentation rate (SR), gamma-glutamyl transferase, glycated haemoglobin A1c, lactate dehydrogenase, potassium, sodium, and total protein). Urine was examined with a dipstick for erythrocytes, glucose, ketone, leucocytes, nitrite, pH, and protein.


**Table 1.** Schedule for the assessment of safety, tolerability and response.

\* Not needed if less than one week after screening. ECP, extracorporeal photopheresis; ECG, electrocardiogram; QoL, quality of life.

Additional blood tests to analyse AST, ALT, bilirubin, and INR were performed to evaluate the effect of ALA-ECP on the liver. Blood analyses were performed at the Department of Clinical Chemistry, St. Olavs Hospital, and reference values were used. Urine was examined using a dipstick test.

Tolerability was observed through the patients' reported adverse events (AEs). Any event during the study period in which the patients felt unwell or different from usual was recorded as an AE. Conceivable AEs of systemic ALA treatment included nausea, vomiting, headache, photosensitivity, and chills. Patients received a diary in which AEs were noted between visits. The seriousness of all events was graded (grade 1: asymptomatic or mild symptoms to grade 5; death related to AE) according to Common Terminology Criteria for Adverse Events (CTCAE) v4.03. The assessment of study continuation was based on the stated protocol criteria. An internal safety review committee (ISRC) was appointed to assess safety and tolerability data and to evaluate the reported AEs.

#### *2.4. Organ, Performance, and Quality of Life Assessments*

Assessments of organ, performance, and quality of life were repeated at approximately 3 month intervals. Investigator-performed evaluations and patient-reported outcome measures were used. The use of immunosuppressive therapy was monitored. Parts of Filipovich and colleagues' clinical organ scoring system were used in the evaluation of skin manifestations [6]. This scoring system combines clinical features with the affected percentage of the body surface area (BSA) (grade 0: no symptoms; grade 1: <18% BSA with disease signs but no sclerotic

features; grade 2: 19–50% BSA or superficial sclerotic features; and grade 3: >50% BSA or deep sclerotic features or impaired mobility, ulceration, or severe pruritus). The affected percentage of BSA was additionally registered in the evaluation of skin disease. Pruritus was assessed using a visual analogue scale (VAS) graded from 1 to 10, with an increasing number corresponding to a higher severity of pruritic manifestation. The diagnostic features of mucosal manifestations were not systematically recorded. Schirmer's test was used to evaluate tear production. Sterile paper strips were placed into the inferior-temporal aspect of the conjunctival sac of both eyes without topical anaesthesia, and the wetted length was measured in millimetres after 5 min. A measurement of ≥15 mm was considered grade 1 (normal), 14–9 mm as grade 2 (mild), 8–4 as grade 3 (moderate), and less than 4 mm as grade 4 (severe significance). Mouth, gastrointestinal, and performance were assessed using Filipovich's clinical system for scoring the severity and extent of cGvHD using a 4-point scale (grade 0: no symptoms, grade 1: mild symptoms, grade 3: moderate symptoms, and grade 4: severe symptoms) [6]. Functional status was assessed using Karnovsky's performance status scale [19]. The patients were given a score on a linear scale between 0 and 100, summarising their ability to perform daily activities: the lower the score was, the worse the status was. Patients were also asked to complete the Skindex-29 and European Organisation for the Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC30) and the Functional Assessment of Cancer Therapy–Bone Marrow Transplantation (FACT–BMT) questionnaires [20–22]. Skindex-29 is designed to measure the overall effect of skin disease on health-related quality of life and is sensitive to minor changes over time. EORTC30 is a widely used measure of cancer-specific health-related quality of life. FACT-BMT is a general measure of cancer-specific health-related quality of life in combination with a module developed specifically for evaluating the quality of life of bone marrow transplant patients.

#### *2.5. Statistical Methods*

Descriptive data are reported as numbers, percentages (%), and mean (min–max.) and standard deviation (±), together with line graphs and box plots (median, 25–75% percentile). IBM SPSS software version 27 was used.

#### **3. Results**

#### *3.1. Patients and Treatments*

Five patients (three women and two men) were included, of which four completed the study. The mean age at entry was 48 years (32–60). Four patients were diagnosed with acute myeloid leukaemia, and one was diagnosed with high-risk myelodysplastic syndrome before undergoing allogeneic bone marrow transplantation. Prior to study inclusion, the patients had received a mean of 25 (10–49) treatment-cycles of 8-MOP-ECP. Three patients received systemic steroids.

During the study period, the patients received a total of 82 single treatments (42 treatmentcycles) with ALA-ECP. One patient missed one treatment due to clotting in the buffy coat. A second patient missed one treatment because of a gastric flu event. The mean volume of the buffy coat collected at treatments was 146 mL (100–251), and the mean irradiation time was 17 min (2–45). As the patients' symptoms of the disease varied, the treatment intervals were individualised. Two patients had an ECP treatment interval of 4 weeks, each receiving 20 treatments within 9 months. The remaining three patients had an interval of 6–8 weeks and received 18, 13, and 11 treatments, respectively. One patient met a protocol-defined discontinuation criterion 8 months into the study when the treatment with systemic prednisolone was increased from 10/5 mg/every other day to 25 mg/day due to the worsening of symptoms. After discharge from the study, the patient received two 8-MOP-ECP treatments before being diagnosed with lung cancer, after which ECP was discontinued.

#### *3.2. Safety and Tolerability*

We did not observe any clinically significant changes in the patients' vital signs. The details of the results are presented in Figure 1.

**Figure 1.** Systolic and diastolic blood pressure, heart rate, and temperature of patients, as measured at baseline and before and after each day in all treatment-cycles. (Boxplot with; median, 25th and 75th percentile, whiskers—value which is not an outlier or an extreme score, ° outlier more than 1.5 box lengths from the box, \* extreme case more than 3 box lengths from the box.) **Figure 1.** Systolic and diastolic blood pressure, heart rate, and temperature of patients, as measured at baseline and before and after each day in all treatment-cycles. (Boxplot with; median, 25th and 75th percentile, whiskers—value which is not an outlier or an extreme score, ◦ outlier more than 1.5 box lengths from the box, \* extreme case more than 3 box lengths from the box.)

Few clinically significant changes in laboratory values were observed. Two patients had a transient increase in CRP values (34 and 31 mg/L) and SR values (31 and 21 mm/h), respectively, compared with the baseline. One patient had transient high values of eosinophil count (15%), absolute eosinophil count (1.98 × 109/L), and lactate dehydrogenase level (239 U/L). No evidence of liver toxicity was observed (Figure 2). One patient had an increased QTc interval value of 467 ms on ECG at the 3-month control compared to 450 ms at baseline (prolonged QTc defined as a value > 450 ms [23]). Consequently, an ECG was performed before and after three consecutive treatment cycles, and the patient underwent cardiological examination with an echocardiogram and stress ECG with no pathological findings. Both the cardiologist and ISRC concluded that the QTc interval in this patient was unlikely to be affected by the study's medicine.

Few clinically significant changes in laboratory values were observed. Two patients had a transient increase in CRP values (34 and 31 mg/L) and SR values (31 and 21 mm/h), respectively, compared with the baseline. One patient had transient high values of eosinophil count (15%), absolute eosinophil count (1.98 <sup>×</sup> <sup>10</sup>9/L), and lactate dehydrogenase level (239 U/L). No evidence of liver toxicity was observed (Figure 2).

Most of the bilirubin and INR measurements showed transiently higher values a few days after treatment. High levels of ALT and AST were confirmed at baseline in one patient (patient two) with a history of elevated liver enzymes. This patient regularly used paracetamol but had no established liver disease. Another patient (patient five) with elevated liver enzyme and bilirubin levels four days after treatment had a known bile disorder. No clinically significant long-term liver effects were observed. Albumin and total protein levels were stable in all the patients throughout the study. Dipstick urinalysis revealed no severe kidney or urinary tract disorders. One test yielded a positive urine culture, resulting in antibiotic treatment. ALA-induced PpIX was determined from 75 buffy coat and 104 plasma samples from the five patients and was detected in all the buffy coat

samples with a mean concentration of 122 (123) nM. No PpIX was found in the plasma samples taken immediately or at 24 h after re-infusion.

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 8 of 14

**Figure 2.** (**a**) Measurements of aspartate transaminase (AST), alkaline phosphatase (ALT), bilirubin, and international normalised ratio (INR) for each patient at baseline and on day 2 of treatment and days 4, 7, and 12 following the first treatmentcycle. (**b**) Measurements of AST, ALT, bilirubin, and INR at baseline and after day 2 of each treatment-cycle. (Boxplot with; median, 25th and 75th percentile, whiskers—value which is not an outlier or an extreme score, ° outlier more than 1.5 box lengths from the box, \* extreme case more than 3 box lengths from the box.) **Figure 2.** (**a**) Measurements of aspartate transaminase (AST), alkaline phosphatase (ALT), bilirubin, and international normalised ratio (INR) for each patient at baseline and on day 2 of treatment and days 4, 7, and 12 following the first treatment-cycle. (**b**) Measurements of AST, ALT, bilirubin, and INR at baseline and after day 2 of each treatment-cycle. (Boxplot with; median, 25th and 75th percentile, whiskers—value which is not an outlier or an extreme score, ◦ outlier more than 1.5 box lengths from the box, \* extreme case more than 3 box lengths from the box.)

Most of the bilirubin and INR measurements showed transiently higher values a few days after treatment. High levels of ALT and AST were confirmed at baseline in one pa-

In total, there were 62 cases of AEs (two serious adverse events (SAEs), 20 AEs, and 40 conceivable AEs). Of the 62 events, two (3%) were severe, 17 (27%) were moderate, and 43 (69%) were mild symptoms (Tables 2 and 3). None of the AEs evaluated by the ISRC were considered to be likely related to the study medication (Table 2). The two SAEs were observed in the same patient. The first event was a pulmonary viral infection occurring 12 days after the second treatment-cycle, and the patient was hospitalised for 14 days. The ISRC evaluated the event as possibly related to the study medication. The second SAE was a migraine headache episode that occurred two days after the sixth treatment-cycle and required two days of hospitalisation. The patient had a history of migraines. The ISRC evaluated the SAE as unlikely to be related to the study medication. The most common grade one and two AEs reported were clotting in the treatment system, cold-like symptoms, and dysuria (Table 2). No grade four or five AEs were reported. The most frequently reported conceivable AEs were nausea and headache (Table 3). Of all the cases, 85% had mild grade one, and 15% had grade two symptoms. No grade 3–5 events were reported.

**Table 2.** Adverse events and Internal Safety Review Committee's assessments of the severity of the events as well as relation to the study medication.


Grade 1 severity: mild; asymptomatic or mild symptoms; clinical or diagnostic observation only; intervention not indicated; Grade 2 severity: moderate; minimal, local, or non-invasive intervention indicated; limiting age-appropriate instrumental activities of daily living; Grade 3 severity: severe or medically significant but not immediately life-threatening; hospitalisation or prolongation of hospitalisation indicated disabling, limiting self-care activities of daily living.

**Table 3.** Frequency and severity of conceivable adverse events among patients.


*Nausea* Grade 1: loss of appetite without alteration in eating habits, Grade 2: Oral intake decreased without significant weight loss, dehydration, or malnutrition; *Vomiting* Grade 1: 1–2 episodes in 24 h, Grade 2: 3–5 episodes in 24 h; *Headache* Grade 1: mild pain, Grade 2: moderate pain, limiting instrumental activity of daily living; *Chills* Grade 1: mild sensation of cold, shivering, chatting of teeth.

#### *3.3. Organ, Performance, and Quality of Life Assessments*

The assessment scores from the baseline and the patients' last control are shown in Table 4. Most of the conditions showed an improvement in scores with the greatest improvement in skin, BSA involvement, and pruritus. The eye assessment showed the best score at baseline. In one patient, the prednisolone dose of 5 mg/every other day was unchanged, and in another, it was reduced from 10/5 mg/every other day at baseline to 2.5 mg/day at the last control.



EORTC30: European Organisation for the Research and Treatment of Cancer Quality of Life Questionnaire Core 30, FACT-G: Functional Assessment of Cancer Therapy—Global, FACT-BMT: FACT—Bone Marrow Transplantation.

#### **4. Discussion**

This paper reports the results of 82 ALA-ECP treatments administered to five patients with cGvHD who were considered to respond inadequately to 8-MOP-ECP. We did not observe any clinically significant changes in vital signs or detect any persistent abnormal laboratory findings. Overall, we registered an improvement in the patients' skin scores during the study period.

The rationale for the clinical use of ALA in ECP is multiple. ALA does not induce carcinogenesis, since ALA-induced PpIX localises to the cellular membrane structures outside the nucleus [12,24]. Transformed or activated T-cells produce considerably more ALA-induced PpIX than normal resting cells [15]. As a result, these cells are highly selectively destroyed after light irradiation, while resting normal T-cells remain undamaged. Furthermore, ALA combined with light-based therapy (ALA-based PDT) induces antitumour immunity [25]. Besides cell necrosis, it also initiates apoptotic cell death through the mitochondrion and ER-Ca2+-pathways [26], which, in turn, can promote immunosuppressive effects, including regulatory T-cell activation [14]. The highly selective photodamage of transformed or activated T-cells by ALA-PDT with the subsequent immunogenic cell death-mediated induction of an individualised and specific immuno-modulatory response may be a major advantage over non-specific immunosuppressive drugs.

This is the first study of ALA administered to patients treated with ECP. We took the precaution of using a dose of ALA corresponding to 11.2 mg/kg if assuming a 75 kg body weight (10mM). When previously used ex vivo for a 24-h incubation, the same dose gave no dark toxicity to leukocytes from cGvHD patients [16]. Moreover, a single oral dose of 20 mg/kg ALA in drinking water has been approved for the fluorescence-guided resection of glioma [10] in humans, while that of 60 mg/kg has been administered for the PDT treatment of Barrett's oesophagus [11].

Common side effects observed from the use of oral ALA are nausea, vomiting, and a transient rise in some liver enzymes and bilirubin, which typically resolve after 48 h [27,28]. Similar effects were observed in the present study. The additional finding of a modest increase in the INR value on day two of treatment may be seen in relation to the heparinisation of the patients' blood in association with ECP. Phototoxicity was not reported as an adverse reaction after treatment and coincides well with the finding of no PpIX in the patients' plasma.

Conventional ECP with 8-MOP is considered a safe treatment modality with side effects that are commonly sporadic and mild, such as nausea, fever, or headache [29]. Of all the AEs reported in this study, clotting in the buffy coat and episodes of infections were most frequently evaluated to likely be related to the study medicine. We are uncertain whether ALA and/or the 1-h incubation period before the photoactivation of the buffy coat contributed, since abnormal clotting is also recognised with conventional 8-MOP- ECP [30]. Although heparin is part of the standard ECP operational system, the use of anticoagulants varies between hospitals and is adjusted according to the patients' conditions [31]. Clotting during 8-MOP-ECP is commonly managed at our hospital by increasing the heparin dose. In the four study patients, the heparin dose (5000 IE/mL) was increased from 1.0 to 1.5 mL, then no clotting was observed. No definite association between treatment and the increased chance of infection using ALA-ECP was concluded, although three reported cases of infection were considered to possibly be related. There is no evidence of an increased risk of systemic infection after oral ALA or after conventional ECP [2,30]. The occurrence of secondary malignancies in patients after allogeneic stem cell transplantation has been reported, with an incidence of 5.6% [32]. Second malignancy is not associated with traditional ECP [30,33] and is also not expected using systemic ALA [12,34]. However, unexpected events must be monitored in future larger clinical ALA-ECP studies, the main limitations of the present study being the open-label design and the small patient sample size.

Although safety was the primary focus of this study, organ, performance, and quality of life assessments were regularly performed. It should be emphasised that our observations only reflect changes that occurred during the study period and cannot be attributed to ALA-ECP treatment alone. Factors such as the fluctuating course of the diseases, the comedication of the patients, and the lack of investigator-blinded controls may have influenced the results. Overall, the improvement in scores appears promising, especially for skin. In contrast, tear production was measured to be greatest at baseline. The presence of keratoconjunctivitis sicca is well known in cGvHD [6] and four of the patients used

artificial teardrops due to dry eyes before entering the study. Information to patients not to use drops before the baseline visit may have been insufficient. This could explain the finding of lower eye moisture at controls. However, we cannot rule out that the treatment may have affected tear production, and this should be further evaluated in future studies.

This study met the aim to evaluate safety and tolerability after multiple treatments with ALA-ECP in patients with cGvHD and lays the basis for designing future clinical ALA-ECP studies.

In conclusion, the results indicate that ALA-ECP is safe. Treatment appears to be tolerated by patients, with most adverse events reported to be in the mild-to-moderate range of severity. Apart from reduced eye moisture, no findings suggestive of organ toxicity were observed. Further research is needed to assess the safety and for the optimisation of the treatment.

**Author Contributions:** Conceptualization: E.C. and Q.P.; formal analysis: E.C. and O.A.F.; funding acquisition: Q.P.; investigation: E.C., I.S., and F.P.; methodology: E.C., P.Q.-P., T.H., P.J., and Q.P.; project administrator: E.C., I.S., and Q.P.; supervision: E.C.; visualisation: E.C. and O.A.F.; writing original draft: E.C., O.A.F., and Q.P.; writing—review and editing: E.C., O.A.F., P.Q.-P., I.S., F.P., T.H., P.J., and Q.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** The South-Eastern Norway Regional Health Authority (project numbers: 2015075, 2016092, 2017058, 2020069) and the Norwegian Cancer Society (project number: 190397).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the regional committee for medical research ethics (REK-Nord 2014/2316) and the Norwegian Medicines Agency (National Regulatory Authority 14/16760-29).

**Informed Consent Statement:** Oral and written informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** No datasets were generated or analysed during the current study.

**Acknowledgments:** The authors wish to thank the patients for their participation in this study, the staff at the Photopheresis Centre, St. Olavs Hospital and Olav Spigset, Emadoldin Feyzi and Ragnhild Telnes (Norwegian University of Science and Technology (NTNU) and St. Olavs Hospital) for their great efforts in assessing the study safety and tolerability data, Robert Knobler (Medical University of Vienna, Austria) and Trond Warloe for their valuable discussions and photonamic GmbH & Co. KG, Germany, for providing Gliolan for this study, and Daniel DiGirolamo and Erika Odlund (Mallinckrodt Pharmaceuticals, USA) for their suggestions on using the Therakos System.

**Conflicts of Interest:** Authors Eidi Christensen, Toril Holien, and Qian Peng are co-inventors of patent number 10695371. The additional authors have no conflict of interest to declare.

#### **References**


## *Review* **Photodynamic Therapy Review: Principles, Photosensitizers, Applications, and Future Directions**

**José H. Correia 1,2 , José A. Rodrigues 2,\* , Sara Pimenta <sup>2</sup> , Tao Dong 1,3 and Zhaochu Yang <sup>1</sup>**


**Abstract:** Photodynamic therapy (PDT) is a minimally invasive therapeutic modality that has gained great attention in the past years as a new therapy for cancer treatment. PDT uses photosensitizers that, after being excited by light at a specific wavelength, react with the molecular oxygen to create reactive oxygen species in the target tissue, resulting in cell death. Compared to conventional therapeutic modalities, PDT presents greater selectivity against tumor cells, due to the use of photosensitizers that are preferably localized in tumor lesions, and the precise light irradiation of these lesions. This paper presents a review of the principles, mechanisms, photosensitizers, and current applications of PDT. Moreover, the future path on the research of new photosensitizers with enhanced tumor selectivity, featuring the improvement of PDT effectiveness, has also been addressed. Finally, new applications of PDT have been covered.

**Keywords:** PDT mechanisms; new photosensitizers; PDT tumor treatment; antimicrobial PDT; non-oncologic applications of PDT; PDT in medical devices

### **1. Introduction**

## *1.1. History of Photodynamic Therapy*

Light has been used in the treatment of several diseases since antiquity [1]. The ancient civilizations, Egyptian, Indian, and Chinese, used the sunlight to treat various skin diseases, such as psoriasis, vitiligo, and skin cancer [2]. Herodotus, a famous Greek physician known as the father of heliotherapy, emphasized the importance of whole-body sun exposure for the restoration of health. In the 18th and 19th centuries, in France, sunlight was used in the treatment of several conditions, such as tuberculosis, rickets, scurvy, rheumatism, paralysis, edema, and muscle weakness [3]. At the beginning of the 20th century, the importance of light in the treatment of diseases was recognized, and the 1903 Nobel Prize in Physiology or Medicine was awarded to Niels Finsen for his contribution in this field. Finsen found that sunlight or light from a carbon arc lamp with a heat filter could be used to treat lupus vulgaris, a tubercular condition of the skin. This discovery marked the beginning of modern phototherapy [1,4,5].

Phototherapy describes the use of light in the treatment of a disease. However, photochemotherapy requires the administration of a photosensitizing agent, which is subsequently activated by light in the tissues, where the agent is localized. This form of therapy also dates back over 3000 years, when the Indians and Egyptians used psoralens from natural plants in the treatment of a variety of skin conditions [3,6].

**Citation:** Correia, J.H.; Rodrigues, J.A.; Pimenta, S.; Dong, T.; Yang, Z. Photodynamic Therapy Review: Principles, Photosensitizers, Applications, and Future Directions. *Pharmaceutics* **2021**, *13*, 1332. https:// doi.org/10.3390/pharmaceutics13091332

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 19 July 2021 Accepted: 16 August 2021 Published: 25 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The concept of cell death induced by the interaction of light and chemicals has been recognized for 100 years. In 1900, a German medical student, O. Raab, first reported cell death induced by the interaction of light with chemicals. While working with Professor H. von Tappeiner in Munich, he described the lethal effect that the combination of light and acridine red had on protozoa [3,4,6]. In subsequent experiments, Raab demonstrated that this lethal effect was greater than with acridine red alone, light alone, or acridine red exposed to light and then added the protozoan. He reported that toxicity occurred as a result of fluorescence caused by the transfer of energy from light to the chemical [6,7]. In the same year, the French neurologist J. Prime discovered that patients with epilepsy who were treated with orally administered eosin developed dermatitis in areas exposed to sunlight. Later, in 1903, H. von Tappeiner and A. Jesionek treated skin tumors with topical applications of eosin and white light [3,4,6]. In 1904, H. von Tappeiner and A. Jodlbauer identified that oxygen is an integral component in photosensitization reactions, and in 1907 they introduced the term "photodynamic action" to describe this phenomenon [1–3,6].

In 1841, H. Scherer first produced hematoporphyrin while investigating the nature of blood. However, its fluorescent properties were not described until 1867, and this substance was only named by hematoporphyrin in 1871. In 1911, W. Hausmann reported the effects of hematoporphyrin and light on protozoa and blood cells, describing skin reactions in a mice exposed to light after being administered with hematoporphyrin [3,4,6]. The first report of human photosensitization was in 1913, when F. Meyer-Betz injected himself with 200 mg of hematoporphyrin to determine if similar effects could be induced in humans. He described prolonged pain and swelling in light-exposed areas [2,3,6]. In 1960, R. Lipson and S. Schwartz initiated the concept of photodynamic therapy (PDT) at the Mayo Clinic, discovering the cancer diagnostic and therapeutic effects by injecting a hematoporphyrin derivative (HpD) [1]. In 1975, a significant breakthrough in PDT occurred, when T. Dougherty reported that administration of HpD and its activation with red light completely eradicated the growth of mammary tumor in mice. In the same year, J. F. Kelly proved the elimination of bladder carcinoma in mice, by activating HpD with light [4]. In 1976, another major event in the development of PDT occurred, when J. F. Kelly and M. E. Snell proceeded to the first human study of the effects of PDT in bladder cancer using HpD [3]. The use of this technique in the treatment of pathologies in the gastrointestinal tract was first performed in 1984 by J. S. McCaughan, who used PDT to treat patients with esophageal cancer. One year later, Y. Hayata used PDT to treat patients with gastric carcinoma [3,4]. Dougherty and his coworkers also purified HpD and produced Photofrin, which was the first photosensitizer molecule (PS) approved by the US Food and Drug Administration (FDA) in 1995 for cancer treatment [2]. Since then, PDT has continued to evolve and its clinical application was extended to other areas besides tumors treatment. Dr. M. Weber, known as the pioneer of modern laser therapy, also applies PDT to treat bacterial, viral, and parasitic diseases, named as antimicrobial PDT. The main advantage of this new approach is to combat multiresistant pathogens. Dr. Weber developed the Weberneedle® technology, which allows to apply highly focused and efficient lasers of different wavelengths to intravenous, interstitial, and intra-articular irradiation [8,9].

#### *1.2. Principles of PDT*

PDT is based on the dynamic interaction between a PS, light with a specific wavelength, and molecular oxygen, promoting the selective destruction of the target tissue [10]. The PDT treatment consists in the administration of a PS (topically or intravenous), which selectively accumulates in the tumor tissue (during a drug–light interval), followed by subsequent exposition to an appropriate wavelength light (generally in the red spectral region, λ ≥ 600 nm; Figure 1) [11]. The PS itself does not react with biomolecules; however, illumination transfers energy from light to molecular oxygen, to generate reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide radical (O<sup>2</sup> −•), hydroxyl radical (HO• ), and hydrogen peroxide (H2O2) [1]. These cytotoxic photoproducts start a cascade of biochemical events, which can induce damage and death of the target tissue [11].

illumination transfers energy from light to molecular oxygen, to generate reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide radical (O2−•), hydroxyl radical (HO•), and hydrogen peroxide (H2O2) [1]. These cytotoxic photoproducts start a cascade of biochemical events, which can induce damage and death of the target tissue [11].

**Figure 1.** Representation of the clinical application of PDT protocol for the treatment of a solid and localized tumor (based on [12]). **Figure 1.** Representation of the clinical application of PDT protocol for the treatment of a solid and localized tumor (based on [12]).

#### **2. Photodynamic Reaction 2. Photodynamic Reaction**

PDT is a therapeutic modality based on the combination of three factors: PS, light with a specific wavelength, and the presence of molecular oxygen [2]. The photodynamic reaction begins with the absorption of light by the PS in the target tissue, which triggers a series of photochemical reactions that lead to the generation of ROS [10]. After light absorption, the PS is transformed from its ground state (singlet state, 1PS) to a short-lived, electronically excited singlet state (a few nanoseconds or less, 1PS\*) [2,4]. This excited state is very unstable and can decay to the ground state, losing the excess of energy through light emission (fluorescence) or heat production (internal conversion) [13]. However, the singlet state can undergo intersystem crossing and progress to a more stable, long-lived, electronically excited state (triplet state, 3PS\*), through spin conversion of the electron in the higher-energy orbital [13]. This excited state can decay to the ground state through light emission (phosphorescence) or undergo two kinds of reactions [4]. The triplet state has a longer lifetime (up to tens of microseconds), which allows sufficient time for direct transfer of energy to molecular oxygen (O2). This energy transfer step leads to the formation of singlet oxygen (1O2) and the fundamental state of the PS, called type II reaction [2,11]. The singlet oxygen is extremely reactive and can interact with a large number of biological substrates, inducing oxidative damage and ultimately cell death [11]. The type I reaction can also occur if the excited state of the PS reacts directly with a substrate, such as cell membrane or a molecule, and undergoes hydrogen atom abstraction or electron transfer reactions, to yield free radicals and radical ions. These radicals react with molecular oxygen, producing ROS, such as O2−•, HO•, and H2O2, which produce oxidative damage that can lead to biological lesions [11]. Figure 2 shows the modified Jablonski diagram PDT is a therapeutic modality based on the combination of three factors: PS, light with a specific wavelength, and the presence of molecular oxygen [2]. The photodynamic reaction begins with the absorption of light by the PS in the target tissue, which triggers a series of photochemical reactions that lead to the generation of ROS [10]. After light absorption, the PS is transformed from its ground state (singlet state, <sup>1</sup>PS) to a short-lived, electronically excited singlet state (a few nanoseconds or less, <sup>1</sup>PS\*) [2,4]. This excited state is very unstable and can decay to the ground state, losing the excess of energy through light emission (fluorescence) or heat production (internal conversion) [13]. However, the singlet state can undergo intersystem crossing and progress to a more stable, long-lived, electronically excited state (triplet state, <sup>3</sup>PS\*), through spin conversion of the electron in the higher-energy orbital [13]. This excited state can decay to the ground state through light emission (phosphorescence) or undergo two kinds of reactions [4]. The triplet state has a longer lifetime (up to tens of microseconds), which allows sufficient time for direct transfer of energy to molecular oxygen (O2). This energy transfer step leads to the formation of singlet oxygen (1O2) and the fundamental state of the PS, called type II reaction [2,11]. The singlet oxygen is extremely reactive and can interact with a large number of biological substrates, inducing oxidative damage and ultimately cell death [11]. The type I reaction can also occur if the excited state of the PS reacts directly with a substrate, such as cell membrane or a molecule, and undergoes hydrogen atom abstraction or electron transfer reactions, to yield free radicals and radical ions. These radicals react with molecular oxygen, producing ROS, such as O<sup>2</sup> −•, HO• , and H2O2, which produce oxidative damage that can lead to biological lesions [11]. Figure 2 shows the modified Jablonski diagram of the PDT action mechanism.

of the PDT action mechanism. The products resulting from type I and type II reactions are responsible for the effect of cell death and therapeutic on PDT. Type I and type II reactions can occur simultaneously and the ratio between these processes depends on the PS, substrate, oxygen concentration, and binding affinity of the sensitizer to the substrate [2,4,13]. However, type II reaction is predominant during PDT, and singlet oxygen is the primary cytotoxic agent responsible for the biological effects [11]. The quantum yield of singlet oxygen formation is one of the most important features of a PS and is determined by the quantum yield and lifetime of its triplet excited state [10]. Due to the high reactivity and short half-life of the ROS, only cells close to the area of the ROS production (areas where the PS is localized) are directly affected by PDT. The extent of damage and cytotoxicity resulting from PDT is

multifactorial, depending on the type of PS, its extracellular and intracellular location and the total dose administered, the dose of light (light fluence) and the light fluence rate, availability of oxygen, and the time between PS administration and light exposure [4]. PDT of deeper and hypoxic tumors is more difficult due to the low oxygen concentration and low light penetration into the tissue (light absorption by the PS and energy transfer to the oxygen). On the other hand, more superficial and more oxygenated tumors allow greater production of ROS and thus a more effective PDT treatment [14]. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 4 of 16 **Figure 2.** Modified Jablonski diagram of the PDT action mechanism (based on [13]). The products resulting from type I and type II reactions are responsible for the effect of cell death and therapeutic on PDT. Type I and type II reactions can occur simultaneously and the ratio between these processes depends on the PS, substrate, oxygen concentration, and binding affinity of the sensitizer to the substrate [2,4,13]. However, type II reaction is predominant during PDT, and singlet oxygen is the primary cytotoxic agent

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 4 of 16

**Figure 2.** Modified Jablonski diagram of the PDT action mechanism (based on [13]). **Figure 2.** Modified Jablonski diagram of the PDT action mechanism (based on [13]). greater production of ROS and thus a more effective PDT treatment [14].

#### **3. PDT-Mediated Action Mechanisms 3. PDT-Mediated Action Mechanisms**

The products resulting from type I and type II reactions are responsible for the effect of cell death and therapeutic on PDT. Type I and type II reactions can occur simultaneously and the ratio between these processes depends on the PS, substrate, oxygen concentration, and binding affinity of the sensitizer to the substrate [2,4,13]. However, type II reaction is predominant during PDT, and singlet oxygen is the primary cytotoxic agent responsible for the biological effects [11]. The quantum yield of singlet oxygen formation is one of the most important features of a PS and is determined by the quantum yield and lifetime of its triplet excited state [10]. Due to the high reactivity and short half-life of the ROS, only cells close to the area of the ROS production (areas where the PS is localized) There are three main mechanisms by which PDT mediates tumor destruction (Figure 3) [4]. The ROS produced by PDT photochemical reactions can directly destroy tumor cells by inducing apoptosis and necrosis. PDT can also cause the destruction of the tumorassociated vasculature and the surrounding healthy vessels, leading to an interruption of oxygen and nutrient supply and, consequently, to indirect cell death due to hypoxia. Finally, PDT can induce an inflammatory response that activates an immune response against the tumor cells [4,15]. The outcome of PDT depends on all of these mechanisms, and the contribution of each one is determined by the treatment regime used [4,10]. There are three main mechanisms by which PDT mediates tumor destruction (Figure 3) [4]. The ROS produced by PDT photochemical reactions can directly destroy tumor cells by inducing apoptosis and necrosis. PDT can also cause the destruction of the tumor-associated vasculature and the surrounding healthy vessels, leading to an interruption of oxygen and nutrient supply and, consequently, to indirect cell death due to hypoxia. Finally, PDT can induce an inflammatory response that activates an immune response against the tumor cells [4,15]. The outcome of PDT depends on all of these mechanisms, and the contribution of each one is determined by the treatment regime used [4,10].

3) [4]. The ROS produced by PDT photochemical reactions can directly destroy tumor cells by inducing apoptosis and necrosis. PDT can also cause the destruction of the tumor-as-**Figure 3. Figure 3.**  PDT mechanisms for tumor destruction (based on [ PDT mechanisms for tumor destruction (based on [12]). 12]).

#### sociated vasculature and the surrounding healthy vessels, leading to an interruption of *3.1. Apoptosis and Necrosis*

oxygen and nutrient supply and, consequently, to indirect cell death due to hypoxia. Finally, PDT can induce an inflammatory response that activates an immune response against the tumor cells [4,15]. The outcome of PDT depends on all of these mechanisms, and the contribution of each one is determined by the treatment regime used [4,10]. Tumor destruction from PDT can occur by both programmed (apoptotic) pathways and non-programmed (necrosis) pathways [11,16]. Generally, when high light intensity is employed, the tumor cells are rapidly ablated by necrosis [11]. Necrosis is generally described as a rapid and relatively broad mechanism of cell death, and it is characterized by vacuolization of the cytoplasm and cell membrane breakdown, resulting in a local inflammatory reaction due to the release of cytoplasmic content and pro-inflammatory mediators in the extracellular medium [10].

**Figure 3.** PDT mechanisms for tumor destruction (based on [12]).

In contrast, apoptotic death may be initiated by PDT, generally when low light doses are employed [11,16]. Apoptosis is described as a mechanism of programmed cell death that is genetically encoded and energy dependent [10]. Morphologically, it is characterized by chromatin condensation, cleavage of chromosomal DNA into internucleosomal fragments, cell shrinkage, membrane wrinkling, and the formation of apoptotic bodies without plasma membrane breakdown [2,7,10]. No effect or immune response is expected as no toxic chemicals are leaked [11,16].

#### *3.2. Vascular Mechanisms*

In addition to the direct destruction of tumor cells, the application of PDT often also leads to the destruction of the tumor microvasculature. Like tumor cells, endothelial cells of the vascular system can concentrate PS to create free radicals when activated by appropriate light. The disrupting of the vascular walls leads to the interruption of the tumor's feeding (i.e., oxygen and nutrients) and, consequently, to the death of the tumor cells. PDT vascular effect has been shown to be very important for the long-term efficacy of PDT [10]. PDT vascular effect can be greatly enhanced by using a short drug–light interval (the time between systemic PS injection and light illumination) when the PS is predominantly localized in the vasculature [1]. Selectivity in vascular PDT protocols is achieved by the precise application of light on the tumor plus a safety margin of the surrounding healthy tissue [10]. Vascular PDT has important advantages in comparison to PDT protocols that require PS accumulation in the tumor cells: it uses photosensitizers that clear rapidly from the organism and minimize skin photosensitivity, gives higher long-term efficacy, and can be performed in one short session [10,17].

#### *3.3. Immunological Mechanisms*

For many years, PDT was considered a localized treatment, affecting only tumor cells and tumor vasculature. More recently, numerous studies have demonstrated that PDT can significantly influence the adaptive immune response in disparate ways, either through stimulation or suppression of the immune response. The efficacy of PDT appears to be dependent on the induction of antitumor immunity [10]. Long-term tumor control is a combination of the direct effects of PDT on the lesion and its vasculature with upregulation of the immune system [11].

Under certain conditions, PDT induces immunosuppression, which has been mainly associated with reactions to topical treatments with high fluence rates and in large areas of irradiation [10]. In contrast, non-topical PDT treatments are often described as immunostimulatory. The oxidative damage inflicted by PDT on the tumor stroma will eventually result in cell death. When PDT induces necrosis of tumors and their vasculature, an immune cascade is also initiated. The change in tissue integrity and homeostasis triggers an acute inflammatory response initiated by the release of pro-inflammatory mediators, which include various cytokines, growth factors, and proteins [10,11]. These mediators attract the host's innate immune cells, such as neutrophils, mast cells, macrophages, and dendritic cells, which infiltrate the damaged tissue to restore homeostasis in the affected region [10]. Upon arrival, macrophages phagocytize PDT-damaged cancer cells and present proteins from these tumors to CD4 helper T lymphocytes, which then activate CD8 cytotoxic T lymphocytes [11]. These cytotoxic T cells can recognize and specifically destroy tumor cells and can circulate throughout the body for long periods, ensuring a systemic antitumor immune response [10].

#### **4. PDT Essential Elements**

### *4.1. Photosensitizers*

Photosensitizers are key elements for PDT. Ideally, these molecules should accumulate preferentially in the tumors, have a high singlet oxygen quantum yield, have low activity in the absence of light, be quickly eliminated from the patient body, have amphiphilicity, and have a light absorption peak between approximately 600 nm and 800 nm [18,19].

There are a variety of molecular structures of photosensitizers that are currently used in PDT, and it is possible to divide photosensitizers into three generations. The porfimer sodium and the HpD are first-generation photosensitizers. The second-generation photosensitizers arise to overcome some drawbacks of the first-generation ones, related to light absorption at a specific spectral region. Some examples of second-generation photosensitizers are the derivates of chlorins, bacteriochlorins, and phthalocyanines, which can have a stronger action on the tumor regions due to their strong absorbance in the deep red region, and consequently, increased light penetration. Finally, the third-generation photosensitizers are molecules with improved selectivity for tumor regions, due to the conjunction of the PS with targeting molecules or its encapsulation into carriers. Thus, photosensitizers progressed towards the improvement of PDT specificity and efficacy. Today, the functionalization of photosensitizers seems to be the best strategy to achieve a high selectivity to the tumor regions, combining photosensitizers with biomolecules or carriers [18–20]. Photosensitizers can be covalently bonded to several biomolecules, which have affinity to tumors. These biomolecules include antibodies, proteins, carbohydrates, and others. Photosensitizers can also be encapsulated into carriers, such as gold nanoparticles, silica nanoparticles, quantum dots, carbon nanotubes, or others carriers, to guide the photosensitizers to tumors [19,21–24].

Table 1 presents the most used photosensitizers in clinical PDT, including their trade name and class, molecular formula, excitation wavelength, quantum yield, extinction coefficient, and main applications [10,18,25–31]. A variety of photosensitizers under clinical trials for approval in clinical PDT are presented in Table 2 [10,18,26,27,32–34].

Consensus protocol for conventional topical PDT recommends some lesion preparation to increase PS absorption and light penetration. The typical PDT topical treatment protocol follows the next steps [35]:


In dermatological indications, PDT is usually performed by topical application of PS, in particular 5-aminolevulinic acid (5-ALA) or its ester methyl aminolevulinate (MAL) [35]. Three photosensitizers are currently approved for topical use in Europe: MAL Metvix®, 5-ALA Ameluz®, and 5-ALA AlaCare®. MAL Metvix® is used along with red light to treat actinic keratosis, Bowen's disease, and superficial basal cell carcinoma (3 h of drug-light interval and 37–75 J/cm<sup>2</sup> of total light dose). 5-ALA Ameluz® is used in combination with red light for the treatment of mild and moderate actinic keratosis and superficial basal cell carcinoma (3 h of drug-light interval and 37–200 J/cm<sup>2</sup> of total light dose). MAL Metvix® and 5-ALA Ameluz® are also used with daylight to treat moderate actinic keratosis (0.5 h of drug-light interval and exposure during 2 h). 5-ALA AlaCare® is approved for the treatment of mild actinic keratosis with red light (4 h of drug-light interval and 37 J/cm<sup>2</sup> of total light dose). A 20% formulation of 5-ALA Levulan® is approved in North America for the treatment of actinic keratosis with blue light (14–18 h of drug-light interval and 10 J/cm<sup>2</sup> of total light dose). Besides, topical PDT is highly recommended for the photorejuvenation and the treatment of acne vulgaris, although these indications currently lack approval for use and the protocols still need to be optimized [35–38].



#### **Table 2.** Photosensitizers under clinical trials for use in PDT.


A strategy that seems to be of great interest in the near future is to increase the PDT selectivity through the development of activatable multifunctional photosensitizers, which become active after receiving a biological or physical stimulus. Biological stimuli include the physiological conditions associated with cancer, such as temperature, pH, and enzymatic activity. For example, the peptidic zipper-based PS was designed to react under acidic conditions. Physical stimuli refer to an artificial agent activation, applying magnetic or electric fields, ultrasounds, two-photon excitation, etc. [18,19]. For example, electroporation can be used to support PDT (Figure 4). Electroporation is reported as an effective method that could be used to increase the transport of a PS into the pathological cells, which could lead to the increase of cytotoxicity and PDT efficacy. Several studies have been performed over the years, using different photosensitizers, the clinically approved Photofrin PS being the most relevant. The results, including a study performed with human cancer cells, conclude, undoubtedly, that PDT with electroporation is an attractive approach to cancer treatment, but detailed studies on the mechanisms of this approach are still required [20,39]. ganic fluorophores as efficient photosensitizers for PDT has also been reported. The transition metal coordinator complexes, such as ruthenium(II) complexes, iridium(III) complexes, and polymetallic complexes, meet several basic needs for PDT. The most relevant feature is their heavy-atom effect, which mediates strong spin–orbital coupling, providing more time for the excited states to interact with molecular oxygen. Between other features, it can be also be stated that they are easily synthetized, including the possibility of finetuning their properties. The organic fluorophores, such as naphthalimides, xanthenes, boron dipyrromethene (BODIPY), and cyanines, can also be designed as photosensitizers for cancer PDT, with high light absorption at relatively long wavelengths and large molar extinction coefficient. Organic fluorophore photosensitizers have low toxicity, good biocompatibility, and long triplet lifetimes, and their fluorescence emission can be used to perform real-time monitoring during PDT treatment [40,41].

Very recently, the possibility of using transition metal coordination complexes or or-

A strategy that seems to be of great interest in the near future is to increase the PDT selectivity through the development of activatable multifunctional photosensitizers, which become active after receiving a biological or physical stimulus. Biological stimuli include the physiological conditions associated with cancer, such as temperature, pH, and enzymatic activity. For example, the peptidic zipper-based PS was designed to react under acidic conditions. Physical stimuli refer to an artificial agent activation, applying magnetic or electric fields, ultrasounds, two-photon excitation, etc. [18,19]. For example, electroporation can be used to support PDT (Figure 4). Electroporation is reported as an effective method that could be used to increase the transport of a PS into the pathological cells, which could lead to the increase of cytotoxicity and PDT efficacy. Several studies have been performed over the years, using different photosensitizers, the clinically approved Photofrin PS being the most relevant. The results, including a study performed with human cancer cells, conclude, undoubtedly, that PDT with electroporation is an attractive approach to cancer treatment, but detailed studies on the mechanisms of this approach

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 8 of 16

are still required [20,39].

*4.2. Light*  PDT has been performed with various light sources, including lasers, incandescent light, and laser-emitting diodes [42]. Laser light sources are usually expensive and require the use of an optical system to expand the light beam for irradiation of a larger tissue area. Very recently, the possibility of using transition metal coordination complexes or organic fluorophores as efficient photosensitizers for PDT has also been reported. The transition metal coordinator complexes, such as ruthenium(II) complexes, iridium(III) complexes, and polymetallic complexes, meet several basic needs for PDT. The most relevant feature is their heavy-atom effect, which mediates strong spin–orbital coupling, providing more time for the excited states to interact with molecular oxygen. Between other features, it can be also be stated that they are easily synthetized, including the possibility of fine-tuning their properties. The organic fluorophores, such as naphthalimides, xanthenes, boron dipyrromethene (BODIPY), and cyanines, can also be designed as photosensitizers for cancer PDT, with high light absorption at relatively long wavelengths and large molar extinction coefficient. Organic fluorophore photosensitizers have low toxicity, good biocompatibility, and long triplet lifetimes, and their fluorescence emission can be used to perform real-time monitoring during PDT treatment [40,41].

#### *4.2. Light*

PDT has been performed with various light sources, including lasers, incandescent light, and laser-emitting diodes [42]. Laser light sources are usually expensive and require the use of an optical system to expand the light beam for irradiation of a larger tissue area. Non-laser light sources (e.g., conventional lamps) can be used with optical fibers to specify the light wavelength for tissue irradiation. However, conventional lamps may have thermal effects, which must be avoided in PDT. Finally, light-emitting diodes (LEDs) have also been used in PDT as light sources. LEDs are less expensive, less hazardous,

thermally non-destructive, and easily available in flexible arrays [43]. Light penetration into tumor tissue is very complex, as it can be reflected, scattered, or absorbed. The extent of these processes depends on the type of tissue and the wavelength of light [44]. Light absorption is mainly due to endogenous chromophores existing in tissues, such as hemoglobin, myoglobin, and cytochromes, which can reduce the photodynamic process by competing with PS in the absorption process [44,45]. Light absorption by tissues decreases with increasing wavelength, so longer wavelengths of light (red light) penetrate more efficiently through tissue. The region between 600 and 1200 nm is often called the "tissue optical window" [12,44]. Shorter wavelengths (<600 nm) have less tissue penetration and are more absorbed, resulting in increased skin photosensitivity. On the other hand, longer wavelengths (>850 nm) do not have enough energy to excite oxygen in its state of singlet and to produce enough reactive oxygen species. Therefore, the highest tissue permeability occurs between 600 and 850 nm. This range, called the "phototherapeutic window," is predominantly used in PDT [10,12,18,20].

As light is an essential component of PDT, clinical efficacy is highly dependent on the accuracy of its delivery to the target tissue and its dose, which translates into light fluence, light fluence rate, light exposure time, and light delivery mode (single or fractionated) [12]. Light fluence is the total energy of exposed light across a sectional area of irradiated spot and is expressed in J/cm<sup>2</sup> . Light fluence rate is the incident energy per second across a sectional area of the irradiated spot and is expressed as W/cm<sup>2</sup> [4,46,47].

Several studies have reported that low light fluence rates are advantageous for PDT [48–50]. The main reason for the lower efficacy of PDT for high light fluence rates is the oxygen depletion in tissues, which leads to a low photo-degradation of the PS. The light fluence rate has also been shown to have an impact on the dominant mechanism of cell death in the PDT. The use of low light fluence rates increases the selective apoptosis of tumor cells, which is more desirable than inflammation and edema that usually occur with the uncontrolled rupturing of cellular content in necrosis [51].

Another relevant light source for PDT is the natural light. The concept of daylight PDT is based on the use of natural light instead of an artificial light source to treat skin lesions, such as actinic keratosis. The main advantages of using daylight PDT are minimal patient discomfort and shorter clinical visits (patients can complete their therapy at home). Moreover, daylight PDT seems to be as effective as conventional PDT for actinic keratosis. For this specific application, the patients expose the sites to daylight for 2 h, after 30 min of PS application. The short PS incubation time in the daylight PDT, compared with conventional PDT (1–3 h required), allows a smaller and more continuous PS activation, leading to lower patient pain intensity associated with PDT [52,53].

#### *4.3. Oxygen*

The third key component in the PDT mechanism is molecular oxygen. Oxygen is crucial for the production of ROS during PDT. Oxygen concentration in the tissues truly affects the effectiveness of the PDT treatment. In fact, oxygen concentration can vary significantly between different tumors and even between different regions of the same tumor, depending on the density of the vasculature. Especially in deeper solid tumors, often characterized by their anoxic microenvironment, lack of oxygen can be a limiting factor. As mentioned above, the irradiation of the tumor with a high light fluence rate can lead to a temporary local oxygen depletion. This leads to interruption of ROS production and reduced treatment effectiveness. Oxygen depletion occurs when the rate of oxygen consumption by the photodynamic reaction is greater than the rate of oxygen diffusion in the irradiated area. In addition, PDT can cause occlusion of the tumor vasculature, reducing blood flow to the tumor tissue, further increasing hypoxia [10,26,46].

Real-time measurement of the tissue oxygen levels, before and during PDT, is one of the main challenges in the near future. This allows to optimize the PDT therapeutic result by adjusting the light fluence rate (increasing the irradiation time to maintain the total light dose) or using fractional light dose. Several sensors have been used to monitor the

oxygen level in biological media, using imaging agents. However, the combination of these imaging agents with PS has been hardly reported [18]. Other methods to increase oxygen availability in the tumor have been tested: indirect introduction of oxygen and direct introduction of oxygen. One indirect way to increase oxygen concentration in tumor cells is using catalase enzyme to decompose the intracellular hydrogen peroxide into oxygen. The direct delivery of oxygen into tumors is achieved by using oxygen carriers, such as perfluorocarbons and hemoglobin, commonly used to overcome tumor hypoxia in the PDT procedure [37].

#### **5. Advantages and Limitations of PDT**

PDT has several advantages over conventional approaches to cancer treatment. Firstgeneration photosensitizers cause increased skin photosensitivity. However, PDT has no long-term side effects when correctly used. It is less invasive than surgical procedures and can be performed on an outpatient basis. In addition to the tumor itself, PDT can also destroy the vasculature associated with it, greatly contributing to tumor death [54]. PDT can be applied directly and accurately in the target tissue, due to its dual selectivity. The two main factors that contribute to the selectivity of PDT are the intrinsic capacity of some photosensitizers to preferentially accumulate in tumor tissue and light irradiation exclusively in the target tissue [10,54]. The selective accumulation of the PS in the tumor is facilitated in the case of topical application, since PS is applied directly and only to the lesions to be treated. When PS is given intravenously, it needs to remain in circulation long enough to reach and accumulate in the tumor [10]. Furthermore, PDT can be repeated several times in the same location, unlike radiation. There is little or no scarring after healing. Finally, it usually costs less than other therapeutic modalities in cancer treatment [54,55].

Like every therapeutic modalities, PDT also has some limitations. The photodynamic effect occurs selectively in the irradiated site, which makes its use in disseminated metastases very difficult with the currently available technology [54]. Tissue oxygenation is crucial for the photodynamic effect to occur, so tumors surrounded by necrotic tissue or dense tumor masses can lead to ineffective PDT. Finally, the accuracy of target tissue irradiation is the most important point when considering PDT as a treatment option. Therefore, deep tumors (not easily accessible without surgical intervention) are difficult to treat due to the low penetration of visible light into the tissue [54,56]. The main advantages and limitations of PDT are summarized in Table 3.


**Table 3.** Summary of the main advantages and disadvantages of PDT.

#### **6. Applications of PDT**

PDT is a minimally invasive procedure that is clinically used in the treatment of several oncologic human diseases, such as skin, esophageal, head and neck, lung, and bladder cancers [57]. However, PDT also has several non-oncologic applications [58], including the treatment of non-cancerous human diseases, such as dermatologic (acne [59], warts [60], photoaging [61], psoriasis [62], vascular malformations [63], hirsutism [64], keloid [50], and alopecia areata [65]), ophthalmologic (central serous chorioretinopathy [66] and corneal neovascularization [67]), cardiovascular (atherosclerosis [68] and esophageal varix [69]), dental (oral lichen planus [70]), neurologic (Alzheimer's disease [71]), skeletal (rheumatoid arthritis [72]), and gastrointestinal (Crohn's disease [73]).

An extension of PDT procedure can be the inactivation of viruses and microorganisms, including bacteria, yeasts, and fungi, named as photodynamic inactivation of microorganisms (PDI). The viruses or microorganisms are inactivated by combining non-toxic dyes (photosensitizers) with harmless visible light. PDI can be used as an alternative to the use of antibiotics and antiviral drugs that usually cause resistance. The application of PDI is possible in several areas, including human and veterinary medicine, agro-food, wastewater treatment, and biosafety. However, PDI in the treatment of infections is easier to perform in vitro, compared with its clinical applicability, due to the low tissue penetration depth of visible light. Light applied intravenously can be a solution during the clinical treatment of infections. Very recently, the use of the PDT procedure to treat patients with COVID-19 has also been discussed [8,57,74–81].

Dr. M. Weber et al. [8] performed a study to evaluate if the PDT procedure with Riboflavin (also known as vitamin B2) and blue light can be used effectively as a therapy for patients infected with acute COVID-19. The study used COVID-19-positive patients who received PDT therapy and COVID-19-positive patients who received conventional care. The patients that received PDT treatment showed significant improvement in clinical symptoms and viral load within 5 days. The control patients had no significant improvement in clinical symptoms or viral load within 5 days. The results prove the potential of PDT procedure to treat patients infected with COVID-19 virus at an early infection stage and with mild to moderate clinical symptoms. This new application of PDT procedure can prevent hospitalization and intensive care treatments.

Finally, PDT can be implemented in a medical device, e.g., endoscopic capsule. In 2016, G. Tortora et al. [82] developed an ingestible capsule for light delivery in PDT treatment of *Helicobacter pylori* infection. *Helicobacter pylori* bacterium is known to be photosensitive without the exogenous assumption of photosensitizers. This bacterium can be killed by exciting the photosensitizers naturally present in it with the appropriate light wavelength. The capsule with 27 mm length and 14 mm diameter has been equipped with 8 LEDs positioned on an electronic board with a magnetic switch (to turn on the capsule's power) and a battery. The capsule light-emitting module was dimensioned considering the required light necessary to kill the bacterium, with blue (405 nm) and red (625 nm) lights. In 2018, J. A. Rodrigues et al. [26,83] studied the photodynamic activity of the mTHPC (Foscan®) on RKO and HCT-15 cell lines to implement PDT in autonomous medical devices, such as endoscopic capsules for clinical treatment of several gastrointestinal tract tumors. Figure 5 envisages the integration of PDT in the endoscopic capsule. Due to the endoscopic capsule dimensions and battery life, the light fluence and fluence rate of the red light must be minimized to reduce the PDT treatment time. The experimental results showed that a small amount of mTHPC (0.15 mg/kg) and light fluence (5–20 J/cm<sup>2</sup> ) is sufficient to obtain significant photodynamic activity. An array of LEDs with peak transmittance at 652 nm was used in the in vitro PDT assays. The experimental results show that decreased cell viability (down to 30%) can be obtained for 1–5 µg/mL of mTHPC concentrations and 2.5 J/cm<sup>2</sup> of light fluence. The use of a minimum light fluence (2.5 J/cm<sup>2</sup> ) and light fluence rate (11 mW/cm<sup>2</sup> ) allowed to reduce the treatment time to just 3 min and 47 s. The PDT endoscopic capsule was designed with two functional sides. The round side is compound of the conventional optical system of the endoscopic capsule, consisting of the CMOS image sensor, four white LEDs, and focal lenses, and the planar side constitutes the therapeutic module, composed of a red light source (array of 8 SMD red LEDs with total fluence rate of 14 mW/cm<sup>2</sup> ) and a magnetic switch for turning the red light on and off. This capsule has magnetic locomotion control, for immobilization of the capsule during the treatment time, and is 31 mm in length and 14 mm in diameter. mTHPC-mediated PDT, using a light fluence of 2.5 J/cm<sup>2</sup> and fluence rate of 14 mW/cm<sup>2</sup> , reduces the PDT treatment time to approximately 3 min. Faster treatment requires less battery capacity and therefore fewer capsules.

battery capacity and therefore fewer capsules.

**Figure 5.** Illustration of PDT integrated in the endoscopic capsule (Adapted with permission from [83], IEEE, 2019). **Figure 5.** Illustration of PDT integrated in the endoscopic capsule (Adapted with permission from [83], IEEE, 2019).

light on and off. This capsule has magnetic locomotion control, for immobilization of the capsule during the treatment time, and is 31 mm in length and 14 mm in diameter. mTHPC-mediated PDT, using a light fluence of 2.5 J/cm2 and fluence rate of 14 mW/cm2, reduces the PDT treatment time to approximately 3 min. Faster treatment requires less

#### **7. Conclusions 7. Conclusions**

PDT is one of the most interesting and promising approaches to treat various oncologic, non-oncologic, and infectious diseases. This review presented the main principles, mechanisms, and crucial elements of PDT. PDT is based on the dynamic interaction between a PS, light with a specific wavelength, and molecular oxygen, to produce ROS that promote selective destruction of the target tissue. The evolution of photosensitizers was also addressed in this paper, including their future trends. Photosensitizers are evolving towards increasing the PDT efficacy and selectivity, and many possibilities are currently under research. One strategy to increase PDT selectivity involves the development of multifunctional photosensitizers that can be activated by a biological or physical stimulus. PDT is one of the most interesting and promising approaches to treat various oncologic, non-oncologic, and infectious diseases. This review presented the main principles, mechanisms, and crucial elements of PDT. PDT is based on the dynamic interaction between a PS, light with a specific wavelength, and molecular oxygen, to produce ROS that promote selective destruction of the target tissue. The evolution of photosensitizers was also addressed in this paper, including their future trends. Photosensitizers are evolving towards increasing the PDT efficacy and selectivity, and many possibilities are currently under research. One strategy to increase PDT selectivity involves the development of multifunctional photosensitizers that can be activated by a biological or physical stimulus.

PDT has been increasingly used in many applications, such as destroying tumor tissues, bacteria, fungi, and viruses (including COVID-19). Moreover, PDT can be integrated in medical devices. A light delivery capsule has been developed for mTHPC-mediated PDT treatment of several gastrointestinal tract tumors. The good photodynamic response at low light fluence and low light fluence rate allows to reduce the treatment time to a few minutes and thus integrate the PDT in autonomous medical devices, such as endoscopic capsules of very small dimensions, to provide them with an advanced therapeutic func-PDT has been increasingly used in many applications, such as destroying tumor tissues, bacteria, fungi, and viruses (including COVID-19). Moreover, PDT can be integrated in medical devices. A light delivery capsule has been developed for mTHPC-mediated PDT treatment of several gastrointestinal tract tumors. The good photodynamic response at low light fluence and low light fluence rate allows to reduce the treatment time to a few minutes and thus integrate the PDT in autonomous medical devices, such as endoscopic capsules of very small dimensions, to provide them with an advanced therapeutic function.

tion. The interdisciplinary uniqueness of PDT inspires physicists, chemists, biologists, and physicians, and its further development and discovery of new applications will only be The interdisciplinary uniqueness of PDT inspires physicists, chemists, biologists, and physicians, and its further development and discovery of new applications will only be limited by their enormous imagination.

limited by their enormous imagination. **Author Contributions:** The work presented in this paper was a collaboration of all authors. J.A.R. and S.P. made the literature analysis and wrote the first draft of the manuscript. J.H.C., T.D. and Z.Y. corrected and revised the manuscript. J.H.C. supervised and directed the manuscript. All au-**Author Contributions:** The work presented in this paper was a collaboration of all authors. J.A.R. and S.P. made the literature analysis and wrote the first draft of the manuscript. J.H.C., T.D. and Z.Y. corrected and revised the manuscript. J.H.C. supervised and directed the manuscript. All authors have read and agreed to the published version of the manuscript.

thors have read and agreed to the published version of the manuscript. **Funding:** The APC was funded by Chongqing Technology and Business University (CTBU).

**Funding:** The APC was funded by Chongqing Technology and Business University (CTBU). **Institutional Review Board Statement:** Not applicable.

**Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was supported by FCT with project OpticalBrain reference PTDC/CTM-REF/28406/2017, operation code POCI-01-0145-FEDER-028406, through the COMPETE 2020; CMEMS-UMinho Strategic Project UIDB/04436/2020; Infrastructures Micro&NanoFabs@PT, operation code NORTE 01-0145-FEDER-022090, POR Norte, Portugal 2020; and MPhotonBiopsy, PTDC/FIS-OTI/1259/2020. The authors thank Biolitec research GmbH (Jena, Germany) for providing the photosensitizer Foscan®.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Review* **Potential of Cyanine Derived Dyes in Photodynamic Therapy**

**Natalia Lange <sup>1</sup> , Wojciech Szlasa <sup>1</sup> , Jolanta Saczko <sup>2</sup> and Agnieszka Chwiłkowska 2,\***


**Abstract:** Photodynamic therapy (PDT) is a method of cancer treatment that leads to the disintegration of cancer cells and has developed significantly in recent years. The clinically used photosensitizers are primarily porphyrin, which absorbs light in the red spectrum and their absorbance maxima are relatively short. This review presents group of compounds and their derivatives that are considered to be potential photosensitizers in PDT. Cyanine dyes are compounds that typically absorb light in the visible to near-infrared-I (NIR-I) spectrum range (750–900 nm). This meta-analysis comprises the current studies on cyanine dye derivatives, such as indocyanine green (so far used solely as a diagnostic agent), heptamethine and pentamethine dyes, squaraine dyes, merocyanines and phthalocyanines. The wide array of the cyanine derivatives arises from their structural modifications (e.g., halogenation, incorporation of metal atoms or organic structures, or synthesis of lactosomes, emulsions or conjugation). All the following modifications aim to increase solubility in aqueous media, enhance phototoxicity, and decrease photobleaching. In addition, the changes introduce new features like pH-sensitivity. The cyanine dyes involved in photodynamic reactions could be incorporated into sets of PDT agents.

**Keywords:** cyanine dyes; photodynamic therapy; cancer therapy; irradiation

## **1. Introduction**

#### *1.1. Basics of Photodynamic Therapy*

Photodynamic therapy (PDT) is a low-invasive therapy, that destroys cancer cells through the generation of reactive oxygen species (ROSs). PDT involves a photosensitizer (PS), administered topically or intravenously, a light source and oxygen in the targeted tissue [1]. In the dark, the PS remains in its base energy state. However, the absorption of light at the appropriate wavelength moves the PS to an excited state. An excess of absorbed energy leads to its release and reaction with the oxygen in the tissue, thus inducing the formation of ROS [2]. The photodynamic reaction might propagate in two main ways [3]. The Type I mechanism of photodynamic reaction includes the production of highly reactive intermediates, mainly hydrogen peroxide H2O<sup>2</sup> and •OH. Type II involves the formation of oxygen in the singlet state <sup>1</sup>O<sup>2</sup> (Figure 1) [4]. The short half-life of free radicals determines the limited diffusion distance of these high cytotoxic molecules [5]. The dysregulation of the homeostasis free radicals induces irreversible oxidation of proteins, nucleic acids, fatty acids, cholesterol and organelles of the tumor cells. The process leads to loss of function and eventually to the death of the irradiated cells depending on the conditions of the therapy, especially the cellular localization of the photosensitizer [5]. Death might also arise from an induced immune response as well as from the destruction of vessels supplying the tumor with nutrition [6].

**Citation:** Lange, N.; Szlasa, W.; Saczko, J.; Chwiłkowska, A. Potential of Cyanine Derived Dyes in Photodynamic Therapy. *Pharmaceutics* **2021**, *13*, 818. https://doi.org/10.3390/ pharmaceutics13060818

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 15 April 2021 Accepted: 28 May 2021 Published: 31 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** The mechanism of a photosensitizer action in photodynamic therapy. **Figure 1.** The mechanism of a photosensitizer action in photodynamic therapy.

#### *1.2. Advantages and Disadvantages of PDT*

*1.2. Advantages and Disadvantages of PDT*  Several advantages of PDT make it a promising treatment against malignancies. Selectivity of the PS towards cancer cells makes it applicable for curative treatment, early-stage tumors, and reducing the size of inoperable tumors. Additionally, as a local low-invasive procedure, PDT has minimal systemic side effects compared with standard chemotherapy or radiotherapy. PDT can also lead to the eradication of the tumor vascu-Several advantages of PDT make it a promising treatment against malignancies. Selectivity of the PS towards cancer cells makes it applicable for curative treatment, early-stage tumors, and reducing the size of inoperable tumors. Additionally, as a local low-invasive procedure, PDT has minimal systemic side effects compared with standard chemotherapy or radiotherapy. PDT can also lead to the eradication of the tumor vasculature [5], which will be described in Section 1.5.

lature [5], which will be described in Section 1.5. Another point in its favor is that PDT may trigger various immune responses to cancerous cells [7]. Primarily, if the irradiated cells undergo necrosis, an acute inflammatory response mediates the removal of the dead tissue [8]. Then, dendritic cells (DCs) absorb the cancer-related antigens through phagocytosis and present it to the im-Another point in its favor is that PDT may trigger various immune responses to cancerous cells [7]. Primarily, if the irradiated cells undergo necrosis, an acute inflammatory response mediates the removal of the dead tissue [8]. Then, dendritic cells (DCs) absorb the cancer-related antigens through phagocytosis and present it to the immune-system cells, thus triggering the systemic inhibitory response to cancer cells [9].

mune-system cells, thus triggering the systemic inhibitory response to cancer cells [9]. The penetration of light at an appropriate wavelength to activate the FDA-approved PSs (Porfimer sodium, Temoporfin, Verteporfin [10]) can be suppressed by the tissue, thus putting deep-seated tumors out of reach. To overcome this problem, PSs, that absorb light from the NIR-I could be applied. Another obstacle is the low oxygenation of the tumor microenvironment. Namely, when cancer cells are in hypoxia [11], PDT may be rendered ineffective due to the lack of substrate for ROS generation. The main advantages and disadvantages of PDT are shown in Figure 2, whereas a broad reference to The penetration of light at an appropriate wavelength to activate the FDA-approved PSs (Porfimer sodium, Temoporfin, Verteporfin [10]) can be suppressed by the tissue, thus putting deep-seated tumors out of reach. To overcome this problem, PSs, that absorb light from the NIR-I could be applied. Another obstacle is the low oxygenation of the tumor microenvironment. Namely, when cancer cells are in hypoxia [11], PDT may be rendered ineffective due to the lack of substrate for ROS generation. The main advantages and disadvantages of PDT are shown in Figure 2, whereas a broad reference to the efficacy and application of the PDT is reviewed by Agostinis et al. [12].

the efficacy and application of the PDT is reviewed by Agostinis et al. [12].

**Figure 2.** Summary of the main advantages and disadvantages of photodynamic therapy. **Figure 2.** Summary of the main advantages and disadvantages of photodynamic therapy.

## *1.3. Desired Characteristics of the Photosensitizer*

*1.3. Desired Characteristics of the Photosensitizer*  A PS should be characterized by low dark toxicity and selective accumulation in the tumor cells. The compound should be highly susceptible to irradiation and generate as many ROS as possible in the tumor environment [13]. Also, an ideal PS should localize outside the nucleus to avoid the pro-cancerous mutations in the DNA [1]. Preferably, an ROS should be generated in the mitochondria or lysosomes, but an especially effective targeting combination involves both of them [14]. Photodamage to the organelles would result in a controlled cell death, like apoptosis or paraptosis [15], rather than a mutagenic process in the nuclei. Controlled cell death results in the lack of tissue necrosis, which, despite its assets, risks an uncontrolled inflammation response [16]. Moreover, the dye should be characterized by good photostability to prevent photobleaching [13], which is a process of decomposition and loss of fluorescence and occurs when the dye's polymethine chains are oxidized by singlet oxygen species [17]. Interestingly, studies by N.S. James [18] showed no quantitative relation between the photobleaching of the PS and effectiveness of the therapy. The phenomena might be explained by the fact that, aside from the photosensitizer itself, the whole tumor microenvironment undergoes irradia-A PS should be characterized by low dark toxicity and selective accumulation in the tumor cells. The compound should be highly susceptible to irradiation and generate as many ROS as possible in the tumor environment [13]. Also, an ideal PS should localize outside the nucleus to avoid the pro-cancerous mutations in the DNA [1]. Preferably, an ROS should be generated in the mitochondria or lysosomes, but an especially effective targeting combination involves both of them [14]. Photodamage to the organelles would result in a controlled cell death, like apoptosis or paraptosis [15], rather than a mutagenic process in the nuclei. Controlled cell death results in the lack of tissue necrosis, which, despite its assets, risks an uncontrolled inflammation response [16]. Moreover, the dye should be characterized by good photostability to prevent photobleaching [13], which is a process of decomposition and loss of fluorescence and occurs when the dye's polymethine chains are oxidized by singlet oxygen species [17]. Interestingly, studies by N.S. James [18] showed no quantitative relation between the photobleaching of the PS and effectiveness of the therapy. The phenomena might be explained by the fact that, aside from the photosensitizer itself, the whole tumor microenvironment undergoes irradiation, leading to the generation of ROS from different sources [19].

#### tion, leading to the generation of ROS from different sources [19]. *1.4. Cellular Effects of PDT*

*1.4. Cellular Effects of PDT*  The localization of the PSs is crucial when considering the efficacy of the PDT. There are agents that target mitochondria, lysosomes, the endoplasmic reticulum, Golgi apparatus, plasma membranes or combinations of these sites and condition various cytotoxic mechanisms. A target location in lysosomes and mitochondria was noticed to be associated with higher PDT efficacy [14]. Interestingly, Kessel et al. demonstrated that autophagy can offer cytoprotection after mitochondrial photodamage. This process can be avoided by targeting the lysosomes, which potentiates apoptosis [14], whereas ER photodamage primarily evokes paraptosis [15,20]. Castano et al. summarized the effects of the photosensitizer: organelle interactions and proved photosensitizers induced cytotoxicity in different ways by targeting the specific cellular compartment [21]. Our study revealed that the cytotoxic and genotoxic effects varied depending on the plasmalemmal or intracellular localization of the PSs [22]. Mainly, irradiation of a culture of melanoma cells right after the administration of curcumin as a drug yielded the highest cytotoxicity, The localization of the PSs is crucial when considering the efficacy of the PDT. There are agents that target mitochondria, lysosomes, the endoplasmic reticulum, Golgi apparatus, plasma membranes or combinations of these sites and condition various cytotoxic mechanisms. A target location in lysosomes and mitochondria was noticed to be associated with higher PDT efficacy [14]. Interestingly, Kessel et al. demonstrated that autophagy can offer cytoprotection after mitochondrial photodamage. This process can be avoided by targeting the lysosomes, which potentiates apoptosis [14], whereas ER photodamage primarily evokes paraptosis [15,20]. Castano et al. summarized the effects of the photosensitizer: organelle interactions and proved photosensitizers induced cytotoxicity in different ways by targeting the specific cellular compartment [21]. Our study revealed that the cytotoxic and genotoxic effects varied depending on the plasmalemmal or intracellular localization of the PSs [22]. Mainly, irradiation of a culture of melanoma cells right after the administration of curcumin as a drug yielded the highest cytotoxicity, yet with the increase in incubation time toxicity to melanoma cells decreased as the drug moved to the intracellular membranous compartments. The mechanism of melanoma cell death, apoptosis, was proved to be mediated by the caspase-12. Surprisingly, even though cytotoxic tendencies

were similar among cancerous and non-cancerous cells, the genotoxicity in the culture of normal human fibroblasts was significantly lower than in melanoma cells. Moreover, the cytotoxic effect of cyanine dyes can differ depending on the resistance of a cell culture to certain chemotherapeutics. Kulbacka et al. showed that the doxorubicin-resistant cell line of human breast adenocarcinoma presented a weaker dye distribution than wild-type cell lines, and after irradiation the therapy was significantly more effective among the resistant cells, suggesting that PDT using such dyes may offer an alternative treatment for multidrug-resistant tumors [23].

Characterization of new photosensitizing agents often involves assessing sites of subcellular localization. Cyanine dyes were also studied in case of cellular localization-related death induction. First, Delaey et al. showed that, depending on the partition coefficient, different cyanine dye subgroups concentrate more (~1.5) or less intracellularly [24]. The study proved that hydrophobic cyanine dyes, like indocyanines, localize less intracellularly. Murakami et al. proved that bichromophoric cyanine dyes localize preferentially in mitochondria. In the study, high phototoxicity to melanoma was proven to be the result of the interaction between cyanine and mitochondria [25]. Other studies also proved the potency of cyanine dyes in targeting mitochondria [26].

## *1.5. Vascular Effects of PDT*

A specific and important trigger for cancer cell death mediated by photodynamic therapy is the anti-vascular effect. It is known that the application of ICG in PDT reduces perfusion by photocoagulating blood vessels [27]. This indirect pathway of cancer eradication additionally enhances the efficacy of the therapy. Shafirstein et al. observed how the vasculature surrounding the tumor tissue was impaired after applying ICG in PDT of a murine mammary carcinoma. Both phototoxic and significant photothermal effects of the therapy resulted in necrosis of the endothelial cells and the deposition of fibrin within the blood vessels [28]. Therefore, an anti-vascular effect is not the result of blood-vessel destruction alone. As the endothelial cells are damaged, they release clotting factors that lead to the formation of thrombi, constriction and occlusion of vessels [29]. In case of large tumors, treatment with PDT might lead to hemorrhaging, and additional side effects of the therapy. These processes deprive tumor cells of necessary nutrients and oxygen. Nonetheless, the resulting hypoxia may also have a negative impact on the therapy. Photosensitizers currently in use require oxygen to create free radicals [30]. Insufficient oxygenation, characteristic of the tumor microenvironment (TME) [31] and intensified by oxygen-dependent PDT [30], directly reduces its efficacy. Moreover, hypoxia stimulates the release of angiogenic growth factors that lead to neovascularization. To inhibit such a process, angiogenesis inhibitors could be combined with PDT to minimize the risk of tumor recurrence [32].

#### *1.6. Interstitial PDT*

Intratumor light delivery by one or more laser fibers inserted into the target tissue (typically tumor and margins (interstitial PDT, I-PDT)) is applied to activate PSs in deeply seated tumors or tumors more than 10 mm in thickness. If a tumor is too large for the light to be delivered into its entire volume, interstitial PDT (iPDT) could destroy it to the margins of healthy tissue [33]. Moreover, the application of fibers under the guidance of ultrasound or MRI could minimize light scattering by healthy tissue, thus allowing the iPDT to reach the target location and facilitate the eradication of a deep-seated tumor [34] as in pancreatic cancer [35]. According to Chang et al. disulfonated aluminum phthalocyanine (AlS2Pc) application in PDT using fibers placed under ultrasound guidance proved to be effective against prostate cancer by creating lesions up to 12 mm wide. Moreover, the basic connective tissue architecture of the organ and the prostate capsule healed properly, proving that PDT is a less-invasive treatment in comparison to radical surgery [36]. The selective delivery of light could simplify therapies in areas where any excess tissue loss could lead to serious disabilities [37]. An example for such a location is the head and neck,

where iPDT using dyes like Foscan [38], or porfimer sodium [37] has already proven to be effective and cause few side effects. the head and neck, where iPDT using dyes like Foscan [38], or porfimer sodium [37] has already proven to be effective and cause few side effects.

Moreover, the basic connective tissue architecture of the organ and the prostate capsule healed properly, proving that PDT is a less-invasive treatment in comparison to radical surgery [36]. The selective delivery of light could simplify therapies in areas where any excess tissue loss could lead to serious disabilities [37]. An example for such a location is

#### **2. Cyanine Derived Dyes 2. Cyanine Derived Dyes**

Cyanine dyes consist of two-terminal heterocyclic units linked by a polymethine bridge core structure (Figure 3) [39]. Over the last few years, the cyanine dyes and their derivatives were widely analyzed for the cytotoxic activity against wide spectrum of tumors. It has been reported they meet most of the requirements for being used in PDT against deep-seated cancers. Each of the main subdivisions of the dyes was described in the following paragraphs. Cyanine dyes consist of two-terminal heterocyclic units linked by a polymethine bridge core structure (Figure 3) [39]. Over the last few years, the cyanine dyes and their derivatives were widely analyzed for the cytotoxic activity against wide spectrum of tumors. It has been reported they meet most of the requirements for being used in PDT against deep-seated cancers. Each of the main subdivisions of the dyes was described in the following paragraphs.

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 5 of 18

**Figure 3.** Cyanine-derived dye division and backbone structures. R1, R2 and R3 represent the organic substituent groups. **Figure 3.** Cyanine-derived dye division and backbone structures. R1, R2 and R3 represent the organic substituent groups.

#### *2.1. Indocyanine Green 2.1. Indocyanine Green*

One of the most promising cyanine dye is a carbocyanine, widely known as indocyanine green (ICG), the only cyanine dye approved by the Food and Drug Administration (FDA). Due to its absorbance maximum in NIR, prominent fluorescent properties and low dark toxicity, the dye is applied to diagnose liver, cardiovascular and sentinel lymph node pathologies [40,41]. ICG has a high affinity for plasma proteins and is cleared from the body through the biliary pathway, which allows for its use in diagnoses of liver and bile duct functions [42]. Several studies have proven ICG's toxicity towards various cancer cells. For instance, ICG-mediated PDT induced a 38% decrease in choroidal melanoma viability in six months [43]. Here, it is mostly retained in the Golgi apparatus, endoplasmic reticulum, mitochondria and lysosomes [44]. Additionally, ICG proved to be an efficient photothermal agent, suppressing tumor growth under repeated NIR-light irradiation. The process not only includes the induction of oxidative stress but One of the most promising cyanine dye is a carbocyanine, widely known as indocyanine green (ICG), the only cyanine dye approved by the Food and Drug Administration (FDA). Due to its absorbance maximum in NIR, prominent fluorescent properties and low dark toxicity, the dye is applied to diagnose liver, cardiovascular and sentinel lymph node pathologies [40,41]. ICG has a high affinity for plasma proteins and is cleared from the body through the biliary pathway, which allows for its use in diagnoses of liver and bile duct functions [42]. Several studies have proven ICG's toxicity towards various cancer cells. For instance, ICG-mediated PDT induced a 38% decrease in choroidal melanoma viability in six months [43]. Here, it is mostly retained in the Golgi apparatus, endoplasmic reticulum, mitochondria and lysosomes [44]. Additionally, ICG proved to be an efficient photothermal agent, suppressing tumor growth under repeated NIR-light irradiation. The process not only includes the induction of oxidative stress but also the production of heat by converting approximately 88% of the absorbed light into radiation [45].

also the production of heat by converting approximately 88% of the absorbed light into radiation [45]. However, the dye also has several disadvantages. These include low photostability, a high level of photobleaching and no specificity towards cancer cells [46]. Of note, the energy yield from the photodynamic reaction is lower than in the PSs characterized by However, the dye also has several disadvantages. These include low photostability, a high level of photobleaching and no specificity towards cancer cells [46]. Of note, the energy yield from the photodynamic reaction is lower than in the PSs characterized by lower absorbance maxima [42,47].

#### lower absorbance maxima [42,47]. *2.2. ICG Lactosomes*

Studies show that combining high-mass lactate-derived polymers with ICG in ICG– lactosomes (a core-shell-type polymeric micelle or "nanocarrier") results in greater toxicity towards cancer cells in PDT than by using ICG alone. This tendency was observed in vivo on BALB/c nude mice transfected with human hepatocellular carcinoma (HCC) cell line

HuH-7 [48] and gallbladder cancer NOZ cell lines [49]. In the latest case, ICG–lactosomes were effective not only as photosensitizers but also as fluorescent diagnostic agents because of their selective accumulation in cancer tissue, strengthened cytotoxicity and relatively high fluorescence.

#### *2.3. Heptamethine Cyanine Dye*

Heptamethine cyanine dye, Cy7 is an ICG derivative that has maximum absorbance in the near-infrared range (NIR) and shows high selectivity for cancer cells. On a cellular level, the dye is transported through organic anion-transporting polypeptides (OATPs). The expression of OATPs is regulated by hypoxia-inducible factor 1α (HIF1α), which may possibly explain its selectivity for tumor cells [50]. Moreover, Usama et al. proved, that Cy7 cyanine dyes form covalent albumin adducts that can generate long-lasting intratumor fluorescence due to their enhanced permeability and retention in the tumor tissue [51]. Heptamethine dyes aggregate primarily in the mitochondria and lysosomes. Such localization favors inducing apoptosis over necrosis, thereby minimizing the risk of uncontrolled immunological reaction provoked by necrosis of the affected tissue [16].

Cy7 is a promising photosensitizer, diagnostic agent and nanocarrier transporter. Under near-IR light this cyanine derivative might transport anti-cancer drugs exclusively to the tumor [52]. Moreover, Jiang et al. observed that Cy7 conjugated with Gemcitabine displayed relatively high residency time in tumor tissue [53]. Namely, Cy7 remains in a tumor 5–20 days in comparison to ICG, which is removed from the body within 24 h [54].

The disadvantages of heptamethine cyanine derivatives include unfavorable hydrophobicity, which leads to its aggregation in body fluids, extensive photobleaching and insufficiency in ROS production similar to ICG.

#### *2.4. Halogenated Cyanine Dyes*

Due to the heavy atom effect, the production of free radicals can be significantly increased by halogenation of the cyanine dyes [55]. Atchison et al. established that iodinated IR-783 derivative presents high efficacy towards BxPC-3 and MIA PaCa-2 pancreatic cancer cell lines [35]. Halogenating the dye resulted in significant suppression of tumor growth. Moreover, the viability of MIA PaCa-2 cells after PDT was less than 10% and BxPc-3 was lower than 40%. Interestingly, after irradiation of the same cell lines with ICG, no photodynamic effect was observed. The potential of the derivative was proven in studies where PDT was more effective than a 5-FU treatment of pancreatic cancer. The compounds may also inhibit the growth of cancer. Namely, in a murine model transfected with a human xenograft of BxPC-3 Luc, the untreated pancreatic tumor grew to about 500% of the initial tumor size, whereas the one irradiated with iodinated cyanine IR-783 increased its volume by only about 39% [35]. The study presents PDT as a prospective neoadjuvant and palliative therapy against highly aggressive tumors.

Cao et al. modified the ICG derivative Cy7 with heavy atom iodine to form the novel NIR dye CyI [56]. They investigated the application of PDT simultaneously with photothermal therapy in the study of HepG2 cancer cells [56]. The iodinated dye enhanced the production of ROS production. In this case, the addition of photothermal therapy (PTT) had a synergistic effect on the treatment and strengthened the suppression of tumor growth.

However, the iodinated ICG derivative CyI proved to have poor solubility and tumortargeting abilities in clinical application [56]. To overcome the problems the dye was modified by PEGylation and the addition of hyaluronic acid, which increased solubility in water [8].

Another iodidinated cyanine dye, IR-780, accumulates preferentially in tumor tissue after an intravenous injection [57]. The derivative displays a significant in vivo ability to target tumors. The process is dependent on the cancer's energetic metabolism, plasma membrane potential and expression of OATPs [58]. Wang et al. showed no significant difference between laser-irradiated and non-irradiated cells treated with IR-780, which is attributed to its inherent toxicity in the dark [59].

By modifying the heptamethine cyanine dye, Noh et al. developed the mitochondriatargeting photodynamic therapeutic agent MitDt-1 [60]. Bromination of the indoline group of the heptamethine dye proved to increase the production of ROS considerably. MitDt-1 accumulates primarily in mitochondria, thus inducing apoptosis. Also, the derivative containing triphenylphosphonium (TPP) and quaternary ammonium enhanced the dye's solubility and selectivity for the mitochondria of the cancer cells. Moreover, the high toxicity of MitDt-1 toward cancer cells was proven in studies on MCF-7 breast cancer cells in vitro and on NCI-H460 lung cancer both in vitro and in vivo [60].

The analog of IR-780 named IR-808 (MHI-148), was designed, synthesized and screened by Tan et al. [61]. Its photostability and photocytotoxicity was evaluated on the human cervical cancer cell line HeLa and Lewis lung carcinoma (LLC) in mouse xenografts. IR-808 displayed selective aggregation in tumor cells, distinct optical properties and high photostability in serum. Both IR-808 and IR-783 accumulate primarily in mitochondria and lysosomes [62]. Furthermore, IR-808 showed a significant dose-dependent phototoxic effect and distinct suppression of tumor growth after irradiation [62]. In the histopathological examination of the experimental mice, no aggregation in systemic circulation and interstitial fluids were detected. Further investigations into IR-808 proved a high selectivity for cancer and thus potential for imaging gastric [63], prostate [62] and kidney cancer [64].

### *2.5. Incorporation of Organic Groups*

Further modifications of IR-808 aimed to increase its water solubility, which included replacing of one of its side chains with (CH2)4SO<sup>3</sup> in DZ-1 derivative [65]. To prove the potential of the new derivative, ICG and the newly synthesized fluorophore were compared. ICG itself has high selectivity for HCC with a high tumor to-background ratio (255:1) [66], making it an important tool for identifying HCC lesions during surgery [67]. However, the fluorescence displayed by DZ-1 was significant and lasted longer in contrast to ICG, which allowed for the identification of smaller tumor regions. Moreover, the uptake of the dye in vivo was assessed on HCC Hep3B-Luc cell line xenografts and male New Zealand rabbits. In this case, DZ-1 displayed no accumulation in liver or lung tissue, proving that DZ-1 has a higher specificity for cancer cells than ICG does.

A study by Yang et al., presented the modified heptamethine dye by the addition of 4-amino-2,2,6,6,-tetramethylpiperidine-N-oxyl [68]. The dye was highly effective in ROS production. Moreover, this derivative, first introduced by Jiao et al. [69], has a long triplet-state lifetime and the incorporation of sulfonic acid improves its water-solubility. Additionally, the dye prompted a significant apoptosis of the HepG2 cells after NIR irradiation and showed low dark toxicity.

#### *2.6. Incorporation of a Heavy Metal Atom*

Incorporation of a heavy metal into the structure of the dye enhances crossing due to the heavy metal effect [70]. Nevertheless, the introduction of the heavy atom in a cyanine dye poses a risk of enhancing dark toxicity [68] and accumulating in healthy tissues [71]. Conjugation of ICG with Au-based nanomaterials sufficiently enhanced the absorption, emission and stability of the fluorophore [72]. It simultaneously induced PTT and enhanced ROS production in PDT, efficiently killing A549 malignant cells [72]. Similarly, the conjugate of ICG with gold–gold sulfide, where gold also acted as an agent for PTT, presented greater stability and improved cytotoxicity towards a HeLa cell line [73]. In addition, silver was incorporated into ICG nanoparticles, resulting in a synergistic interaction between the photothermal effect and PDT. In the study performed by Tan et al., PEGylated silver nanoparticles with a polyaniline shell acted as nanocarriers for ICG and efficiently induced hyperthermia in HeLa cancer cells. In this case, standalone ICG was responsible for fluorescence and phototoxicity [74].

The platinum (II) complex of heptamethine cyanine, IR797-Platin, proved to have extremely high cytotoxicity under NIR-light conditions towards C-33 A (cervical cancer) and MCF-7 breast cancer cell lines [75]. The drug's cytotoxicity was shown by the photosensitivity of IR797 and the inhibition of DNA transcription and replication by platinum [76].

Zhao et al. synthesized the CYBF2 agent by incorporating: boron difluoride (BF2) into the core structure of cyanine [17]. BF2 reduced the dye's electron density, resulting in the enhanced photostability. The drug accumulated in mitochondria, induced apoptosis and was efficiently absorbed by MCF-7 cells. Furthermore, the compound presented low dark toxicity and a high level of ROS burst induction.

#### *2.7. pH-Sensitive Cyanine Dyes*

Heptamethine cyanine dyes were further modified to be pH sensitive, which eventually increased their tumor specificity [39]. Apart from a relatively high cancer cell selectivity the dye induced fluorescence only after it was placed in an acidic environment. Since extracellular cancer fluid has a high concentration of lactic acid, such a modification made it possible to visualize cancer cells and destroy them more accurately. One of the studied dyes, IR2, incorporated a dimethylamine group that worked as an intramolecular charge transporter. Selectivity was proven by a higher uptake in cancer (HepG2 and HeLa) cell lines than normal cells. Also, the apoptosis following irradiation with the drug was much higher among cancer (80%) than normal (HL-7702) cells (9.4%). Interestingly, cell death was mostly the effect of hyperthermia, not photodynamic reaction.

Siriwibool et al. synthesized a pH switchable dye I2-IR783-Mpip [77] composed of IR783 and N-methylpiperazine. In acidic conditions, the color changes from blue to red, but only the red dye can absorb LED light and displays high toxicity towards HepG2 cells. Cancer cell viability in a neutral environment was about 30% and in an acidic environment decreased to 10%. In this case, death was primarily the result of a free radical production, not the photothermal effect.

Another pH switchable agent was presented by Meng et al. who conjugated 5' carboxyrhodamines (Rho) and heptamethine cyanine IR765 (Cy) [78]. The newly synthesized conjugate (RhoSSCy) had enhanced fluorescence in a decreased pH value and displayed high stability in pH ranging from 5 to 9. The dye accumulated specifically in tumor cells, presenting a significant fluorescence. In the xenograft studies, the phototoxicity towards cancer was high, considerably increasing the survival rate of mice transfected with MCF-7 cells.

#### *2.8. Near-Infrared II Dyes*

Further studies of cyanine dyes were aimed at a red-shifted long-wavelength absorption maximum. Unlike the most dyes currently used in PDT, the PSs that absorb energy into the NIR I range show enhanced tissue penetration. Dyes activated with NIR-II light (1000–1700 nm) were analyzed to lessen the scattering of the light by the tissue, enhance the image contrast and improve the deep-seated tumor detection. The NIR-II dyes, which emit light at a wavelength longer than 1000 nm, were first reported by Antaris et al. [79], followed by the discovery of cyanine dye far-red emissions by Zhu et al. [80]. ICG and IRDye800 were shown to possess emission across NIR-I and NIR-II, marking them as promising and highly specific fluorophores for surgical procedures [42]. The photophysical mechanism of NIR-II emission relies on twisted intramolecular charge transfer (TICT). Further, Starolski et al. observed that the contrast-to-noise ratio (CNR) of the ICG window is twice as high in NIR-II than in NIR-I [81]. Ge et al. studied the efficacy of NIR II-emitting polymer nanoparticles: AuNR vesicles (Ru-complex and a cyanine dye (IR 1061)) [82]. Irradiation by NIR II light induced the release of the Ru complex and generated cytotoxic <sup>1</sup>O2. The therapy efficiently killed the MCF-7 breast cancer cells both in vitro and in vivo.

#### **3. Pentamethine Cyanine Dyes**

Pentamethine cyanine fluorophores were designed to have tissue-specificity and localize primarily in the adrenal and pituitary glands, pancreas and lymph nodes. Their synthesis aimed to give a promising contrast agent for intraoperative imaging of glands [83]. Newly obtained symmetrical penthametine cyanine dyes, based on a benzoindoleninic ring were tried on a human fibrosarcoma cell line (HT-1080) by Ciubini et al. [84]. The dyes remained active at relatively low concentrations (10 nM), and the drugs were rapidly internalized by cancer cells, which led to high ROS burst. Surprisingly, brominating the benzoindolenine did not produce any increase in free radicals.

2-quinolinium pentamethine carbocyanines were analyzed by Ahoulu et al. on an ES2 ovarian carcinoma cell line [85]. The dye, which was brominated at the mesocarbon remained highly phototoxic and reduced cell line viability from 100 ± 10% to 14 ± 1% after irradiation at a 694 nm wavelength. The compound was characterized by great stability, little dark toxicity and displayed DNA-cleavage. Dye localized mainly in the cytosol and perinuclear regions, whereupon it generated hydroxyl radicals after irradiation.

#### **4. Carbocyanines against Drug-Resistant Cancer Cells**

PDT may act as a prominent treatment for tumors with multiple drug resistance (MDR). Kulbacka et al. investigated four different cyanine dyes: two carbocyanines: HM-118, KF-570, merocyanine FBF-749 and pyridine-thiazolidine ER-139 combined with PDT against malignant breast adenocarcinoma cell lines. One of the latter was resistant to doxorubicin (MCF/DX), but the other wasn't (MCF-WT) [86]. Carbocyanines had the greatest phototoxicity. After irradiation with HM-118, 100% of apoptotic cells were detected in both cell lines. When using KF-570, 98% of the wild-type cells and 95% of the doxorubicinresistant cells remained apoptotic. Other dyes had a much lower apoptotic effect, and their phototoxicity was insufficient. After irradiation with HM-118, overexpression of AIF, a protein involved in caspase-independent cell death, was detected in MCF-7/WT cells [87]. Conversely, in doxorubicin-resistant cells, the protein was absent.

#### **5. Squaraine Dyes**

Squaraine dyes possess several unique properties, such as significant fluorescence, distinct stability and absorbance wavelength maxima of 600–800 nm. The disadvantages of the dyes involve low solubility and low ROS production following PDT. Fernandes et al. incorporated the sulfur atom into the indolenine-based squaraine core and assessed the potency of the drug in combination with PDT on HepG2 and Caco-2 cell lines [88]. ROS production was enhanced after dithiosquaraine dyes aided PDT. Conversely, monothiosquaraine dyes turned out to be ineffective. Despite high phytotoxicity of dithiosquaraine dyes, the compounds degraded easily and aggregated in aqueous media.

#### *5.1. Dicyanomethylene Squaraine Dyes*

Martins et al. proved the significant phototoxicity of dicyanomethylene squaraine cyanine dyes against Caco-2 and HepG2 cancer cells in vitro [89]. Despite low singlet oxygen production and moderate light-stability, the chemicals still remained effective. However, further modification of their structure by Wei et al. resulted in dicyanomethylenesubstituted benzothiazole squaraines [90]. Among four synthesized dyes, a squaraine derivative with two methyl butyrate sidechains named CSBE showed an excellent phototoxic effect in vitro against seven different cancer cell lines (PC-3, MCF-7, HCT-8, A549, A549T, K562, and LoVo). Negligible dark toxicity was also advantageous. CSBE effectiveness was assessed in xenograft studies, and irradiation following the injection of the dye induced tumor growth suppression. During a histological examination of the liver and kidney, no harm to healthy cells was detected.

Soumya et al. evaluated the potency of symmetrical diiodinated benzothiazolium squaraine (SQDI) dyes in vitro on Ehrlich's Ascites Carcinoma (EAC) cells [91]. The maximum absorbance of the dye was beyond the NIR of 535 nm. A low concentration of the dye (0.2 mg/mL) induced 100% cytotoxicity after irradiation. Moreover, the dye had no dark toxicity. An in vivo study on Swiss albino mice included the measurement of serum biochemical parameters such as SGPT, SGOT, LDH, CK and ALP after the administration of the dye through the intraperitoneal cavity. None of the parameters increased, meaning that the dye did not extend any toxicity to healthy organs.

#### *5.2. Halogenation*

Halogenated squaraine dyes were analyzed by Serpe et al. Their efficacy in PDT was tested in vitro on a human fibrosarcoma (HT-1080) tumor cell line [92]. Both brominated and iodinated squaraine dyes proved to induce a significant ROS generation in the first few minutes after irradiation. Despite high initial release of cytochrome c, a drastic reduction was observed 3 h after irradiation. Therefore, in this case necrosis was the main cell death type.

#### *5.3. Aminosquaraine Dyes*

A study by Lima et al. evaluated the potency of indolenine-based aminosquaraine cyanine dyes as photosensitizers on several cell lines: Caco-2, MCF-7, PC-3. Non-tumor cell lines (NHDF and N27) were controls [93]. Study revealed that, the zwitterionic dye showed high selectivity for the PC-3 cell line in comparison to the normal human cell line. Almost all aminosquaraine dyes, were specifically cytotoxic towards cancer cells and aggregated in mitochondria.

Magalhães et al. have been evaluated the efficacy of several modified zwitterionic dyes on different cancer cell lines (MCF-7, NCI-H460, HeLa, HepG2) and non-tumor porcine liver primary cell culture (PLP2) in vitro [94]. Modifications to the zwitterionic dyes were designed to increase cellular uptake by enhancing their cationic character, increase the red-shift of the dye's absorption maximum and boost hydrophilicity. All the dyes displayed high phototoxicity towards cancer cell lines, particularly HeLa and MCF-7 cell lines, which showed the highest susceptibility to aminosquaraines. Nevertheless, all dyes showed cytotoxicity against PLP2 cells and a relative inhibition of growth. The use of the aminosquaraine analogues of benzoselenazole was also effective in cancer therapy [95], namely, the inclusion of a heavy metal, selenium, enhanced free radical production while decreasing the dye's fluorescence emission [96]. The absorbance maximum of the modified dyes was in the 665–685 nm range, and its stability improved significantly. The derivatives were more phototoxic than the benzothiazole analogues, but their toxicity in the absence of light was generally higher as well.

To increase the redshift of unsymmetrical squaraine dyes, Lima et al. incorporated quinoline units into the core structure. Some of the new dyes displayed a wavelength of a maximum absorption at in the far-red spectrum (733 nm) [97]. Despite their limitations (i.e., aggregation in aqueous solution and low ROS synthesis) the new derivatives decreased their dark toxicity and presented a higher cellular uptake because of their cationic character. Although production of singlet oxygen was relatively weak, the dyes showed substantial phototherapeutic activity against breast cancer cell lines (MCF-7 and BT-474). These results were comparable to previously studied indolenine-based aminosquaraine dyes. Further modifications to them that would strengthen their singlet oxygen generation and may lead to their successful application in PDT.

#### **6. Merocyanines**

Merocyanines raised hope for low-invasive lymphoma [98], leukemia [99] and neuroblastoma [100] treatments as they exhibited high specificity for cancer cells. The compounds are currently undergoing preclinical studies as a treatment for leukemia [99]. The exceptional permeability of the MC540 dye to leukemic leukocytes and immature hemopoietic precursors led to its extensive analysis.

The drawbacks of the group involve maximum absorbance of light outside of the NIR spectrum (556 nm) and preferential peroxidation of phospholipids in the membrane [101]. The latter leads to the induction of necrosis and constraints on the utility of the drugs in vivo. Additionally, their use in PDT against melanoma on the Cloudman S91 cell line turned out to be less effective than the currently applied porphyrin dyes [102]. Nonetheless, the research on merocyanines as a potential hematological cancer treatment hasn't stopped. A rhodamine complex of merocyanine underwent an in vitro study on K562 leukemia cells and revealed a decrease of cancer cell viability [103]. Apart from cancer treatment, merocyanines are also evaluated as an antimicrobial therapy on *Staphylococcus aureus* [104].

#### *Immunoregulatory Agent*

It has been shown that merocyanines exhibit immunoregulatory properties. The compound group can regulate an immune response by inhibiting T-lymphocyte proliferation and B-cell differentiation. Also, T-cell helper activity can be stimulated [105]. Therefore, merocyanines reveal the potency against leukemia and lymphoma. The drugs may also may find application in graft-versus-host disease prophylaxis and treatment of several autoimmune diseases. Traul et al. evaluated the application of PDT with MHC540 in reducing GVHD in murine models of allogeneic hematopoietic stem cell transplantation [106]. Prior studies reported, that the sensitivity of cells to merocyanines is determined by the dye's binding to the targeted cells [107]. The binding affinity is reduced in mature lymphocytes and elevated in hematopoietic stem cells. Irradiated lymphocytes displayed no proliferative response after treatment with ConA (concanavalin A), LPS (lipopolysaccharide), PHA (phytohemagglutinin) and IL-2 (interleukin-2). Also the survival of MHC540 treated cells increased by 50–80% [106].

#### **7. Phthalocyanines**

Phthalocyanines are successful photosensitizers in the therapy of skin malignancies like basal cell carcinoma and diseases like psoriasis [108]. The compounds belong to the group of second-generation photosensitizers present some similarities to porphyrins. In contrast to the latter, they have absorption maxima in the range of 670–780 nm [109].

#### *7.1. Incorporation of a Metal Atom*

The substitution of the phthalocyanine central atom of with zinc (II), aluminum (III), gallium (III) or silicon results in increased cytotoxicity in biological studies of the drugs [110]. The advantages include high stability, fluorescence [111] and low dark toxicity [112]. Their weakness, however, is hydrophobicity, which leads to aggregation in aqueous media [109]. Zinc (II) phthalocyanine Pc13 induced apoptosis and necrosis triggered by free radical production in B16Fo melanoma cells [113]. The level of apoptosis regulators (Bcl-2, Bcl-xL and Bid) increased after irradiation with the drugs. In addition, permeability of the mitochondria towards the inner-derived ROS was detected. The necrotic pathway resulted from an increase in lactate dehydrogenase concentration in extracellular compartments [113].

To enhance water solubility of the zinc phthalocyanine, sulfonic [114], phosphoric [115] and carboxylic group [116] substituents were introduced. The exposure of cervical cancer cells (HeLa) to sulphated zinc (II) phthalocyanines and irradiation with 673 nm diode laser resulted in DNA fragmentation, membrane damage and effective cytotoxicity. This led to a 25% decrease in cell viability [117]. Aniogo et al. combined sulfonated zinc (II) phthalocyanine with doxorubicin and observed a synergistic cytotoxic effect on MCF-7 cell lines [118]. To improve cytotoxicity and penetration of phthalocyanine derivatives into cancer cells, the cyanines might be conjugated with chemotherapeutical drugs. For instance, Al (III) phthalocyanine chloride tetrasulfonic acid (AlPcS4) with different chemotherapeutic agents was studied on gastric cancer cells [119], and zinc phthalocyanine with doxorubicin acted against SK-MEL-3 melanoma cells [120].

#### *7.2. Nanoemulsions*

Because phthalocyanines are lipophilic, L.A. Muehlmann developed nanoemulsions that can transport aluminum-phthalocyanine chloride (AIPc) to cancer tissue. The approach prevented aggregation in aqueous media [121]. The nanoemulsions are mostly composed of castor oil, Cremophor ELP® and a monodisperse population of nanodroplets. Cell viability of mammary MCF-7 adenocarcinoma cells significantly decreased and the production of LDH excessively increased. Conversely, without such emulsions AIPc was ineffective. The complex dye aggregated mostly in the cytoplasm and outside the nucleus, thus inducing no damage to the DNA and preventing genome modifications. Subsequently, nanoemulsions of aluminum-phthalocyanine were studied in vivo in a PDT against 4T1 breast adenocarcinoma tumor [122]. The primary breast tumors were eradicated after application of PDT, and contrary to the untreated mice group, metastases to the lungs were not observed.

#### **8. Conclusions**

The absorbance maxima of cyanine dyes lie within the NIR-I spectrum and the light used to activate them penetrates deeper into the tissue. They exhibit significant fluorescent properties and thus are applied in photodynamic diagnostics. ICG is an FDA approved dye which has for decades been clinically used as a diagnostic agent. Heptamethine dye is exceptional for cancer cells selectivity. It is transported through organic anion-transporting polypeptides (OATPs) the production of which increases in cancer cells. Certain cyanine dyes (ICG and IRDye800) can emit light at an NIR-II wavelength of 1000–1700 nm. Such a characteristic could provide a better contrast-to-noise ratio (CNR) diagnostic image and higher specificity for tumors [81].

Cyanine dyes do have their weaknesses: poor water solubility and low ROS generation. Nonetheless, the ROS production can be increased by incorporating certain organic groups, heavy atoms, halogenation or metal atoms [55,56,60,72,73,92,96]. This review of the use of cyanines and their derivatives as potential photosensitizers indicates that they could be efficiently activated by light, causing the death of target cells [35,48,49,56,62,68,82,85,89,122]. Cyanine dyes provoke cell death primarily through apoptosis [2,17,69], which benefits the therapy because it prevents an excessive inflammatory response. To overcome the problem of poor water solubility the dyes can be further modified by, for example, incorporating organic [8,69] or other groups [114–116]. Specificity for cancer tissue can be improved by sensitizing cyanine dyes to pH and enhancing their phototoxicity in an acidic environment, which is characteristic for extracellular cancer fluid [39,78].

Moreover, merocyanines, are especially worth mentioning for their unique immunoregulatory properties, namely, the ability to interact with lymphocytes [105]. Currently, merocyanines are in preclinical studies focused on treating leukemia [99].

Current studies involving numerous cyanine dyes show that they may enrich the stock of the permanently used pool of agents in PDT. Considering the very high cost of introducing new agents, it is important to show persuasive evidence of their new and remarkable properties. Further studies to exploit the advantages of cyanine dyes for therapeutic possibilities in low-invasive cancer treatments include absorbance within the NIR, low dark toxicity and fluorescent properties, combined with the application of optical fibers.

**Funding:** The research was supported Statutory Subsidy Funds of the Department of Molecular and Cellular Biology no. SUB.D260.21.095 (PI: J.Saczko).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors are very grateful to Julita Kulbacka from Wroclaw Medical University for the scientific impact with this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Review* **Latest Innovations and Nanotechnologies with Curcumin as a Nature-Inspired Photosensitizer Applied in the Photodynamic Therapy of Cancer**

**Laura Marinela Ailioaie <sup>1</sup> , Constantin Ailioaie <sup>1</sup> and Gerhard Litscher 2,\***


**Abstract:** In the context of the high incidence of cancer worldwide, state-of-the-art photodynamic therapy (PDT) has entered as a usual protocol of attempting to eradicate cancer as a minimally invasive procedure, along with pharmacological resources and radiation therapy. The photosensitizer (PS) excited at certain wavelengths of the applied light source, in the presence of oxygen releases several free radicals and various oxidation products with high cytotoxic potential, which will lead to cell death in irradiated cancerous tissues. Current research focuses on the potential of natural products as a superior generation of photosensitizers, which through the latest nanotechnologies target tumors better, are less toxic to neighboring tissues, but at the same time, have improved light absorption for the more aggressive and widespread forms of cancer. Curcumin incorporated into nanotechnologies has a higher intracellular absorption, a higher targeting rate, increased toxicity to tumor cells, accelerates the activity of caspases and DNA cleavage, decreases the mitochondrial activity of cancer cells, decreases their viability and proliferation, decreases angiogenesis, and finally induces apoptosis. It reduces the size of the primary tumor, reverses multidrug resistance in chemotherapy and decreases resistance to radiation therapy in neoplasms. Current research has shown that the use of PDT and nanoformulations of curcumin has a modulating effect on ROS generation, so light or laser irradiation will lead to excessive ROS growth, while nanocurcumin will reduce the activation of ROS-producing enzymes or will determine the quick removal of ROS, seemingly opposite but synergistic phenomena by inducing neoplasm apoptosis, but at the same time, accelerating the repair of nearby tissue. The latest curcumin nanoformulations have a huge potential to optimize PDT, to overcome major side effects, resistance to chemotherapy, relapses and metastases. All the studies reviewed and presented revealed great potential for the applicability of nanoformulations of curcumin and PDT in cancer therapy.

**Keywords:** light; malignant tumors; nanomedicine; natural photosensitizer; photobiomodulation (PBM); photodynamic therapy (PDT); cancer; curcumin

## **1. Introduction**

Light as a treatment dates back to ancient times, but modern photodynamic therapy (PDT) has advanced following the accidental rediscovery in the early twentieth century of the light-mediated killing effect in the presence of molecular oxygen on acridine-incubated *Paramecium caudatum* [1].

Exactly in the same direction, concerning the use of light in medicine, the first Nobel Prize for outstanding applications of phototherapy was won in 1903 by Niels Finsen for smallpox and skin tuberculosis treatments [2,3].

**Citation:** Ailioaie, L.M.; Ailioaie, C.; Litscher, G. Latest Innovations and Nanotechnologies with Curcumin as a Nature-Inspired Photosensitizer Applied in the Photodynamic Therapy of Cancer. *Pharmaceutics* **2021**, *13*, 1562. https://doi.org/ 10.3390/pharmaceutics13101562

Academic Editor: Nejat Düzgüne¸s

Received: 29 August 2021 Accepted: 22 September 2021 Published: 26 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Herman von Tappeiner defined "photodynamic action" [4] and gave it the name known today as photodynamic therapy, a contemporary and non-invasive form of anticancer therapy, well studied in present also for infections, non-oncological disorders, and in some countries, as a standardized protocol, along with radiotherapy and chemotherapy in anti-cancer treatments [5,6]. known today as photodynamic therapy, a contemporary and non-invasive form of anticancer therapy, well studied in present also for infections, non-oncological disorders, and in some countries, as a standardized protocol, along with radiotherapy and chemotherapy in anti-cancer treatments [5,6]. The first goal of this review was to present and discuss the latest nanotechnologies

Exactly in the same direction, concerning the use of light in medicine, the first Nobel Prize for outstanding applications of phototherapy was won in 1903 by Niels Finsen for

Herman von Tappeiner defined "photodynamic action" [4] and gave it the name

The first goal of this review was to present and discuss the latest nanotechnologies in relation to curcumin as photosensitizer (PS) to optimize photodynamic therapy (PDT), to overcome major side effects, resistance to chemotherapy, relapses and metastases. in relation to curcumin as photosensitizer (PS) to optimize photodynamic therapy (PDT), to overcome major side effects, resistance to chemotherapy, relapses and metastases. The second objective was to reveal the state of the art of photodynamic therapy as a

The second objective was to reveal the state of the art of photodynamic therapy as a field of continuous effervescent research and medical applications in direct connection with nanoformulations of curcumin. field of continuous effervescent research and medical applications in direct connection with nanoformulations of curcumin. The third aim was to analyze the molecular and cellular mechanisms, targeted deliv-

The third aim was to analyze the molecular and cellular mechanisms, targeted delivery and localization of nanoparticles in the tumor, as well as the interconnected parts related to PDT, to increase the antiproliferative and apoptotic activity and minimize toxicity in nearby healthy cells. ery and localization of nanoparticles in the tumor, as well as the interconnected parts related to PDT, to increase the antiproliferative and apoptotic activity and minimize toxicity in nearby healthy cells. The fourth goal was to push forward the field of study of natural photosensitizers,

The fourth goal was to push forward the field of study of natural photosensitizers, especially curcumin- and PDT-related nanotechnologies, providing an up-to-date high-quality evidence base for researchers and scientists to accelerate and rethink new experiments and innovations in cancer. especially curcumin- and PDT-related nanotechnologies, providing an up-to-date highquality evidence base for researchers and scientists to accelerate and rethink new experiments and innovations in cancer.

#### **2. Photosensitizers and Photodynamic Therapy 2. Photosensitizers and Photodynamic Therapy**

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 2 of 39

smallpox and skin tuberculosis treatments [2,3].

Photosensitizers (PSs) are molecules unchanged before and after energy exchange, that can absorb electromagnetic radiation from infra-red, visible and UV range and trigger the physicochemical change of a neighboring molecule by yielding an electron to the substrate or by extracting an atom from it, and finally, the PS returns to its ground state, where it remains unchanged until it absorbs radiation again [6,7]. Photosensitizers (PSs) are molecules unchanged before and after energy exchange, that can absorb electromagnetic radiation from infra-red, visible and UV range and trigger the physicochemical change of a neighboring molecule by yielding an electron to the substrate or by extracting an atom from it, and finally, the PS returns to its ground state, where it remains unchanged until it absorbs radiation again [6,7].

When incident photons are absorbed, PS advances an electron in a single excited state, which, through an intrinsic spin rotation, can pass into an excited triplet state with a longer lifetime, thus increasing the probability of PS interaction with next-door molecules, and finally, conducting to the selective death of diseased cells through the generation of cytotoxic oxidation species (Figure 1). Depending on the internal structure of the PS, they have different efficiencies when interacting with diverse wavelengths [6,8]. When incident photons are absorbed, PS advances an electron in a single excited state, which, through an intrinsic spin rotation, can pass into an excited triplet state with a longer lifetime, thus increasing the probability of PS interaction with next-door molecules, and finally, conducting to the selective death of diseased cells through the generation of cytotoxic oxidation species (Figure 1). Depending on the internal structure of the PS, they have different efficiencies when interacting with diverse wavelengths [6,8].

PS had an inherent basis in nature through the green pigment chlorophyll, existing in all green plants and in cyanobacteria, absorbing light to deliver the photosynthesis' energy, as well as other light-sensitive molecules in the plant kingdom.

PDT implies the selective sensitization of tissues to light and the first studies on PS started at the beginning of the last century, when researchers noticed this phenomenon and undertook investigation of it in malignant tumors [9].

In the middle of the last century, Figge et al. showed that exogenous porphyrins accumulated selectively in murine tumors [10] and the results were afterwards applied to cancer patients by first injecting raw hematoporphyrins [11], followed by improvement using a hematoporphyrin "derivative" which highlighted the enhanced selective fluorescence of the neoplasms [12,13].

However, we could consider that the contemporary version of PDT was born in the 1970s in the United States due to the activity of Dr. T.J. Dougherty and collaborators, who later used the more refined version of the "hematoporphyrin derivative", called Photofrin, the most implemented PS worldwide, even nowadays, despite the many difficulties (longterm photosensitivity of the skin of cancer patients treated, reduced absorption in large tumors, due to the limited penetration of photons etc.) [14].

In a paper published 25 years later by Thomas J. Dougherty et al., PDT is well defined as involving the management of a PS, which may also need a metabolic combination (a prodrug), followed by exposure to light with a certain wavelength for maximum absorption. The effect is an irreversible succession of photochemical and photobiological reactions with the permanent photodeterioration of malignant cells. Preclinical and clinical research has established PDT as a useful care procedure in early or advanced stages for lung, digestive, genitourinary cancer, etc. [15].

The merit of Thomas Dougherty's pre-clinical and clinical trials and efforts, led to FDA approval of PDT in modern clinical practice, and paved the way for current and future advances in PDT, coupled nowadays with nanotechnologies and subsequent drug discoveries [16].

Without Dougherty's endeavor, it is questionable that PDT would not have remained just "a minor biomedical curiosity" [17].

PDT is a therapy that comprises light, a chemical compound that makes cells abnormally sensitive or reactive to light, and in the presence of tissue oxygen induces cell death by generated reactive oxygen species (ROS), i.e., through phototoxicity. Today, this technology is extensively used for certain conditions, incorporating the latest applications in antiviral treatments and cancers that tend to metastasize. For example, in practically applied PDT, the light with specific wavelengths is usually guided by optical fibers to the patient's tumor, which has been given a photoactive drug, which will attach intensely to abnormal cells. The light will stimulate the substance used for treatment, will generate cytotoxic species in the presence of oxygen, and thus, the malignant cells could be destroyed, procedure considered to be invasively negligible and, to a lesser extent, toxic. A disadvantage is the long-term photosensitization, unpleasant and annoying for patients, but counterbalanced by the diminished necessity for fine operations, shortening recovery time and reducing cicatrices or deformities to the smallest possible size [6,18].

PDT practice implies: the PS, the light irradiation system (L) and the molecular oxygen of the tissue (O2). Wavelength of L must be quantum suited to move PS into action to generate free radicals (type I reaction), as a result of electronic extraction or relocation to an underlying molecule and/or reactive oxygen species, especially singlet oxygen (type II reaction), an extremely reactive species. PDT is composed of several consecutive phases. First and foremost, PS is applied without light, either systemically (intravenous administration), or by topical application. Thereafter, when enough PS is fused into the unhealthy cells to be destroyed, the PS is stimulated by setting the light for a well-defined time interval. The applied dose of electromagnetic radiation provides enough energy to advance PS to higher excitation energy states, but not sufficient to deteriorate the adjacent healthy cells. In the next step of relaxation are generated reactive oxygen species that will put to death the targeted tissue. Unlike other molecules that are normally in a singlet state, molecular oxygen in the atmosphere and in living cells exists in a triplet state, but because quantum physics interdicts reactions between triplets and singlets, this postulates molecular oxygen as inactive in normal states. However, the PS used in medical applications can undergo a process of intersection with oxygen during excitation, a process during which, from an excited singlet state, it will pass to an excited triplet state at the point where the two potential energy curves crosses, as presented in Figure 1 (intersystem crossing), giving rise also to phosphorescence, and consequently, the extremely cytotoxic singlet oxygen will be generated, which will attack any organic substance with which it comes into contact, being meanwhile eliminated very quickly in less than 3 microseconds from the illuminated cells [7,19,20].

When excited in type II reactions, PS provides its excess energy during the process of interacting with molecular triplet oxygen (3O2) and produces singlet oxygen (1O2), a highly reactive species that reacts with the substrate to generate other oxidized products that will attack cellular constituents, leading to the targeted killing of cells in the supplied light field.

PSs contain chromophores, i.e., they are photosensitizers. A part of a molecule responsible for its color, is that part of the molecule in which the energy gap between two different molecular orbitals is in the visible area of the spectrum and, when it meets light and absorbs a photon, an electron from its ground state will pass into an excited state, and a conformational change of the molecule will occur. Once stimulated, the photosensitizer passes from the baseline S0 (ground state) into the short-lived excited state, with lots of vibrational sub-levels, it can decrease its energy by rapidly dropping these sub-levels via inner adjustment to populate the first excited singlet state S1, before it quickly relaxes back to S0 (see Figure 1). The transition from S1 to S0 is via fluorescence, with very short lifespans (10−9–10−<sup>6</sup> s). From S1, it can pass through spin switch via intersystem crossing and can occupy the first excited triplet state, and after that to go downhill to baseline via phosphorescence, with a much longer lifespan (10−<sup>3</sup> <sup>−</sup> 1 s); enough to enable the PS in excited triplet state to act on circumambient bio-compounds via type I and type II reactions [7,21].

Singlet oxygen produces effects at 10–55 nm from its origin in about 10–320 ns and can diffuse up to approximately 300 nm in vivo [7,21,22]. All time sequences involved in PS photoactivation and in type I and II reactions play a key role in disrupting the cellular machine, but type II reactions are deemed to be most efficient for cell impair, leading to the ultimate goal of killing unhealthy photo irradiated cells. However, in real practice, PSs with a triplet state life of less than 20 ns could still prove to be productive photodynamic agents [21].

There are a lot of photosensitizers for PDT, classified into porphyrins, chlorins, dyes (e.g., phenothiazinium salts, rose bengal, squaraines), and recently, nature-inspired PSs.

In radiotherapy, the cellular DNA is the target, but almost all PSs will deteriorate other distinct subcellular entities, which makes the distinction between various PSs applied in PDT.

The perfect PS should accumulate, especially in unhealthy cells, and when light is applied, it should produce toxic species that will destroy the target tissue. Characteristics of an ideal PS are the following: high absorption with maximum absorption coefficient in longer wavelengths, close to the red/infrared spectrum with deeper penetration, to make it possible to deal with grand tumors; better adhesion and fixation in unhealthy tissues than in normal ones; reliable and easy dissoluble in biological systems, permitting intravenous management and fast elimination from the organism after PDT, without residual sensitivity of the skin to light or long-term photosensitization; very good chemical balance and insignificant toxic effect on cells in the absence of light or in low darkness; long triplet lifespan and high triplet state effect, as well as high efficiency for triplet formation and singlet oxygen generation; reduced photobleaching and genuine fluorescence, low production price [6,23–26].

During development, there were several generations of PS, such as:



During development, there were several generations of PS, such as:

**Figure 2.** Some examples of second-generation photosensitizers (PSs). **Figure 2.** Some examples of second-generation photosensitizers (PSs).

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 5 of 39

Third generation PSs include antibody-directed photosensitizers, such as, for example, the monoclonal antibodies for rising specificity and enhanced capacity of action, better pharmacokinetics, improved function, selective targeted and delivery etc., and support Third generation PSs include antibody-directed photosensitizers, such as, for example, the monoclonal antibodies for rising specificity and enhanced capacity of action, better pharmacokinetics, improved function, selective targeted and delivery etc., and support the hope for a future better featured PDT [28,30].

the hope for a future better featured PDT [28,30]. The use of natural compounds has been approached as a new trend in photodynamic therapy, and the natural photosensitizers investigated coupled with the latest nanotech-The use of natural compounds has been approached as a new trend in photodynamic therapy, and the natural photosensitizers investigated coupled with the latest nanotechnologies have been proven to be effective in the clinical practice, as is the case with curcumin. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 39

nologies have been proven to be effective in the clinical practice, as is the case with curcumin. An illustrative diagram of photodynamic therapy using curcumin as a photosensi-An illustrative diagram of photodynamic therapy using curcumin as a photosensitizer is shown in Figure 3.

The use of nanotechnologies in recent years has brought significant improvements in

There are already many techniques for nano formulations, biomaterials and types of nanoparticles suitable for loading curcumin, to increase its therapeutic efficacy and to

Liposomes are spherical vesicles composed of simple or multiple layers that surround aqueous units and have become ideal delivery systems for biologically active substances, because they offer high biocompatibility and biodegradability, high solubility, stability and flexibility, low toxicity, preparation-controlled distribution with specific cell targeting. PEGylated nanoliposomes (attached strands of polyethylene glycol (PEG) to nanoliposomes) were the first nanoparticles approved for nano-drugs by the FDA.

Curcumin is a bioactive agent isolated from *Curcuma longa* rhizomes that, in the last three decades, has reached the peak of research for its biological functions; anti-inflammatory, antioxidant, antimicrobial, antiviral, antimutagenic, antitumor and anti-angiogenic. Liposomes solubilize curcumin and allow its distribution on the aqueous medium

It has been shown experimentally that blue light stops the multiplication of cancer cells, induces cell death by activating caspase and increasing intracellular reactive oxygen species. A recent study investigated the *in vitro* effects of curcumin-loaded liposomes at concentrations of 0–100 µmol/L in combination with PDT at 457 nm (blue) and the fluence of 220.2 W/m2 on three papilloma virus-associated cell lines, and proved that after 24 h, the blue light activation of curcumin-loaded liposomes led to "a significant reduction in colony formation and migratory abilities, as well as to an increase in tumor cell death"

Curcumin-loaded liposomes (Lipo-cur) and blue light distributed by PDT can

Polymeric micelles have been used as nanodrugs for over 40 years; they are structured on a hydrophilic shell, and hydrophobic core that can be loaded with hydrophobic

and increase its effect for the treatment of various cancers and other diseases.

achieve excellent bioactivity and strong anticancer activity [31–33].

drugs. Recent studies have shown another way to use curcumin to increase its bioavaila-

bility, plasma concentration and ability to penetrate and concentrate inside cells.

**Figure 3.** PDT with blue light and curcumin. **Figure 3.** PDT with blue light and curcumin.

avoid its possible side effects, as follows:

*2.1. Curcumin-Loaded Liposomes (Lipo-Cur)* 

[31].

drugs [34].

*2.2. Cur-Loaded Polymeric Micelles* 

The use of nanotechnologies in recent years has brought significant improvements in the pharmaceutical industry through the discovery of nanoparticles, extraordinary innovations have been made for the production and delivery of the active principles of new drugs. Recent studies have shown another way to use curcumin to increase its bioavailability, plasma concentration and ability to penetrate and concentrate inside cells.

There are already many techniques for nano formulations, biomaterials and types of nanoparticles suitable for loading curcumin, to increase its therapeutic efficacy and to avoid its possible side effects, as follows:

#### *2.1. Curcumin-Loaded Liposomes (Lipo-Cur)*

Liposomes are spherical vesicles composed of simple or multiple layers that surround aqueous units and have become ideal delivery systems for biologically active substances, because they offer high biocompatibility and biodegradability, high solubility, stability and flexibility, low toxicity, preparation-controlled distribution with specific cell targeting. PE-Gylated nanoliposomes (attached strands of polyethylene glycol (PEG) to nanoliposomes) were the first nanoparticles approved for nano-drugs by the FDA.

Curcumin is a bioactive agent isolated from *Curcuma longa* rhizomes that, in the last three decades, has reached the peak of research for its biological functions; antiinflammatory, antioxidant, antimicrobial, antiviral, antimutagenic, antitumor and antiangiogenic. Liposomes solubilize curcumin and allow its distribution on the aqueous medium and increase its effect for the treatment of various cancers and other diseases.

It has been shown experimentally that blue light stops the multiplication of cancer cells, induces cell death by activating caspase and increasing intracellular reactive oxygen species. A recent study investigated the in vitro effects of curcumin-loaded liposomes at concentrations of 0–100 µmol/L in combination with PDT at 457 nm (blue) and the fluence of 220.2 W/m<sup>2</sup> on three papilloma virus-associated cell lines, and proved that after 24 h, the blue light activation of curcumin-loaded liposomes led to "a significant reduction in colony formation and migratory abilities, as well as to an increase in tumor cell death" [31].

Curcumin-loaded liposomes (Lipo-cur) and blue light distributed by PDT can achieve excellent bioactivity and strong anticancer activity [31–33].

#### *2.2. Cur-Loaded Polymeric Micelles*

Polymeric micelles have been used as nanodrugs for over 40 years; they are structured on a hydrophilic shell, and hydrophobic core that can be loaded with hydrophobic drugs [34].

Chang et al. studied how Cur-loaded polymeric micelles influenced endocytosis and exocytosis in colon carcinoma cells and proved that curcumin-loaded micelles had an increased stability compared to the unloaded micelles and were rapidly incorporated by the cells within minutes, becoming cytotoxic after 72 h of exposure.

In this experiment, the micelles applied to encapsulate curcumin had a diameter of about 16–46 nm, and when loading curcumin, their size was approximately doubled, with a loading efficiency of 58%. Results showed the importance of micelle size and loading on drug delivery and cytotoxicity [35].

A wide variety of amphiphilic polymers (diblock, triblock, grafted copolymers, etc.), hydrophobic materials and vitamin E, have been produced for the preparation of mycelium, but the most used are polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and chitosan [36,37].

Liu et al. have developed curcumin-loaded polymeric micelles to surmount the reduced ability of curcumin to be dissolved in water and to achieve better intravenous administration. Cur-loaded polymeric micelles had a very strong antitumor effect, inhibiting tumor growth and spontaneous lung metastases in a model of breast tumor in mice, proving to be a very good option for breast cancer [37].

In order to easily penetrate the cells and increase the therapeutic intake, the micellar surface was modified by adding ligands (epidermal growth factor receptor, folate, etc.) that more quickly recognize the receptors expressed on the surface by cancer cells [38].

#### *2.3. Cur-Loaded Polymeric NPs*

Nanoparticles are designed at the atomic or molecular level, have a diameter 1000 times smaller than a medium-sized cell in our body and are very valuable for the administration of drugs, because their physical, chemical and biological properties are unique. Curcumin can be encapsulated in a wide variety of nanoparticles based on polymers, solid, magnetic, gold and albumin-based lipids, which increase its solubility, pharmacokinetics, controlled release capacity and exact cell strike [39–42].

#### *2.4. Cur-Loaded Mesoporous Silica NPs*

Nanotechnology has overcome conventional concepts and ideas in the pharmaceutical industry. More than 30 years ago, Mobil Corporation first produced mesoporous silica nanoparticles (MSNs). The mesoporous structure is unique, provides chemical stability and high drug loading capacity, biocompatibility, large pore volume, large surface area, controlled release at the target, and low toxicity. In cancer, there are large disorders of the normal structures of the lymphatic and vascular system, so that MSN nanoparticles can more easily penetrate cancer cells through the process of phagocytosis and pinocytosis. The nanoencapsulation of curcumin with silica and chitosan increases the stability of curcumin and increases its cytotoxic activity on cell carcinoma cells [43,44].

### *2.5. Cur-Loaded Protein-Based NPs*

Protein-based nanoparticles are widely used as natural biomaterials in the biomedical field because they meet the special qualities of biocompatibility, biodegradability and non-immunogenicity. Albumin from chicken serum or eggs was the most studied protein for obtaining drug-releasing nanostructures. Recently, many types of Cur-loaded protein nanoparticles have emerged, such as bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin (OVA), zein, casein and curcumin–silk fibroin nanoparticles (CM–SF NPs) [32,45,46].

### *2.6. Cur-Loaded Solid Lipid NPs*

Solid lipid nanoparticles (SLNs) are colloidal systems made of biodegradable solid lipids used in the pharmaceutical industry, because they perform through physical stability, high biocompatibility and the controlled release of embedded drugs. Cur-loaded solid lipid NPs were investigated in vitro and in vivo, demonstrating good stability and bioavailability, increased absorption in cells, and high anti-cancer efficacy [47–49].

#### *2.7. Cur-Loaded CDs NPs*

Cyclodextrins (CDs) comprise a family of cyclic glucose oligomers, which have excellent biocompatibility properties, complexation with lipophilic structures, very low toxicity, non-immunogenicity and targeted drug release. Cur-loaded CDs NPs have been shown to be highly effective in antioxidant activity, to reduce the oxidative stress associated with various cancer and a preventive role for nosocomial infections [50–52].

#### *2.8. Cur-Loaded Nanogels*

The proposed nanogel formulation of curcumin has several advantages over other delivery systems, because it ensures a higher concentration of the drug at the target sites and can reduce the exposure of curcumin to serum proteins and biological degradation after systemic administration. Multifunctional hybrid nanogels, through their ability to emit fluorescence, can be used for imaging, cell monitoring and, because they have high absorption in the near infrared range, are useful in photothermal conversion, and loaded with curcumin, are aimed at suppressing drug-resistant tumors [53–55].

#### *2.9. Cur-Loaded Nanocrystals*

Nanocrystals, although small particles have a large surface area for loading the hydrophobic drug, and due to their high solubility and saturation capacity, they increase bioavailability and biodistribution.

For example, in a recent study, Wang et al. have prepared curcumin nanocrystals (CNs) with a mean diameter of 15 nm by the quick emulsion freeze-drying method. CNs proved to be effective in drug delivery to selectively deliver Curcumin to cancer cells. CNs were prepared via the oil-in-water emulsion freeze-dried method: firstly, they prepared the emulsion, the oil phase (O) was dichloromethane solution containing 40 mg/mL curcumin, and the water phase (W) was containing 1 wt% Pluronic®F-127-COOH aqueous solution, at a volume ratio O/W fixed at 1:20, and the blend was mixed through ultrasonic (100 w, 10 min) emulsification to obtain the emulsion (O/W). Secondly, the above emulsion was further added into 20-fold the volume of the water phase, and the mixture was frozen by liquid nitrogen, followed by freeze-drying using a vacuum freeze-dryer. Finally, CNs suspension was obtained when lyophilized products were dispersed into the water, and free surfactants were removed by ultracentrifugation [56].

Curcumin nanocrystals (CNs), by the ability to avoid absorption in the reticuloendothelial system, prolong the circulation time of curcumin, increase the capacity of permeation and retention, accumulating in large quantities in the tumor [56,57].

#### *2.10. Cur-Metal Oxide NPs*

Inorganic nanomaterials used as metal nanoparticles (carbon, nanotubes, graphene, minerals and metal oxides) that carry drugs would be more advantageous than organic ones, because it possesses bioavailability, tolerance towards most organic solvents and better stability, increased surface area and porosity, better loading and dispensing capacity of drugs, with lower toxic effects [58].

Nanoconjugated zinc oxide nanoparticles (ZnONPCS) with curcumin (ZnONPCS-Cur) showed higher cytotoxicity in several cancer cell lines (breast, cervix, osteosarcoma and myeloma) and had anticancer and anti-inflammatory activity in vitro, and therefore, they could be used as possible therapeutic nanoconjugates for future cancer treatments [59].

#### **3. Curcumin and Latest Cancer Applications**

On 14 December 2020, International Agency for Research Cancer (IARC) released Globocan 2020, which published the latest data on the incidence of cancer, which rose to 19.3 million new cases and 10 million cancer deaths in 2020. According to Globocan 2020, which is a statistical database for IARC on incidence and mortality in 185 countries for 36 types of cancers, it has been estimated that cancer had 19.3 million new cases per year, of which breast cancer was in first place, with about 11.7% new cases, followed by lung cancer 11.4%, colorectal 10%, prostate 7.3%, and followed by stomach cancer at 5.6% [60,61].

The field of cancer research is a dynamic and evolving domain, with a multitude of models and scenarios proposed for examination over the decades on the hidden cause behind tumors development (genetic mutations, microorganisms, metabolic changes and so on), with fluctuating evidence or achievements, whose major goal remains the discovery of new methods and drugs for stopping disease progression and even eradicative therapeutics.

On this line, the cancer stem cell (CSC) model encompasses unusual immortal cells, such as those existing in tumors or blood cancers, similar to regular stem cells, but capable of generating the full range of cells from a given cancer specimen, which, through selfrenewal and differentiation into multiple types of cancer cells, are tumorigenic, i.e., they generate recurrences and metastases, and so, supplementary malignancies [62,63].

In cancers that pursue the CSC model, some intracellular pathways may be attacked with natural compounds, such as curcumin or drugs, to overcome the danger of the development of new tumors at a distance. Promoting appropriate CCS-oriented treatments could improve the survival and quality of the life of patients with metastases [25,63–66].

Very recently, research based on this model shows the effectiveness of curcumin in various forms of cancer [67].

Even though it has reduced bioavailability, being insoluble in water, curcumin has been intensively studied as an authentic polyphenol and practically the main constituent of *Curcuma longa*, for its multiple beneficial effects in the treatment of various inflammatory, auto-immune, degenerative diseases, etc., and going to important applications in cancer, not only for the protective effect, but especially by destroying malignant cells. To overcome this drawback, various water-soluble mixtures have been imagined, such as liposomes or the incorporation of curcumin into micelles at the nanometer scale, with raised assimilations appropriate for cancer studies. The first formulations of curcumin in organic solvents proved to be toxic to living cells, and even with genotoxic capabilities. No investigation with curcumin embedded in the micelles has been planned until not long ago. In a recent experiment, Beltzig et al. comparatively investigated the cytotoxic and genotoxic action of genuine curcumin dissolved in ethanol (Cur-E), or integrated into micelles (Cur-M), and evaluated cell killing, apoptosis, necrosis, senolysis and genotoxicity, on a multitude of elementary and settled cell lines, proving that both formulations reduced viability for all cells in the same dose interval. Cur-E and Cur-M induced apoptosis as a function of dose, without senolytic action. Genotoxic repercussions disappeared in the absence of curcumin, denoting a prompt and full repair of DNA. In every experiment, Cur-E and Cur-M were, to the same extent, dynamic, and had important cytotoxic and genotoxic action, starting with 10 µM. Micelles without curcumin content were fully inoperative. The results proved similar in terms of cytotoxicity and genotoxicity for micellar curcumin as the native one, so the administration of micellar curcumin as a dietary supplement is safe and paves the way for new applications [68].

Major goals of pharmaceutic investigations are the innovative transport/delivery systems of drugs in cancer treatments. Zarrabi et al. have researched the manufacture of a new intelligent biocompatible stealth-nanoliposome to supply curcumin in cancer therapies. Four distinct classes of liposomes (plus or minus pH-sensitive polymeric film) were obtained by the Mozafari process, and then investigated by multiple trials. The embarkation and deliverance of curcumin were assessed at two different pH values, 7.4 and 6.6, but also the cytotoxicity of the specimens. The optimal average size for the smart stealthliposome was 40 nm, and the efficacy of the drug's catch was about 84%, comparatively with 50 nm and only 74% performance by uncovered liposomes. Nano-carrier discharge from the stealth-liposome was better directed than in the uncovered. Experiments have shown the toxicity of drug's nanocarriers on malignancies. We could conclude that soon, the pH-sensitive intelligent stealth nanoliposome may become a true aspirant in cancer treatments [69].

Resistance to medicine and bad outcome in some cancer cases is often due to the hyperactivation of NRF2, a group of transcription factors, i.e., the nuclear factor erythroid 2 p45, detected in some tumors. It was demonstrated that curcumin can induce either cytoprotection or tumor growth by activating NRF2, as a function of the phase of the malignancy. Garufi et al. highlighted the anticancer effects through manifold molecular processes related to curcumin, and recently explored the fundamental molecular sequence of steps linked to making operative NRF2 by the zinc-curcumin [Zn (II) -curc] complex. Indeed, the therapy with Zn (II)–curc raised the NRF2 proteins concentrations and their connections, the heme oxygenase-1 (HO-1) and p62/SQSTM1, while particularly decreased the levels of Keap1 (Kelch-like ECH-associated protein 1), which stopped the NRF2 in all the investigated malignant cell lines. The inhibition of NRF2 or p62/SQSTM1 with distinctive siRNA proved the existence of the communication channel between the two molecules, and that the easily disassemble of any molecule surged the killing of cancer cells by Zn (II)–curc, a fact that could be implemented in the future to improve the receptivity to tumors treatment by this method [70].

Curcumin displays multiple proven effects on cells; for example, an inhibitive action on thrombocytes, but not known if it is owed to thrombocyte apoptosis or to pro-coagulant platelet organization.

Recently, Rukoyatkina et al. reported that curcumin did not initiate caspase 3—relying on the apoptosis of human thrombocytes, but led to the organization of pro-coagulant thrombocytes. At 5 µM concentration, the effect increased, but at ten times' higher concentration, the thrombocytes apoptosis was stopped by the suppression of ABT-737 (small molecule drug that inhibits Bcl-2 and Bcl-xL) that was interfered with thrombin production.

Curcumin did not alter thrombocytes' ability to survive at low concentrations but decreased it by 17% at higher concentrations. Autophagy caused by curcumin in human thrombocytes was accompanied by the operative configuration of adenosine monophosphate kinase (AMP), and the cessation of protein kinase B function. Curcumin could block the P-glycoprotein (P-gp) in tumors, and therefore defeat the manifold medicines resistance, and likewise could also stop the thrombocytes P-gp action. The effects of curcumin on human thrombocytes are due to complex processes through pro-coagulant thrombocytes organization, and so it can support pro—or against caspase—subordinate thrombocyte's death, but only in distinctive cases [71].

Systematically checking the abnormal functioning of cells, tissues or organs in the inceptive steps of the initiation of malignant processes and the monitorization of the cellular oxygenation is of maximum significance, both for the fundamental applications, but also in the practical medical ones.

A non-invasive modality for both the in vivo and in vitro assessment of cellular oxygenation is evaluating the lifespan of the luminescence of molecular sensors, but still very difficult in the case of increased oxidative stress.

Molecular probes, such as mitochondrial probes or [Ru (Phen)3]2+ state-of-the-art, intact cell phosphorescence imaging technologies applied by Huntosova et al., in a model of chorioallantoic membrane (CAM), offer reduced phototoxicities and could also be applied in curcumin cancer therapy in tumors originating from the neuroglia of the brain or spinal cord. These results could be useful and universalized for the evaluation of tissue oxygenation as an advanced and original method, based on the analogies between diverse interacting biological factors, especially in cancer therapies that deal with metabolic or oxygen changes, glucose and lipid loss, and so on [72].

Important attempts to improve the potency of targeted drug carriers in the lung cancer were made, but the prognosis is still very poor, with only 15% survivors, 5 years after identification. The best choice for the direct administration of chemotherapy to the lungs would be the inhalation formulation.

Currently, this type of formulation to accomplish successfully, at the same time, a significant dose of specific chemicals that are selectively destructive to malignant cells and tissues in the solid tumor and to function with reduced local lung toxicity, is still an aim, as only 10–30% of lung chemotherapy nowadays already has the quality of being toxic [73].

Lee et al. imagined a dry powder easy to inhale (DPI) holding a chemotherapeutic agent (paclitaxel, PTX) and the native antioxidant curcumin (CUR) that defends the healthy cells to be damaged during direct lung transfer chemotherapy. Grinding CUR and PTX in co-jet as aerosol formulation, with more than 60% of very fine particles and a fit mass median aerodynamic diameter, exhibits an important cytotoxic effect for lung tumors, giving rise to apoptosis/necrotic cell killing, extending mitochondrial oxidative stress (ROS), depolarizing the mitochondria membranes, and decreasing the ATP in malignant cells. Incorporating CUR is decisive for correcting the cytotoxic effects of PTX against healthy cells and depends on dose, providing an easy and efficient DPI formulation with special discriminating cytotoxicity in lung malignancies [73].

In another experiment concerning lung cancer, Wan Mohd Tajuddin et al. studied the diarylpentanoid (DAP), changed the structure analog from the genuine curcumin, and proved to improve anticancer effects in different forms of malignancies, by comparing the outcomes (toxic impact, proliferative and apoptotic action) on two subtypes of non-

small cell lung cancer (NSCLC) cells: the squamous cell carcinoma (NCI-H520) and the adenocarcinoma (NCI-H23). The gene expression to reveal the main signaling pathways, the targeted genes, the cytotoxicity screening, the anti-proliferative action, as well as the apoptosis linked to the rise in caspase-3 activity and decrease in Bcl-2 protein concentration were investigated and proved to be function of dose and time in all studied cells. This new compound, derived from curcumin, should be henceforth investigated as a possible representative anticancer drug for NSCLC cancer treatment [74].

#### **4. Effects of Curcumin and PDT in Various Forms of Cancer**

#### *4.1. Breast Cancer*

Breast cancer at onset has no clinical symptoms, because it initially affects the glandular epithelium of the ducts or lobe structure, but over the years, it can progress and invade the surrounding breast tissue and lymph nodes or various organs; therefore, if a woman dies, the cause is multiple distant metastases. Breast cancer can occur in women right after puberty, or at different stages of life, and the number of cases has increased year on year, becoming the most common form of cancer worldwide. According to World Health Organization (WHO) publications, as of 2020, 2.3 million women have been confirmed with breast cancer, of which 685,000 deaths have occurred worldwide, and an impressive number have been left with various disabilities in daily life [75].

It is currently known that breast cancer can be classified into three subtypes:


Although the breast cancer therapy available today (anticancer chemotherapeutic drugs, antihormones, biologics and radiation therapy) has very good results on early detected primary tumor, many deaths still occur due to recurrences and multisystemic metastases. In this pathology, it has been shown that a type of cancer cells, like normal stem cells, has a strong potential for multiplication, self-renewal, differentiation, and their oncogenicity can underlie recurrences and metastases [77].

Radiation therapy is one of the standard treatment methods for breast cancer; applied in the early stages, it can decrease mastectomy surgery; in later stages, it can reduce the risks of recurrence, and in advanced stages, it can prolong the patient's life [75].

However, this method still has many disadvantages, in that the tumor may become radioresistant, and cancer and metastases may recur.

Targeting breast cancer stem cells is the clue for ameliorating the results of breast cancer radiotherapy.

Yang et al. investigated the effects of curcumin combined with glucose nano-gold particles (Glu-GNPs), for a decrease in radiotherapy resistance activity, by targeting MCF-7 and MDA-MB-231 breast cancer stem-like cells (BCSCs), in order to increase apoptotic, colony-forming and antiproliferative activity. Irradiation was carried out with a 6 MV X-ray at a total dose of 0 and 4 Gy; the dose rate was 2 Gy/min, for two minutes, the depth of penetration was 2 cm, and the irradiation distance of 50 cm. The authors demonstrated that curcumin, combined with Glu-GNPs, increased the ROS level of mammals MCF-7 and MDA-MB-231 in hypoxic conditions by inhibiting the factor-1alpha (HIF-1alpha) (an oxygen-sensitive transcriptional activator) and the heat shock protein 90 (HSP90) activities, ameliorating the apoptotic activity of tumor stem cells, and thus, it has increased the sensitivity to radiation therapy [77].

Minafra et al. performed a study to evaluate in vitro the bioavailability and the radiosensitizing effects of curcumin-loaded solid nanoparticles (Cur-SLN) on three breast cell lines, non-tumorigenic MCF10A and tumorigenic cell lines MCF7 and MDA-MB-231 BC, exposed to irradiation. A multi- "omic" assay was used to clarify the radiosensitizing action of Cur-SLN by microarray and metabolomic exploration techniques. The authors demonstrated the antioxidant, radiosensitizing and anti-tumor capacity of Cur-SLN through a transcriptomic and metabolomic analysis [78].

The conventional treatment protocol for breast cancer includes breast removal surgery, X-ray therapy, and the administration of specific drugs, i.e., chemotherapy. Drugs administered for these cancers have many side effects, negatively affecting patients' quality of life, which has required the discovery of new effective, but less toxic treatments. In recent decades there has been a growing trend to find natural remedies with anti-tumor potential, including curcumin and its derivatives.

Research to date has shown that curcumin has anti-neoplastic properties through various mechanisms, including the following: inhibition of endothelial growth factor [79], lipoxygenase enzyme (LOX) pathway [80], blocking the activity of NF-kB and Wnt signaling pathways [81–83], stops cell cycle and p53-dependent apoptosis, disrupts the expression of signaling protein kinase B (Akt) and phosphatidylinositol 3-kinase (PI3K) [84,85].

Experimental studies have shown that curcumin can inhibit EZH2 (enhancer of zest homolog-2), which is a histone methyltransferase that catalyzes the trimethylation of histone H3 in lys 27 (H3K27me3), is found in an increased amount in human cancer, has an oncogenic, metastatic role, and influences drug resistance. It was postulated that, on the EZH2, miR-375, FOXO1 and p53 axis, special processes with direct impact in the proliferation of the breast tumor could take place [86,87].

Gallardo et al. showed, in a study on MCF-10F and MDA-MB-231 cell lines of human breast cancer, that curcumin can modulate the expression of miR-34a and Rho-A, which reduces cancer progression, metastasis, and increases the sensitivity of anti-cancer drugs [88].

The chemical structure of curcumin is responsible for many of its biological and pharmacological activities, as low solubility in aqueous media, poor bioactive absorption, physicochemical instability, rapid metabolism, sensitivity to alkaline environment, which restricts its clinical scope [89,90].

With the patenting of curcumin nanoencapsulations, these impediments have been overcome. There are currently several nanoencapsulation techniques, but ionic gelation and antisolvent precipitation are in vogue. The products used for nanoformulations currently include: liposomes, polymers, nanoparticles, conjugates, solid dispersions, cyclodextrins, micelles, nanospheres and microcapsules and other various nanoformulations [42,90,91].

Due to the maximum wavelength of blue light absorption (408–434 nm), curcumin is used as a natural photosensitizer for PDT applications in various medical, antimicrobial, antiviral and antitumor fields [92,93].

The mechanisms by which PDT can eradicate tumor tissue are synthesized in the following modes of action: initially, it will locate and activate PS in the tumor area, which will release ROS with the potential to destroy neoplastic cells; the second mechanism would be that PDT disrupts the usual supply of oxygen and nutrients by compromising vascularity; the last postulated mechanism is the activation of the immune system, which triggers an inflammatory process against tumor structures.

Sun et al. studied experimentally, on 4T1 mouse breast cells, the effects of carrier-free curcumin nanodrugs (Cur NDs) used in conjunction with PDT administered by a device that emitted blue light at a power of 640 mW on a wavelength of 450 nm. Cur NDs were prepared without using any toxic solvents through an easy and green reprecipitation method, as follows: 1 mg/mL Cur was dissolved in ethyl alcohol; afterwards, 1 mL of the solution was rapidly injected into 20 mL of high-purity water under robust stirring. NDs were purified and concentrated by ultrafiltration, and at the end, the Cur NDs were

achieved by lyophilization. In vitro irradiation (λ = 450 nm, 640 mW, 1 min) of 4T1 cells after 24 h of incubation significantly increased the expression of p-JNK, Bax and cleaved caspase-3, with the generation of a large amount of ROS, activation of MAPKs with induction of apoptosis, and reduced the cells viability. The study demonstrates that Cur NDs is a valuable photosensitizer for PDT in eradicating breast cancer, and with very good prospects for use in the clinical practice [94].

Because curcumin has a low solubility, nanoemulsions and microemulsions as administration systems with dimensions between 100–300 nm facilitate an expansion of bioavailability, the therapeutic window, and even a controlled delivery to the desired area [95–97].

Machado et al. investigated the effect of curcumin-nanoemulsion (CNE), a new and well-designed drug delivery system (DDS+) molecule, as a novel photosensitizer in the photodynamic therapy of breast cancer on MCF-7 cell model. CNE was achieved by interfacial pre-polymer deposition and spontaneous nanoemulsification. Curcumin was set up in an oil phase at 0.1 mg/mL. Organic phase (acetone) was obtained from medium-chain-triglycerides, natural soy phospholipids. This was added into the aqueous phase containing an anionic surfactant, poloxamer 188. Organic solvent was taken off through rota-evaporation. The authors revealed that curcumin encapsulation in lipid nanoparticles increased the concentration of this compound, its biological effects, solubility and bioavailability, without substantially affecting the cell viability of HFF-1 and MCF-7 cells. After incubating HFF-1 cells and MCF-7 cells and applying two doses of 80 J/cm<sup>2</sup> with a laser device at 440 nm, the mortality rate of breast carcinoma cells was 65% and 90%, respectively, demonstrating the value of CNE as photosensitizer [98].

Curcumin, a component derived from the dried rhizomes of *Curcuma Longa*, is the most requested phytochemical component for cancer therapy, and is therefore considered the "magic molecule" [99,100].

Remote spread of primary malignancy is the cause of multiple cancer deaths. To date, the entire arsenal of available drugs and treatments are still insufficient to manage the metastases that are forming. Therefore, maximum attention is directed to the prevention of metastases by targeting circulating metastatic tumor cells.

A lot of circulating tumor cells (CTCs) will perish in the blood circulating through the body, but a small number will most likely generate distant metastases, and thus a negative prognosis for the patient. These stem-type CTCs can escape immune surveillance and show a high ability to withstand treatment. The latest strategies should address metastatic CTCs and their progenitors based on their molecular and cellular characteristics, to expand current plans for the successful prevention of metastases [101].

In a recent study in this significant direction, Raschpichler et al. advanced an in vitro exploratory model to mimic the circulation of CTCs, and to render them inactive by PDT and curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles. Researchers obtained after blue laser irradiation (30 min, λ = 447 nm, output power 100 mW), an important decrease in the viability of MDA-MB-231 breast cancer cells under flow conditions. Accumulation of curcumin on cell membranes and high fluorescence signal after irradiation was detected by laser scanning confocal microscopy. PDT determined alterations in the cancer cells, i.e., apoptosis and prompt necrosis, as a function of time, and during laser activation of curcumin nanoparticles, a change in blue absorption spectra and a decrease in total curcumin occurred. Findings are incentive for the extension of these in vitro CTCs studies to in vivo experiments, so that PDT becomes an advanced action plan for future clinical applications for metastasis prevention [102].

The anti-cancer activity of curcumin by formulation as nanostructured lipid carriers (NLCs) using solid lipids (glyceryl monooleate, glyceryl dibehenate, glyceryl distearate) and olive oil as a natural liquid lipid, was tested in vitro on a breast cancer cell line. The curcumin thus prepared has been used as a photosensitizer for PDT in MCF-7 breast cancer cells. The loading of CUR into NLCs increased the penetrating power into cancer cells and the cytotoxic potential in both dark and light conditions [103].

Approaching cancer therapy with current means brings a major drawback, namely the deterioration of healthy tissue in the vicinity of the tumor. To avoid these major problems, the association between PDT and photothermal therapy (PTT) has aroused the interest of researchers in the therapy of a wide range of diseases, including cancer. Very good results with reduced invasive effect and negligible side effects were obtained by combining PTT that releases heat, and PDT that not only produces ROS, but also promotes the release of products with multiple antitumor immunity capacity, in order to dissipate tumors. Phototherapy using nanoparticles can target and destroy undetectable metastatic tumors, restore the anti-tumor capacity of drugs and modulate the immune system in the tumor microenvironment [104–107].

The association between PTT and PDT has the advantage of an excellent curative result by eliminating tumor cells, as well as by immunomodulating the apoptotic and inflammatory responses of tumor cells.

An additional barrier of the immune control point to the photo treatment could increase the antitumor effects, generating more CD4 + and CD8 + T cells in the tumor [108].

An in vivo experiment on Female Balb/c mice tumorized with 4T1 cells has investigated the antitumor effects of curcumin loaded with Fe3O4–SiO<sup>2</sup> nanoparticles, i.e., a dualfunctioned nanocomposite (NC), and also PDT and PTT. After tumorization, the mice were divided into six groups: control group injected with phosphate buffer saline (PBS); group II with CUR and irradiation with blue diode laser at 450 nm, intensity of 150 mW/cm<sup>2</sup> for 3 min (CUR and PDT group); group III, PBS injection plus irradiation with blue diode laser for 3 min followed by NIR laser with intensity of 0.5 W/cm<sup>2</sup> for 7 min, i.e., the blue + NIR (near-infrared) laser group; group IV, nanocomposite injection (NC group); group V with NC plus irradiation with NIR laser at 808 nm for 7 min (NC and PTT); and the last group with NC + PDT + PTT. The treatment protocol combining NC and PDT together with PTT on the highly invasive triple-negative breast cancer model used in this study could be considered for the replacement of chemotherapy in these types of cancer.

Results of the study revealed that in the group treated with NC and PDT together with PTT, the tumor volume decreased significantly, and the expression of proapoptotic proteins Bax and Caspase 3 increased meaningfully compared to the control group, without weight loss, and no adverse effects on vital organs (liver and lung) in the mice autopsy image [109].

In an in vitro study, Halevas et al. reported that the gallium curcumin complex used as a photosensitizer in photodynamic therapy on human breast MCF-7 adenocarcinoma cells, increased intracellular ROS levels, and significantly decreased the number of cancer cells compared to simple curcumin. New curcumin-Ga complex did not show dark toxicity at low concentrations against MCF-7 breast cancer cells, and the decrease in cell survival was dependent on the laser dose [110].

Table 1 summarizes aspects of recent research that have shown that curcumin nano formulations, together with PDT, can contribute to the treatment of breast cancer by strong penetration into cancer cells, high cytotoxic potential, decreased tumor volume, prevention of metastases, without weight loss, and without adverse effects on vital organs (liver and lungs) in experimental animals, with prospects for future clinical applications [94,98,102,103,109,110].

**Table 1.** Curcumin nanoformulations and PDT in breast cancer.



#### **Table 1.** *Cont.*

curcumin.

#### *4.2. Gynecologic Cancers*

Cancer worldwide is a major health problem, and one of the most common causes of death [111]. According to statistics from the last two decades, the incidence of cancer in women has been steadily increasing by about 0.5% per year for breast cancers, and about 1% for uterine cancer, due to the reduction in the number of fertile women and weight gain [112–114].

Over the past three decades, the survival rate of cancer patients has increased, except for the cancers of the uterine cervix and of the uterine corpus [115].

Cervical cancer, with almost 0.6 million cases and 0.3 million deaths per year in 2018, ranked fourth in the world in terms of incidence and mortality caused by cancer after breast cancer (2.1 million cases), colorectal cancer (0.8 million cases) and lung cancer (0.7 million cases) [116].

Today, special efforts are being made to prevent, detect early and implement new therapeutic strategies to improve the efficacy and safety of chemotherapy and radiotherapy, prevent recurrences, metastases and increase patient survival. PDT, together with various curcumin nanoformulations, are promising treatment modalities for a wide range of cancers, such as cervical cancer.

Nanoemulsion-curcumin behaved as a photosensitizing drug in PDT, generating a high phototoxic effect, with less than 5% of viability in the experiment with cervical carcinoma cell lines (CasKi and SiHa) and human keratinocytes spontaneously immortalized cell line (HaCa). In an experiment done by de Matos et al., increased activity of the enzymes caspase 3 and caspase 4 was observed, suggesting that cell death occurred by apoptosis. The authors propose the use of curcumin-nanoemulsion and PDT through an in situ optic fiber, as an alternative treatment in cervical cancer [117].

A very important issue in PDT is to avoid the accumulation of PS in healthy tissues and prevent unwanted effects, which can be solved by using encapsulated PS in polymeric nanoparticles, in which the active product is protected from degradation by the physiological environment; an example is the case of poly (lactic-co-glycolic acid) (PLGA), recognized as one of the most valuable drug-carrying polymers (DCs) that is used to obtain high quality products. PLGA polymer is approved as a biocompatible and biodegradable polymer by the FDA and the European Medicines Agency (EMA) [118–120].

Duse et al. published the results of in vitro irradiation research (457 nm LED with a radiation flux of 8.6J/cm<sup>2</sup> ) on the human ovarian adenocarcinoma cell line SK-OV-3, using a photosensitizer with biodegradable PLGA nanoparticles loaded with curcumin (CUR-NP). In this study, we analyzed the effect of nanoformulation on human erythrocytes, by haemolysis test and blood clotting, which showed that there was a slight increase in haemolysis and improved serum stability, assuming that there is an interaction of curcumin with intrinsic proteins or coagulation factors. The authors showed that the nanoformulation allowed the use of higher amounts of curcumin, which showed cytotoxic effects on tumor cells after the administration of PDT at low intensity, thus selectively inhibiting tumor growth, and believe that LEDs provide an economic and technical advantage over laser devices [121].

Curcumin, as a special low-toxicity photosensitizer, has higher pro-apoptotic potency when combined with PDT. However, the mechanisms of action are still poorly understood. He et al. investigated the results of the combination of different concentrations of curcumin mediated PDT with/without Notch receptor blocker (DAPT), after 180 s of irradiation with 445nm laser at a dose of 100 J/cm<sup>2</sup> , used together with curcumin, on the survival rate of cervical cancer Me180 cells in female BALB/c mouse. Following the administration of curcumin with PDT plus DAPT, a synergistic interaction occurred that significantly increased the overall rate of cell mortality. The authors claim that this combination could inhibit Notch-1 expression and downstream protein synthesis in in vitro and in vivo cervical cancer. Notch-1, an advanced preserved signaling pathway that controls interactions between adjoining cells and NF-κB, could be the targets of the curcumin-PDT combination

in the success of cervical cancer therapy in women. This can be explained by the fact that Notch-1 activation is associated with the onset and development of cervical cancer [122].

Figuratively speaking of Notch, we can consider that it belongs to the group of arbitrators who decide the fate of the cell; in fact, it modulates the balance between differentiation and multiplication of cells. The Notch pathway has a very important role in the evolution of breast, cervix, ovary, and uterine endometrium epithelial tissues, and is frequently involved in the appearance and expansion of cancers [123].

#### *4.3. Skin Cancer*

Human skin consists of three layers of cells superimposed from depth to the surface, as follows: epidermis, dermis, and hypodermis. The epidermis consists of five types of overlapping cells, one on top of the other, from the depth to the surface in the basal or germinal layer—spiny, granular, lucidum and horny. All the layers come from the germinal one, whose cells, as they multiply, are pushed to the surface, changing in 26–28 days their shape and structure, and then they will be eliminated as dead cells in the form of barely visible scales. Melanocytes are dermal cells that secrete melanin, a pigment that tans the skin under the action of the sun, and the corpuscles Meissner, Ruffini, Krause, Vater-Pacini and Merkel give the skin the ability to perceive and transmit the proprioceptive information to the brain. If these cells undergo pathological changes, they will give rise to various forms of skin cancer. Each type of skin cancer has a potential for severity, so it must be detected early and treated accordingly. The most common types of skin cancer include the following: basal cell carcinoma, recurrent basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, and other types of rare skin cancers [124].

According to data published by the American Cancer Society, about 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US, of which approximately 3.3 million American patients have basal cell carcinoma, meaning that 8 out of 10 have the malignant cells in the basal layer.

Basal cell carcinoma develops in the basal layer of the epidermis, has a tropism for the neck and head regions that are more frequently exposed to the sun, but can evolve elsewhere and over time metastasize to the loco-regional ganglia [125].

Basal cell carcinoma can recur in the same area as before, or in another zone of the body; it has a risk of recurrence of up to 50% after 5 years of diagnosed primary cancer. The risk of recurrence is higher in patients with a personal history of eczema, dry skin, prolonged exposure to the sun or tanning devices with UV light, who have had deep skin carcinoma or a size greater than 2 cm.

Squamous cell skin cancer grows slowly, metastasizes less often, but can invade deep into the skin. This type of skin cancer develops from the flat squamous cells that form the superficial layer of the epidermis, and occurs mainly in the region of the neck, face, ears, external genitalia, dorsal area of the hands, etc. [123].

The exact number of the most common types of basal cell and squamous cell cancer (i.e., keratinocyte carcinoma or KC), known as non-melanoma skin cancer, is not known exactly, because it should not be reported in cancer registries [126].

Among Caucasian populations, squamous cell carcinoma (SCC) of the skin, also known as cutaneous squamous cell carcinoma (cSCC), accounts for 20% of all malignant skin tumors [127].

The incidence of squamous cell carcinoma (SCC) increased in recent decades by 10% per year, so today, the ratio is 1:1 compared to basal cell carcinoma (BCC) in populations in Australia and the US. The most important risk factors for disease or recurrence are chronic sun exposure, ultraviolet A (UVA) radiation, patients using immunosuppressive drugs, those with organ transplants, and the HIV-positive. The disease is more common in the elderly and men. The diagnosis of cSCC can be made especially based on the clinical aspect, which must be confirmed by histopathological examination to anticipate the correct prognosis and the management of therapy. The first intention treatment is the one of complete surgical excision, with histopathological control of the excision edges. When

lymph nodes are contained by cSCC, a regional dissection of the lymph nodes will be performed, followed by radiotherapy and various chemotherapeutic agents, and more recently, photodynamic therapy and epidermal growth factor receptor (EGFR) inhibitors [128–132].

Response to treatment is usually good; however, a subtype in the cSCC category needs special monitoring because it has a much higher risk of local recurrence, locoregional metastasis, or distant metastasis and death [133,134].

Although primary cSCC is not a fatal tumor, it can raise major cosmetic problems caused by localization in the face area (surgery can be disfiguring), increased morbidity, and high costs, which will place a significant burden on the public health system [124,131].

The prognosis can be very good, with a 90% survival rate for 10 years, when the disease is in its early stages; if the cancer is extensive or metastasized, survival is reduced to 15.3 months on average, making it the second leading cause of death from cSCC after melanoma in skin cancers [135,136].

Surgical treatment in this type of cancer is the first choice; however, if the tumor is enlarged, its resection will lead to an unsightly scar with a strong psychological impact. Chemotherapy has major disadvantages due to haematological, gastrointestinal, hepatic, or renal side effects, the emergence of drug resistance, and necrosis, fibrosis, secondary tumor formation on long-term administration; after radiotherapy, free radicals can be released that cause local inflammations, dermatitis, ulcers, etc. [137,138].

Given these aspects, it is very important to be found and select effective and safe means of treatment in skin cancer. In recent decades, PDT has established its position in the treatment protocol with predilection for skin cancer, including basal cell carcinoma, recurrent basal cell carcinoma, squamous cell carcinoma, Bowen's disease, actinic keratosis, etc. Depending on the photosensitizer and the light source, PDT triggers oxidative stress by generating ROS in cancer cells and produces their apoptosis [139–144].

Xin Y. et al. investigated the apoptotic effects and molecular mechanisms of action of a treatment combination of ultraviolet B radiation and demethoxycurcumin (DMC) in vitro on A431 and HaCaT cells. The association between demethoxycurcumin as a photosensitizer and ultraviolet B radiation induced apoptosis in vitro in A431 and HaCaT cells, by activating p53 and caspase pathways, increasing Bax and p-p65 expression and suppressing Bcl-2, Mcl-1 and NF-κB pathway. At the same time, high levels of reactive oxygen species were observed, along with the significant depolarization of the mitochondrial membrane [145].

Abdel Fadeel et al. demonstrated the beneficial effects of PDT with violet light (410 nm) and Cur-loaded PEGylated lipid nanoparticles in an in vitro study with increased cytotoxicity in cell culture, human squamous cell carcinoma cell line (A431), and in vivo skin carcinoma in mice [146].

The hypothesis of this study is supported by other researchers [147,148].

Cur can produce higher cytotoxicity when charged on a nanocarrier and, in addition, this quality can be increased by exposure to blue light radiation, therefore, together they will generate ROS that affect the activity of components cell and mitochondrial membrane that will lead to the apoptosis of cancer cells.

In patients with advanced cancer who are malnourished and stay in bed longer, but also in other categories of people who do not mobilize, especially in the elderly, the skin may degrade due to local compression, ischemic and reperfusion disorders [149].

Skin in that area initially becomes erythematous, then necrosis follows and bed-sores or pressure ulcers form. A recently published meta-analysis showed that, in adults, pressure ulcer (PU) has a prevalence of 12.8%, and the incidence rate is 5.4% per 10,000 patients per day, and the rate of PU acquired in hospital was 8.4% [150].

In addition to the high costs for patients and health services, these injuries can even be fatal through local and systemic infections. The conventional treatments with antibiotics, surgical measures, ultrasound, electromagnetic or genetic therapies have failed to completely cure PU in an advanced stage of evolution.

To avoid all these aspects, it seems that a much more beneficial option would be that of the concomitant use of laser radiation, together with curcumin.

The results of studies published so far on the concomitant use of curcumin and laser radiation demonstrate two interesting aspects, namely: laser light [151,152], can excessively increase ROS production, while curcumin is able to inhibit oxidative stress by reducing the activation of ROS-generating enzymes or by eliminating free radicals; these phenomena are very useful for the purpose of the therapy, when it is desired to induce apoptosis in skin cancer or to accelerate the healing process of PU [153,154].

Ebrahiminaseri et al., in an in vitro experiment on mouse MEFs cells using the association between dendrosomal nanocurcumin (DNC) and laser therapy at a dose of 0.95 J/cm<sup>2</sup> , demonstrated the significant migration of MEF cells in the denuded area, increase in the S-phase cell population and the growth factors (TGF-β, VEGF) and decrease in the cell population in the GF/G1 phase and of the pro-inflammatory cytokines (TNF-α, IL-6). This combined therapy (DNC + PDT) also highlighted the modulating role of nanocurcumin on the production and excessive accumulation of ROS generated by the action of the laser. In conclusion, the authors consider that additional in vivo studies are needed to confirm the hypothesis that the combined treatment of DNC simultaneously with PDT (450 nm) may favor the wound healing process [155].

Malignant melanoma is an aggressive form of cancer that results from the degeneration of melanocytes, cells of neuroectodermal origin with the role of melanin secretion as a protective response of the skin against the harmful action of ultraviolet sunlight.

Incidence of melanoma [156], mainly metastatic melanoma, is constantly increasing, especially after global warming.

Although current treatment is well established by surgical excision followed by chemotherapy, immunotherapy, recently based on inhibitors of immune checkpoints, small molecule-targeted therapy and oncolytic viral therapy, which have led to an increase in the survival rate of patients at 5 years; however, the incidence of metastases, the high number of deaths and the side effects of drugs require studies to find new therapeutic ways [157,158].

Szlasa et al., in an in vitro study focusing on the anticancer effect of PDT together with curcumin, revealed an increase in the number of apoptotic and necrotic cells by the overexpression of caspase-3, DNA cleavage and reorganization of the actin cytoskeleton, compared to incubation without irradiation. Fibroblasts have been less influenced by this treatment, which seems beneficial for the increased ability to regenerate irradiated skin areas. PDT, together with curcumin, can be an effective way to induce apoptosis in melanoma [159].

Nanoparticles of selenium-polyethylene glycol-curcumin (Se-PEG-Cur) used in an experimental study on melanoma cancer cells as a photosensitizer for phototherapy and sonotherapy greatly increased intracellular ROS levels and cytotoxicity and decreased cell viability [160].

The liposomal formulation of curcumin has been shown to be more effective in killing malignant cells in several studies. The increase in ROS generation and the photodestructive effects of PDT, together with turmeric tetraether liposomes on microvasculature, were observed by Duse et al. in an in vivo study using the chick chorioallantoic membrane model [121].

Research group led by Vetha et al. reported apoptosis of A549 cancer cells in vitro, by generating singlet oxygen, increasing intracellular ROS levels and activating Caspase-3, by using small CUR molecules, encapsulated in liposome nanoparticles (LIP-CUR) coupled with PDT via emitting diodes blue light (BLED) [161].

In an experimental study, Wo´zniak et al. demonstrated a significant improvement in the bioavailability and stability of encapsulated liposomal curcumin as a potent apoptotic photosensitizer in squamous cell carcinoma (SCC-25) and melanoma (MugMel2); at the same time, low phototoxicity was observed in normal cutaneous keratinocyte HaCaT cells after PDT treatment [162].

These results promote liposomal curcumin as a potential natural photosensitizer that can improve its absorption, safety and efficacy in photodynamic therapy in human cancers.

Table 2 highlights some of the effects of PDT and curcumin nanoformulations in skin cancers, by significantly improving the bioavailability and stability of nanocurcumin, increasing cytotoxicity on malignant cells, but with low phototoxicity on normal keratinocytes, the apoptotic effects and molecular mechanisms by generating high levels of ROS, along with the significant depolarization of mitochondrial membranes, while other in vitro experiments on mice demonstrated significant cell migration in the denuded area and highlighted the modulatory role of nanocurcumin on excessive production and the accumulation of ROS generated by laser irradiation [145,146,155,159,160,162].


**Table 2.** Nanocurcumin in skin cancer therapy.


#### **Table 2.** *Cont.*

#### *4.4. Gastrointestinal Cancers*

Incidence of gastrointestinal cancer varies over time, from short intervals of 8 years to longer intervals of 20 years, suggesting that it is under the influence of a predestined epigenetic program. It is assumed that, until the onset of cancer, there are several stages that include accumulations of precursor events, which makes the incidence of cancer higher after the age of 40 years [163].

In the natural process of human evolution from youth to old age, inherited native stem cells age and are replaced by new stem cells that are phenotypically labile and prone to turn into cancer cells. New, unstable stem cells can degenerate malign in a short time through a process of methylation in the so-called early exponential phase of accelerated carcinogenesis. In the colon, stem cells in glandular structures are replaced every 8 years [164–166].

Colorectal cancer has a very high incidence worldwide, ranking third and second in terms of cancer mortality. The incidence varies a lot depending on the level of socioeconomic development, geographical regions, food, cultural level, etc. The incidence is lower in the countries of Africa and South-Central Asia and much higher in Europe, Australia, and North America [167].

By expanding and deepening knowledge about the molecular biology and pathogenesis of cancer, the treatment of various tumors has greatly improved today. However, chemoresistance, recurrence rate and mortality are still far from being resolved. Therefore, ways of prevention, and especially new means of treatment with increased efficiency, are constantly being sought. Treatment for colon, gastric and many other cancers usually involves surgery to remove pathological tissue and locoregional lymph nodes, followed by radiotherapy and chemotherapy with conventional drugs and/or immunotherapy.

Vetha et al., in an in vitro experimental study on a CT26 murine colorectal carcinoma cell line, used 450 nm blue light diode-induced photodynamic therapy (BLED-PDT) at a power of 2.4 mW/cm<sup>2</sup> for 30 min (6.3 J/cm<sup>2</sup> ), together with F127-CUR micelles.

The authors report that cell line carcinoma treated with F127-CUR micelles together with BLED significantly reduced cell density and anticipate that this treatment may be promising for cancer eradication [168].

¸Sueki et al. have tested curcumin, which is a non-toxic compound that has antitumor properties, to check if it can increase the effectiveness of PDT on resistant cancer cells. For this purpose, PC-3 cancer line of prostate cancer cells and the Caco-2 cell lines of colon cancer were used, which were previously identified as non-resistant and resistant to PDT, respectively. Finally, the authors reported that 5-aminolevulinic acid (5-ALA) -mediated PDT, combined with curcumin, has improved the antitumor efficacy of PDT on cells Caco-2, which is considered a highly resistant cancer cell line [169].

In this scenario, the provision of new drugs and scientific treatment methods based on natural products is part of the research conducted by the group led by de Freitas et al., who studied in vitro effects on Caco-2 intestinal cancer cells treated with curcumin-conjugated silver nanoparticles (CUR-AgNPs) and PDT. The results of the study showed that PDT, in the presence of CUR incorporated into hydrogels consisting of CHT and CS, natural biopolymers, capable of the controlled release of CUR-AgNPs, led to the inhibition of human Caco-2 colon cancer cells [170].

Another attempt to obtain a superior photosensitizer in cancer cell therapy was performed by Tsai et al., which encapsulated curcumin, by crosslinking with chitosan, tripolyphosphate (TPP) and conjugation with epidermal growth factor to target the epidermal growth factor receptor (EGFR), overexpressed on cancer cells. The research targeted two cell lines that were used in this study, including a human gastric cancer cell line (MKN45) and the human gastric (non-cancerous) epithelial mucosa (GHG) cell line, which were irradiated with a light-emitting blue diode (460 nm, 5 ± 0.1 mW, measured on the sample surface) for 30 min, at a dose of 9 J/cm<sup>2</sup> . Curcumin-encapsulated chitosan/TPP nanoparticles showed a superior PDT effect in the cancer cells, with a four-fold decrease in IC50. This mode of therapy is promising against cancers that overexpress EGFR [171].

Human hepatoblastoma (HB) is the most common form of liver cancer in infants and children under 5 years of age [172].

To reduce the tumor, but especially to eradicate circulating tumor cells, chemotherapy is used before and after surgery. Despite all the positive results, some cases with extensive or recurrent tumors are a major problem, due to the emergence of drug resistance. Ellerkamp et al. investigated in vitro hepatoblastoma cell lines (HuH6, HepT1) and hepatocellular carcinoma cell lines (HepG2, HC-AFW1) treated with curcumin and exposed to blue light (480 nm, 300 W, for 10 s), and revealed decreased cell viability and a significantly increased level of ROS [173].

The emergence of multidrug resistance (MDR) is a major impediment to the long-term success of therapy against various cancers. It is already known that P-glycoprotein (P-gp) is a membrane transporter, which is ATP-dependent, and it has the function to drain the drug molecules from the cancer cell, so that chemotherapy is less effective. In their adaptive evolution, cancer cells protect themselves by increasing P-gp expression, thus avoiding chemotherapy-induced cellular degradation [174,175].

PDT has become an attractive method of treatment for hepatocellular carcinoma, because it is easy to administer and does not affect normal tissues.

To avoid the effects of chemoresistance, Li et al. have developed a new ICG & Cur @ MoS2 system (ICG and Cur represent indocyanine green and curcumin respectively) nanoplatform, which can perform photothermal-photodynamic therapy and inhibit P-gp efficiently and safely. The researchers used HepG-2 cells (human hepatoma cells) cultured in vitro with ICG and Cur @ MoS2 and irradiated with an 808 nm NIR laser at 2.0 W/cm<sup>2</sup> for 5 min to evaluate the photothermal effect. Acute toxicity was investigated in vivo in female mice, in which the tumor was irradiated with an 808 nm (1.2 W/cm<sup>2</sup> ) NIR laser for 5 min. Cell apoptosis was significant in the ICG @ MoS2 and NIR group, indicating that it was induced by heat and ROS. MRNA decreased significantly in the ICG and Cur @ MoS2 group, indicating that ICG and Cur @ MoS2 inhibited MDR1 transcription. In conclusion, the ICG and Cur @ MoS2 nanoparticles, under the action of PTT-PDT, have inhibited P-gp effectively, and may have great potential in the treatment of hepatocellular carcinoma [176].

#### *4.5. Lung Cancer*

Globally, lung cancer is one of the leading causes of death in both men and women. The small cell form represents about 80% of all types of lung cancer, and the one through which deaths are more common, because the diagnosis is generally made when the disease is quite advanced. After the approval of modern biological therapy, the number of deaths was substantially reduced. However, there are special problems because the disease continues to progress in most cases, due to the development of drug resistance [177–179].

As in other forms of cancer due to these problems, research continues to discover drugs or alternative ways to overcome the phenomenon of drug resistance.

Jiang et al. investigated the effects of the new Cur-SLN photosensitizer on A549 cells of small lung cancer cells irradiated with a 430 nm light-emitting diode (LED; power density 50 mW/cm<sup>2</sup> ) for 20 min. Cur-SLNs were obtained by emulsification and solidification at low temperature. The organic phase with 0.1 g lecithin, 0.15 g Cur and 0.2 g stearic acid was dissolved in 10 mL of chloroform. Moreover, 0.2 g of polyoxyethylene stearate (40) (Myrj52) dissolved in 30 mL of deionized water formed the aqueous phase. The organic phase was injected into the aqueous phase and stirred until the organic solvent disappeared. Then, in an environment at 0–2 ◦C, 10 mL of cold water were added and stirred at 1200 rpm. The supernatant was removed by centrifugation and the precipitate was washed twice with deionized water, resuspended in ultrapure water, and refrigerated at −80 ◦C, and finally lyophilized. The results revealed that Cur-SLN increased the expression of caspase-3, caspase-9, and promoted the Bax/Bcl-2 ratio, which demonstrated that this new Cur photosensitizer significantly induced apoptosis in A549 cells for this type of lung cancer [180].

In the last decade, the delivery agents of photosensitizing nano-carriers have been developed a lot, and those who respond very well to near-infrared (NIR) lasers have received special attention because they absorb light very strongly from 700–900 nm.

Local temperature released from the interaction of NIR light with the photosensitizer deeply destroys cancer cells, which gave high hopes for the beneficial effect of eradicating tumors; however, the damage to nearby tissues is still an unresolved issue.

Its outstanding optical properties and biocompatibility have helped Indocyanine Green (ICG) to be FDA approved as a photosensitizer for NIR in clinical use [25,181,182].

Huang et al. developed an antitumor product consisting of a green photosensitizer with indocyanine (ICG) that was co-encapsulated with curcumin (CUR) in liposomes (LPs), followed by conjugation of the GE11 peptide to provide targeting effects to cancer cells that express on their surface epidermal growth factor receptor (EGFR). A fresh medium containing CUR/ICG, free CUR/ICG-LPs and GE11-CUR/ICG-LPs was added to A549 non-small lung cancer cells line, HeLa human cervical cancer cells and LO2 human normal liver cells, incubated for 24 h, followed by 808 nm NIR laser irradiation, (1W/cm<sup>2</sup> ) for 0, 5, and 10 min. In this study, after the administration of PDT, the ICG photosensitizer generated an increase in temperature in the tumor area and, on the other hand, the GE11- CUR/ICG-LPs complex slowly released CUR, obtaining strong anticancer effects. The research results showed that GE11-CUR/ICG-LPs, together with PDT, could induce the apoptosis of cancer cells by promoting ROS generation and cell cytoskeleton disruption by the increased stimulation of apoptotic signaling pathways and the inhibition of the EGFR-mediated PI3K/AKT pathway [183].

Another research study to obtain a photosensitizer that is easier to administer and with more effective properties was completed by Baghdan et al. They obtained curcuminloaded PLGA nanoparticles (PLGA.CUR.NPs) as nano-in-microparticles for inhalation administration. In vitro irradiation results on human lung epithelial carcinoma cells (A549) showed a significant increase in cellular phototoxicity, but dependent on the radiation dose used by the 457 nm LED device. In short, the authors argue that nano-in-microparticles are promising carriers of drugs for the photodynamic therapy of lung cancer [184].

#### *4.6. Other Cancers*

Glioblastoma multiforme accounts for approximately 80% of primary malignant brain tumors. Despite current innovations in cancer treatment, this type of tumor has a disappointing prognosis [185].

From the results of several randomized international studies reported so far, it can be seen that they have failed to achieve convincing results with long-term survival after immunotherapy [186,187].

Because there is no reasonable treatment in glioblastoma multiforme, the use of PDT with different CUR nanoparticles is being tested experimentally in vitro. In a recent research study, Kielbik et al. analyzed the cytotoxic effects of CUR alone and as a photosensitizer on glioblastoma SNB-19 cells. After incubation with CUR and irradiation with blue light (6 J/cm<sup>2</sup> ), over 90% of glioblastoma SNB-19 cells underwent apoptosis [188].

Jamali et al. used biodegradable polymeric nanoparticles (PLGA NPs) conjugated with the anti-EGFRvIII monoclonal antibody (MAb-CUR-PLGA NPs) to enhance the photodynamic action of CUR on overexpressed EGFRvIII cells (DKMG/EGFRvIII cells) of glioblastoma tumors. The results demonstrated in vitro that following PDT, the photocytotoxicity of MAb-CUR-PLGA NPs was significantly higher than that of CUR-PLGA NPs in the DKMG/EGFRvIII glioma cells. The authors propose that anti-EGFRvIII MAb-CUR-PLGA NPs should be used as a PDT-specific photosensitizer in overexpressed EGFRvIII tumor cells [189].

Curcumin has been used as a PS in PDT and has been the subject of numerous studies, due to its valuable antiviral, antimicrobial, and especially antineoplastic effects in various forms of human cancer [190–192].

of cancer.

Kazantzis et al. investigated the natural characteristics of bisdemethoxy curcumin regarding their efficacy together with PDT in vitro on prostate cancer cells. In these experiments, all four curcuminoids released enough ROS that produced cytotoxicity after PDT, with a significant reduction in prostate cancer LNCaP cells [193]. *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 28 of 39 **5. Final Remarks and Conclusions** 

#### **5. Final Remarks and Conclusions** As revealed by all the above-mentioned revised studies, the latest nanoformulations

As revealed by all the above-mentioned revised studies, the latest nanoformulations of curcumin applied in PDT illustrate its complex actions on tumor cells, as shown in Figure 4. of curcumin applied in PDT illustrate its complex actions on tumor cells, as shown in Figure 4.

**Figure 4.** Effects of latest innovations and nanotechnologies with curcumin applied in the photodynamic therapy (PDT) **Figure 4.** Effects of latest innovations and nanotechnologies with curcumin applied in the photodynamic therapy (PDT) of cancer.

Although curcumin is a polyphenol that has been used in Ayurvedic medicine for many years, as well as a spice for food fragrance, food coloring or adjuvant, its pharmacological complex, anti-inflammatory, antiviral, anti-bacterial, antifungal, anti-neurodegenerative, and its anti-cancer properties have been intensively investigated in recent years. Therapeutic applications of curcumin have been limited, due to its extremely low solubility, instability in body fluids and rapid metabolism. Nanomedicine and the latest nanotechnologies have shown excellent potential to improve the solubility, biocompati-Although curcumin is a polyphenol that has been used in Ayurvedic medicine for many years, as well as a spice for food fragrance, food coloring or adjuvant, its pharmacological complex, anti-inflammatory, antiviral, anti-bacterial, antifungal, antineurodegenerative, and its anti-cancer properties have been intensively investigated in recent years. Therapeutic applications of curcumin have been limited, due to its extremely low solubility, instability in body fluids and rapid metabolism. Nanomedicine and the latest nanotechnologies have shown excellent potential to improve the solubility, biocompatibility and therapeutic effects of curcumin.

bility and therapeutic effects of curcumin. State-of-the-art research presented here in the photodynamic therapy of various forms of cancer has been able to highlight the significant anticancer potential of curcumin State-of-the-art research presented here in the photodynamic therapy of various forms of cancer has been able to highlight the significant anticancer potential of curcumin as a valuable photosensitizer when it is combined with nanotechnologies.

as a valuable photosensitizer when it is combined with nanotechnologies. In all *in vitro* or *in vivo* experiments performed for the study of curcumin nanoformulations, it has been shown to have a high encapsulation efficiency and long-term controlled release, increasing its solubility, bioavailability and biocompatibility, as well as In all in vitro or in vivo experiments performed for the study of curcumin nanoformulations, it has been shown to have a high encapsulation efficiency and long-term controlled release, increasing its solubility, bioavailability and biocompatibility, as well as stability and long-down circulation.

stability and long-down circulation. Curcumin incorporated into nanotechnologies has a higher intracellular absorption, a superior targeting rate, increased toxicity to cancer cells, accelerates the activity of caspa-Curcumin incorporated into nanotechnologies has a higher intracellular absorption, a superior targeting rate, increased toxicity to cancer cells, accelerates the activity of caspases and DNA cleavage, and ultimately induces apoptosis.

ses and DNA cleavage, and ultimately induces apoptosis. PDT applied together with nanoformulations of curcumin, diminishes the mitochondrial activity of cancer cells, decreases the viability and proliferation of cancer cells, drops PDT applied together with nanoformulations of curcumin, diminishes the mitochondrial activity of cancer cells, decreases the viability and proliferation of cancer cells, drops the angiogenesis, the cancer cells' mobility and metastases.

> The experiments included in this review showed that PDT and nano-curcumin reduce the size of the primary tumor and invert the multidrug resistance in chemotherapy

the angiogenesis, the cancer cells' mobility and metastases.

The experiments included in this review showed that PDT and nano-curcumin reduce the size of the primary tumor and invert the multidrug resistance in chemotherapy and decrease the resistance to radiotherapy in neoplasms.

PDT applied with curcumin nanoformulations has been shown to have no significant phototoxic effects on healthy cells near the treated tumor.

Regarding breast cancer that came first in terms of incidence, recent experimental research has shown that nanoformulations of curcumin together with PDT can contribute to its treatment by deep infiltration into cancer cells, high cytotoxic action, shrinking tumor volume and stopping metastases.

Current research has shown that the use of PDT and curcumin nanoformulations have a modulating effect on ROS generation, so light or laser irradiation will lead to excessive ROS growth, while nano-curcumin will reduce the activation of ROS-producing enzymes, or will determine the elimination of ROS, seemingly opposite but synergistic phenomena by inducing neoplasm apoptosis, but at the same time, accelerating nearby tissue repair.

All studies reviewed and presented revealed the great potential for applicability of PDT and curcumin nanoformulations in cancer therapy.

This review paves the way for further investigations and advances in this field.

**Author Contributions:** Conceptualization, L.M.A. and C.A.; writing and original draft preparation, L.M.A.; review and editing, G.L., L.M.A., and C.A.; scientific organizational manuscript issues G.L. All authors (L.M.A., C.A. and G.L.) have read and agreed to the published version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The references used to support the findings of this study are available from the first (L.M.A.) and the corresponding (G.L.) authors upon request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**




### **References**


## *Review* **Liposome Photosensitizer Formulations for Effective Cancer Photodynamic Therapy**

**Sherif Ashraf Fahmy 1,2 , Hassan Mohamed El-Said Azzazy 1,\* and Jens Schaefer 3,\***


**Abstract:** Photodynamic therapy (PDT) is a promising non-invasive strategy in the fight against that which circumvents the systemic toxic effects of chemotherapeutics. It relies on photosensitizers (PSs), which are photoactivated by light irradiation and interaction with molecular oxygen. This generates highly reactive oxygen species (such as <sup>1</sup>O<sup>2</sup> , H2O<sup>2</sup> , O<sup>2</sup> , ·OH), which kill cancer cells by necrosis or apoptosis. Despite the promising effects of PDT in cancer treatment, it still suffers from several shortcomings, such as poor biodistribution of hydrophobic PSs, low cellular uptake, and low efficacy in treating bulky or deep tumors. Hence, various nanoplatforms have been developed to increase PDT treatment effectiveness and minimize off-target adverse effects. Liposomes showed great potential in accommodating different PSs, chemotherapeutic drugs, and other therapeutically active molecules. Here, we review the state-of-the-art in encapsulating PSs alone or combined with other chemotherapeutic drugs into liposomes for effective tumor PDT.

**Keywords:** photodynamic therapy; photosensitizers; liposomes; stealth liposomes; thermosensitive liposomes; tetraether lipids; cancer

## **1. Introduction**

One of the fundamental challenges in designing successful tumor-targeting approaches is the selective delivery of anticancer drugs to cancerous cells. Although both the cancerous and healthy tissues are impacted by the cytotoxic effects of anticancer drugs, most targeting approaches depend on the fact that the rapidly proliferating cancer cells would be more affected by chemotherapeutics than healthy ones [1–4]. This warrants the development of novel targeted delivery systems capable of selectively eradicating cancerous cells. Various drug delivery vehicles and nanocarriers have been developed in this context, including polymeric and metal nanoparticles, supramolecular nanocapsules, liposomes, host-guest complexes, and nanofibers [1,2,5,6]. Multimodal systems were designed by exploiting the capability of the nanosystems to co-deliver chemotherapeutic drugs and targeting entities, resulting in more efficient treatment.

Photodynamic therapy (PDT) employs pharmacologically inactive photosensitizers activated upon exposure to light in the presence of oxygen. It is a non-invasive therapeutic approach that does not require sophisticated equipment or setup and has been employed to treat cancer, cardiovascular, and skin diseases [5–15]. This review presents the basic principles and current challenges of using PDT in cancer therapy and state-of-the-art approaches in formulating liposome photosensitizers to improve the therapeutic significance of PDT.

**Citation:** Fahmy, S.A.; Azzazy, H.M.E.-S.; Schaefer, J. Liposome Photosensitizer Formulations for Effective Cancer Photodynamic Therapy. *Pharmaceutics* **2021**, *13*, 1345. https://doi.org/10.3390/ pharmaceutics13091345

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 20 July 2021 Accepted: 24 August 2021 Published: 27 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Photodynamic Therapy in Treating Cancer**

PDT was approved in several countries for treating cancer after its approval for recurrent bladder cancer treatment by the Canadian Health Protection Branch [1]. PDT depends on the interaction of a photosensitizer (PS), delivered to target tissues, and with the light of a specific wavelength in the presence of molecular oxygen dissolved in the cytoplasm [16–22]. Upon light absorption, the PS molecules are transferred from the ground state to an excited singlet state and then to a triplet excited state via the intersystem crossing. In the triplet excited state, the PS undergoes two simultaneous reactions (1) Type I electron transfer reactions which involve the direct reaction of PS with cell components forming anionic or cationic radicals that react with molecular oxygen generating ROS, and (2) Type II energy transfer reactions which involve direct reaction of PS with molecular oxygen producing singlet oxygen (1O2) (Figure 1). The generated highly reactive ROS (such as <sup>1</sup>O2, H2O2, O2, ·OH) exert their cytotoxic effects via irreversible oxidation of the cellular and subcellular organelles and induce apoptosis or necrosis, leading to cell death. ROS can also induce autophagy by several mechanisms, leading to cytoprotective and cell killing responses [23–25].

**Figure 1.** Mechanism of action of PDT demonstrating Type I and Type II reactions. PSEs, PS excited singlet state; PSEt, PS excited triplet state; ROS, reactive oxygen species. Reprinted with permission from ref. [26]. 2016 Calixto et al.

Many parameters influence the effectiveness of PDT. These include the type and dose of PS, route of administration, intensity of light used, type of tumor, and concentration of dissolved cytoplasmic oxygen. The ideal PSs should be pharmacologically inactive in the absence of light irradiation, pure, water-soluble, and selectively present in tumor cells. It should have an absorption spectrum preferably between 650 and 800 nm and rapid elimination rates. Two classes of PSs exist: Porphyrin PSs (three generations) and nonporphyrin PSs [27]. First-generation PSs (such as hematoporphyrins) have been developed and undergone clinical trials for more than 40 years [16]. However, they suffered various drawbacks, including (i) poor tissue penetration, (ii) low chemical stability, (iii) activation when irradiated with wavelengths below 650 nm, (iv) causing skin hypersensitivity reactions, and (v) low elimination rates and long half-lives. Most of the issues attributed to hematoporphyrins refer mainly to Photofrin. This is a complex mixture of porphyrin dimers and higher oligomers, some of which persist in the skin and result in skin photosensitization. Second-generation PSs (such as metalloporphyrins, porphycenes, purpurins, chlorins, protoporphyrins) was developed to overcome most of these shortcomings [17]. 5- Aminolevulinic acid (ALA), methyl aminolevulinate (MAL; Metvix), and Hexvix/Cysview are precursors of protoporphyrin IX, which absorbs at 630 nm [28,29]. They were granted FDA approvals and are used to treat glioblastoma, basal cell carcinoma, Bowen disease, prostate, bladder, and colon cancers [30–32]. Meta-tetrahydroxy phenyl chlorin (m-THPC,

temoporfin), excitation wavelength of ~652 nm, is approved in the EU to treat biliary and pancreatic cancers and breast cancer metastases. Verteporfin (Visudyne), a benzoporphyrin derivative (BPD), with an excitation wavelength of 690 nm, has been granted FDA approval to treat choroidal hemangioma and gastric cancer. Additionally, hypericin (excitation wavelength of 570–650 nm) is an FDA-approved PS for treating skin cancers (Figure 2) [33–35]. Recently, third-generation PSs have been developed by conjugation of second-generation PSs to biological targeting moieties, such as carbohydrates, peptides, or antibodies. This would enhance the selectivity of PSs and minimize undesired adverse effects [29,36]. Non-porphyrin PSs include psoralens, anthracyclines, chalcogenopyrylium dyes, cyanines, and phenothiazinium dyes. Despite the promising effects of PDT in cancer treatment, it still suffers from several shortcomings. These include poor biodistribution of hydrophobic PSs, poor cellular uptake, difficulty applying PDT to deep cancer tissues (hindering light penetration), and low selectivity to cancer cells [37,38]. In addition, cancer cells' oxygenation is essential to achieving effective PDT. Because cancer tissues are bordered by necrotic cells, and compact tumor masses, it is challenging to treat deep cancers using the traditional PDT. Because of the low tissue penetration of visible light, PDT is only effective for treating superficial, and skin tumors [26]. Furthermore, using photofrin may cause long-lasting photosensitivity reactions. Consequently, encapsulation of PSs within different nanocarriers has been proposed to overcome traditional PDT's shortcomings, such as improving water solubility, bioavailability, and selective targeting of PSs [39].

**Figure 2.** (**A**) General principle of photodynamic therapy; (**B**) FDA and/or EU-approved photosensitizers in cancer photodynamic therapy; (**C**) Chemical structures of approved photosensitizers. PS, photosensitizer; PS\*, photosensitizer excited state.

#### **3. Liposomal Photosensitizer Formulations for Tumor Photodynamic Therapy**

PSs have entirely or partially been encapsulated, conjugated, or immobilized onto different nanocarriers. Multimodal integration of nanocarriers, such as polymeric nanoparticles, silica nanoparticles, and nanofibers, with PSs, offers several advantages compared to traditional PDT [1–4]. These include: (i) increasing the solubility of hydrophobic PSs, and hence, improving their biodistribution, pharmacokinetics, and cellular uptake; (ii) maintaining constant release rates of PSs at the targeted tumor cells; (iii) the ability to selectively target high loading capacity enabled by the substantial surface-to-volume ratios of nanocarriers which facilitate their surface decoration with particular ligands that can target the overexpressed receptors and proteins in tumor tissues without off-target toxic effects; (iv) boosting the preferential accumulation of PSs into cancer cells via the enhanced permeability and retention (EPR) effect; and (v) expanding the clinical applications of PDT to include additional types of cancer [40–48].

Liposomes are promising nanoplatforms that could be integrated with PDT to enhance the eradication of cancer cells without affecting healthy ones. They are spherical vesicles that consist of a hydrophilic head and a hydrophobic tail. They are self-assembled in an aqueous medium, with the assistance of hydrophobic interaction, developing a sealed structure formed of one or more lamella having the hydrophilic heads oriented towards the outer surfaces of the lamella and the hydrophobic tails founding the lamella interiors. This forms an aqueous core inside the liposomes (Figure 3) [49,50]. Liposomes are prepared using diverse types of naturally occurring phospholipids, which are biocompatible and biodegradable (Table 1). The lipid composition plays a vital role in the stability of liposomes in the systemic circulation, drugs encapsulation efficiencies, and drug release at tumor sites. For instance, the use of DSPC in the liposomal formulation results in improved encapsulation efficiency and stability compared to EPC and DPPC. This is because of the lengthy fatty acid chain of DSPC and the rigidity of the acyl chains of DSPC. Moreover, the use of cholesterol enhances liposomal stability and inhibits the undesirable drug in the systemic circulation. The inclusion of DPPE mPEG5000 in the lipid composition prolongs the blood circulation time of the liposomes, due to the additional steric hindrance created. This reduces liposomal uptake by the reticuloendothelial system. Moreover, the fusogenic features of some lipids (such as DPPG) in the liposomal membrane are reported to improve the ability of liposomes to cross the cancer cell membrane [51,52].

**Figure 3.** Liposomes are spherical vesicles with an aqueous inner core enclosed by one or more phospholipid bilayers which permit the conjugation of various functional groups. Reprinted with permission from ref. [49]. 2020 Almeida et al.


**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations. **Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations. **Lipid Name Chemical Structure Charge Lipid Name Chemical Structure Charge Lipid Name Chemical Structure Charge Lipid Name Chemical Structure Charge Lipid Name Chemical Structure Charge Lipid Name Chemical Structure Charge Lipid Name Chemical Structure Charge** 

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 19

**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations.

**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations.

**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations.

**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations.

**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations.

**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations.

**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations.

**Table 1.** Chemical structure and charge of the most common lipids used in liposomal formulations.

glycol)-2000] (sodium salt) Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, Liposomes have unique biological and physicochemical properties setting them apart from other nanoparticles. They are prepared by the self-assembly of phospholipids and/or tetraethers lipids which are biocompatible and biodegradable [44]. Moreover, liposomes can: (i) Accommodate hydrophilic and hydrophobic agents (such as proteins, nucleic acids, chemotherapeutics, and various PSs); (ii) be decorated and bioconjugated with various functional groups and surface targeting moieties (such as proteins, polymers, and peptides), which would improve their physicochemical properties, drug loading, clearance and/or trigger the release of their cargos into the target tissue; (iii) minimize the opsonization phe-

nomenon by being less identifiable by the reticuloendothelial system leading to prolonging the half-life of the host molecules in the systemic circulation (>48 h) and facilitating the preferential passive accumulation inside tumor tissues; and (iv) widen the therapeutic index for most drugs (Figure 3) [44–49]. Liposomes can enter the cancer cells either by endocytosis or via membrane fusion. For instance, liposomes containing anionic phospholipids display faster endocytosis which augments their intracellular uptake. Moreover, liposomes containing fusogenic lipids demonstrate the ability to fuse and penetrate the cancer cell membrane. A recent publication examined the pathways of cellular internalization of liposomes [50].

It is worth mentioning that proteins in the circulation adsorb to liposomes administered systemically, leading to the formation of a protein corona that interacts with immunoglobulins, complement proteins, and phagocytes in the circulation. This would stimulate cytokine production in the tumor microenvironment leading to adaptive antitumor immunity. Moreover, the interaction of liposomes with serum proteins (specially opsonins) plays an essential role in the rapid clearance of liposomal by phagocytes in the blood, liver, and spleen. PEGylation slows the release of liposomes leading to prolongation of their half-lives [53,54].

Various liposomal formulations which improved the physicochemical properties and pharmacokinetics of cancer drugs have received FDA approval (Table 2). Similar liposomal formulations encapsulating PSs can be investigated for improved cancer PDT.


**Table 2.** FDA-approved liposomal-based formulations in cancer treatment [52].

#### *3.1. Liposomes for Photodynamic Tumor Therapy*

Several liposomes encapsulating anticancer drugs and carrying targeting moieties (such as mab, glutathione, EndoTAG-1, and transferrin) are currently in clinical trials [55]. In PDT, liposomes can target the loaded PSs to cancer cells via active or passive mechanisms. Active targeting is achieved by decorating the liposomal surface with ligands (such as antibodies) that recognize and bind to overexpressed receptors and proteins in tumor tissues, such as folate, estrogen, spermine, and galactose receptors. Passive targeting takes place via the EPR effect. Blood vessels in healthy tissues are organized and tightly packed, which prevents the extravasation of liposomes. On the other hand, the blood vessels of tumor tissues are disorganized, due to the rapid proliferation of the vascular endothelium

inside the tumor tissues [44–56]. Furthermore, the lymphatic drainage is impaired inside the tumor tissue, resulting in the overexpression of permeability mediators, such as bradykinin, nitric oxide, and prostaglandins, which increase EPR [56]. Consequently, nanoliposomes (100–300 nm) can passively cross the loose tumor endothelial barrier through the small open junctions and accumulate inside tumor tissues by the EPR effect [44,56].

Several recent studies have reported the design, optimization, and use of various types of liposomes as nanocarriers for PSs alone or combined with other chemotherapeutic agents, for effective cancer PDT [38,39].

#### 3.1.1. Tetraether Lipid-Based Liposomes

Tetraether lipids have longer lipophilic chains that contain ethers bonds, and hence, show high stability and lower susceptibility to oxidation. Thus, the use of tetraether lipid-based liposomes can increase liposomal membrane stability and integrity [45,56].

Curcumin, with an absorption spectrum of 300–500 nm, is a promising natural PS that could be used safely (at doses up to 12 g/kg/day) in PDT of superficial tumors. Reported studies demonstrate the ability of cancer cell lines to preferentially uptake a curcumin composite compared to normal cell lines. Upon its photoactivation, curcumin (in micromolar concentrations) produces capable of killing cancer cells to treat local superficial infections and cancers [57,58]. Duse et al. developed tetraether lipid-based (TELs) liposomes loaded with curcumin to kill cancer cells selectively [59]. Liposomes were designed by preparing a molar ratio of 90 DSPC:10 TELs using the thin-film hydration method followed by sonication at 56 ◦C. The curcumin-loaded liposomes had a size of about 208 nm, a zeta potential of −5.9 ± mV, % encapsulation efficiency (%EE) of 91%, and pronounced stability attributed to the tetraether lipids, which are reported to increase membrane stability and integrity. Furthermore, the curcumin liposomes showed the highest photocytotoxicity on SK-OV-3 ovarian carcinoma cells with a radiation fluence of 13.2 J/cm<sup>2</sup> for the light-induced PDT (IC50 of 8.7 µM). Moreover, the prepared curcumin-loaded liposomes were found to have a minimal hemolytic effect (<40%) and a coagulation time of only 9.7 s compared to 112 s in the case of free curcumin [59].

The in vitro anticancer activities of the prepared curcumin-TEL liposomes were further investigated against cervical cancer cells and papillomavirus-related cancer cell lines [60]. The cancer cell lines were incubated with the designed liposomes at various concentrations ranging from 0 to 100 µmol/L for 4 h followed by LED irradiation at 457 nm for 45, 136, and 227 s at a fluence of 1, 3, and 5 J/cm<sup>2</sup> . The cytotoxic effects were then evaluated using the MTT, SYTO9/PI (propidium iodide), Annexin V-FITC (fluorescein isothiocyanate), clonogenic survival, and scratch (wound closure) assays against three different cancer cell lines (HeLa, UD-SCC-2, and VX2). The findings showed enhanced cytotoxicity at a light fluence of 3 J/cm<sup>2</sup> (IC50 values of 9.52 µmol/L, 7.88 µmol/L, and 20.70 µmol/L against HeLa, UD-SCC-2, and VX2 cancer cell lines, respectively), reduced colony formation, proliferation, and cell migration rates. Liposomes loaded with curcumin was suggested as an efficient treatment of papillomavirus-associated cancers.

Plenagl et al. reported tetraether liposomes prepared with a molar ratio of 90 DPPC: 10 TELs encapsulating either hypericin (Hyp-TEL) using thin-film hydration method or hypericin-hydroxypropyl-β-cyclodextrin inclusion complex (HPCD-Hyp-TEL) through dehydration-rehydration vesicle technique [61]. The inclusion of hypericin in hydroxypropyl-β-cyclodextrin in liposomes prevents its photodegradation. The designed Hyp-TEL had an average particle size of 127 ± 14 nm, a zeta potential of −2 ± 1 mV, % encapsulation efficiency (%EE) of 82.5 ± 2.8. At the same time, the HPCD-Hyp-TEL had an average particle size of about 212 ± 13 nm, a zeta potential of −56 ± 1 mV, % encapsulation efficiency (%EE) of 81.6 ± 3.2. The %EE of hypericin was enhanced by increasing the liposomal membrane stability upon the addition of TEL [62]. The phototoxic effect of the prepared liposomes was evaluated on SK-OV-3 cancer cells using an LED fluence of 12.4 J/cm<sup>2</sup> . The IC50 values of Hyp-TEL and HPCD-Hyp-TEL were found to be 48 and 136 nM, respectively. Furthermore, considerable amounts of hypericin were uptaken by

cancer cells as supported by confocal laser scanning microscopy results. The designed liposomes were hemocompatible and showed a coagulation time range of 5–12 s. These results support the use of integrated liposomal/PDT in cancer-targeted therapy.

Tetraether liposomes were also used to encapsulate protoporphyrin IX PS for vascular targeting and cancer treatment using PDT [63]. The liposomes, designed using 62 mol% TELs, had an average size of 170 nm and an average zeta potential −42 mV. A significant improvement of protoporphyrin IX phototoxicity (IC50 of 5 µM), when loaded in TEL liposomes and tested against SK-OV-3 cancer cells (irradiated with LED light of 672 mJ/cm<sup>2</sup> ). On the other hand, the protoporphyrin IX-loaded TEL liposomes showed lower phototoxicity (IC50 of 12 µM) when tested on mouse fibroblasts.

Ali et al. reported developing different liposomal formulations accommodating temoporfin (second-generation, synthetic, effective PS) with enhanced phototoxicity against SK-OV-3 cancer cells [64]. Three liposomal formulations loaded with temoporfin (mTHPC) were prepared with molar ratios of 90 DPPC: 10 Cholesterol (DPPC/Chol/T), 95 DPPC: 5 DPPE–mPEG5000 (DPPC/DPPE mPEG5000/T), and 90 DPPC: 10 TEL (DPPC/TEL/T). The prepared liposomes had an average size range of 115 nm and zeta potentials ranging from −6.0 to −13.7 mV. The %EE was 78 ± 4, 81.7 ± 3, and 90 ± 3 % for DPPC/Chol/T, DPPC/DPPE mPEG5000/T and DPPC/TEL/T, respectively. The cell viability of SK-OV-3 cancer cells was reduced to 20% in the three liposomal formulations loaded with temoporfin when exposed to LED light of 10 J/cm<sup>2</sup> . Moreover, all the designed liposomal formulations exhibited hemocompatibility (<10% hemolysis) and a coagulation time <40 s.

#### 3.1.2. Stealth Liposomes

Stealth liposomes (PEGylated liposomes) are developed by the adsorption of PEGylated lipids (e.g., DPPE mPEG5000) on the liposomal surface, which prolongs the half-life of liposomes in circulation [65]. Steric hindrance occurs due to PEG reduces liposomal uptake by the reticuloendothelial system and their elimination via renal globular filtration. Moreover, the inclusion of the PEGylated lipids in the liposomal formulations improves their hydrophilicity, and hence, their shelf life [65].

In a different study, curcumin was loaded into liposomes comprising 9.5 HSPC: 0.5 DPPE mPEG5000 HCPC inhibits the leakage of curcumin outside the lipid membranes by imparting rigidity to the liposomal bilayer membrane [66]. The prepared liposomes exhibited significant phototoxic activity against skin cancer melanoma cells (MUG-Mel2) and squamous cell carcinoma (SCC-25) compared to free curcumin after exposure to LED light fluence of 2.5 J/cm<sup>2</sup> . The cytotoxicities were 53% (against MUG-Mel2) and 58% (against SCC-25) for curcumin-loaded liposomal mediated PDT compared to 27% (against MUG-Mel2) and 34% (against SCC-25) for free curcumin (10 µM) mediated PDT. The cytotoxic activity of the liposomal preparation against normal keratinocyte cells (HaCaT) was 11%, highlighting the safety of the designed liposomes. Flow cytometry showed that integration of PDT with liposome encapsulating curcumin increased apoptosis to 30% and 40% in MUG-Mel2 and SCC-25 cancer cells, respectively.

A study conducted by Corato et al. loaded iron oxide nanoparticles in the aqueous liposomal core and mTHPC PS in the lipid bilayer [67]. The dual-loaded liposomes were prepared with a molar ratio of 85 DPPC: 10 DSPC: 5 DPPE mPEG5000, which was then mixed with 3.3 mg/mL mTHPC and 0.7 M iron oxide NPs utilizing the reverse-phase evaporation approach. The prepared magneto-photoresponsive liposomes had a spherical structure, an average particle diameter of 150 nm, and one-month stability at 4 ◦C. The in vitro cell viability was assessed against SK-OV-3 cancer cells after exposure to either magnetic hyperthermia alone, PDT alone, or combined therapies. The % cell viabilities were 10% after exposure to magnetic hyperthermia alone, 5 and 1% after exposure to PDT alone (at 5 and 10 J, respectively), 0.2 and 0% after treatment with the liposomes loaded with iron oxide in the core and mTHPC in the aqueous layer/PDT (at 5 and 10 J, respectively). As supported by proteomic analysis, the dual-loaded liposomes seem to activate intrinsic apoptotic pathways by increasing ROS levels and mitochondrial damage.

Fisher et al. reported the design of lapatinib-loaded PEGylated liposomes for low-dose PDT of the invasive and resistant glioma [68]. Lapatinib is an antineoplastic, clinically approved PS that acts as an epidermal growth factor receptor (EGFR) inhibitor; however, it has poor penetration into glioma cells. PEGylated liposomes were prepared by mixing DPPC: DOTAP (1,2-dioleoyl-3-trimethylammonium-propane): cholesterol: DSPE-mPEG<sup>2000</sup> at a molar ratio of 0.6:0.079:0.289:0.031. The prepared liposomes had an average particle diameter of 132 ± 9 nm, a zeta potential of 14.3 ± 0.8 mV, and %EE of 64.5 ± 8%. The moderately positive charge aids electrostatic attraction of liposomes to the anionic cells leading to increased lapatinib uptake. The phototoxicity of the nanoformulation loaded with lapatinib and ALA (the latter is metabolized in vivo to protoporphyrin IX) was assessed against human glioblastoma cancer cell lines (U87 and U87vIII) irradiated with 635 nm light provided by a laser at a light dose of 65 mW/cm<sup>2</sup> . A remarkable reduction (46%) in the LD50 in the case of lapatinib-loaded liposomes compared to the free lapatinib was observed.

Peng et al. designed long-circulating dual-loaded PEGylated liposomes loaded with Chlorin e6 PS and cisplatin, a first-generation platinum-based chemotherapeutic drug (PL-Ce6-Cis) [69]. In this context, the hydrophilic cisplatin was loaded in the aqueous liposomal core, while hydrophobic Ce6 was incorporated in the outer lipid bilayer. The dual-loaded PEGylated liposomes were prepared with a molar ratio of 10 DSPC:0.2 DSPE-PEG2000:5 cholesterol using the ethanol injection approach. A single dose of the dualloaded liposomes integrated with light irradiation with a fluence of 100 J/cm<sup>2</sup> could kill 80% of C26 colon cancer cells (compared to only 20% without irradiation), while maintaining minimal adverse toxic effects.

Another recent study reported the use of combined chemotherapy and PDT using dual-loaded PL-Ce6-Cis to treat malignant peripheral nerve sheath cancer (MPNST) [70]. In this regard, the cytotoxicity assessment was conducted against three different MPNST cancer cell lines (T265, ST8814, and S462-TY), derived from NF1 patients using a 662 nm diode laser with a power intensity of 95 mW. The cell viability was reduced significantly (<40%) for PL-Ce6-Cis compared to PL-Ce6 or PL-Cis. Moreover, the PL-Ce6-Cis dualmodal formulation was found to exert minimal neurotoxicity.

#### 3.1.3. Thermosensitive Liposomes (TSLs)

Thermosensitive liposomes (TSLs) are used for triggered release delivery into solid tumors, with at least one formulation currently under phase III clinical trials [71–74]. Exposure to mild hyperthermia (39–43 ◦C), causes a phase change of the liposomal lipid bilayer from a solid gel well-arranged phase (Lβ) to a fluid tangled phase (Lα). This results in enhanced membrane permeability, thus facilitating the release of the liposomal cargo into cancer cells [75–77]. It is of note that the lipid composition of liposomes is key in the TSLs temperature-triggered drug release [78]. Drug release occurs at temperatures equal or lower (by 1–2 ◦C) to the melting phase transition temperature (Tm) of lipids where structural deformations in the lipid membrane increase the membrane permeability of the lipid bilayer, facilitating the release of the liposomal drug content [79,80]. DPPC, for instance, has a Tm slightly higher than normal body temperature (about 41 ◦C), and is, thus, used for TSL systems [78].

Shemesh et al. developed an integrated thermosensitive liposomes (TSLs)/PDT encapsulating indocyanine green PS (ICG) to treat triple-negative breast cancer [81]. The TSLs are designed by mixing a molar ratio of 100 DPPC:50 SoyPC (l-α-phosphatidylcholine):30 Chol:0.5 DSPE-PEG 2000 using the thin-film hydration method. The prepared TSLs had an average particle diameter of 71 ± 10 nm and could encapsulate ~500 µM of ICG. The TSLs loaded with ICG (37.5 µM) demonstrated significant cytotoxicity (cell viability < 20%) against MDA-MB-468 and HCC-1806 cells at laser radiation of 14 J/cm<sup>2</sup> compared to free ICG [81].

Meng et al. combined chemotherapy, immunotherapy, and PDT to treat gastric cancer [82]. Thermosensitive liposomes were loaded with IR820 PS, paclitaxel anticancer drug,

and imiquimod (R837) (Figure 4). IR820 PS is derived from ICG and is more stable, less expensive, and suitable for photothermal and photodynamic therapies [83,84]. Imiquimod (R837), a dendritic cell activator, is an agonist to TLR7 and has immunomodulatory activities [85]. Mouse fore-stomach carcinoma cell (MFC) treated with the designed liposomes and irradiated with 808 nm light provided by a laser at a light dose of 2.5 W/cm<sup>2</sup> showed that the cell viability decreased to 10% (Figure 5).

**Figure 4.** Thermosensitive liposomes loaded with IR820 PS, paclitaxel, and imiquimod (R837) [81]. DPPC, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2-Dipalmitoyl-snglycero-3-phosphoglycerol, sodium salt; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (ammonium salt); R827, imiquimod; PTX, Paclitaxel; IR820, photosensitizer. Reprinted with permission from ref. [82]. 2019 Meng et al.

#### 3.1.4. Miscellaneous Liposomes

A study reported developing a novel multimodal delivery system, lipopolyplexes (LPPs) loaded with curcumin, combined with PDT for improving gene delivery to SK-OV-3 cancer cells [86]. LPPs used comprised cationic polyethyleneimine and luciferaseexpressing pCMV-luc plasmid with anionic liposomes in a ratio of 1:0.5. Liposomes were formulated with a molar ratio of 70 DOPE:15 DPPC:15 cholesterol. The designed LPPs loaded with curcumin were spherical and had a particle size of 200 nm and zeta potential of +8.6 mV ± 1.7 mV. Treatment of SK-OV-3 cells with LPPs loaded with curcumin and irradiation with LED light at 457 nm (irradiation fluence of 1 J/cm<sup>2</sup> ) resulted in a significant increase in luciferase expression. The designed LPPs had minimal hemolytic effects and plasma coagulation time of 32 s.

Another recent study reported PDT combined with curcumin-loaded magnetic/ photoresponsive liposomes (MPLs) in treating papillomavirus-related cancers (Figure 5) [87]. Citric acid-coated iron oxide (CMNPs) magnetic nanoparticles are synthesized employing the chemical precipitation method [88]. Liposomes were prepared using thin-film hydration technique and lipid mixtures in a molar ratio of 5 DSPC: 3 Chol: 1 DDAB (1,2 stearoyl-sn-glycero-3-phosphocholine, dimethyl octadecyl ammonium bromide). In the hydration step, the thin film was hydrated with 0.3 mg/mL CMNPs, 50 µg/mL ICG, producing the ICG-loaded magnetic liposomes. Liposomes were then coated with hyaluronic acid-polyethylene glycol (HA-PEG) PS via sonication. Interactions between anionic HA and the cationic DDAB were responsible for the self-assembly of HA-PEG on the surface of MPLs. HA binds to the over-expressed CD44 receptors on the cell surface of human primary glioblastoma cancer cells (U87MG). The designed HA-PEG MPLs had an average

particle size of about 220 nm and exhibited significant cytotoxicity (cell viability of ~30% at a concentration of 2 mg/mL) against U87MG after exposure to near-infrared laser radiation of 2 W/cm<sup>2</sup> . Moreover, a xenograft tumor model designed by subcutaneous implantation of U87MG cells in nude mice showed the accumulation of HA-PEG MPLs in cancerous tissues. Following laser irradiation, the tumor size was reduced by 13% compared to the control (Figure 5).

**Figure 5.** Schematic illustration of HA-PEG coated magnetic/photoresponsive liposomes for combined photothermal/photodynamic cancer therapy. ICG, Indocyanin green PS; HA-PEG, hyaluronic acid-polyethylene glycol; MPLs, magnetic/photoresponsive liposomes; PTT, photothermal therapy; ROS, reactive oxygen species. Reprinted with permission from ref. [87]. 2019 Meng et al.

Another recent study reported the encapsulation of ICG in chitosan-coated liposomes for PDT of melanoma. Chitosan coating has imparted stability to liposomes and improved their cellular uptake into B16-F10 melanoma cancer cells [89]. The liposomes were designed with a molar ratio of 16 DMPC: 3 Chol using the modified freeze-drying method. The positively charged chitosan (0.1%) coated the anionic liposomal surface through electrostatic interactions. The prepared chitosan-coated liposomes loaded with ICG had a mean droplet size of 1983 ± 270 nm, a zeta potential of 43.2 ± 1.2, and a loaded ICG concentration of 1.19 ± 0.03 mg/mL. Cellular uptake of ICG was dramatically increased (owing to the cationic nature of chitosan coats). Moreover, the prepared liposomes showed remarkable phototoxicity against B16-F10 cancer cells after exposure to laser (775 nm) at a power of 0.23 mW for 2.5 min.

A study conducted by Wu et al. reported the preparation of zwitterionic liposomes encapsulating methylene blue (MB) PS that can generate ROS after light exposure and causing cancer cell apoptosis [90]. MB has poor penetration into cancer cells; hence, this study aimed to encapsulate MB into zwitterionic liposomes to protect loaded drugs from degradation, prolong systemic circulation, and enhance cellular uptake. A zwitterionic polymer-lipid poly(12-(methacryloyloxy) dodecyl phosphorylcholine) was self-assembled with DSPC in a molar ratio of 1:4. The prepared zwitterionic liposomes had an average particle size of 150 nm and fast release rates (about 90% of the MB was released over 8 h). A significant increase in the ROS release was observed in the case of MB-loaded liposomes compared to the unloaded MB. Increased cytotoxicity of the MB-loaded zwitterionic liposomes was observed against breast cancer cells (4T1 cells) compared to unloaded MB

irradiated with LED light (660 nm) of 165 mW for 6 min. Annexin V-FITC assay showed that cytotoxicity took place via apoptosis.

Finally, Rizvi et al. prepared two liposomal formulations encapsulating Visudyne or a lipid conjugate of BPD. These formulations were combined to localize the PSs and cause photodamage (following irradiation with a 690 nm diode laser at a light dose of 50 mW/cm<sup>2</sup> ) to mitochondria, endoplasmic reticulum, and lysosomes in a 3D model of ovarian cancer [91]. The recent studies in integrating liposomes with PDT in cancer treatment are summarized in Table 3.


**Table 3.** Encapsulation of PSs into liposomes in cancer PDT.


**Table 3.** *Cont.*

#### **4. Conclusions**

In this paper, we reviewed the most recent state-of-the-art studies concerning the use of integrated liposomes/PDT for effective cancer PDT. The ability of liposomes to accommodate hydrophilic and hydrophobic photosensitizers in the aqueous interior and the outer lipid bilayer, respectively, make them top candidates for the delivery of PSs in cancer PDT. Besides, liposomes can target the loaded PSs to cancer cells via active or passive targeting. Moreover, liposome surfaces could be decorated with particular ligands that recognize and bind to overexpressed receptors and proteins in tumor tissues, such as folate, estrogen, spermine, and galactose receptors. Liposomes were also found to improve the selective targeting of PS alone or combined with other chemotherapeutic drugs, hence minimizing their toxic effects. Several studies have reported the enhanced effectiveness of PDT upon encapsulation of PSs in different liposomal formulations. Therefore, these combinatorial/multimodal platforms are likely promising candidates that can improve the effectiveness of cancer PDT.

**Author Contributions:** Conceptualization, S.A.F., H.M.E.-S.A. and J.S.; methodology, S.A.F.; data curation, S.A.F.; writing—original draft preparation, S.A.F.; writing—review and editing, S.A.F., H.M.E.-S.A. and J.S.; supervision, H.M.E.-S.A. and J.S.; project administration, H.M.E.-S.A. and J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been funded by a grant from the American University in Cairo to H.M.E.-S.A.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Combination-Based Strategies for the Treatment of Actinic Keratoses with Photodynamic Therapy: An Evidence-Based Review**

**Stefano Piaserico \* , Roberto Mazzetto , Emma Sartor and Carlotta Bortoletti**

Dermatology Unit, Department of Medicine, University of Padua, 35121 Padua, Italy **\*** Correspondence: stefano.piaserico@unipd.it; Tel.: +39-049-8212914; Fax: +39-049-8212502

**Abstract:** Photodynamic therapy (PDT) is a highly effective and widely adopted treatment strategy for many skin diseases, particularly for multiple actinic keratoses (AKs). However, PDT is ineffective in some cases, especially if AKs occur in the acral part of the body. Several methods to improve the efficacy of PDT without significantly increasing the risks of side effects have been proposed. In this study, we reviewed the combination-based PDT treatments described in the literature for treating AKs; both post-treatment and pretreatment were considered including topical (i.e., diclofenac, imiquimod, adapalene, 5-fluorouracil, and calcitriol), systemic (i.e., acitretin, methotrexate, and polypodium leucotomos), and mechanical–physical (i.e., radiofrequency, thermomechanical fractional injury, microneedling, microdermabrasion, and laser) treatment strategies. Topical pretreatments with imiquimod, adapalene, 5-fluorouracil, and calcipotriol were more successful than PDT alone in treating AKs, while the effect of diclofenac gel was less clear. Both mechanical laser treatment with CO<sup>2</sup> and Er:YAG (Erbium:Yttrium–Aluminum–Garnet) as well as systemic treatment with Polypodium leucotomos were also effective. Different approaches were relatively more effective in particular situations such as in immunosuppressed patients, AKs in the extremities, or thicker AKs. Conclusions: Several studies showed that a combination-based approach enhanced the effectiveness of PDT. However, more studies are needed to further understand the effectiveness of combination therapy in clinical practice and to investigate the role of acitretin, methotrexate, vitamin D, thermomechanical fractional injury, and microdermabrasion in humans.

**Keywords:** photodynamic therapy (PDT); actinic keratoses; combination; topical; systemic; laser

## **1. Introduction**

Actinic keratoses (AKs) are a very common skin disease caused by chronic sun damage. More than 75% of AKs arise in chronically sun-exposed areas such as the face, scalp, neck, and back of the hands/forearms. The rates of malignant transformation vary from 0.025% to 16%, and the risk of progression of squamous cell carcinoma (SCC) increases in patients with multiple AKs [1].

Photodynamic therapy (PDT) is a highly effective and well-tolerated local treatment based on the interaction between a photosensitizer, an appropriate wavelength of light, and oxygen (Figure 1).

It is generally administered with a topical photosensitizer drug, such as 5-aminolevulinic acid (ALA), or derivatives such as methyl aminolevulinate (MAL) [2]. These precursors of the heme biosynthetic pathway are converted within target cells into photoactivatable porphyrins, especially protoporphyrin IX (PpIX). Protoporphyrin IX has its largest absorption peak in the blue region at 410 nm with smaller absorption peaks at 505, 540, 580, and 630 nm<sup>2</sup> . Most light sources for PDT target the 630 nm absorption peak in the red region, associated with greater tissue penetration, although a blue, fluorescent lamp (peak emission 417 nm) is sometimes used with ALA-PDT [3].

**Citation:** Piaserico, S.; Mazzetto, R.; Sartor, E.; Bortoletti, C. Combination-Based Strategies for the Treatment of Actinic Keratoses with Photodynamic Therapy: An Evidence-Based Review. *Pharmaceutics* **2022**, *14*, 1726. https://doi.org/ 10.3390/pharmaceutics14081726

Academic Editor: Jun Dai

Received: 6 July 2022 Accepted: 12 August 2022 Published: 18 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Schematic representation of PDT's mechanism of action. **Figure 1.** Schematic representation of PDT's mechanism of action.

It is generally administered with a topical photosensitizer drug, such as 5-aminolevulinic acid (ALA), or derivatives such as methyl aminolevulinate (MAL) [2]. These precursors of the heme biosynthetic pathway are converted within target cells into photoactivatable porphyrins, especially protoporphyrin IX (PpIX). Protoporphyrin IX has its The antitumor effects of PDT are based on three principal mechanisms: direct cytotoxic effects on cancer cells, indirect effects due to the fact of tumor vasculature damage, and the activation of the immune response (Figure 1) [4]. PDT with 5-ALA or MAL showed lesion clearance rates of 81–92% for thin and moderately thick AKs on the face and scalp [5]. PDT short-term (3 months) and long-term (12 months) efficacy is comparable or superior to imiquimod 5%, 5-fluorouracil 0.5%, and cryosurgery [6–9].

largest absorption peak in the blue region at 410 nm with smaller absorption peaks at 505, 540, 580, and 630 nm<sup>2</sup> . Most light sources for PDT target the 630 nm absorption peak in the red region, associated with greater tissue penetration, although a blue, fluorescent lamp (peak emission 417 nm) is sometimes used with ALA-PDT [3]. The main limitation of PDT compared to other treatments is pain, which may be the reason for stopping the treatment and may induce the patient to decline further treatment. Nerve block, subcutaneous infiltration anesthesia, and cold analgesia are associated with less PDT-related pain [10]. Lower irradiance (as in daylight PDT) and possibly shorter application times are associated with decreased discomfort without loss of PDT efficacy [3].

The antitumor effects of PDT are based on three principal mechanisms: direct cytotoxic effects on cancer cells, indirect effects due to the fact of tumor vasculature damage, and the activation of the immune response (Figure 1) [4]. PDT with 5-ALA or MAL Nonetheless, PDT is generally considered to give good cosmetic outcomes [6]. This might be of importance when treating lesions localized on the face or the scalp and may explain patients' preference for and satisfaction with PDT compared to a physically destructive method such as cryotherapy.

showed lesion clearance rates of 81–92% for thin and moderately thick AKs on the face and scalp [5]. PDT short-term (3 months) and long-term (12 months) efficacy is comparable or superior to imiquimod 5%, 5-fluorouracil 0.5%, and cryosurgery [6–9]. The main limitation of PDT compared to other treatments is pain, which may be the However, PDT efficacy may be limited by the reduced ALA/MAL penetration depth and subsequently insufficient PpIX production in lesional skin. Indeed, thicker lesions in some other areas (especially in the upper and lower limbs) are less responsive to this treatment, and some patients show treatment failure or recurrence.

reason for stopping the treatment and may induce the patient to decline further treatment. Therefore, AK treatment by combining PDT with other methods of therapy might be more efficacious, although the evidence for such procedures is limited [11].

Nerve block, subcutaneous infiltration anesthesia, and cold analgesia are associated with less PDT-related pain [10]. Lower irradiance (as in daylight PDT) and possibly shorter application times are associated with decreased discomfort without loss of PDT efficacy [3]. Nonetheless, PDT is generally considered to give good cosmetic outcomes [6]. This In this review, we discuss evidence-based in vitro experiments and clinical trials regarding the administration of other therapeutic methods before or after PDT. Specifically, we assessed the effectiveness of topical (i.e., diclofenac, imiquimod, adapalene, 5-fluorouracil, and calcitriol), systemic (i.e., acitretin, methotrexate, vitamin D, and polypodium leucotomos), and mechanical–physical (i.e., radiofrequency, thermomechanical fractional injury ((TMFI)), microdermabrasion, microneedling, and laser) treatment strategies (Figure 2).

more efficacious, although the evidence for such procedures is limited [11].

might be of importance when treating lesions localized on the face or the scalp and may explain patients' preference for and satisfaction with PDT compared to a physically de-

and subsequently insufficient PpIX production in lesional skin. Indeed, thicker lesions in

However, PDT efficacy may be limited by the reduced ALA/MAL penetration depth

Therefore, AK treatment by combining PDT with other methods of therapy might be

In this review, we discuss evidence-based in vitro experiments and clinical trials regarding the administration of other therapeutic methods before or after PDT. Specifically, we assessed the effectiveness of topical (i.e., diclofenac, imiquimod, adapalene, 5-fluorouracil, and calcitriol), systemic (i.e., acitretin, methotrexate, vitamin D, and polypodium

structive method such as cryotherapy.

**Figure 2.** Combination-based strategies for the treatment of actinic keratoses with PDT. **Figure 2.** Combination-based strategies for the treatment of actinic keratoses with PDT.

leucotomos), and mechanical–physical (i.e., radiofrequency, thermomechanical fractional injury ((TMFI)), microdermabrasion, microneedling, and laser) treatment strategies (Fig-

### **2. Topical Treatments Combined with PDT**

#### **2. Topical Treatments Combined with PDT** *2.1. Diclofenac*

ure 2).

*2.1. Diclofenac* Diclofenac is a nonsteroidal anti-inflammatory drug that reduces the production of prostaglandins by inhibiting inducible COX-2 (cyclo-oxygenase-2) [12]. It is used for treating AKs, considering that arachidonic acid metabolites promote epithelial tumor growth by stimulating angiogenesis, inhibiting apoptosis, and increasing the invasiveness of tu-Diclofenac is a nonsteroidal anti-inflammatory drug that reduces the production of prostaglandins by inhibiting inducible COX-2 (cyclo-oxygenase-2) [12]. It is used for treating AKs, considering that arachidonic acid metabolites promote epithelial tumor growth by stimulating angiogenesis, inhibiting apoptosis, and increasing the invasiveness of tumor cells [12].

mor cells [12]. Van Der Geer et al. evaluated the effectiveness of 3% diclofenac, applied for four weeks, twice a day, before administering ALA-PDT. The treatment of AKs on the dorsum of the hands of 10 patients had a slightly better long-term outcome (though not significant). Upon evaluating the pain scores during treatment, unbearable pain was detected to Van Der Geer et al. evaluated the effectiveness of 3% diclofenac, applied for four weeks, twice a day, before administering ALA-PDT. The treatment of AKs on the dorsum of the hands of 10 patients had a slightly better long-term outcome (though not significant). Upon evaluating the pain scores during treatment, unbearable pain was detected to a greater extent in the patients of the diclofenac group [13].

a greater extent in the patients of the diclofenac group [13]. However, since 3% diclofenac gel with hyaluronic acid showed better outcomes with longer applications (i.e., 60 and 90 days of administration showed total clearances of 30% and 50%, respectively, in the patients [14]), the effectiveness of PDT treatment in combination with diclofenac and alone should be compared after administration for more than However, since 3% diclofenac gel with hyaluronic acid showed better outcomes with longer applications (i.e., 60 and 90 days of administration showed total clearances of 30% and 50%, respectively, in the patients [14]), the effectiveness of PDT treatment in combination with diclofenac and alone should be compared after administration for more than four weeks [15].

#### four weeks [15]. *2.2. Imiquimod*

*2.2. Imiquimod* Imiquimod 5% cream stimulates the innate immune response through the interaction Imiquimod 5% cream stimulates the innate immune response through the interaction with Toll-like receptor 7 (TLR7).

with Toll-like receptor 7 (TLR7). Shaffelburg [16] compared sequential treatment with ALA-PDT versus ALA-PDT alone. Patients with at least 10 actinic keratoses on the face were treated with photodynamic therapy with aminolevulinic acid (20%) at baseline and month 1. At month 2, imiquimod cream (5%) was applied to half of the face, and the vehicle was applied to the Shaffelburg [16] compared sequential treatment with ALA-PDT versus ALA-PDT alone. Patients with at least 10 actinic keratoses on the face were treated with photodynamic therapy with aminolevulinic acid (20%) at baseline and month 1. At month 2, imiquimod cream (5%) was applied to half of the face, and the vehicle was applied to the other half twice a week for 16 weeks.

other half twice a week for 16 weeks. At month 12, the median reduction in the lesion was 89.9% and 74.5% (*p* = 0.0023) on the parts of the face treated with the combination of the imiquimod cream with ALA-PDT and ALA-PDT alone, respectively. Furthermore, tolerance was similar in patients treated At month 12, the median reduction in the lesion was 89.9% and 74.5% (*p* = 0.0023) on the parts of the face treated with the combination of the imiquimod cream with ALA-PDT and ALA-PDT alone, respectively. Furthermore, tolerance was similar in patients treated with imiquimod and those treated with placebo. Serra et al. [17] compared the effectiveness of PDT alone, imiquimod (5%) alone, and the combined administration in 105 patients (PDT followed by imiquimod twice a week for 16 weeks).

They reported a complete clinic-pathologic response (*p* = 0.038) in 10% of the patients in the PDT-alone group, 27% of the patients in the imiquimod-alone group, and 34% of the patients in the PDT + imiquimod group. Moreover, a significantly greater percentage of patients who received PDT were extremely satisfied with the treatment than those who received only imiquimod (90% PDT vs. 61% imiquimod). Another study with an openlabel parallel cohort design investigated the effectiveness of the treatment with imiquimod cream (3.75%), combined with photodynamic therapy (ALA-PDT) vs. imiquimod cream (3.75%) alone [18]. The former group received imiquimod (3.75%) daily for two weeks in a two-week cycle, separated by two weeks of no treatment and followed by one session of photodynamic therapy of the entire face with ALA and blue light. In the latter group, the treatment was administered daily for two weeks in a two-week cycle, separated by two weeks of no treatment. A mean reduction rate of 81% was found for the group treated with PDT, and 83% for the group treated only with imiquimod (the mean difference between the combination treatment and treatment with only imiquimod cream (3.75%) was 2%). However, five out of nine patients who received the combination treatment showed complete clearance of AKs, while only two out of nine patients showed this outcome in the other group. The rates of adverse events observed between the two groups were similar.

#### *2.3. Topical Retinoids*

Retinoid drugs are also used widely for the treatment of AKs, as they can disrupt gene expression, modify cell differentiation, and modulate cell proliferation or hyperplasia [19].

Currently, three generations of synthetic retinoids exist. First-generation retinoids include tretinoin (all-trans RA), isotretinoin (13-cis-retinoic acid), and alitretinoin (9-cis RA). Second-generation retinoids include etretinate and acitretin. Third-generation retinoids include adapalene, tazarotene, and bexarotene [1,2]. The application of tazarotene gel (0.1%) twice daily on AKs of the upper extremities one week before the administration of ALA-PDT (20%) increased the reduction in the lesion count by ≥50% (*p* = 0.0547) [20].

Galitzer [21] studied the effectiveness of adapalene gel (0.1%) as a pretreatment topical drug for the treatment of AKs on the dorsal hands and forearms by administering a single dose of ALA-PDT. A 10% ALA gel was used instead of a 20% ALA gel, and a red light was used for illumination instead of blue light. The difference between the two groups was significant (*p* = 0.0164), with a median lesion count reduction of 79% in the adapalene pretreated group compared to 57% in the standard therapy group. The discomfort under red light was slightly greater in patients of the standard therapy group, but the difference was not significant.

#### *2.4. 5-Fluorouracil Cream*

Topical 5-fluorouracil (5-FU) has greater efficacy than ALA and MAL-PDT [22].

It is converted in the cell to a thymidylate synthase inhibitor that interrupts the synthesis of thymidine, causing growth arrest and apoptosis [23].

In mouse models of SCC (SKH-1 and A431 mice), pretreatment with 5-FU for three days followed by ALA for 4 h caused a considerable, tumor-selective increase in PpIX levels and enhanced cell death upon illumination. The effect might be related to a change in the expression of the heme enzyme (upregulating coproporphyrinogen oxidase and downregulating ferrochelatase) and to a substantial, tumor-selective increase in apoptosis [24].

Likewise, an increase in the PpIX levels (two-fold to three-fold) in 5-FU-pretreated lesions versus controls was also reported in patients with AKs [25].

The clearance rates after treatment with MAL-PDT, with or without six days of 5-FU pretreatment, were 75% and 45% at three months and 67% and 39% at six months, respectively [26].

Niessen et al. [27] found that pretreatment with 5-FU for a week, twice daily before treatment with daylight (DL)-PDT, was more effective for AKs on the dorsal side of hands compared to treatment with DL-PDT alone. At the three-month follow-up, the overall lesion response rate was significantly higher for the combination treatment (62.7%) than for the treatment with DL-PDT alone (51.8%), while no difference was found in the degree of erythema one day after PDT between the treatment groups (pretreatment with 5-FU only caused minimal erythema in a few patients before illumination).

Other studies confirmed that the combined treatment was more effective for AKs on the upper limbs or the face [28–30].

#### *2.5. Topical Calcitriol/Calcipotriol*

Vitamin D is a differentiation-promoting hormone, and calcitriol is the most potent and active form of the hormone, the receptor of which is also present in cancer cells [22].

Furthermore, it induces a marked increase in protoporphyrin IX accumulation at the site of the tumor and promotes the activation of the extrinsic apoptotic pathway [31]. Therefore, cell damage after light irradiation is considerably higher in calcitriol-pretreated cells [32–34].

Galimberti first proposed pretreatment with calcipotriol (50 mcg/g) during the treatment of actinic keratoses with MAL DL-PDT [35]. Three months after the treatment, the complete response rate was 85% after calcipotriol pretreatment, while it was 70% for the DL-PDT alone group. Calcipotriol/DL-PDT showed a stronger association with erythema than DL-PDT alone. Some patients preferred DL-PDT alone due to the inconvenience caused by the daily application of calcipotriol and the related erythema and desquamation.

Torezan et al. confirmed these findings and showed improvement in keratinocyte atypia following treatment with MAL-PDT, with slightly more improvement on the parts pretreated with calcipotriol. However, erythema, edema, crusting, and postulation represented the most common adverse events and occurred more frequently in the group treated with the topical vitamin D3 analog [36].

Moreover, a long-term study on treatment with MAL-PDT with prior application of topical calcipotriol or conventional MAL-PDT for AKs on the scalp showed an overall AK clearance of 92% and 82% after three months for CAL-PDT and conventional PDT, respectively (*p* < 0.001). Similar results were found at 6 and 12 months, i.e., 92% vs. 81% (*p* < 0.001) and 90% vs. 77% (*p* < 0.001) for CAL-PDT and conventional PDT, respectively [37].

Another intra-individual, randomized trial [38] was conducted with patients with AKs on the dorsum of the hands or forearms, who were treated with another vitamin D analog, calcitriol (3 mg/g). Calcitriol or a placebo was applied once daily for 14 consecutive days. On day 15, the first administration of MAL DL-PDT was performed, and the second one took place after one week. A higher efficacy was found for grade II and grade III AKs, with a response rate of 55.24% for the group pretreated with calcitriol and 39.58% for the control group (*p* = 0.038).

The administration of topical vitamin D analogs might be more effective than treatment with PDT alone, particularly for thicker and difficult-to-treat AKs on the upper extremities and the scalp. However, the administration of vitamin D analogs might be associated with an increase in local skin reactions.

#### **3. Systemic Treatment Combined with PDT**

#### *3.1. Acitretin*

Acitretin is a systemic retinoid drug. Although the on-label instruction for acitretin only recommend its use for psoriasis, its off-label use in keratinization disorders is very common [39].

Acitretin induces normalization of differentiation and proliferation as well as the modification of inflammatory responses and neutrophil function in the skin. Moreover, some studies have shown the effectiveness of this drug in preventing non-melanoma skin tumors and AKs [40].

In a nonclinical setting, retinoic acid preconditioning before PDT can induce a moderate (though nonsignificant) increase in ALA–PpIX levels in prostate cancer cells. However, Hasan et al. found a dramatic increase in PDT response after pretreatment with retinoids due to the increase in the production of protoporphyrin IX (PpIX) in the target cells [41,42].

Preclinical studies support the hypothesis that induction of keratinocyte differentiation can increase intracellular PpIX accumulation in cells treated with ALA before light exposure in mice [41]. Treatment with a combination of acitretin and ALA-PDT can significantly increase the apoptosis rate and the mortality rate of SCL-1 cells compared to treatment with acitretin or ALA-PDT alone [43].

However, we found no studies regarding the effectiveness of combining PDT with acitretin in clinical trials or observational studies. This might be an important treatment strategy, considering the encouraging results reported in vitro.

#### *3.2. Methotrexate*

Methotrexate (MTX) is widely used for the treatment of psoriasis and other conditions characterized by cell hyperproliferation and lack of differentiation. MTX is a chemotherapeutic agent that inhibits cell proliferation and triggers cellular differentiation [44].

Pretreating human SCC13 cells, HEK1 cells, and normal keratinocytes with MTX for 72 h enhanced the PpIX levels by two-fold to four-fold in cancerous cells relative to the PpIX levels in nontumoral keratinocytes [45]. This was probably because MTX modified the expression of certain enzymes involved in the porphyrin metabolic pathway (a four-fold increase in coproporphyrinogen oxidase and a stable or slight decrease in the expression of ferrochelatase) and induced the expression of differentiation markers (i.e., E-cadherin, involucrin, and filaggrin) in cancer cells [45]. Photodynamic cell killing was thus synergistically enhanced by the combined therapy compared to the effectiveness of PDT alone.

However, the oral administration of MTX before PDT in patients with AKs, specifically in the elderly and those with several comorbidities, might not be possible, since the risks could exceed the benefits compared to the risks with other topical or systemic drugs.

#### *3.3. Vitamin D*

We previously discussed the ability of vitamin D derivatives and their prodifferentiating effects to increase protoporphyrin IX accumulation and enhance ALA-PDT-mediated cell death.

Systemic delivery of calcitriol is easily performed in mice and increases the tumoral accumulation of PpIX up to 10-fold, while in humans, high calcitriol levels are associated with the risk of developing hypercalcemia. Interestingly, the deficiency of serum vitamin D is associated with poorer responses of AKs to MAL-PDT [46] and, thus, further studies are needed to investigate its role in the treatment of AKs.

#### *3.4. Polypodium Leucotomos*

Polypodium leucotomos (PL) is an effective systemic photoprotective agent that strongly protects the skin against UV radiation [47].

PL is extracted from ferns grown in Central America and has been used for centuries by Native Americans for treating malignant tumors. Studies have shown that PL has antioxidative properties, immunomodulatory properties, and antitumoral activity [48].

Specifically, PL extract supplementation induces p53 activation and reduces acute inflammation by inhibiting the Cox-2 enzyme and increasing the removal of cyclobutane pyrimidine dimers [49,50].

In clinical settings, PL supplementation at a dose of 960 mg per day for one month and then 480 mg per day for five months decreased the recurrence rate of AKs at six months after two sessions of MAL-PDT, administered one week apart. MAL-PDT + PL supplementation showed a median reduction of 87.5% in scalp AKs compared to a reduction of 62.5% in the group treated only with MAL-PDT (*p* = 0.040). Moreover, no major side effects were recorded in either group [51].

Therefore, using PDT-MAL combined with PL might be an effective treatment for AKs due to the fact of its effect on reducing immunosuppression in the human skin not only after UV irradiation but also after PDT treatment, thus reducing the recurrence of AKs in high-risk individuals.

#### **4. Physical and Mechanical Treatments Combined with PDT**

Most of the aforementioned topical and systemic drugs improve PDT efficacy by preferentially enhancing PpIX production in AK cells, acting as differentiation-promoting agents.

However, PDT is mostly limited by the poor ALA/MAL penetration within lesional skin. The skin barrier is a lipid layer along which molecules can migrate through diffusion; they can enter through transcellular pathways, intercellular spaces, and the trans-appendageal pathway that includes hair follicles, sebaceous glands, and sweat glands [52,53].

One extensively investigated strategy to enhance drug delivery through the skin is by adding chemical enhancers such as fatty acids, surfactants, esters, and alcohols [54,55].

However, mechanical, physical, and active transport techniques also enhance skin penetration [56]. Some of these treatment methods have been assessed in combination with PDT for AK treatment. The disruption and ablation of the stratum corneum (the primary barrier to the delivery of topical drugs) by using lasers, radiofrequency (RF), thermomechanical fractional injury (TMFI), microdermabrasion (MD), or microneedling can enhance PDT treatment.

#### *4.1. Laser-Assisted PDT*

Different types of light sources are used for topical PDT including fluorescent lamps, light-emitting diodes (LEDs), and lasers [2]. However, lasers are not widely used because of their side effects, including pain and discomfort during treatment, as well as erythema, blistering, crusting, and pigmentation after treatment. Moreover, their application is limited while treating larger fields [57].

Due to the advancement in technology, fractional ablative lasers have been developed for promoting topical/transdermal drug delivery. Er:YAG (erbium:yttrium–aluminum– garnet) and CO<sup>2</sup> (carbon dioxide) lasers produce microscopic vertical channels in the skin that enhance the penetration of photosensitizers [58].

Thus, lasers can be used during pretreatment to facilitate the enrichment of ALA or MAL in dysplastic cells, and this approach is also known as laser-assisted drug delivery.

#### 4.1.1. Er:YAG (Erbium:Yttrium–Aluminium–Garnet) Lasers

Shen et al. studied the in vivo kinetics of protoporphyrin IX (PpIX) after topical ALA application, enhanced by an Er:YAG laser; the increase in the ratio of PpIX with laser-treated murine skin ranged from 1.7 to 4.9-times compared to the PpIX levels in the control group [59].

Gellén et al. [60] conducted an intra-patient randomized study and showed that fractional laser pretreatment leads to significantly higher clearance at three months compared to the clearance with PDT alone. However, no difference in recurrence rates was found during follow up after 12 months. In every patient, two random treatment areas received conventional PDT or Er:YAG-ablative fractional laser-assisted PDT (AFL-PT). The number of AKs decreased by 87.5 ± 17.3% and 82.5 ± 16.5% (*p* = 0.039) three months after Er:YAG-AFL PDT and cPDT, respectively. Furthermore, upon assessing the immune infiltration, a reduction in Ki67-positive cells and CD8+ T cells was found three months after Er:YAG-assisted PDT [60].

Togsverd-Bo et al. [61] examined difficult-to-treat AKs in organ-transplant recipients (OTRs) using fractional Er:YAG laser-assisted DL-PDT. At three months, the clearance rates were 74% after AFL-dPDT, 46% after dPDT, 50% after cPDT, and 5% after AFL (*p* < 0.001). AFL-dPDT had excellent tolerability, even if the pain was higher in areas treated with AFL-assisted DL-PDT than in areas treated with DL-PDT only.

Another prospective randomized nonblinded trial [62] was conducted to evaluate patients who underwent one session of MAL-PDT using a red light-emitting diode lamp at 37 J/cm<sup>2</sup> , and the patients were randomly assigned to receive fractional Er:YAG laser (AFL-PDT) pretreatment. AFL-PDT was significantly more effective than MAL-PDT for treating patients with AKs of all grades (86.9% vs. 61.2%; *p* < 0.001), although the efficacy of AFL-PDT was the most pronounced in treating Olsen grade III AKs (69.4% vs. 32.5%; *p* = 0.001). AFL-PDT also showed a lower lesion recurrence rate than MAL-PDT (9.7% vs. 26.6%; *p* = 0.004), and an excellent or good cosmetic outcome was reported in >90% of the cases. Erythema and hyperpigmentation intensities were higher but not significant in the AFL-PDT group, while the side effects were mild but more frequent in the AFL-PDT group, although the result was not statistically significant [62].

## 4.1.2. CO<sup>2</sup> (Carbon Dioxide) Laser

Haedersdal et al. evaluated drug delivery of ALA and MAL using CO<sup>2</sup> ablative fractional laser (AFXL). They found that AFXL increased topical uptake of porphyrin precursors when it was followed by the topical application of MAL for 3 h. This pretreatment with laser generated 3 mm microchannels in the skin and, consequently, enabled a more homogeneous distribution of the porphyrins throughout the skin [63].

Syed et al. [64] studied 12 OTRs with multiple (>5) AKs of identical size in two areas on the scalp and/or the forehead. The treatment areas, randomized to AFL-assisted DL-PDT, were first treated with a carbon dioxide laser (eCO2) targeting single AK lesions, followed by treatment of the whole field with the methyl aminolevulinate (MAL) cream applied to both treatment areas. After 30 min, both treatment areas were exposed to sunlight for 2 h. At the four-month follow up, the overall complete response was 75.5% in areas treated with AFL-assisted DL-PDT and 64.0% in areas treated with DL-PDT. There was a significant interaction between a complete response and the AKs' grade (*p* = 0.001), as well as between a partial response and the AKs grade (*p* = 0.007). Patient-reported pain was significantly higher in areas treated with AFL-assisted PDT in the first two days (*p* = 0.008) but not after five days (*p* = 0.11) [64].

Togsved-Bo et al. [65] found that at the three-month follow up, AFL-PDT was significantly more effective than PDT for all grades of AKs. Complete lesion response of grade II-III AKs was 88% after AFL-PDT and 59% after PDT (*p* = 0.02). In grade I AKs, 100% of the lesions were cleared after AFL-PDT, while 80% of the lesions were cleared after PDT (*p* = 0.04). The AFXL-PDT-treated skin showed significantly fewer new AK lesions (*p* = 0.04) and better photoaging (*p* = 0.007) than the PDT-treated skin. The pain scores during illumination (*p* = 0.02), erythema, and crusting were higher. Inflammatory reactions were more intense in AFL-PDT-treated skin than in PDT-treated skin in 83% of the patients and were of equal intensity in 17% of the patients. PpIX fluorescence was higher in AFL-pretreated skin (*p* = 0.003) [65] and supported the clinical superiority of the combination treatment.

Paasch et al. evaluated the effectiveness and safety of CO<sup>2</sup> AFL-LAD combined with indoor daylight (IDL) ALA-PDT for treating skin field cancerization associated with AKs. All patients showed remission (complete: 71.7%; partial: 28.3%), suggesting that AFL-LAD combined with IDL-PDT is an exceptionally effective treatment approach. However, the high pain score associated with this combined approach is a limiting factor [66].

Jang et al. [67] showed that laser-assisted PDT might reduce the photosensitizer incubation time or the number of sessions required for complete response (efficacy of 70.6% in three sessions).

In conclusion, several studies showed a reduction in the number of AKs and an improvement in the recurrence rate in the approach combining laser treatment and PDT. A recent meta-analysis [30] reported an overall significantly high clearance rates for laserassisted PDT than for PDT alone (RR: 1.33, 95% CI: 1.24–1.42); however, the evidence for this outcome was graded as low (GRADE ++- -). Furthermore, the pain was similar for both treatments (mean difference of 0.31, 95% CI: 0.12–0.74, low quality of evidence, GRADE ++- -).

#### *4.2. Radiofrequency and Thermomechanical Fractional Injury (TMFI)*

Fractional radiofrequency (RF) creates plasma close to the skin and provokes plasma sparks on the skin's surface that induce epidermal ablation and produce microchannels that perforate the dermis, thus improving drug delivery. Additionally, sonophoresis facilitates the movement of molecules through the intact skin under the influence of an ultrasonic perturbation. Low-frequency ultrasound (frequencies below 100 kHz) can highly enhance transdermal transport [68].

Park et al. demonstrated that fractional RF with sonophoresis effectively enhanced ALA penetration in swine skin [69]. Prefractional RF followed by post-treatment with sonophoresis is a promising therapeutic combination for ALA-PDT to enhance ALA uptake [67]. However, no studies on humans have been conducted yet.

Shavit et al. [70] evaluated the efficacy of pretreatment by thermomechanical fractional injury (TMFI) at low-energy settings in five healthy subjects to increase the permeability of the skin to four topical preparations. TMFI, (Tixel®;Novoxel®, Netanya, Israel), is a thermomechanical system developed for providing fractional treatment. The system is designed for treating soft tissues by direct conduction of heat, which allows water to evaporate rapidly while causing low thermal damage to the surrounding tissue.

The authors evaluated the permeability of 20% ALA gel, 10% ALA microemulsion gel, 16.8% MAL cream, and 20% ALA hydroalcoholic solution. Pretreatment with low-energy TMFI at a pulse duration of 6 milliseconds increased the percutaneous permeation of ALA when the 20% gel was used.

Interestingly, after 2 h and 3 h of treatment, the TMFI-treated sites exhibited a higher hourly rate of PpIX fluorescence intensity, which was 156–176% higher than that in the control (*p* ≤ 0.004). Thus, TMFI is a powerful method to enhance the transdermal drug delivery of ALA and its derivatives. Further studies are needed to investigate its role in improving PDT treatment.

#### *4.3. Microdermabrasion (MD)*

Microdermabrasion (MD) is an easily accessible and cost-effective technique that can be used to increase the efficacy of PDT as a pretreatment method.

Bay et al. compared PpIX accumulation after a range of standardized pretreatments and found significantly higher median intracutaneous PpIX fluorescence intensities after AFL compared to that with alternative physical modalities such as MD [71].

The clinical efficacy of PDT was evaluated after pretreatment with either Er:YAG AFL or MD in a side-by-side trial [72].

MD pretreatment was performed using a pad with particles that had a diameter of 58.5 µm (Ambu® Skin Prep Pads 2121M; Ambu A/S, Ballerup, Denmark). Two large areas were randomly selected in each individual, and a single treatment was administered with AFL + DL-PDT or microdermabrasion + DL-PDT. Interestingly, AFL-dPDT resulted in a significantly higher AK clearance (81% vs. 60%, *p* < 0.001), led to fewer new AKs (*p* < 0.001), and showed higher improvement in dyspigmentation (*p* = 0.003) and skin texture (*p* = 0.001), which was related to microneedling-assisted DL-PDT (MD-dPDT). However, laser-assisted PDT caused more local skin reactions than microdermabrasion pretreatment [72].

#### *4.4. Microneedling-Assisted PDT*

Microneedling represents another pretreatment option to improve the effects of PDT. A microneedle consists of tiny needles on a roller or a stamp that punctures the skin and forms microchannels.

In vivo experiments using nude mice showed that microneedle punctures could reduce the application time and ALA dose required to induce high levels of the photosensitizer protoporphyrin IX in the skin. This is beneficial for clinical practice, as shorter application times are more convenient for patients and clinicians [73].

The Microneedling Photodynamic Therapy II (MNPDT-II) study revealed a statistically significant difference in the clearance of AKs for the 20 min incubation arm between the microneedling side and the PDT alone side (76% and 58%, respectively). The microneedle device consisted of a single-use sterile array of microneedles (200 µm long), while the sham treatment consisted of the applicator roller without microneedles. Immediately after sham and microneedle pretreatment, topical ALA was applied to the entire face, followed by exposure to blue-light PDT [74].

Likewise, Spencer and Freeman reported that microneedling pretreatment induced a significantly greater reduction in the AK count compared to that after PDT alone (89.3% and 69.5%, respectively); the occurrence of side effects was similar [75].

Furthermore, a comparative study conducted by Chen et al. to evaluate ALA-PDT revealed that plum-blossom needling (a method of shallow insertion of multiple needles into the skin) caused a broader diffusion of ALA than that with CO<sup>2</sup> AFXL, and the technique had a similar clinical effect at a considerably lower cost. The needle-pretreatedlesion had a stronger surface fluorescence intensity than the laser-pretreated-lesion [76].

However, Lev-Tov et al. [77] conducted a randomized controlled evaluator-blind trial and found no significant differences in the efficacy between microneedling-PDT and ALA-PDT (with a short incubation time of 60 min), while the microneedling side showed significantly higher pain scores. The authors stated that this conflicting result was due to the use of shorter microneedles, which only reached the epidermis (and not the dermis), than those used in other studies.

#### **5. Other Physical and Chemical Treatments**

Other methods have been used to increase the effectiveness of PDT. However, no studies have compared them to conventional-PDT or to daylight-PDT for the treatment of AKs.

In vivo studies showed that the incorporation of a thermogenic and vasodilating substances, such as methyl nicotinate, in the MAL cream increased the amount of PpIX produced in the tissue, causing a greater effect on the epidermis after PDT as well as reducing the cream's incubation time [78].

Iontophoresis, a physical treatment aimed at facilitating transport through the skin of ionic species using a voltage-gradient applied to an electrolytic formulation [23], was reported to reduce the incubation time by 1 h in a study comparing iontophoresis-assisted AFL-PDT with AFL-PDT [79].

Photobiomodulation consists of the illumination of tissues with subthermal radiometric conditions (red or near-infrared), stimulating cell metabolism and enhancing the production of PpIX and the effectiveness of PDT in vitro and in vivo tumors (human glioma cells) [80].

Another possible approach to increase 5-ALA penetration and efficiency is the addition of glycolic acid (GA) to 5-ALA for the treatment of patients with SCC [81] and ethylenendiamine-tetra-acetic-acid (EDTA) and dimethylsulphoxid (DMSO) to 5-ALA in order to improve protoporphyrin IX accumulation in certain subtypes of BCC, SCC, and Bowen's disease [82].

Therefore, iontophoresis, photobiomodulation, and the addition of methyl nicotinate, GA, or DMSO in ALA-based topicals represent encouraging possibilities to enhance conventional-PDT or daylight-PDT efficacy and should be further studied to assess their role in treating AKs as well.

#### **6. Conclusions**

Our review showed that combination treatment may significantly improve the effectiveness of PDT in reducing AKs (Table 1). Pretreatment with imiquimod, adapalene, 5-FU, and calcipotriol showed greater effectiveness in treating AKs than PDT alone, while evidence supporting the effectiveness of microneedling and diclofenac gel was poor.




#### **Table 1.** *Cont.*


#### **Table 1.** *Cont.*

\* Settimo, L.; Bellman, K.; Knegte, R.M.A. Comparison of the Accuracy of Experimental and Predicted pKa Values of Basic and Acidic Compounds. *Pharm. Res*. 2014, 31, 1082–1095; https://doi.org/10.1007/s11095-013-1232-z, (accessed on 7 August 2022); \*\* US Environmental Protection Agency. Comptox Chemicals Dashboard. https://comptox. epa.gov/dashboard/chemical/details/DTXSID3037208 (accessed on 7 August 2022); § https://chemaxon.com/products/ calculators-and-predictors#pka, (accessed on 7 August 2022); ◦ Computed by XLogP3 3.0 (PubChem release 7 May 2021); <sup>ϕ</sup> Sangster J; LOGKOW Databank. Sangster Res. Lab., Montreal Quebec, Canada (1994).

Pretreatment with 5-FU, calcitriol, and adapalene can substantially increase the effectiveness of PDT in the extremities (10.9%, 15.7%, and 22% more than PDT alone, respectively). Immunosuppressed patients may benefit from the administration of 5-FU cream pre-PDT or by the combination of PDT with laser. In particular, Er:YAG laser and CO<sup>2</sup> laser-assisted PDT showed greater efficacy than PDT alone (28% and 11.5%, respectively). Treatment of thicker AKs with PDT can be improved by combination with Er:YAG laser-PDT (69.4% effectiveness, 36.9% more than PDT) even though a slight increase in the incidence of side effects was found and should be considered. AKs of higher grade may also be pretreated with higher effectiveness with CO<sup>2</sup> laser-PDT or with calcitriol (3 mg/g).

Therefore, combining treatments may greatly enhance PDT's ability to reduce AKs. However, more studies are needed to further understand the effectiveness of combination therapy in clinical practice and to investigate the role of systemic drugs, such as acitretin, methotrexate, and vitamin D, in humans.

**Author Contributions:** Conceptualization, S.P.; Methodology, S.P., R.M., E.S. and C.B.; Writing– Original Draft Preparation, S.P., R.M., E.S. and C.B.; Writing—Review and Editing, S.P. and R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **List of Abbreviations**


## **References**


## *Review* **Effectiveness of Antimicrobial Photodynamic Therapy in the Treatment of Periodontitis: A Systematic Review and Meta-Analysis of In Vivo Human Randomized Controlled Clinical Trials**

**Snehal Dalvi 1,2,\* , Stefano Benedicenti <sup>1</sup> , Tudor Sălăgean 3,\*, Ioana Roxana Bordea 4,† and Reem Hanna 1,5,†**


**Abstract:** This systematic review and meta-analysis evaluated antimicrobial photodynamic therapy (aPDT) efficacy in periodontitis. The review protocol was conducted in accordance with PRISMA statements, Cochrane Collaboration recommendations and is registered in PROSPERO (CRD 42020161516). Electronic and hand search strategies were undertaken to gather data on in vivo human RCTs followed by qualitative analysis. Differences in probing pocket depth (PPD) and clinical attachment level (CAL) were calculated with 95% confidence intervals and pooled in random effects model at three and six months. Heterogeneity was analyzed, using Q and I<sup>2</sup> tests. Publication bias was assessed by visual examination of the funnel plot symmetry. Sixty percent of 31 eligible studies showed a high risk of bias. Meta-analysis on 18 studies showed no additional benefit in split mouth studies in terms of PPD reduction (SMD 0.166; 95% CI −0.278 to 0.611; *P* = 0.463) and CAL gain (SMD 0.092; 95% CI −0.013 to 0.198; *P* = 0.088). Similar findings noted for parallel group studies; PPD reduction (SMD 0.076; 95% CI −0.420 to 0.573; *P* = 0.763) and CAL gain (SMD 0.056; 95% CI −0.408 to 0.552; *P* = 0.745). Sensitivity analysis minimized heterogeneity for both outcome variables; however, intergroup differences were not statistically significant. Future research should aim for well-designed RCTs in order to determine the effectiveness of aPDT.

**Keywords:** antimicrobial photodynamic therapy; periodontitis; scaling and root planing; systematic review; meta-analysis

## **Highlights**


**Citation:** Dalvi, S.; Benedicenti, S.; S˘al˘agean, T.; Bordea, I.R.; Hanna, R. Effectiveness of Antimicrobial Photodynamic Therapy in the Treatment of Periodontitis: A Systematic Review and Meta-Analysis of In Vivo Human Randomized Controlled Clinical Trials. *Pharmaceutics* **2021**, *13*, 836. https://doi.org/10.3390/ pharmaceutics13060836

Academic Editors: Maria Nowakowska and Nejat Düzgünes

Received: 17 April 2021 Accepted: 2 June 2021 Published: 4 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

5. The efficacy of aPDT in improving treatment outcomes when it is utilized in the non-surgical management of periodontitis remains debatable.

#### **1. Introduction**

Antimicrobial Photodynamic therapy (aPDT) involves photo-excitation, which occurs when a photosensitizer (PS) dye is illuminated by a light of a matched wavelength, resulting in its activation and stimulation of a phototoxic response in the presence of ambient oxygen [1]. It has been persistently observed that bacterial recolonizations of *Aggregatibacter actinomycetemcomitans (A.a)* occur in periodontal pockets even after scaling and root planing (SRP) [2]. Aggressive periodontitis (AgP) is frequently associated with fewer local etiologic factors; therefore, it is believed that the affected patients are more likely to benefit from the antimicrobial effect of aPDT [3]. In contrast, chronic periodontitis (CP) patients usually have complex and thick deposits of polymicrobial communities on the affected root surfaces [4]. This may hamper penetration of PS, thereby reducing its effect and leading to an increase in the 'red complex' bacterial counts within a short period of time, resulting in a disease relapse [5]. Hence, the concept of replacing conventional SRP with aPDT is a controversial one with several imperative demerits, as enlisted above.

Utilization of adjunctive aPDT and its comparison with the gold standard SRP is a concept that has been studied extensively in both CP and AgP patients [6–16]. While SRP can quantitively lower the biomass of bacteria, aPDT has a more qualitative approach of a non-invasive nature, by creating alterations in cell membranes or Deoxyribonucleic Acid (DNA) damage [5]. Hence, a combination of these two therapies can be vouched for, since their mechanisms of action on microbiota and role in the periodontal repair process is distinct from the other and thus might have synergistic effects [17].

Distant sites of infection such as tonsils or base of tongue, which are affected due to the spread of tissue penetrating periopathogens, can be successfully reduced with local or systemic antibiotics (AB) [18]. Nonetheless, many clinicians often conduct NSPT without adjunctive AB, which is only used when initial treatment has failed [19]. In AgP, evidence suggests that SRP+AB therapy does not show satisfactory long-term results, unless reinstrumentation of affected sites is performed, as an additional step in the maintenance phase [20]. Furthermore, owing to the development of antibiotic resistant strains, it has been suggested that AB usage should be restricted to those with a highly active disease or a specific microbiological profile [21]. In order to maintain an adequate mean inhibitory concentration (MIC) of any antimicrobial drug, either a sustained-release carrier medium is required or, conversely, a prompt bactericidal approach is needed to overcome the problem of physical displacement from the sulcus [22]. The aPDT falls into the latter category, demonstrating a 4–6-fold logarithmic bacterial reduction within a time frame of 60 s along with repeated applications [23]. A comprehensive assessment to evaluate the impact of these new trials on the role of aPDT in the treatment of periodontitis is unresolved, owing to the diversity in the methodology and results of existing scientific evidence [6–16].

In lieu of the prevailing pertinent literature, the present systematic review and metaanalysis aimed to provide a systematic evaluation of available scientific evidence to determine the efficacy of aPDT in the treatment of periodontitis. The objectives of this critical review were to evaluate the outcomes of this treatment strategy through various PS-laser wavelength combinations, as well as the laser parameters, in order to deduce an ideal PS-laser wavelength combination and treatment protocol for future scientific research.

#### **2. Materials and Methods**

#### *2.1. Protocol and Registration*

The present systematic review was reported based on the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) Statement and Cochrane Collaboration recommendations (Supplementary file 1) [24,25]. The review protocol is published in the Prospective Register of Systematic Reviews (PROSPERO); ref CRD 42020161516.


#### *2.3. Focused Research Question*

Is aPDT effective as a primary mode of treatment or as an adjunct to SRP compared to SRP alone or in combination with local or systemic antibiotics (AB), in terms of clinical or microbiological or immunological profiles, in patients with Periodontitis?

#### *2.4. Search Strategy*

The search strategy only included terms relating to or describing the study's domain and intervention. The use of relevant free text keywords and medical subject heading (Mesh) terms, which were logically connected with the help of Cochrane MEDLINE filters for controlled trials of interventions, was implemented. Individual search algorithms were developed for the following databases: MEDLINE (NCBI PubMed and PMC), Cochrane Central Register of Controlled Trials (CCRCT), Scopus, ScienceDirect, Google Scholar, EM-BASE and EBSCO. Electronic search databases were searched thoroughly from their earliest records until 31 December 2019. The following journals were manually searched: Journal of Periodontology, Photomedicine and Laser Surgery, Clinical Oral Investigation, Journal of Clinical Periodontology, Journal of Dental Research, Lasers in Medical Science, Journal of Photochemistry and Photobiology and Photodiagnosis and Photodynamic Therapy. Related review articles and reference lists of all identified articles were searched through for further studies. Abstracts of the American Academy of Periodontology (AAP) and the European Federation of Periodontology (EFP) as well as sources for grey literature were screened to detect unpublished studies. In some instances, an attempt was made to establish a communication with the corresponding author in an attempt to obtain additional information related to the study; however, the attempts were unsuccessful. Search strategy was performed by two blinded, independent reviewers (S.D. and R.H.). In order to assess inter-reviewer reliability analysis, Kappa (κ) statistics were performed and a minimum value of 0.8 was considered acceptable [27]. In case of any disagreements, reviewers would discuss the discrepancies with a third author (S.B.), if necessary.

#### *2.5. Search Algorithms*

"Photodynamic therapy" **OR** "photochemotherapy"

## **AND**

"Scaling" **OR** "Root planing" **OR** "non-surgical periodontal therapy"

**AND**

"Periodontitis" **OR** "Chronic Periodontitis" **OR** "Aggressive Periodontitis" **OR** "Early Onset Periodontitis"

## *2.6. Eligibility Criteria*

2.6.1. Inclusion Criteria


#### 2.6.2. Exclusion Criteria


Changes in PPD and CAL from baseline up to the end of follow-up.

#### 2.7.2. Secondary Outcome Measures

Changes in GR, BOP, PI, GI, microbiological and immunological profile from baseline up to the end of follow-up.

#### *2.8. Data Extraction*

Two reviewers independently (S.D. and R.H.) selected eligible studies from the search. They performed the review, assessment and data extraction for each eligible study. Each study received an identification with the name of the first author, year of publication and origin. A tabular representation of additional relevant information such as impact factor of journal, study design, sample size, demographics of the participants, baseline characteristics, intervention and comparator groups, type of photosensitizer used and dosage, laser parameters utilized, number of aPDT sessions performed, follow-up duration, statistical tests performed and results and conclusions, were gathered from each eligible study.

#### *2.9. Qualitative Analysis*

A qualitative assessment for each study was performed using the Revised Cochrane Risk-of-Bias (RoB) tool for Randomized trials, Version 2.0 (RoB 2) by two independent reviewers (S.D. and R.H.) [28–30]. Detailed assessment under the following headings was performed: 1. Bias arising from the randomization process; 2. Bias due to deviations from intended interventions; 3. Bias due to missing outcome data; 4. Bias in measurement of the outcome; 5. Bias in selection of the reported result. Depending upon fulfilment of above-mentioned criteria, the studies were determined as low, moderate or high RoB. Disagreements between the reviewers were resolved by discussion with a third author (S.B.) as well as use of 'discrepancy check' feature in RoB 2, in order to obtain consensus.

#### *2.10. Statistical Analysis of Data*

When appropriate and quantifiable data of interest were extracted from the eligible studies and combined for meta-analyses, using Stata version 15.1 software (StataCorp, Pyrmont, Australia), random effects meta-analyses were conducted to reflect the expected

heterogeneity. As continuous outcomes were expected, overall treatment effects were calculated through pooled standardized mean differences (SMDs) with associated 95% confidence intervals (95% CIs) for PPD and CAL. When information was presented in median and inter-quartile ranges, means and SDs were estimated [31]. Results from SM and PG studies were pooled separately at 3 and 6 months, respectively. A pooled overall effect was considered statistically significant when *p* < 0.05. Consequently, statistical heterogeneity to identify outlier studies was performed by visual inspection of forest plots. Additionally, the Cochran Q test was conducted to assess statistical heterogeneity (*p* < 0.10) [32]. *I* 2 statistics for homogeneity was expressed in a range of 0–100%, with the following interpretation; 0% = no evidence of heterogeneity; 30–60% = moderate heterogeneity; 75–100% = high heterogeneity [33]. Sensitivity analysis was conducted to negate the effect of heterogeneity in between included studies by identifying the outlier studies by visual inspection of forest plots [34]. Publication bias was evaluated by visual assessment of funnel plot symmetry.

### **3. Results**

#### *3.1. Study Selection*

Four hundred and sixty-two study titles were obtained from a combined electronic and manual search. Four study titles were obtained from cross-references. Therefore, a total of 466 study titles were included from all databases in the preliminary screening (inter-reviewer agreement, κ = 0.9). Three hundred and eighty-seven articles were excluded, due to duplication and the remaining 79 records were further evaluated (inter-reviewer agreement, κ = 0.94). Twelve articles were excluded based on their titles and abstracts, mainly due to an inappropriate study design (inter-reviewer agreement, κ = 0.92). Thus, 67 articles were assessed for their eligibility. These articles were evaluated based on eligibility criteria. Additionally, 36 studies were excluded due to following reasons: Smokers were included or smoking details were not provided in 12 studies [23,35–45]; Laser or LLLT was utilized, as an adjunct to SRP in eight studies [46–53]; LED-aPDT was performed in seven studies [54–60]; aPDT was used in management of residual pockets in four studies [61–64] and as an adjunct to supportive periodontal therapy in two studies [65,66]; patients with systemic diseases were included in two studies [67,68], whereas one study did not perform a follow-up assessment [69] (inter-reviewer agreement, κ = 1). Hence, out of 67 full text articles, 31 articles were included and analyzed in the present systematic review [2,3,5,17,70–96]. All included articles were in vivo human studies. A meta-analysis on 18 out of 31 studies which assessed efficacy of SRP+aPDT was conducted [17,71,73–76,80,81,84–86,88–93,96] (inter-reviewer agreement, κ = 1). Figure 1 depicts the PRISMA flow diagram for search strategy utilized in the present systematic review and meta-analysis.

#### *3.2. Study Characteristics*

#### 3.2.1. Country of Origin

A substantial diversity in the country of origin was noted amongst included papers (Table 1). Distribution of studies was as follows: 11 in Brazil [2,3,5,17,71,82,83,87,92,95,96], 6 in India [78,81,88,89,91,93], 4 in Germany [76,77,80,94], 4 in Iran [70,85,86,90], 3 in Poland [72–74], whereas there is 1 study each, in the following countries; Spain [75], Japan [79], Thailand [84].

**Figure 1.** PRISMA flow diagram of the study selection criteria. **Figure 1.** PRISMA flow diagram of the study selection criteria.

#### *3.2. Study Characteristics*  3.2.2. Study Design

3.2.1. Country of Origin A substantial diversity in the country of origin was noted amongst included papers (Table 1). Distribution of studies was as follows: 11 in Brazil [2,3,5,17,71,82,83,87,92,95,96], Twenty studies were conducted using a SM study design [2,3,5,17,70,71,77,80–82, 84–87,89,90,92–95], whereas a PG study design was utilized in the remaining 11 studies [72–76,78,79,83,88,91,96] (Table 1).

#### 6 in India [78,81,88,89,91,93], 4 in Germany [76,77,80,94], 4 in Iran [70,85,86,90], 3 in Poland 3.2.3. Selection Criteria

76,78,79,83,88,91,96] (Table 1).

3.2.3. Selection Criteria

AgP [2,3,5,71–74,83,85,87].

3.2.4. Documentation of Laser Parameters

[72–74], whereas there is 1 study each, in the following countries; Spain [75], Japan [79], Thailand [84]. 3.2.2. Study Design Several inconsistencies were observed amongst the included studies [2,3,5,17,70–96], which have been outlined in Table 1, in which 21 out of 31 studies included patients with CP [17,70,75–82,84,86,88–96], whereas the remaining 10 studies included patients with AgP [2,3,5,71–74,83,85,87].

Twenty studies were conducted using a SM study design [2,3,5,17,70,71,77,80–82,84– 87,89,90,92–95], whereas a PG study design was utilized in the remaining 11 studies [72–

Several inconsistencies were observed amongst the included studies [2,3,5,17,70–96], which have been outlined in Table 1, in which 21 out of 31 studies included patients with CP [17,70,75–82,84,86,88–96], whereas the remaining 10 studies included patients with

Table 2 describes various dye laser combinations, as well as laser dosimetry that was utilized to perform aPDT in all eligible studies. Twenty-six out of 31 studies utilized a laser wavelength in the range of 630–690 nm [2,3,5,17,70–77,79,81–88,91,92,94–96] to perform aPDT. While four studies utilized a laser wavelength in the range of 808–810 nm

**Table 1.** Tabular representation of eligible in vivo human RCTs in terms of demography, study design, intervention groups, methods of assessment, evaluation period and outcomes. Refer to Supplementary file 2 for list of abbreviations.










#### 3.2.4. Documentation of Laser Parameters with the periodontal pocket in order to perform aPDT [2,3,5,78,81,82,84–86,88–96]. Only 5

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Table 2 describes various dye laser combinations, as well as laser dosimetry that was utilized to perform aPDT in all eligible studies. Twenty-six out of 31 studies utilized a laser wavelength in the range of 630–690 nm [2,3,5,17,70–77,79,81–88,91,92,94–96] to perform aPDT. While four studies utilized a laser wavelength in the range of 808–810 nm [78,80,90,93], one of the included studies utilized a 980 nm diode laser wavelength to perform aPDT [89] (Table 2) (Figure 2). Emission mode was reported only in five studies [78,88,90,92,93], in which four of them utilized a continuous wave emission mode [78,88,90,92], whilst the remaining one study utilized a gated continuous wave emission mode [93]. Eighteen out of the 31 eligible studies used the laser fibre tip in 'contact mode' with the periodontal pocket in order to perform aPDT [2,3,5,78,81,82,84–86,88–96]. Only 5 studies reported total energy, and it ranged from 1.5–9 J [82,90,92,93,95,96]. Only 19 studies reported power output in the range of 30 mW–1 W [71,75–80,82–85,87,89–96], whereas the use of a power meter to measure the therapeutic power output, reaching the target tissues was not performed in any of the included studies. Spot size was reported in only four studies [5,85,92,96] ranging from 0.02–0.07 cm<sup>2</sup> . Ten out of the 31 studies reported the diameter of fibre tip, [2,3,5,80–82,88,89,93,94] ranging from 200–600 µm. The energy density (fluence) was calculated in 18 out of 31 studies [5,17,70–74,78–80,82,83,86,87,92,93,95,96], and its value ranged from 0.01–2829 J/cm<sup>2</sup> , whereas the power density (irradiance) values ranged from 60 mW–4 W/cm<sup>2</sup> and were calculated in 13 studies [2,3,5,17,71–74,81,88,92,94,95]. Finally, the exposure time for laser irradiation was mentioned in all included studies except one study [80], and the values ranged from 10–120 s/site amongst included studies. studies reported total energy, and it ranged from 1.5–9 J [82,90,92,93,95,96]. Only 19 studies reported power output in the range of 30 mW–1 W [71,75–80,82–85,87,89–96], whereas the use of a power meter to measure the therapeutic power output, reaching the target tissues was not performed in any of the included studies. Spot size was reported in only four studies [5,85,92,96] ranging from 0.02–0.07 cm2. Ten out of the 31 studies reported the diameter of fibre tip, [2,3,5,80–82,88,89,93,94] ranging from 200–600 μm. The energy density (fluence) was calculated in 18 out of 31 studies [5,17,70–74,78– 80,82,83,86,87,92,93,95,96], and its value ranged from 0.01–2829 J/cm2, whereas the power density (irradiance) values ranged from 60 mW–4 W/cm2 and were calculated in 13 studies [2,3,5,17,71–74,81,88,92,94,95]. Finally, the exposure time for laser irradiation was mentioned in all included studies except one study [80], and the values ranged from 10–120 s/site amongst included studies. 3.2.5. PS Utilized Type of PS varied amongst eligible clinical trials. Eleven studies utilized phenothiazine chloride [2,3,5,71–74,76,81,84,94] while 10 employed methylene blue [17,75,77,79,82,87–89,96]. Five studies utilized toluidine blue O [70,85,86,91,95], four studies used indocyanine green [78,80,90,93], whereas chloro-aluminum phthalocyanine was utilized in one study [92] (Figure 3). Interestingly, 18 out of 31 studies specified the concentration of the PS [2,3,17,71,75,77–80,82,83,87–90,92,95,96], while 13 studies failed to report the same [5,70,72–74,76,81,84–86,91,93,94] (Table 2).

[78,80,90,93], one of the included studies utilized a 980 nm diode laser wavelength to perform aPDT [89] (Table 2) (Figure 2). Emission mode was reported only in five studies [78,88,90,92,93], in which four of them utilized a continuous wave emission mode [78,88,90,92], whilst the remaining one study utilized a gated continuous wave emission mode [93]. Eighteen out of the 31 eligible studies used the laser fibre tip in 'contact mode'

**Figure 2.** 3D pie diagram illustrating the percentage-wise distribution of predominant laser wavelengths utilized for aPDT in the included studies. **Figure 2.** 3D pie diagram illustrating the percentage-wise distribution of predominant laser wavelengths utilized for aPDT in the included studies.

#### 3.2.5. PS Utilized

Type of PS varied amongst eligible clinical trials. Eleven studies utilized phenothiazine chloride [2,3,5,71–74,76,81,84,94] while 10 employed methylene blue [17,75,77,79,82,87– 89,96]. Five studies utilized toluidine blue O [70,85,86,91,95], four studies used indocyanine green [78,80,90,93], whereas chloro-aluminum phthalocyanine was utilized in one study [92] (Figure 3). Interestingly, 18 out of 31 studies specified the concentration of the PS [2,3,17,71,75,77–80,82,83,87–90,92,95,96], while 13 studies failed to report the same [5,70,72–74,76,81,84–86,91,93,94] (Table 2).







**Figure 3.** 3D pie diagram illustrating the percentage-wise distribution of predominant photosensitizers utilized for aPDT in the included studies. **Figure 3.** 3D pie diagram illustrating the percentage-wise distribution of predominant photosensitizers utilized for aPDT in the included studies.

3.2.6. Utilization of aPDT as a Mono-Therapeutic or an Adjunctive Therapeutic Agent 3.2.6. Utilization of aPDT as a Mono-Therapeutic or an Adjunctive Therapeutic Agent

While 28 out of the 31 eligible studies utilized SRP+aPDT, aPDT monotherapy was performed in three studies [2,3,5] (Table 1). While 28 out of the 31 eligible studies utilized SRP+aPDT, aPDT monotherapy was performed in three studies [2,3,5] (Table 1).

#### 3.2.7. Comparison in between SRP+ aPDT versus SRP+AB 3.2.7. Comparison in between SRP+ aPDT versus SRP+AB

Six out of the 31 eligible studies compared efficacy of SRP+aPDT versus SRP+AB [72– 74,79,83,96] (Table 1). Six out of the 31 eligible studies compared efficacy of SRP+aPDT versus SRP+ AB [72–74,79,83,96] (Table 1).

#### 3.2.8. Number of aPDT Sessions 3.2.8. Number of aPDT Sessions

While a single session of aPDT was applied in 22 out of the 31 included studies [2,3,5,70,76–78,80–89,91–95], multiple aPDT sessions were performed in nine studies [17,71–75,79,90,96]. None of the eligible studies compared single versus multiple sessions of aPDT (Table 2). While a single session of aPDT was applied in 22 out of the 31 included studies [2,3,5,70,76–78,80–89,91–95], multiple aPDT sessions were performed in nine studies [17,71–75,79,90,96]. None of the eligible studies compared single versus multiple sessions of aPDT (Table 2).

#### 3.2.9. Follow-Up Assessment

3.2.9. Follow-Up Assessment A follow-up assessment at three months from the baseline visit was performed in 18 out of the 31 eligible studies [2,3,5,17,70,71,74,76,81,85–87,90–94,96], whereas 12 studies conducted a longer follow-up assessment at six months [72,73,75,77,78,80,82–84,88,89,95]. Only one study performed a follow-up assessment at one month from the baseline visit A follow-up assessment at three months from the baseline visit was performed in 18 out of the 31 eligible studies [2,3,5,17,70,71,74,76,81,85–87,90–94,96], whereas 12 studies conducted a longer follow-up assessment at six months [72,73,75,77,78,80,82–84,88,89,95]. Only one study performed a follow-up assessment at one month from the baseline visit [79]. A long-term follow-up of a minimum one year from baseline visit lacked in all eligible studies.

[79]. A long-term follow-up of a minimum one year from baseline visit lacked in all eligi-

#### ble studies. *3.3. Qualitative Assessment*

*3.3. Qualitative Assessment*  Qualitative assessment was performed using the RoB 2 tool, designed for in vivo human RCTs, as depicted in Figures 4 and 5. The most recent version of this tool was utilized to perform a qualitative assessment for both randomized PG and SM human RCTs [29,30]. Figure 4 represents a risk of bias assessment summary of all eligible studies. Figure 5 is a Qualitative assessment was performed using the RoB 2 tool, designed for in vivo human RCTs, as depicted in Figures 4 and 5. The most recent version of this tool was utilized to perform a qualitative assessment for both randomized PG and SM human RCTs [29,30]. Figure 4 represents a risk of bias assessment summary of all eligible studies. Figure 5 is a graphical representation of percentage RoB score for each risk domain, which has been evaluated, using the abovementioned tool. Furthermore, 53.1% of included trials

were at a high risk of inadequate randomization, whereas 40.6% and 6.3% of included trials were at a low risk or had some concerns, respectively. In addition, 50% of included studies were at a high risk of deviations from intended interventions, whereas 43.7% and 6.3% of them were at a low risk or had some concerns, respectively. All included papers reported substantial evidence (100%) for reporting missing outcome data and, hence, were at a low risk. Although a majority of studies were free of bias arising from reporting outcome measurement (71.9%), 28.1% were at a high risk. In terms of selective reporting of the results, inferences are as follows: 59.4% studies were at a high risk, 37.5% studies were at a low risk, and 3.1% studies had some concerns. Overall, 60% studies reported a high risk of bias, while 35% studies had a low risk of bias, and the final 5% studies had some concerns. It should be noted that information provided in these figures represents the consensual answers verified using the 'Discrepancy check' feature of RoB 2 tool, across two independent reviewers (S.D. and R.H.) (inter-reviewer agreement, κ = 0.94), and, in case of any disagreements, a third author (S.B.) was consulted. risk of inadequate randomization, whereas 40.6% and 6.3% of included trials were at a low risk or had some concerns, respectively. In addition, 50% of included studies were at a high risk of deviations from intended interventions, whereas 43.7% and 6.3% of them were at a low risk or had some concerns, respectively. All included papers reported substantial evidence (100%) for reporting missing outcome data and, hence, were at a low risk. Although a majority of studies were free of bias arising from reporting outcome measurement (71.9%), 28.1% were at a high risk. In terms of selective reporting of the results, inferences are as follows: 59.4% studies were at a high risk, 37.5% studies were at a low risk, and 3.1% studies had some concerns. Overall, 60% studies reported a high risk of bias, while 35% studies had a low risk of bias, and the final 5% studies had some concerns. It should be noted that information provided in these figures represents the consensual answers verified using the 'Discrepancy check' feature of RoB 2 tool, across two independent reviewers (S.D. and R.H.) (inter-reviewer agreement, κ = 0.94), and, in case of any disagreements, a third author (S.B.) was consulted.

graphical representation of percentage RoB score for each risk domain, which has been evaluated, using the abovementioned tool. Furthermore, 53.1% of included trials were at a high


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**Figure 4.** Risk of Bias assessment summary of the included studies based on the consensual answers across two individual assessors (S.D. and R.H.). **Figure 4.** Risk of Bias assessment summary of the included studies based on the consensual answers across two individual assessors (S.D. and R.H.).

**Figure 5.** Risk of Bias assessment graph of the included studies expressed as percentages, based on the consensual answers across two individual assessors (S.D. and R.H.). **Figure 5.** Risk of Bias assessment graph of the included studies expressed as percentages, based on the consensual answers across two individual assessors (S.D. and R.H.).

#### *3.4. Quantitative Assessment 3.4. Quantitative Assessment*

#### 3.4.1. Outcome Variables 3.4.1. Outcome Variables

Primary outcomes of 18 out of 31 studies, which have assessed efficacy of SRP+aPDT in the management of periodontitis, contributed to this meta-analysis [17,71,73– 76,80,81,84–86,88–93,96]. Data were pooled separately for SM and PG studies for differences in PPD and CAL respectively at three and six months, respectively. At three months, the mean difference in PPD reduction was not statistically significant for SM studies (SMD 0.166; 95% CI −0.278 to 0.611; *P* = 0.463) and PG studies (SMD 0.076; 95% CI −0.420 to 0.573; *P* = 0.763) along with a high heterogeneity for SM studies (Q = 15.81; *P* = 0.0001; I2 = 91.21%) and moderate heterogeneity (Q = 11.87; *P* = 0.018; I2 = 66.31%) for PG studies (Table 3). The mean difference in PPD reduction at six months did not show a statistically significant difference for SM studies (SMD 0.005; 95% CI –0.126 to 0.136; *P* = 0.935) as well as PG studies (SMD 0.141; 95% CI −1.007 to 1.288; *P* = 0.809) although contrasting findings were noted in terms of level of heterogeneity which was not evident for SM studies (Q = 0.06; *P* = 0.99; I2 = 0.00%) and high for PG studies (Q = 18.71; P = 0.0001; I2 = 89.31%) (Table 4). CAL gain at three months was not statistically significant in SM studies (SMD 0.092; 95% CI −0.013 to 0.198; *P* = 0.088) with no evident heterogeneity (Q = 8.74; *P* = 0.655; I2 = 0.00%) as well as in PG studies (SMD 0.056; 95% CI −0.408 to 0.552; *P* = 0.745) with moderate heterogeneity (Q = 8.95; *P* = 0.028; I2 = 70.31%) (Table 3). At six months, results for SM studies were not statistically significant (SMD −0.013; 95% CI −0.148 to 0.121; *P* = 0.846) with no evident heterogeneity (Q = 0.03; *P* = 0.984; I2 = 0.00%), whereas, for PG studies, the findings were statistically significant (SMD -0.441; 95% CI −0.805 to −0.075; *P* = 0.018) with no evidence of heterogeneity (Q = 1.70; *P* = 0.42; I2 = 0.00%) but favoring control group (Table 4). Assessment of secondary outcome variables was conducted in the majority of in-Primary outcomes of 18 out of 31 studies, which have assessed efficacy of SRP+aPDT in the management of periodontitis, contributed to this meta-analysis [17,71,73–76,80,81,84– 86,88–93,96]. Data were pooled separately for SM and PG studies for differences in PPD and CAL respectively at three and six months, respectively. At three months, the mean difference in PPD reduction was not statistically significant for SM studies (SMD 0.166; 95% CI −0.278 to 0.611; *P* = 0.463) and PG studies (SMD 0.076; 95% CI −0.420 to 0.573; *P* = 0.763) along with a high heterogeneity for SM studies (Q = 15.81; *P* = 0.0001; I<sup>2</sup> = 91.21%) and moderate heterogeneity (Q = 11.87; *P* = 0.018; I<sup>2</sup> = 66.31%) for PG studies (Table 3). The mean difference in PPD reduction at six months did not show a statistically significant difference for SM studies (SMD 0.005; 95% CI –0.126 to 0.136; *P* = 0.935) as well as PG studies (SMD 0.141; 95% CI −1.007 to 1.288; *P* = 0.809) although contrasting findings were noted in terms of level of heterogeneity which was not evident for SM studies (Q = 0.06; *P* = 0.99; I<sup>2</sup> = 0.00%) and high for PG studies (Q = 18.71; P = 0.0001; I<sup>2</sup> = 89.31%) (Table 4). CAL gain at three months was not statistically significant in SM studies (SMD 0.092; 95% CI <sup>−</sup>0.013 to 0.198; *<sup>P</sup>* = 0.088) with no evident heterogeneity (Q = 8.74; *<sup>P</sup>* = 0.655; I<sup>2</sup> = 0.00%) as well as in PG studies (SMD 0.056; 95% CI −0.408 to 0.552; *P* = 0.745) with moderate heterogeneity (Q = 8.95; *P* = 0.028; I<sup>2</sup> = 70.31%) (Table 3). At six months, results for SM studies were not statistically significant (SMD −0.013; 95% CI −0.148 to 0.121; *P* = 0.846) with no evident heterogeneity (Q = 0.03; *P* = 0.984; I<sup>2</sup> = 0.00%), whereas, for PG studies, the findings were statistically significant (SMD -0.441; 95% CI −0.805 to −0.075; *P* = 0.018) with no evidence of heterogeneity (Q = 1.70; *P* = 0.42; I<sup>2</sup> = 0.00%) but favoring control group (Table 4).

cluded studies, which are as follows: Changes in GR, BOP, PI and GI in 28 studies [3,17,70,71,73–96], microbiological analysis in 11 studies [5,71,75,78–80,85,86,91,94,95], and immuno-histological in seven studies [2,17,70–72,75,79]. Table 5 provides an overview of clinical parameters which have been assessed in 28 of 31 included studies along with corresponding level of significance, in accordance to data provided in Table 1. Eleven studies performed a microbiological analysis [5,71,75,78–80,85,86,91,94,95], out of which

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**Overall CAL Gain for SM Studies at 3 Months** 

**Overall CAL Gain for SM Studies at 3 Months** 

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**Table 4.** Forest plots illustrating the overall PPD reduction and CAL gain at 6 months. Refer to Supplementary file 2 for a list of abbreviations.

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**Overall PPD Reduction for PG Studies at 6 Months**  Study SMD SE 95% CI Weight (%) *P* = 0.809 Arweiler et al., 2014 −0.722 0.342 −1.417 to −0.027 33.04 Vidal et al., 2017 −0.109 0.322 −0.763 to 0.545 33.47 Raut et al., 2018 1.241 0.321 0.594 to 1.888 33.49 Total (random effects) 0.141 0.579 −1.007 to 1.288 100.00 **Overall PPD Reduction for PG Studies at 6 Months** Study SMD SE 95% CI Weight (%) *P* = 0.809 **Overall PPD Reduction for PG Studies at 6 Months**  Study SMD SE 95% CI Weight (%) *P* = 0.809 Arweiler et al., 2014 −0.722 0.342 −1.417 to −0.027 33.04 Vidal et al., 2017 −0.109 0.322 −0.763 to 0.545 33.47 Raut et al., 2018 1.241 0.321 0.594 to 1.888 33.49 Total (random effects) 0.141 0.579 −1.007 to 1.288 100.00 Arweiler et al., 2014 −0.722 0.342 −1.417 to −0.027 33.04 Vidal et al., 2017 −0.109 0.322 −0.763 to 0.545 33.47 Raut et al., 2018 1.241 0.321 0.594 to 1.888 33.49 Total (random effects) 0.141 0.579 −1.007 to 1.288 100.00

Heterogeneity: Q = 18.71; DF = 2; *P* = 0.0001; I2 = 89.31% Heterogeneity: Q = 18.71; DF = 2; *P* = 0.0001; I2 = 89.31% Heterogeneity: Q = 18.71; DF = 2; *P* = 0.0001; I2 = 89.31% **Overall CAL Gain for SM Studies at 6 Months** 

**Overall CAL Gain for SM Studies at 6 Months** 

*P* = 0.846

*P* = 0.846

Study SMD SE 95% CI Weight (%)

Study SMD SE 95% CI Weight (%)

Malgikar et al., 2015 −0.057 0.284 −0.629 to 0.515 5.82 Bundidpun et al., 2017 0.025 0.310 −0.602 to 0.653 4.88 Hill et al., 2019 −0.012 0.072 −0.155 to 0.130 89.30 Total (random effects) −0.013 0.068 −0.148 to 0.121 100.00

Malgikar et al., 2015 −0.057 0.284 −0.629 to 0.515 5.82 Bundidpun et al., 2017 0.025 0.310 −0.602 to 0.653 4.88 Hill et al., 2019 −0.012 0.072 −0.155 to 0.130 89.30 Total (random effects) −0.013 0.068 −0.148 to 0.121 100.00


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447

Heterogeneity: Q = 1.70; DF = 2; *P* = 0.42; I2 = 0.00%

Heterogeneity: Q = 1.70; DF = 2; *P* = 0.42; I2 = 0.00%

Heterogeneity: Q = 1.70; DF = 2; *P* = 0.42; I2 = 0.00%

Assessment of secondary outcome variables was conducted in the majority of included studies, which are as follows: Changes in GR, BOP, PI and GI in 28 studies [3,17,70,71,73–96], microbiological analysis in 11 studies [5,71,75,78–80,85,86,91,94,95], and immuno-histological in seven studies [2,17,70–72,75,79]. Table 5 provides an overview of clinical parameters which have been assessed in 28 of 31 included studies along with corresponding level of significance, in accordance to data provided in Table 1. Eleven studies performed a microbiological analysis [5,71,75,78–80,85,86,91,94,95], out of which five studies reported that aPDT therapy could significantly reduce periopathogenic burden [71,75,78,91,94] and six studies failed to achieve this outcome [5,79,80,85,86,95]. In terms of immune-histological analysis, seven studies [2,17,70–72,75,79] assessed various pro-inflammatory cytokines and growth factors such as; IL-1β, IL-10, IF-γ, TNF-α, MMP-8, MMP-9, RANK, RANK-L, OPG and FGF-2 (Table 1). Biomarkers for assessment of bone resorption (RANK, RANK-L, OPG) were assessed in three studies [2,17,75]. Two studies [17,75] assessed efficacy of SRP+aPDT in comparison to conventional SRP alone, and showed that SRP+aPDT successfully suppressed the bone resorption process. Levels of IL-1β, IL-10, IF-γ, and TNF-α were assessed in three studies [70,71,79], of which two studies have confirmed immunological benefits of aPDT [70,71], whereas one study [79] failed to show any advantage of aPDT for the same. It should, however, be noted that, while the former two studies [70,71] have compared the efficacy of SRP+aPDT to conventional SRP alone, the latter study [79] has compared SRP+aPDT to SRP+AB and demonstrated the advantages of AB over aPDT. Additionally, SRP+aPDT showed an increased expression of FGF-2, which plays a role in tissue repair as compared to SRP alone, and was assessed in only one study [17]. A meta-analysis on secondary outcomes was not possible due to disparity in scoring methodology, incomplete, or incomparable data.

### 3.4.2. Sensitivity Analysis

A sensitivity analysis was conducted due to the noteworthy heterogeneity arising from outlier studies which were detected upon visual inspection of Forest plots [74,86,90,92,93] (Tables 3 and 4). This analysis was conducted only for the three-month follow-up due to unavailability of data in included studies (Table 6). In terms of PPD reduction, SM studies (SMD 0.282; 95% CI −0.286 to 0.624; *P* = 0.153) as well as PG studies (SMD 0.257; 95% CI −0.230 to 0.683; *P* = 0.361) did not report a statistically significant improvement. No evident heterogeneity (Q = 9.14; *P* = 0.7; I<sup>2</sup> = 0.00%) in SM studies and in PG studies (Q = 8.87; *P* = 0.22; I<sup>2</sup> = 0.00%) was noted (Table 6). Although improvement in CAL gain was noted after omitting outlier studies, this difference was statistically not significant in both SM (SMD 0.162; 95% CI −0.326 to 0.406; *P* = 0.166) and PG studies (SMD 0.227; 95% CI <sup>−</sup>0.420 to 0.673; *<sup>P</sup>* = 0.234) with no evident heterogeneity (Q = 8.40; *<sup>P</sup>* = 0.625; I<sup>2</sup> = 0.00%) in SM studies as well as in PG studies (Q = 9.7; *P* = 0.22; I<sup>2</sup> = 0.00%) (Table 6).





**Table 6.** Forest plots based on sensitivity analysis illustrating the overall PPD reduction and CAL gain at 3 months without outlier studies. Refer to Supplementary file 2 for a list of abbreviations. **Table 6.** Forest plots based on sensitivity analysis illustrating the overall PPD reduction and CAL gain at 3 months without outlier studies. Refer to Supplementary file 2 for a list of abbreviations.

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451

**Overall CAL Gain for SM Studies at 3 Months** 

**Overall CAL Gain for SM Studies at 3 Months** 

*P* = 0.166

*P* = 0.166

Heterogeneity: Q = 8.40; DF = 7; *P* = 0.625; I2 = 0.00%

Study SMD SE 95% CI Weight (%)

Heterogeneity: Q = 8.40; DF = 7; *P* = 0.625; I2 = 0.00%

Raj et al., 2016 0.669 0.441 −0.258 to 1.595 18.89 Vidal et al., 2017 −0.102 0.372 −0.514 to 0.793 25.59 Theodoro et al., 2017 −0.106 0.374 −0.997 to 0.743 22.43 Joseph et al., 2014 0.456 0.255 −0.120 to 0.673 33.19 Total (random effects) 0.227 0.352 −0.420 to 0.673 100.00

Study SMD SE 95% CI Weight (%)

Raj et al., 2016 0.669 0.441 −0.258 to 1.595 18.89 Vidal et al., 2017 −0.102 0.372 −0.514 to 0.793 25.59 Theodoro et al., 2017 −0.106 0.374 −0.997 to 0.743 22.43 Joseph et al., 2014 0.456 0.255 −0.120 to 0.673 33.19 Total (random effects) 0.227 0.352 −0.420 to 0.673 100.00

**Overall CAL Gain for PG Studies at 3 Months** 

**Overall CAL Gain for PG Studies at 3 Months** 

*P* = 0.234

*P* = 0.234

Study SMD SE 95% CI Weight (%)

Heterogeneity: Q = 8.87; DF = 3; *P* = 0.22; I2 = 0.00%

Heterogeneity: Q = 8.87; DF = 3; *P* = 0.22; I2 = 0.00%

Chitsazi et al., 2014 0.439 0.287 −0.140 to 1.017 5.52 Moreira et al., 2015 −0.040 0.310 −0.667 to 0.588 5.02 Franco et al., 2014 0.601 0.364 −0.144 to 1.346 4.20 Malgikar et al., 2015 −0.048 0.284 −0.620 to 0.523 5.60 Ahad et al., 2016 0.158 0.199 −0.237 to 0.552 10.35 Bundidpun et al., 2017 0.032 0.310 −0.595 to 0.660 5.02 Hill et al., 2019 0.019 0.072 −0.123 to 0.161 59.29 Braun et al., 2008 0.161 0.310 −0.468 to 0.789 5.01 Total (random effects) 0.162 0.253 −0.326 to 0.406 100.00

Study SMD SE 95% CI Weight (%)

Chitsazi et al., 2014 0.439 0.287 −0.140 to 1.017 5.52 Moreira et al., 2015 −0.040 0.310 −0.667 to 0.588 5.02 Franco et al., 2014 0.601 0.364 −0.144 to 1.346 4.20 Malgikar et al., 2015 −0.048 0.284 −0.620 to 0.523 5.60 Ahad et al., 2016 0.158 0.199 −0.237 to 0.552 10.35 Bundidpun et al., 2017 0.032 0.310 −0.595 to 0.660 5.02 Hill et al., 2019 0.019 0.072 −0.123 to 0.161 59.29 Braun et al., 2008 0.161 0.310 −0.468 to 0.789 5.01 Total (random effects) 0.162 0.253 −0.326 to 0.406 100.00


**Figure 6.** (**a**,**b**) Funnel plots illustrating the publication bias in overall PPD reduction in SM and PG studies, respectively; (**c**,**d**) funnel plots illustrating the publication bias in overall CAL gain in SM and PG studies, respectively. Each circle represents a single included study, the y-axis and x-axis represent the standard error of the effect estimate and the results of the study respectively and the graphical plot resembles an inverted funnel with scatter due to sampling variations.

**4. Discussion** 

Based on the hypothesis that aPDT monotherapy or as an adjunct to NSPT can enhance the clinical or microbiological or immunological profile in comparison to conventional SRP, or SRP+AB, a critical appraisal of the available scientific evidence was conducted. After meticulous scrutiny, 31 studies were included in the present systematic review [2,3,5,17,70–96]. Owing to the methodological discrepancies, only 18 out 31 studies were eligible for a meta-analysis [17,71,73–76,80,81,84–86,88–93,96]. This report is the first to evaluate the role of SRP+aPDT compared to SRP alone or SRP+AB in SM and PG studies in AgP as well as in CP patients. The results of this meta-analysis indicated that, in comparison to SRP alone or SRP+AB, SRP+aPDT failed to show any additional benefit in the management of periodontitis up to six months. A significant heterogeneity was reported, arising from confounders in aPDT protocols. Subsequently, after omitting outlier studies [74,86,90,92,93], sensitivity analysis was able to eliminate heterogeneity completely but

Heterogeneity: Q = 8.87; DF = 3; *P* = 0.22; I2 = 0.00%

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**Table 6.** Forest plots based on sensitivity analysis illustrating the overall PPD reduction and CAL gain at 3 months without

**Overall PPD Reduction for SM Studies at 3 Months** 

*P* = 0.153

Heterogeneity: Q = 9.14; DF = 7; *P* = 0.71; I2 = 0.00%

Study SMD SE 95% CI Weight (%)

Raj et al., 2016 0.669 0.441 −0.258 to 1.595 18.89 Vidal et al., 2017 −0.060 0.322 −0.714 to 0.593 25.59 Theodoro et al., 2017 −0.127 0.374 −0.897 to 0.643 22.43 Joseph et al., 2014 0.556 0.215 0.127 to 0.984 33.19 Total (random effects) 0.257 0.278 −0.230 to 0.683 100.00

**Overall PPD Reduction for PG Studies at 3 Months** 

*P* = 0.361

outlier studies. Refer to Supplementary file 2 for a list of abbreviations.

Study SMD SE 95% CI Weight (%)

Chitsazi et al., 2014 0.525 0.289 −0.056 to 1.106 12.28 Moreira et al., 2015 0.205 0.311 −0.425 to 0.834 12.11 Franco et al., 2014 0.631 0.364 −0.115 to 1.378 11.67 Malgikar et al., 2015 0.119 0.284 −0.453 to 0.691 12.31 Ahad et al., 2016 0.639 0.204 0.235 to 1.043 12.88 Bundidpun et al., 2017 0.007 0.310 −0.620 to 0.635 12.11 Hill et al., 2019 0.039 0.072 −0.103 to 0.181 14.47 Braun et al., 2008 0.139 0.310 −0.490 to 0.767 12.11 Total (random effects) 0.282 0.234 −0.286 to 0.624 100.00

#### 3.4.3. Publication Bias

Heterogeneity: Q = 9.7; DF = 3; *P* = 0.22; I2 = 0.00%

Visual inspection of funnel plots revealed noticeable asymmetry in SM-study analysis for PPD reduction indicating a probable risk of publication bias in SM studies included in this meta-analysis. However, slight asymmetries for PG studies were noted in corresponding funnel plots suggestive of a low risk of publication bias in the same (Figure 6).

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**Figure 6.** (**a**,**b**) Funnel plots illustrating the publication bias in overall PPD reduction in SM and PG studies, respectively; (**c**,**d**) funnel plots illustrating the publication bias in overall CAL gain in SM and PG studies, respectively. Each circle represents a single included study, the y-axis and x-axis represent the standard error of the effect estimate and the results of the study respectively and the graphical plot resembles an inverted funnel with scatter due to sampling variations. **Figure 6.** (**a**,**b**) Funnel plots illustrating the publication bias in overall PPD reduction in SM and PG studies, respectively;(**c**,**d**) funnel plots illustrating the publication bias in overall CAL gain in SM and PG studies, respectively. Each circle represents a single included study, the y-axis and x-axis represent the standard error of the effect estimate and the results of the study respectively and the graphical plot resembles an inverted funnel with scatter due to sampling variations.

#### **4. Discussion 4. Discussion**

Based on the hypothesis that aPDT monotherapy or as an adjunct to NSPT can enhance the clinical or microbiological or immunological profile in comparison to conventional SRP, or SRP+AB, a critical appraisal of the available scientific evidence was conducted. After meticulous scrutiny, 31 studies were included in the present systematic review [2,3,5,17,70–96]. Owing to the methodological discrepancies, only 18 out 31 studies were eligible for a meta-analysis [17,71,73–76,80,81,84–86,88–93,96]. This report is the first to evaluate the role of SRP+aPDT compared to SRP alone or SRP+AB in SM and PG studies in AgP as well as in CP patients. The results of this meta-analysis indicated that, in comparison to SRP alone or SRP+AB, SRP+aPDT failed to show any additional benefit in the management of periodontitis up to six months. A significant heterogeneity was reported, arising from confounders in aPDT protocols. Subsequently, after omitting outlier studies [74,86,90,92,93], sensitivity analysis was able to eliminate heterogeneity completely but Based on the hypothesis that aPDT monotherapy or as an adjunct to NSPT can enhance the clinical or microbiological or immunological profile in comparison to conventional SRP, or SRP+AB, a critical appraisal of the available scientific evidence was conducted. After meticulous scrutiny, 31 studies were included in the present systematic review [2,3,5,17,70–96]. Owing to the methodological discrepancies, only 18 out 31 studies were eligible for a meta-analysis [17,71,73–76,80,81,84–86,88–93,96]. This report is the first to evaluate the role of SRP+aPDT compared to SRP alone or SRP+AB in SM and PG studies in AgP as well as in CP patients. The results of this meta-analysis indicated that, in comparison to SRP alone or SRP+AB, SRP+aPDT failed to show any additional benefit in the management of periodontitis up to six months. A significant heterogeneity was reported, arising from confounders in aPDT protocols. Subsequently, after omitting outlier studies [74,86,90,92,93], sensitivity analysis was able to eliminate heterogeneity completely but failed to report statistically significant improvements in primary outcome variables. Furthermore, risk of publication bias was reported indicating a possible selective outcome reporting in eligible published studies. In some instances, missing outcomes could not be detected by comparing the published report with the respective study protocol due to unavailability of the latter. Until now, seven meta-analyses have been reported to assess the role of aPDT in periodontitis [6–12]. The present review protocol is in accordance with the existing reviews [8,10–12]. Azaripour et al. is the only other systematic review

and meta-analysis that has assessed the efficacy of SRP+aPDT as compared to SRP alone for SM and PG studies [8]. While three reviews report short-term benefits of aPDT up to 6 months [7–9], four have reported otherwise [6,10–12]. Our findings are in accordance with findings of the latter scientific reports. In order to gain an insight on merits and inadequacies of each included study, a comprehensive and systematic investigation was performed as follows:

#### *4.1. Role of Baseline Characteristics*

A key feature of RCTs is the application of balanced baseline characteristics in treatment arms of the trial in order to achieve unbiased treatment outcomes [97]. Most often, researchers provide a tabular representation of relevant variables to confirm an impartial baseline evaluation. In case of missing information on baseline characteristics, a 'selection bias' can be suspected [98]. All eligible studies have provided this vital information and were free from any kind of 'selection bias'. Additionally, evidence-based studies have suggested the potential harmful effects of smoking on the onset and progression of periodontitis, for which smokers were excluded [99,100]. Likewise, the inter-relationship of periodontitis and its systemic manifestations are well-established, resulting in the exclusion of patients with systemic diseases [101,102]. Utilization of 'placebo/sham' procedures to enhance clinical outcomes of the trial is an evidence-based verified concept [103]. In the present systematic review, only six out of 31 studies [71,78,92–94,96] have utilized a 'placebo/sham' procedure as an adjunct to conventional SRP. Furthermore, the role of SRP+aPDT+AB, compared to SRP+AB and SRP+aPDT as well the efficacy of SRP+PS compared to the conventional SRP and SRP+ aPDT have been assessed in this review [83,95]. Differences in study designs were apparent and the majority of clinicians have utilized the SM study design in oral health research [104]. Hence, this meta-analysis included both SM and PG studies in order to assess whether the estimated intervention effect has differed between them.

#### *4.2. Assessment Methods for Various Parameters and Their Inferences to Determine aPDT Efficacy*

A decrease in periodontal inflammation is directly proportional to a decrease in the incidence of BOP and detectable plaque levels [105]. Furthermore, the endpoints of a comprehensive periodontal therapy include PPD reduction and CAL gain, both of which are crucial for determining treatment success [106]. Clinical evidence has proven that there is a direct correlation between initial severity of PPD and CAL values and the amount of post-operative differences [107–109]. In terms of disease severity at baseline evaluation, a lack of homogeneity in pre-treatment values of PPD and CAL in the data extracted from various studies was observed (Table 1). Therefore, variations in level of significance across clinical parameters were noted, thus making it difficult to provide a cumulative result (Table 5). Utilization of a narrow laser optic fibre tip in deep periodontal pockets facilitates easy and atraumatic periodontal pocket probing ultimately resulting in the PPD and GR reduction post-operatively [3,88]. Furthermore, evidence-based research has proven that clinical outcome remains unaffected by the type of instrumentation utilized in SRP [76,110], which was observed to vary across all eligible studies (Table 1).

An imbalance amongst the local etiological factors such as dental plaque and calculus, inflammation, and a host defense system can have a great impact on the disease severity and progression [17]. Hence, it is essential to monitor the microbiological and the molecular changes of various growth factors and proinflammatory cytokines. De Oliveira and coworkers have demonstrated, through their studies, the importance of SRP in reducing bacterial load from tooth surface and the failure of aPDT monotherapy in reducing the bacterial counts of *A.a*. periodontal disease activity as well as bone resorption [2,5].

#### *4.3. Representation of the Treatment Outcomes*

Positive outcomes of any treatment strategy are governed by several factors such as evaluation of disease status at carefully planned follow-up visits, signs of unevent-

ful healing, role of supportive periodontal therapy (SPT) and patient compliance with treatment [111–113]. The majority of included studies performed follow-up assessment for up to 3 or 6 months, whereas results of a longer follow-up ranging up to 1–2 years was lacking. Collective data obtained from longitudinal studies have confirmed the role of long-term follow up visits in greater reduction of clinical parameters. These findings have been confirmed by three studies included in the present review [73,77,95]. With regard to healing outcomes, 24 out of 31 studies reported no uneventful healing associated with the absence of any postoperative complications, after application of aPDT [2,3,5,70–75,77–81,83–90,92,95]. While six studies have failed to provide any information on healing outcomes [17,76,82,91,93,94], one study [96] has reported a gastrointestinal complication in one patient of the aPDT group. This happens to be unique evidence that has not been registered elsewhere. The presence of residual plaque and calculus resulting in a relapse of periodontal disease severity being inevitable with aPDT monotherapy. Hence, the role of SPT is quintessential. In the present systematic review, apart from the three sequential studies conducted by De Oliveira and co-workers [2,3,5] which utilized aPDT monotherapy, all of the other studies have utilized SRP+aPDT. In the former group of studies, a supragingival professional tooth cleaning was performed 14 days prior to the application of aPDT monotherapy in the treatment sites, which have extensively improved the post-operative clinical findings. Likewise, amongst the studies that have assessed the efficacy of SRP+aPDT, only seven studies [71,75,82,84,90,92,95] have mentioned a planned SPT protocol, which was implemented throughout their study period. Furthermore, information on oral hygiene instructions tailored according to respective study criteria has been specified in all included studies. Co-relation of patient's compliance in adhering to hygiene instructions cannot be overlooked, since it is vital for treatment success and prevention of disease recurrence [114]. Quantitative measurement of plaque by means various indices can aid in monitoring compliance towards therapy. In connection to this, the assessment of plaque levels was performed in 21 studies included in this review [3,17,71,73–75,77–79,81,82,84,85,88–94]. Therefore, an inconsistency in the representation of treatment outcomes in this review is evident.

#### *4.4. Role of Laser Parameters*

Apart from study methodology, laser parameters are crucial in determining treatment outcome. Calibration of therapeutic power output with a power-meter can aid in regulating low output power for achieving the desired aPDT effect at treatment sites [115]. This technique can also prevent any inadvertent damage caused by utilization of high output power, resulting in a photothermal effect. However, none of the included studies have utilized a power-meter to calibrate the power output. Furthermore, some other parameters that have been overlooked were: emission mode, contact/non-contact mode, energy/treated site, power output, spot size/fibre diameter, fluence and irradiance. Diode lasers have a high affinity to pigment, which, in the case of aPDT, is the PS. However, inflamed periodontal tissues are rich in blood and high levels of proteins, which are also rich in pigments. Traumatic instrumentation can lacerate the sulcus lining and elicit bleeding [23]. Consequently, the overall aPDT effect, which could be achieved by an effective PS dye-laser wavelength combination, will be compromised [23,116]. Additionally, placement of the fibre tip inside the gingival sulcus needs to be performed judiciously, in order to avoid further trauma to inflamed gingival sulcus, serving as a niche for plaque accumulation and favoring disease relapse [3,88].

Bacterial re-colonization after SRP has been proven to occur after three weeks [117] and, hence, multiple aPDT sessions can help to delay this pathological process. Annaji et al., 2016 [46] have compared the efficacy of three sessions (0, 7th and 21st day) versus a single session (0 day) of aPDT in the management of CP, and concluded that the group receiving multiple sessions demonstrated superior treatment outcomes. Nine studies included in this review conducted multiple aPDT sessions [17,71–75,79,90,96], and have unanimously concluded that the utilization of multiple aPDT sessions has positive healing effects. However, the frequency of aPDT application varies in all these studies (Table 2). As a result, a conclusion on the ideal number of sessions and frequency of application of aPDT cannot be drawn.

Additionally, PS concentration and pre-irradiation time (wash-out or PS incubation time) are governed by the binding capacity of PS to target cells [118]. It was observed in 13 out of 31 studies, whereby the PS concentration was not reported [5,70,72–74,76,81,84– 86,91,93,94], whereas a range of PS concentrations have been utilized in the remaining eligible studies. Furthermore, four studies have failed to mention PS pre-irradiation time [5,75,77,90], whereas the remaining 27 studies have reported this time, which ranged from 1–5 min for different PS. An in vitro study by Fumes et al. [119] evaluated the effect of different PS pre-irradiation time periods (1, 2 and 5 min) in aPDT on the biofilms formed by *Streptococcus mutans* and *Candida albicans,* by monitoring the microbial load and have successfully demonstrated that the efficacy of all pre-irradiation times was equal. They emphasized patient discomfort associated with longer pre-irradiation times and thus have advised the use of shorter pre-irradiation times (1 min) in future studies. Up until now, there have been no studies that have determined the minimal duration of PS incubation as well as its role against periopathogens. Hence, further studies on this subject should be sought after. Moreover, discrepancies in the reported data, in terms of the number of sites receiving aPDT application per tooth as well the irradiation time, were noticed amongst the eligible studies (Table 2). These voids have raised concerns regarding the reliability of existing literature, which lacks a reproducible methodology and ultimately hampers the rational use of aPDT in non-surgical management of periodontitis.

### *4.5. Role of RoB Assessment*

A vast majority of bias were raised from inadequate randomization, deviations from intended interventions and selective reporting of results (Figures 4 and 5). Another key finding of this systematic review was the presence of a potential conflict of interest in 10 out of the 31 studies [2,3,5,72–74,76,79,80,94]. Therefore, the results of the included studies are questionable, and their biased methodology cannot be relied upon.

#### *4.6. Limitations of the Present Systematic Review*

The majority of the included studies have assessed the efficacy of SRP+aPDT in comparison to aPDT monotherapy resulting in a lack of meta-analysis on the latter. Utilization of a limited number of teeth (mostly anterior teeth) or on specific sites (interproximal sites in case of deep pockets) or on any two quadrants of the dental arch that could facilitate cross contamination from the untreated sites and overshadow the putative benefits of aPDT was noted. In this review, efficacy of aPDT was monitored in systemically healthy non-smokers only, and its benefits in their immunocompromised counterparts were not established. A lack of long-term follow-up in order to determine the stability in healing after aPDT was also observed. The number of studies included in the meta-analysis was nearly half of the studies eligible for a systematic review due to paucity and inconsistency in available literature. Owing to the aforesaid drawbacks, the objective of this review could not be accomplished.

#### *4.7. Future Scope*

Future investigations should compare aPDT monotherapy versus SRP+aPDT and provide details of all appropriate laser and PS parameters. Efficacy of aPDT in smokers with various systemic diseases in CP and AgP should be established. The role of patient related outcomes and SPT should be emphasized upon. Nevertheless, future RCTs must have a robust methodology with balanced baseline characteristics, performed by experienced, masked and calibrated clinicians, which will reduce bias. Owing to the evident benefits of a PG-study design, clinicians should prefer its utilization in order to minimize potential 'carry-over' effects. Additionally, researchers should conduct a full mouth study protocol with a long-term follow-up assessment of minimum 6 months duration, consisting of, but

not limited to, a vast range of clinical, microbiological, radiographic as well as, immunehistological profiles.

#### **5. Conclusions**

Within the limits of the present systematic review and meta-analysis, it can be concluded that the efficacy of aPDT in improving treatment outcomes, when it is utilized in the non-surgical management of periodontitis, remains debatable. However, the results of a majority of included studies have demonstrated the effectiveness of aPDT, and this role is more pronounced for SRP+aPDT rather than aPDT monotherapy. A careful and critical appraisal was performed which helped to obtain a qualitative assessment of eligible studies, thus highlighting the substantial flaws that prevent a reproducible methodology. Data on standardized aPDT study protocol, ideal PS dye-laser combination, optimal laser and PS parameters remain inconsistent and inconclusive amongst the prevalent literature owing to a highly inferior RoB in many studies. Finally, future research should aim for well-designed, robust and preferably PG-RCTs that will overcome the abovementioned limitations and confounders, in order to achieve palpable progress in this field of research while ensuring the use of an appropriate local laser safety protocol.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics13060836/s1, PRISMA Checklist (Supplementary file 1), List of abbreviations (Supplementary file 2).

**Author Contributions:** Conceptualization, S.D., R.H.; methodology, S.D., R.H.; software, I.R.B., T.S.; validation, S.D., S.B., R.H., I.R.B.; formal analysis, S.B., I.R.B.; investigation, S.D., R.H.; resources, S.D., R.H., S.B., T.S., I.R.B.; data curation, S.D., R.H.; writing: original draft preparation, S.D., R.H.; writing: review and editing, S.D., R.H.; visualization, S.B., I.R.B., T.S.; supervision, S.D., R.H., S.B., T.S., I.R.B.; project administration, T.S., I.R.B.; funding acquisition, T.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Antimicrobial Photodynamic Therapy: Latest Developments with a Focus on Combinatory Strategies**

**Raphaëlle Youf <sup>1</sup> , Max Müller <sup>2</sup> , Ali Balasini <sup>3</sup> , Franck Thétiot 4,\*, Mareike Müller <sup>2</sup> , Alizé Hascoët <sup>1</sup> , Ulrich Jonas <sup>3</sup> , Holger Schönherr 2,\* , Gilles Lemercier 5,\* , Tristan Montier 1,6 and Tony Le Gall 1,\***


**Abstract:** Antimicrobial photodynamic therapy (aPDT) has become a fundamental tool in modern therapeutics, notably due to the expanding versatility of photosensitizers (PSs) and the numerous possibilities to combine aPDT with other antimicrobial treatments to combat localized infections. After revisiting the basic principles of aPDT, this review first highlights the current state of the art of curative or preventive aPDT applications with relevant clinical trials. In addition, the most recent developments in photochemistry and photophysics as well as advanced carrier systems in the context of aPDT are provided, with a focus on the latest generations of efficient and versatile PSs and the progress towards hybrid-multicomponent systems. In particular, deeper insight into combinatory aPDT approaches is afforded, involving non-radiative or other light-based modalities. Selected aPDT perspectives are outlined, pointing out new strategies to target and treat microorganisms. Finally, the review works out the evolution of the conceptually simple PDT methodology towards a much more sophisticated, integrated, and innovative technology as an important element of potent antimicrobial strategies.

**Keywords:** antimicrobials; ROS; combinatory strategies; photodynamic therapy; multidrug resistance; nanoparticles; photosensitizers

## **1. Introduction**

Antimicrobial resistance (AMR) occurring in bacteria, viruses, fungi, and parasites is a global health and development threat, declared by the WHO as one of the top 10 global public health threats facing humanity. The misuse and overuse of antimicrobials make almost all disease-causing microbes resistant to drugs commonly used to treat them [1]. Multidrug resistance (MDR) to critical classes of antibiotics has gradually increased in nosocomial pathogens, including *Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, and *Enterobacter* spp. (which are gathered in the so-called ESKAPE group) [2]. Currently, in Europe, AMR is estimated to be responsible for 33,000 deaths every year and the annual economic toll, covering extra

**Citation:** Youf, R.; Müller, M.; Balasini, A.; Thétiot, F.; Müller, M.; Hascoët, A.; Jonas, U.; Schönherr, H.; Lemercier, G.; Montier, T.; et al. Antimicrobial Photodynamic Therapy: Latest Developments with a Focus on Combinatory Strategies. *Pharmaceutics* **2021**, *13*, 1995. https:// doi.org/10.3390/pharmaceutics13121995

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 7 October 2021 Accepted: 17 November 2021 Published: 24 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

healthcare costs and productivity losses, amounts to at least EUR 1.5 billion [3,4]. Unless adequately tackled, 10 million people a year will die from drug-resistant infections by 2050, according to the predictions of the government-commissioned O'Neill report [5].

With the decline in the discovery of new antimicrobials since 1970s, the mainstream approach for the development of new drugs to combat emerging and re-emerging resistant pathogens has focused on the modification of existing compounds. However, one key recommendation encourages stimulation of early stage drug discovery [6]. Among emerging antimicrobial therapeutic alternatives, light-based approaches show particular promise [7]. Traditionally, phototherapy was already a common practice in ancient Greece, Egypt, and India to treat skin diseases [8]. At the beginning of the 20th century, Oscar Raab first described the phototoxicity of the dye acridine red against *Paramecium caudatum,* and Tappenier and Jesionek reported the photodynamic effects of eosin suitable for treating diverse cutaneous diseases. Since then, PDT was established as the administration of a non-toxic photosensitizer (PS) followed by exposure to light irradiation at an appropriate wavelength focused on an area to treat [7]. While anti-cancer PDT is a clinical reality for 25 years [9], PDT as an antimicrobial treatment was demonstrated for the first time against drug-resistant infections in the healthcare sector in the early 1990s, leading to a "photo-antimicrobial renaissance era" [7]. Major MDR bacteria have been found susceptible to antimicrobial PDT (aPDT), independently of their drug-resistance profiles [10,11]. To date, resistance to aPDT is rarely reported, indicating that the possibility for microbes to adapt and escape this treatment can occur but is still contained. More effective aPDT systems are continuously developed, notably via combinatory approaches using multiple chemical systems and/or modalities. At the current stage of development, aPDT cannot address systemic infections but it holds great promise for treating localized infections and to fight AMR.

While excellent earlier authoritative reviews provide a detailed description of aPDT [12–15], the present review focuses on most recent developments in the field for the last 5 years, with a focus on aPDT combinatory strategies. It should be noticed that aPDT is sometimes referred to as photodynamic antimicrobial chemotherapy, light-based antimicrobial therapy, photo-controlled antimicrobial therapy, or antimicrobial photo-inactivation. In this review, all these synonyms were considered to present (i) the current state of aPDT applications in preclinical and clinical settings, (ii) a state of the art with recent developments in photosystems, (iii) the implementation of multicomponent nanotechnologies and recent molecular engineering, and (iv) the exploration of combinatory aPDT approaches towards possible future therapeutic innovations.

#### **2. PDT: General Presentation and Features**

#### *2.1. Photochemical Pathways and Reactive Oxygen Species Production*

Generally speaking, a given PS has the potential to produce reactive oxygen species (ROS) under specific conditions (Figure 1A). Typically, it possesses a stable electronic configuration called ground state level. Following irradiation and absorption of a photon, the PS is converted from a low (fundamental) energy level (1PS) to a "Frank Condon" short-lived, very reactive, excited singlet state <sup>1</sup>PS\* [16–18]. Subsequently, the PS can lose energy by emitting fluorescence or heat via internal conversion (IC), thereby returning to its initial ground state level; alternatively, it can be converted by a so-called inter-system crossing (ISC) to a longer-living excited triplet state <sup>3</sup>PS\*. From this state, two types of chemical reaction pathways can occur, known as Type I electron transfer and Type II energy transfer, which can take place simultaneously [19]. In the Type I reaction, the <sup>3</sup>PS\* captures an electron (e−) from a reducing molecule (R) in its vicinity, which induces an electron transfer producing the superoxide anion radical (O<sup>2</sup> •−) and, after the subsequent reduction, leads to the generation of more cytotoxic ROS including hydrogen peroxide (H2O2) and hydroxyl radical (HO• ). In the Type II reaction, a direct energy transfer occurs from the <sup>3</sup>PS\* to the ground state molecular oxygen (3O2) that is then converted to singlet oxygen ( <sup>1</sup>O2). The ROS thus produced encompass O<sup>2</sup> •−, H2O2, HO• , and <sup>1</sup>O2, the last two being

the most reactive and most cytotoxic species but also those with the shortest diffusion distance. One PS molecule can generate thousands of molecules of <sup>1</sup>O2, depending notably on its <sup>1</sup>O<sup>2</sup> quantum yield, the surrounding environment, and the respective occurrence of Type I and Type II mechanisms [12,17,20]. most reactive and most cytotoxic species but also those with the shortest diffusion distance. One PS molecule can generate thousands of molecules of 1O2, depending notably on its 1O2 quantum yield, the surrounding environment, and the respective occurrence of Type I and Type II mechanisms [12,17,20].

●−, H2O2, HO●, and 1O2, the last two being the

leads to the generation of more cytotoxic ROS including hydrogen peroxide (H2O2) and hydroxyl radical (HO●). In the Type II reaction, a direct energy transfer occurs from the 3PS\* to the ground state molecular oxygen (3O2) that is then converted to singlet oxygen

*Pharmaceutics* **2021**, *13*, x 3 of 60

(1O2). The ROS thus produced encompass O2

**Figure 1.** (**A**) Modified Jablonski diagram describing the photochemical and photophysical mechanisms leading to ROS production during PDT. (**B**) Overview of aPDT already applied to the critical category of pathogens, as defined by the NIAID (https://www.niaid.nih.gov/research/emerging-infectious-diseases-pathogens, accessed date: 1 September 2021). For each category, the chart specifies the number and the proportion (percent of pathogens already assayed in at least one aPDT study either before or after 2015). **Figure 1.** (**A**) Modified Jablonski diagram describing the photochemical and photophysical mechanisms leading to ROS production during PDT. (**B**) Overview of aPDT already applied to the critical category of pathogens, as defined by the NIAID (https://www.niaid.nih.gov/research/emerginginfectious-diseases-pathogens, accessed date: 1 September 2021). For each category, the chart specifies the number and the proportion (percent of pathogens already assayed in at least one aPDT study either before or after 2015).

In addition to the above-mentioned Type I and Type II mechanisms, Hamblin et al. recently proposed introduction of a Type III photochemical pathway, following which the radical anion PS•− and/or inorganic radicals formed in absence of oxygen could also lead to photoinactivation [21]. These authors indeed identified several circumstances in which oxygen-independent photoinactivation of bacteria using specific PSs can be obtained. In addition to the above-mentioned Type I and Type II mechanisms, Hamblin et al. recently proposed introduction of a Type III photochemical pathway, following which the radical anion PS•− and/or inorganic radicals formed in absence of oxygen could also lead to photoinactivation [21]. These authors indeed identified several circumstances in which oxygen-independent photoinactivation of bacteria using specific PSs can be obtained.

#### *2.2. Biological Effects of aPDT: Potential Targets and Related Mechanisms 2.2. Biological Effects of aPDT: Potential Targets and Related Mechanisms*

The main first targets of aPDT are external microbial structures, i.e., the cell wall, cell membrane, or virus capsid and envelope [22,23]. Photodynamic inactivation (PDI) can be The main first targets of aPDT are external microbial structures, i.e., the cell wall, cell membrane, or virus capsid and envelope [22,23]. Photodynamic inactivation (PDI) can beobtained against microorganisms growing as planktonic cells and/or in biofilms [13]. In biofilm matrices, the diffusion of PSs can be delayed or PSs can be sequestered, in spite of photodamage induced on various components such as polysaccharides and extracellular

DNA [24,25]. The diffusion potential of ROS depends on (i) the maximal time-limited diffusion length, especially for <sup>1</sup>O<sup>2</sup> that possesses a shorter half-life compared with other ROS, (ii) the photostability in a given environmental medium, and (iii) the chemical properties of PSs (e.g., molecular size, charge, lipophilicity, stability), which influence the interactions of the latter with target microorganisms [26]. Photoinactivation of Gram(+) bacteria can be obtained with a given PS, irrespective of its charge, whereas that of Gram(−) bacteria generally requires a cationic PS, or a combination of a neutral PS with membranedisrupting agents [27].

Internalization of PSs in prokaryotic or eukaryotic cells can also occur, thus causing various intracellular oxidative damage (such as in organelles in eukaryotic cells, e.g., nucleus and mitochondria in fungal cells) [28]. To protect their intracellular components, microbial cells can induce the production of antioxidant defenses such as protective enzymes (such as superoxide dismutase (SOD), catalase and glutathion (GSH)-peroxidase) or pigments (such as carotenoids acting as nonphotochemical <sup>1</sup>O<sup>2</sup> quenchers). Nevertheless, these mechanisms can be insufficient to thwart aPDT-induced oxidative stress because intracellular components (including antioxidant defenses) can be also irreversibly photodamaged by ROS [29]. The latter can act on the DNA level through two mechanisms, i.e., alteration or modulation. Breaks in single-strand and double-strand DNA, and the disappearance of the super-coiled form of plasmid DNA have been reported in both Gram(+) and Gram(−) species. Indeed, PSs can interact with nucleic acids via electrostatic interactions and induce reduction of guanine residues causing DNA cleavage [30]. Again, microorganisms can induce the overproduction of proteins involved in the repair of photodamaged DNA; however, some bacteria, such as *Helicobacter pylori*, possess only a few of such protective repair mechanisms [31]. Upon PDT treatment, ROS and <sup>1</sup>O<sup>2</sup> can modify bacterial gene expression profiles by modulating (i) the quorum sensing pathway, therefore inhibiting biofilm formation as shown in in vitro models, and/or (ii) the anti-virulence activities by reducing the gene expression of virulence factors in diverse clinical pathogens [32–35].

Given the multi-targeted nature of aPDT, the possibility for microorganisms to develop resistance is supposed to be very limited [36]. However, they may be able to respond to aPDT in different ways. For instance, light response adaptation can occur in some microorganisms, such as *E. coli* upon exposure to blue light inducing the production of a biofilm polysaccharide colonic acid [37]. Moreover, some PSs are substrates of efflux systems that may be overproduced; specific inhibitors of the latter can be used to restore phototoxicity [38–40]. After sublethal aPDT, the biofilm-forming ability of bacteria can increase, making them less susceptible to the same treatment [41]. Each strategy should thus be carefully examined with regard to the ability of target microorganisms to adapt and escape treatments. The latter may be noticeably reduced when using at the same time multiple antimicrobial molecular partners and modalities (read below).

#### *2.3. Important Parameters and Requirements for an "Ideal" aPDT*

According to Cieplik et al. [12], an "ideal" aPDT system should meet the following set of general requirements: (i) PS physicochemical properties: efficient PSs for aPDT possess most frequently a high hydrophilicity index and at least one cationic charge promoting interactions with pathogens, especially Gram(−) bacteria; (ii) PS photosensitivity: following irradiation, a good PS produces a high rate of cytotoxic oxygen species (Figure 1A), depending notably on its <sup>1</sup>O<sup>2</sup> quantum yield, its stability, and the environmental media; (iii) light source parameters: efficient irradiation of PSs must take into account a coherent light exposure ensuring a good transmittance efficiency with no side effects; (iv) safety: the PS has to be specific to target microorganism(s), inducing insignificant or only a few side effects for the host, including no or few immunity responses; and (v) ease for implementation in clinical practice: aPDT has to be relatively easy to use (due to the rapid, non-aggressive, and non-invasive light application), cost-effective, and accessible. It is obvious that the detailed specification of requirements can vary depending on the target applications. The

improvement of PSs is an ongoing challenge that implies moving towards more rational chemical engineering and biological investigations [11,42], as discussed in this review.

#### **3. Positioning of aPDT in Current Human Healthcare Treatments**

Over the past years, the number of studies dealing with aPDT has dramatically increased, emphasizing the potential of this therapeutic approach to treat a broad spectrum of microorganisms including bacteria, fungi, viruses, and parasites (Figure 1B). In this part, recent in vitro screening studies, preclinical (using animal models) and clinical investigations are briefly reviewed. Details about the PSs involved and their structures are provided in Section 4 "State of the art with recent photo(nano)system developments".

### *3.1. Curative Preclinical aPDT*

#### 3.1.1. Treatment of Bacterial Infections

PDT is a promising alternative approach to antibiotherapy for photoinactivating a broad spectrum of bacterial pathogens, either Gram(+) or Gram(−), responsible for diverse infections in humans. The antibacterial versatility of PDT can be highlighted in different ways, notably by considering lists of critically important human pathogens. First, in recent years, more attention has been paid to the potential of aPDT to fight against bacteria involved in hard-to-treat infections, especially those forming the ESKAPE group [2,43]. Second, other critical pathogen lists can be considered, such as the NIAID emerging infectious diseases/pathogens category that includes biodefense research and additional emerging infectious diseases/pathogens. To our knowledge, the susceptibility of more than 50% of the bacteria in the NIAID critical pathogen list has been considered in at least one aPDT study. In other words, bacteria causing the worst endemic infections including anthrax, botulism, melioidosis, cholerae, plague, and tuberculosis have already been considered. On the opposite, vector-borne diseases transmitted by human parasites, such as *Borrelia mayonii* and *Bartonella henselae* have not been addressed in that regard yet. Multiple experimental settings have been considered to demonstrate the potential of aPDT to photoinactivate pathogenic bacteria, growing as planktonic forms, but also in biofilm matrices, and using diverse animal models [44,45]. Among human pathogens, bacteria implicated in oral infections, especially cariogenic, periodontic, and endodontic injuries, have probably been the most intensely investigated [46]. Although less considered, other indications have also been evaluated with aPDT, including osteomyelitis, meningitis, pneumonia, lung abscess, and emphysema [47,48].

#### 3.1.2. Treatment of Fungal Infections

Fungal infections caused by invasive candidiasis are widely recognized as a major cause of morbidity and mortality in the health care environment [49]. In addition to the opportunistic features of some fungi, resistance to first-line antifungals (such as echinocandins and fluconazole) is spreading, compromising the efficiency of conventional antifungal therapies. Despite the fact that yeasts are naturally more resistant to PDT than bacteria, noticeable in vitro and in vivo effects against fungal infections have been reported, including germ load reduction, biofilm inhibition and clearance, and eradication of persistent colonization [50–53]. Furthermore, antifungal PDT has demonstrated its potential as an adjunctive (potentially synergistic) treatment procedure to the conventional fungicide Nystatin [54]. Chen et al. identified and summarized other fungal infections that may be treated with aPDT, including *onychomycosis*, *tinea cruris*, *pityriasis versicolor*, *chromoblastomycosis*, and the cutaneous *sporothricosis* [55]. Recently, other fungal infections, such as fungi associated to *mucormycosis* (recognized as emerging critical pathogens in the NIAID) were successfully treated with PDT [56].

#### 3.1.3. Treatment of Viral Infections

Although vaccines have drastically reduced the spreading of some of the most virulent viruses around the world, antiviral research and development remain a healthcare priority notably due to emerging viral infectious diseases [57]. PDT holds promises to help treat the latter, as well as other viruses implicated in complications of some cancers. The oldest, but also the most current, application of antiviral PDT concerns the decontamination of blood products potentially containing hepatitis B/C or West Nile virus [23,58]. The PDI of viral infections was explored in many studies considering other various viruses including arbovirus, SV40, poliovirus, encephalitis virus, phages, and HSV [59,60]. In addition, emerging viruses such as Zika, Ebola, or Tickborne hemorrhagic fever viruses have been considered [23], as well as viruses responsible for epidemic/pandemic crises such as influenza virus, SARS-CoV-2 virus, and its mutants/variants [61,62].

#### 3.1.4. Treatment of Parasite Infections

Drug resistance is also rapidly spreading in parasites. For example, resistance to artemisinin (which is used to treat plasmodium infections causing malaria) increases drastically, even when combined with other drugs (WHO, https://www.who.int/news-room/ fact-sheets/detail/antimicrobial-resistance, accessed date: 1 September 2021). Antiparasital effect of PDT was demonstrated toward critical parasites in public health such as tropical pathogens including Leishmania, African trypanosoma, and Plasmodium [63,64]. Another way to limit the propagation of the vectors is to rely on photoinductible biolarvicides. Such an approach was investigated in order to control Aedes, Anopheles, and Culex, which are tropical disease-carrying species [65,66]. This was also investigated in Lyme disease using safranin-PDT for reducing the reproduction of ticks [67].

#### 3.1.5. Treatment of Polymicrobial Infections

Quite recently, interest has grown to explore the potential of aPDT against polymicrobial infections involving multispecies pathogens. A study demonstrated that the susceptibility to PDI of *S. aureus* and *C. albicans* growing in mixed biofilms is lower compared with single-species biofilms, which may be due to the difference in the chemical composition and viscosity of the composed matrix [24]. Nevertheless, aPDT applications are of interest regarding hard-to-treat infections due to polymicrobial biofilms colonization, such as chronic rhinosinusitis. They could effectively be eradicated by aPDT in a maxillary sinus cavity model [68]. Moreover, Biel et al. showed that aPDT can eradicate polymicrobial biofilms in the endotracheal tubes, which are factors leading to ventilator-associated pneumonia [69]. More recently, aPDT was shown capable of significantly improving wound healing in mice with polymicrobial infections [70]. However, such an approach remains a challenge due to the respective affinity of PSs to each species being usually reduced in polymicrobial systems.

#### *3.2. Current Clinical aPDT Practices*

Many clinical trials have been done for evaluating aPDT approaches in the treatment of bacterial/fungal oral infections. This is facilitated by the development of easy to use light sources in dentistry. On the opposite, it can be compromised by the development of persistent (multispecies) biofilms. Skin infections such as *Acnes vulgaris,* caused by *Propionibacterium acnes*, was one of the first microbial infections to reach the stage of aPDT clinical trial. A few clinical trials demonstrated that onychomycosis, such as *tinea cruris*, *tinea pedis*, and interdigital mycoses, could be treated with aPDT. Results demonstrated that aPDT is effective and well-tolerated, but infections can recur frequently [71,72]. Among cutaneous infections, non-healing chronic wounds in patients with chronic leg and/or foot ulcers were efficiently treated with aPDT, inducing a significant reduction in microbial load (even immediately after the treatment), a better wound healing, and no safety issues [73,74]. Osteomyelitis in patients with chronic ulcers can be treated with aPDT to prevent gangrene and amputations in the extremities of diabetic patients [75]. Clinical studies for treating *H. pylori* in gastric ulcers can be conducted using phototherapy without any PS administration; *H. pylori* naturally accumulates intracellular PSs (porphyrins) and therefore could be inactivated by phototherapy thanks to an ingenious blue/violet light delivery system [76]. A list of recently closed aPDT clinical trials is provided in Table 1; many other trials (not shown here) are still ongoing.


#### *3.3. Toward Preventive/Prophylactic Treatments*

Beside curative antimicrobial treatments, PDT may be also used to decontaminate medical equipment and tools in hospitals, for preventive/prophylactic aims [27]. For example, photoantimicrobial textiles were reported to efficiently photoinactivate bacteria and viruses, suggesting that self-sterilizing medical gowns could be developed [77]. PDT can also decontaminate medical tools similarly to conventional chemical agents, as demonstrated in a recent comparative study [78]. Its application for the decontamination of routine informatics tools, office equipment, or packing materials demonstrates sterilization potential that could be useful to avoid hospital-acquired infections and to protect healthcare workers. Furthermore, photodisinfection of water and photoinactivation of food-borne pathogens can bring substantial benefits to people's daily lives [65].

#### **4. State of the Art with Recent Photo(nano)System Developments**

#### *4.1. Single PSs*

#### 4.1.1. Organic PSs and Their Derivatives

Organic PSs used in aPDT have been well described in some recent reviews [79–81] (Figure 2A). Briefly, since the first use of eosin in 1904, various PSs were investigated, especially in the phenothiazinium group, which includes methylene blue (MB) and toluidine blue O (TBO). Thanks to an absorption spectrum in the red region of light, these PSs can be effective in tissues while being less toxic than other PSs. Their aPDT properties are mostly due to a high ROS production following Type I mechanism (Figure 1A). Structural derivatives have been also reported, including new MB and dimethyl-MB [42]. Another group gathers compounds featuring a macrocyclic structure composed of pyrroles, such as porphyrins and its precursor 5-aminolevulinic acid (5-ALA), phthalocyanines, and chlorins. Macrocyclic compounds are generally hydrophilic, positively charged, and they exhibit a good singlet oxygen quantum yield. Modifications of their chemical structure were intensively studied, especially for porphyrins and phthalocyanines [82–87]. The halogenated xanthenes gather PSs with a structure similar to that of fluorescein. Among them, eosin Y, erythrosine, and rose bengal (RB) were the most studied [88–90]. These compounds are anionic, which can limit their interaction with bacterial cells and their aPDT effect in spite of good singlet oxygen quantum yields. Natural compounds, including coumarins, furanocoumarins, benzofurans, anthraquinones, and flavin derivatives are often found in plants and other organisms. They are characterized by an absorption spectrum in white light or UVA. The most used are curcumin, riboflavin, and hypericin [7,80]. Nanostructures such as fullerenes are interesting PSs because of their ability to modulate Type I and Type II reactions (Figure 1A), depending on the near environment and the light source applied [91,92]. In this family, some quantum dots (QDs) can act as photoantimicrobials [80]. Other synthetic fluorescent dyes such as organoboron compounds (e.g., boron–dipyrromethene (BODIPY)), and cyanine dyes (e.g., indocyanine green (ICG)) are known for their high photostability, high extinction coefficients, and high fluorescence quantum yields [93].

Over the past five years, some new organic PSs were reported. Among them, optimized natural PSs such as anthraquinones and diacethylcurcumin can be listed. Others include derivatives of synthetic dyes such as monobrominated neutral red or azure B [79–81] (Figure 2A).

**Figure 2.** Representative compounds in various classes of PSs used in aPDT. (**A**) Examples of some organic PSs and their derivatives. (**B**) Examples of metallic-based PSs. (**C**) Different types of polymer-based PS carriers, which can be functionalized with ligands for specific target delivery (Adapted from [94], published by MDPI, 2020). **Figure 2.** Representative compounds in various classes of PSs used in aPDT. (**A**) Examples of some organic PSs and their derivatives. (**B**) Examples of metallic-based PSs. (**C**) Different types of polymerbased PS carriers, which can be functionalized with ligands for specific target delivery (Adapted from [94], published by MDPI, 2020).

#### 4.1.2. Coordination and Organometallic Complexes-Based PSs 4.1.2. Coordination and Organometallic Complexes-Based PSs

Distinctly from metal nanoparticles (NPs; see Section 4.2.1 "Metal-based systems"), metal complexes, either coordination or organometallic complexes, are of increasing interest as PSs in PDT [95] (Figure 2B). They generally consist of a central metal core combined with ligands, involving coordinate covalent bonds (in coordination compounds) or at least one metal–carbon bond (in organometallic compounds). Compared with organic compounds, metal complexes have been notably much less considered and still remain largely underexploited regarding their potential use as new antibiotics [96]. Frei et al. recently reported on the antimicrobial activity of 906 metal-containing compounds. They Distinctly from metal nanoparticles (NPs; see Section 4.2.1 "Metal-based systems"), metal complexes, either coordination or organometallic complexes, are of increasing interest as PSs in PDT [95] (Figure 2B). They generally consist of a central metal core combined with ligands, involving coordinate covalent bonds (in coordination compounds) or at least one metal–carbon bond (in organometallic compounds). Compared with organic compounds, metal complexes have been notably much less considered and still remain largely underexploited regarding their potential use as new antibiotics [96]. Frei et al. recently reported on the antimicrobial activity of 906 metal-containing compounds. They showed that, considering antimicrobial activity against critical antibiotic-resistant pathogens, metal-bearing compounds displayed hit rates about 10 times higher than purely organic molecules [97].

Metal complexes display a panel of specific properties that make them promising PSs candidates. The variety of metal ions and ligands can be assembled in scaffolds featuring very diverse geometries [97,98]. Whereas most organic PSs are linear or planar molecules, metal complexes can exhibit much more complex—three-dimensional—geometries, which can improve interaction and molecular recognition with cellular targets, enlarge the spectrum of activity, and impact on biological fate [98–100]. Furthermore, the modulation of the design of metal complexes allows to fine tune their hydrophilic–lipophilic balance, solubility, photophysical properties, and eventual "dark toxicity" (i.e., the toxicity in the absence of specific irradiation). Metal complexes can display many excited-state electronic configurations associated with the central metal, the ligands, or involving both the metal and the ligand(s) in charge-transfer states (metal-to-ligand charge transfer or ligand-tometal charge transfer). Although it is not always considered, the investigation of excited states of metal complexes (Figure 1A) is however of prime importance. Triplet states can be more easily accessed due to the enhanced spin-orbit coupling induced by the presence of the heavy metallic atom. Compared with natural PSs, metal complexes can act, besides ROS generation, via other mechanisms including redox activation, ligand exchange, and depletion of substrates involved in vital cellular processes [96,97,101].

Besides their first intended development as anticancer compounds, metal complexes have also been envisaged as potential "metallo-antibiotics", benefiting from the knowledge accumulated about their chemical properties and biological behaviors [102]. Quite recently, several metal compounds were characterized for their activity in aPDT. For instance, platinum(II), molybdenum(II), ruthenium(II), cobalt(II), and iridium(III) were proposed as new classes of stable photo-activatable metal complexes capable of combating AMR [11,103–108]. In particular, many mononuclear and polynuclear Ru(II/III) complexes have been considered as potential antibiotics, antifungals, antiparasitics, or antivirals, which have been recently extensively reviewed [107]. It is worth noticing here that, within a series of 906 metal compounds, ruthenium was the most frequent element in active antimicrobial compounds that are nontoxic to eukaryotic cells, followed by silver, palladium, and iridium [97]. Ru(II) polypyridyl complexes were assayed in several aPDT studies. For instance, Ru(DIP)2(bdt) and Ru(dqpCO2Me)2(ptpy)2+ (DIP = 4,7-diphenyl-1,10-phenanthroline, bdt = 1,2-benzenedithiolate, dqpCO2Me = 4-methylcarboxy-2,6-di(quinolin-8-yl)pyridine) and ptpy = 40 -phenyl-2,20 :60 , 2" -terpyridine) were tested with *S. aureus* and *E. coli* [109]. The complexes were found active against the Gram(+) strain and to a lesser extent against the Gram(−) strain, such difference in susceptibility being commonly reported in studies using other PSs [110] (Figure 2B). This observation was further detailed and rationalized by us when investigating a collection of 17 metal-bearing derivatives; two neutral Ru(II) complexes (Ru(phen)2Cl<sup>2</sup> and Ru(phen-Fluorene)2Cl2) as well as a mono-cationic Ir(III) complex (Ir(phen-Fluorene)(ppy)2+; ppy = phenypyridyl ligand) were found almost inactive, whereas a dicationic Ru(II) (Ru(phen-Fluorene)(phen)<sup>2</sup> 2+) was found to be the most active against a panel of clinical bacterial strains [11]. More recently, Sun et al. described a Ru(II) complex bearing photolabile ligands; they showed its ability to safely photoinactivate intracellular MRSA while inducing only negligible resistance after bacterial exposure for up to 700 generations [111]. Although the precise mechanism(s) of action is not well-established in every case, Ru(II/III) compounds are also increasingly considered for their potential anti-parasitic activity for combating neglected tropical diseases such as malaria, Chagas' disease, and leishmaniasis. Moreover, potent antiviral activities have been noted for the ruthenium complex BOLD-100, particularly against HIV and SARS-CoV-2 [112]; importantly, this compound appears to retain its activity on all mutant strains of the SARS-CoV-2 [107]. All combined, metal complexes—especially ruthenium-based compounds—can display antimicrobial activity via multiple, likely synergistic, mechanisms, involving notably their ability to produce ROS. Therefore, they are of major interest for a wide range of aPDT-related applications.

#### *4.2. Multicomponent PSs and Nanoscale Implementation: Extension to Nanoedifices with PSs*

The eventual limitations in the use of singular photoactive molecules as PSs for aPDT applications reside notably in the recurrent lack of solubility and stability in the target media (typically leading to aggregates and/or PS quenching), biocompatibility ("immune stealth") and bioavailability, but also in the relative absence of selectivity for a prospected target (e.g., efficiency in the interactions with a defined target, stimuli-responsive or alternate triggers for controlled release). Thus, the widespread nanoscale implementation into the development progress of upgraded PSs has indubitably provided significant flexibility to first address these drawbacks, and implicitly contribute to the optimization of the aPDT activity via an extensive panel of mainly exclusive features specific to nano entities; the latter typically include an advantageous surface to volume ratio (with a high PS per mass content), access to unique chemical/physical/biological properties (e.g., optical properties with QDs or magnetic ones with superparamagnetic iron oxide NPs (SPIONs)), and almost inexhaustible synthetic options in the design of nanoplatforms [113–115].

Due to their paramount structural diversity and intrinsic variety of properties, the classification of the so-called "PS nanosystems" or "nano-PSs"—which partly include and overlap with "conjugated systems" and "combinatorial strategies"—remains rather challenging; however, we can usually identify the following criteria as the main pillars to rationalize and compare these aPDT nanomaterials [27,80,116]:


quench the aPDT activity. Meanwhile, with surfaces of nano-objects decorated with PSs, the PSs may then contribute to some extent as an interface with the biological medium or the target.


Thus, in the overview presentation of the different PS nanosystems hereafter, the chemical features (i) and (ii) have been conventionally defined as the main criteria for the classification. Alternatively, the nature of the aPDT applications has also been used as the main criterion for classification in some references [120]. Another approach consists of systemizing all the nanosystems dedicated to a given PS (e.g., curcumin) [121].

Within the extensive collection of aPDT nanomaterials reported to date, the majority belongs to the category labeled as "PS nanocarriers" with the nanocomponent acting as a delivery system for PS molecules; however, increasing examples involving PS-active nano building blocks have emerged as well. Overall, this multipurpose role of the nanomoiety may include avoiding aggregation (e.g., dimerization, trimerization) and correlated PS quenching, enhancing "solubility" (i.e., dispersibility), stability and bioavailability, allowing "biological stealth", on-demand release and target specificity, and ultimately triggering eventual synergistic aPDT activity with the complementary or ameliorative intrinsic properties of the nanocomponent; although irrevocably confirmed in many nano-PSs, the mechanisms involved in the synergy may differ from one system to another, and often remain partly or integrally unresolved due to the complexity of these tacit multiparameter contextures [113]. It is noteworthy that most systems comprise "classic"/"traditional" organic PSs (natural or synthetic, e.g., curcumin, MB) with fewer examples involving metallated PS molecules such as the recent review from Jain et al. dedicated to ruthenium-based photoactive metalloantibiotics [108]. Among aPDT nanomaterials, we can thus identify various families of nanoplatforms based on the nature of the nanocore, starting here with the inorganic vectors followed by the organic templates; as an indication, in the common cases of "multi-component nano-PSs", the classification has been defined hereafter according to the main/prominent nano building block involved in the composition, i.e., metal-based systems, silicon-based systems, carbon-based systems, lipid-based systems, and polymer-based systems. Regarding the following presentation of aPDT nanosystems, it is important to specify that it is not exhaustive, but instead provides a panorama of the main categories of nanosystems—either colloids or surfaces [27,122]—and their related specificities, with an emphasis on recent developments.

#### 4.2.1. Metal-Based Systems

Metal-based nanostructures have been extensively investigated—both as "PS cargo" and PS active entities—through the exploration of the richness and diversity of the respective subcategories related to this class of compounds, as detailed below. Each of the below-mentioned inorganic classes presents distinct specificities of relevance for aPDT applications, with a choice to be defined on a case-by-case basis according to the target, the nature of the PSs involved (with possible preferential affinity), or the anticipated complemental or synergistic role of the selected nano entity (based upon its chemical, physical,

and/or biological features, with the eventual PS molecules located either at the surface of the NPs or within, when present). It is important to notice that the nanocores from each subcategory can be either further implemented, with silica or polymer coating for example, or combined (multicomponent nano-PSs) in order to adequately optimize the efficiency of the systems [117]; however, the outcomes of these hybridity processes are complex to anticipate with systematic rationality, with either enhancement or quenching of the properties observed depending on the composition of the combinations.

#### Metal NPs

Metal NPs—mainly gold, but also silver—maybe considered among the "gold standards" in nanomaterials through the history and expansion of nanosciences in terms of dedicated publications and vastness of related possible applications [123]. As already well documented for Au and Ag NPs, the reasons are numerous and reside notably in their relatively easy accessibility with low-cost and highly reproducible (large scale) biogenic and chemical synthetic routes, in addition to the flexibility to finely tailor the properties via a refined size and shape control (with narrow size distribution and diverse shapes), and the facility for functionalization with various types of molecules. Moreover, gold NPs display biocompatibility, low toxicity, and immunogenicity, almost chemical inertness (distinctly from their inherent catalytic properties), while silver nanomaterials present intrinsic antimicrobial activity against a broad spectrum of microorganisms and related MDR infections (e.g., towards Gram(−) and Gram(+) mature biofilms of MRSA), and disruption of biofilm formations while being safe for mammalian cells [124–126].Ultimately, both Au and Ag NPs share nano features specific to noble metal systems, i.e., localized surface plasmon resonance (LSPR, arising from their resonant oscillation of their free electrons upon light exposure) and resonance energy transfer (RET), with subsequent optical and photothermal properties of enhancing appositeness in aPDT applications and PDI efficacy (e.g., ROS production) [127–129]. For the most part, Au and Ag NPs of various shapes (e.g., spheres, rods, cubes) are combined with organic PS molecules such as RB and MB [128–136], but also with metallated PSs such as ruthenium complexes, metallophthalocyanines, and metalloporphyrins [108,137,138]; the corresponding PS nanovehicles can also be labeled as "conjugates" but they strictly differ from the "mixtures" involving metal NPs and PS molecules [139]. Other noble metal NPs, viz., platinum, have also been employed in aPDT applications due to their multitarget action to inactivate microbes, although to a lower degree up to now owing to synthetic limitations [27,140]. Alternately, redox-active copper NPs are typically less costly and easier to access and present unique features among which the faculty to generate oxidative stress to various microbes through the genesis of ROS [141], such as in the recently developed copper–cysteamine (Cu–Cy) nano-PS that can be activated either by UV, X-ray, microwave, or ultrasound, to produce ROS against cancer cells and bacteria [142,143]. More unwontedly, approaches to treat subcutaneous abscesses lead to the use of acetylcholine (Ach) ruthenium composite NPs (Ach@RuNPs) as an effective appealing PDT/PTT dual-modal phototherapeutic killing agent of pathogenic bacteria, with Ach playing a role in targeting the bacteria and promoting the entry into the bacterial cells [108]. While belonging to the same category, each metal displays particular specificities; consequently, with the objective of optimization, nano-PSs resulting from alloys or multimetallic NPs have been further designed such as the Au@AgNP@SiO2@PS and AA@Ru@HA-MoS<sup>2</sup> (AA: ascorbic acid, HA: hyaluronic acid) nanocomposites [117,118].

#### Metal Oxides

Similar to gold and silver, metal oxides such as iron oxide, titanium oxide and zinc oxide have been cornerstone contributors in the global evolution of applied nanomaterials (particularly in medicine), due notably to intrinsic magnetic and optical nanoscale features, with the latter typically available at "room temperature" and commonly finely tunable via shape, size, and crystal structure parameters [144]. Indeed, specific single-domain superparamagnetic iron oxide NPs (SPIONs)—either magnetite (Fe3O4) or maghemite

(γ-Fe2O3) of various shapes and sizes, including ferrite or doped derivatives—exhibit an outstanding magnetization behavior with no remanent or coercive responses upon exposure to a magnetic field. As a result, such magnetic NPs have legitimately generated interest and use as magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) agents, as well as magnetic fluid hyperthermia (MFH), magnetic cell separators [145], or drug delivery conveyers with the possibility to guide the NPs to the targeted area via external magnetic fields [146]. More recently, iron oxide nano-objects proved to be also of pertinence for aPDT applications not only as a magnetic "nanocargo" for various organic and inorganic PSs such as curcumin, MB, ICG, BODIPYs, porphyrins, metallophtalocyanines, or ruthenium derivatives among others [27,108,147–155], but also with peroxidase-like activity to enhance the cleavage of biological macromolecules for biofilm elimination [156]. Extension in the design of more elaborate multicomponent architectures involving hybrid iron oxide nanocore led *inter alia* to Ag/Fe3O4, Ag/CuFe2O4, CoFe2O4, and Fe3O4/MnO<sup>2</sup> NPs conjugated with different PSs [27,117,147,150,151,157–160].

Other oxides have also drawn heavy attention, in particular zinc oxide and titanium oxide, as the photophysical properties of these wide bandgap semiconducting nanomaterials efficiently translate into a multi-level antimicrobial activity including PS vessel and/or PS active agent (with possible coupled aPDT response), and/or membrane disruptor [161–164]. Thus, ZnO and TiO<sup>2</sup> nanoplatforms possess the ability to alter microbes' integrity—through alternate mechanisms involving ROS and/or metallic ions—in the dark or via photoactivation [165]. The latter is customarily triggered by UV or X-ray irradiation, with the eventual possibility to adequately shift to other wavelengths such as visible light irradiation in virtue of diverse modification methods of the oxides encompassing notably: doping or surface alteration (e.g., F-doped ZnO, coatings or oxygen deficiency), coupling with other bandgap semiconductors (e.g., ZnO/TiO2) or sensitizing dyes, and composites [121,156,164,166–171]. Furthermore, beside the size and shape of the NPs, the crystallographic phase appears to be a tuning parameter of particular importance for the antimicrobial effects of some oxides, especially for TiO<sup>2</sup> with the distinction between the anatase, rutile, and brookite structures [172–175]. Although reported to a lesser extent, the list of alternate oxides exhibiting potential in aPDT applications comprises CuO/Cu2O, MnO2, and rare earth oxides to mention just a few [141,176–180].

#### QDs and Metal Chalcogenide Nanomaterials

Aside from zinc/titanium oxides, distinct semiconductors such as QDs and metal chalcogenide (e.g., metal sulfide) nanomaterials (involving elements from different groups in the periodic table) proved to be efficient disruptors against various multi-drug-resistant microorganisms. Due to their smaller size of a few nanometers (ca. up to 10 nm), QDs differ from other nano-objects with physical and optoelectronic properties governed by the rules of quantum mechanics, high chemical stability and resistance to photobleaching, and near-infrared (NIR) emission (typically above 700 nm) notably allowing for deep-tissue imaging [181]. Appositely comparable, metal chalcogenide likewise reveals unorthodox physio and physicochemical properties, accordingly garnering a legit interest for antimicrobial applications [182]. Consequently, not only can these nano building blocks carry PS molecules and alter the integrity of microbial walls/membranes or gene expression, but they may as well act as PSs; when coupled with other PSs, synergistic interactions in the QD-PS edifices might occur resulting from mechanisms such as Förster resonance energy transfer (FRET, non-radiative energy transfer from QD donors to PS acceptors) to generate free radicals and ROS. Among examples of such QDs and metal chalcogenide aPDT systems can be cited CdTe QDs and related CdTe-PS conjugates, CdSe/ZnS QDs combined with PSs, InP and InP-PS, Mn-doped ZnS, MoS2, but also CuS and CdS nanocrystals, with ultimately hybrid systems involving for instance CoZnO/MoS<sup>2</sup> or AgBiS2–TiO<sup>2</sup> composites [123,183–196].

Metal–Organic Framework (MOF) Nanoscaffolds, Upconversion Nanomaterials and Other Metal Ion Nanostructures

Although less investigated than the above-mentioned alternatives, other original metal-based nanostructures identified as MOF nanostructures, upconversion nanoplatforms, and alternate metal ion nanomaterials tend to further consolidate their promising potential for aPDT applications. Considering their towering surface area and porous ordered structure with substantial loading capacity (e.g., adsorption of O<sup>2</sup> and ensuing photocatalytic production of <sup>1</sup>O<sup>2</sup> via a heterogeneous process), stable versions of colloidal nano-MOFs—or less common covalent organic frameworks (COFs), i.e., reticulation variants typically defined by non-metal "nodes" instead of metal ones—have emerged as efficient heterogeneous photosensitizers—with frameworks acting as PSs or entrapping PSs—towards antimicrobial applications (e.g., enhanced penetration for bacterial biofilms eradication), with porphyrin-based or porphyrin-containing MOFs and COFs, or Cu-based MOFs embedded with CuS NPs for rapid NIR sterilization among recently reported solutions [197–205].

Moreover, upconversion NPs (UCNPs) generally involve actinide- or lanthanidedoped transition metals and refer to the nonlinear process of photon upconversion, viz., a sequential absorption of two or more photons resulting in an anti-Stokes type emission (i.e., emission of light at a shorter wavelength than the excitation wavelength); when translated into biomedical context, UCNPs can be typically activated by NIR light—characterized by deeper tissue penetration and reduced autofluorescence, phototoxicity, and photodamage when compared with UV or blue light—and produce high energy photons for optical imaging or more recently aPDT when combined with PSs [206–209]. Intrinsically limited by low upconversion quantum yield, the current focus consists of developing hybrid UCNPs to improve aPDT efficiency; thus, auspicious progress has been achieved with examples such as {UCNPs (NaYF4:Mn/Yb/Er)/MB/CuS-chitosan)} multicomponent nanostructured system revealing a superior antibacterial activity with the UCNPs enhancing the energy transfer to MB, the CuS triggering synergistic PDT/PTT effects, and chitosan assuring stability and biocompatibility [156]. Other examples also include silica coating β-NaYF4:Yb, Er@NaYF<sup>4</sup> UCNPs loaded with MB as PS and lysozyme as a natural protein-inducing bacterial autolysis, Fe3O4@NaGdF4:Yb:Er combined with the photo/sonosensitizer hematoporphyrin monomethyl ether (HMME), UCNPs@TiO2, N-octyl chitosan (OC) coated UCNP loaded with the photosensitizer zinc phthalocyanine (OC-UCNP-ZnPc), or the UCNPs-CPZ-PVP system (CPZ: β-carboxy-phthalocyanine zinc, PVP: polyvinylpyrrolidone) to name but a few [206,210–213].

Alternatively, more disparate metal-ion aPDT systems have been reported such as PS encapsulated dual-functional metallocatanionic vesicles against drug-resistant bacteria involving copper-based cationic metallosurfactant, or self-assembled porphyrin nanoparticle PSs ZnTPyP@NO using zinc meso-tetra(4-pyridyl)porphyrin (ZnTPyP) and nitric oxide (NO) [214–216].

#### 4.2.2. Silicon-Based NPs

This category will be divided hereafter into two main subclasses—porous silicon (pSi) and (mesoporous) silica (SiO2)—which differ in the oxidation state of the silicon and display distinctive properties, more specifically different quenching behavior and photodynamic activity (singlet oxygen quantum yield under irradiation).

Porous silicon NPs (pSi NPs) are among the most promising types of inorganic nanocarriers for biomedical applications and have been intensively investigated since the first publication by Sailor et al. in 2009 regarding their application for in vivo treatment of ovarian cancer. Composed of pure silicon, pSi NPs indeed exhibit relevant features encompassing not only pores with large capacity for drug loading combined with specific surface area allowing for implemental functionalization, but also degradability in an aqueous environment, and biocompatibility [217]. Moreover, porous silicon particles are known to be photodynamically active with related inherent antimicrobial properties by generation

of ROS under irradiation with light of a specific wavelength [218,219]. Because of the low quantum yield of singlet oxygen production from porous silicon itself, particles can be grafted with additional PSs, such as porphyrins, to enhance the yield of singlet oxygen generation and thereby antimicrobial properties for PDT applications [219,220]. Consequently, several silicon-based systems have been reported in recent years, mainly for PDT applications [221,222]. Furthermore, pSi NPs display intrinsic fluorescence, which can be applied for imaging and real-time diagnostics regardless of any surface functionalization [220,223].

As previously mentioned, pSi has a low singlet oxygen quantum yield due to quenching, which makes silica particles in comparison a more suitable substitutional system combining similar biocompatibility with improved optical properties. On the other hand, one of the pivotal advantages to use silica (SiO2) conjugates with PSs is to achieve a better "solubilization"—or more accurately, dispersibility—of hydrophobic dyes and a better photostabilization, thus limiting the self-photobleaching of PSs. Further advantages to name as the most important features of silica are high biocompatibility, antimicrobial properties, and high surface area for mesoporous silica that can be synthesized easily from commercially available precursors [27,224,225]. Furthermore, SiO<sup>2</sup> exhibits an effective PS-grafting capacity [226]; the latter can be accomplished via adsorption, covalent bonding, binding to the hydroxyl groups from silica surface, and entrapment during formation in silica particles or matrix [27]. Recently, Dube et al. reported about the photo-physicochemical behavior of silica NPs with (3-aminopropyl)triethoxysilane (APTES), and subsequently PS-modified surfaces for aPDT [150]. In addition, silica coatings have also been reported to prevent the degradation of nanocarriers (magnetite) and prolong the stability and functionality of PS systems [227]. Interestingly, coupling PSs to silica or Merrifield resin leads to distinct advantages; indeed, immobilized Ce6 notably displays significantly higher aPDT efficiency in comparison with the free form, which is probably due to an enhancement of the adhesion of PSs to bacterial cells resulting in a stronger cell wall disorganization [228,229]. Unsurprisingly, many approaches with encapsulated PSs in silica NPs for potential aPDT applications have been reported in recent years [229–232].

Distinctly, combinatory approaches involving other nanocomponents, such as silicacontaining core-shell particles or silica-coated inorganic NPs, have emerged to further implement the properties of silica with the specific features (e.g., magnetic, photoactive, or antimicrobial properties) from other nanomaterials of relevance [118]. Thus, since the surface of silica can be easily grafted with PSs and is highly biocompatible, the surface modification of silica particles using metallic NPs (e.g., Ag NPs) to enhance the antibacterial photodynamic activity, has been developed for improved aPDT [233] while combinations with carbon quantum dots have been assessed for imaging-guided aPDT [234]. Another example refers to sufficient aPDT/PDI systems, more precisely to mesoporous silica-coated NaYF4:Yb:Er NPs with the PSs (silicon 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine dihydroxide) loaded in the silica shell to enhance bacterial targeting of *E. coli* and *S. aureus* [235].

In addition to silica-based NPs or silica-containing core-shell particles, lesser-known silica nanofibers also proved to be suitable substrates for potential aPDT, PDT, and PDI applications. As an illustration, Mapukata et al. [236] recently reported silver NP-modified silica nanofibers with embedded zinc phthalocyanine as PS for aPDT applications. The nanofiber-based substrates offer the advantage of fast removal after application, which can allow limiting any dark toxicity [236–238].

Silica NPs and fibrous or dendritic fibrous nanosilica have also been reported for the formation of nanocomposites to create antimicrobial photodynamically active surfaces for aPDT or PDI; to create such surfaces, silica NPs can be embedded into polymeric matrices for enhanced biocompatibility and complementary surface properties from the selected polymer [239].

Additionally, silica substrates and nanoconjugates offer a suitable platform for combinatory approaches since they can be easily modified. For example, Zhao et al. described polyelectrolyte-coated silica NPs modified with Ce6 [240]. These complexes could be

extracted, by bacteria, from silica NPs to form stable binding on the bacterial surface, changing the aggregation state of Ce6 and leading to both the recovery of PS fluorescence and <sup>1</sup>O<sup>2</sup> generation. Such bacteria-responsive multifunctional nanomaterials allowed for simultaneous sensing and treating of MRSA. Another approach is illustrated by the photoinduced antibacterial activity of amino- and mannose-decorated silica NPs loaded with MB against *E. coli* and *P. aeruginosa* strains [241]. The modification of silica substrates with mannose led to an increased targeting of *P. aeruginosa* and reduced dark toxicity of the systems.

#### 4.2.3. Carbon-Based Nanomaterials

Akin to silicon-based nano-objects, the notable diversity of allotropic customizable carbon-based nanostructures—either conveyers for traditional PS molecules or intrinsically PS active—legitimizes their distinct consideration in the actual classification of nano-PSs [242–244], with the following subcategories.

#### Fullerenes, Carbon Nanotubes (CNTs), and Nanodiamonds

In the evolution of carbon-based nano-objects, fullerenes and carbon nanotubes derivatives may be chronologically introduced as the "first generation". Discovered in the mid-1980s, the proper C<sup>n</sup> (*n* = 60–100) spheroidal "soccer ball" π-conjugated structures of the fullerenes yield tremendous chemical modularity and electrochemical and physical properties, including photostability, the propensity to act as a PS via Type I or Type II pathways (Figure 1A) with high ROS quantum yield, and oxygen-independent photokilling by electron transfer. Despite their intrinsic hydrophobicity typically requiring surface functionalization for biocompatibility and related dispersibility, they have proven even nowadays their effectiveness as broad-spectrum photodynamic antimicrobial agents, with photoactive antimicrobial coating based on a PEDOT-fullerene C<sup>60</sup> polymeric dyad (PEDOT: poly(3,4-ethylenedioxythiophene)), BODIPY-fullerene C60, diketopyrrolopyrrole– fullerene C60, and cationic fullerene derivatives among recent examples [80,92,156,245–251]. Mainly developed a few years later, carbon nanotubes (CNTs)—either single wall CNTs (SWCNTs) describable simply as a single-layer sheet of a hexagonal arrangement of hybridized carbon atoms (graphene) rolled up into a hollow cylindrical nanostructure, or multiwall CNTs (MWCNTs) consisting of nested SWCNTs—unveil both independent capacities to produce ROS upon irradiation and high surface area for decoration with PS molecules [156]. Neoteric specimen of PS-CNTs encompass toluidine blue, polypyrrole, malachite green, MB, RB, and porphyrins [156,252–257]. Although purportedly older since it was discovered in the 1960s, diamond NPs or nanodiamonds seemingly remain the lesser known carbon-based nanomaterials to date; nevertheless, the latter dispose of legit aPDT arguments with their fluorescence, photostability, proclivity for conjugation with diverse PSs such as porphyrins or metallated phthalocyanines and silver NPs, but also inherent antibacterial activity [120,258–262].

#### Carbon QDs (CQDs)

As the next momentum in the blossoming of "nano-carbon" era, the carbon QDs were discovered in the early 2000s [263,264]. The physical and chemical properties of these fluorescent particles, commonly quasi-spherical with less than 10 nm in diameter, can be finely tuned upon size/shape variations or doping with heteroatoms (e.g., B, N, O, P, S) [263,265]. By virtue of their biocompatibility and dispersibility, photostability, low toxicity and related eco-friendliness, good quantum yield and conductivity, CQDs have been investigated for various applications, and more recently as antimicrobial agents; withal, their environment-friendly features combined with low cost and rather ecological biogenic or synthetic routes (from natural or synthetic precursors) place them advantageously as a viable scalable photocatalytic disinfection material compared with alternate nano-PSs. Late cases involve doped or hybrid CQDs or more conventional conjugates of CQDs with PSs [121,266–271].

## Graphene, Graphene QDs (GQDs), and Graphene Oxide (GO) Nanostructures

In a similar timescale to CQDs is the quantum leap discovery and blooming of graphene and graphene oxide materials. Graphene can be defined as a 2D allotrope of carbon, more accurately a monolayer of atoms with a hexagonal lattice structure (or single-layered graphite) and identifiable as the "building block" for the discrete fullerenes, 1D carbon nanotubes, and 3D graphite. Despite its stunning mechanical/electronic properties and chemical inertness, the limitations of graphene, such as zero bandgap and low absorptivity, lead to the ulterior conversion of the 2D graphene into "0D" GQDs [272,273]. Due to quantum confinement and edge effects, GQDs exhibit different chemical and physical properties when compared with other carbon-based materials, as well as a non-zero bandgap, good dispersibility, and propensity for functionalization and doping. Structurally, GQDs differ from CQDs because they comprise graphene nanosheets with a plane size less than 100 nm [272,274]. Likewise, graphene oxide (GO) is the oxidized form of graphene i.e., a single atomic sheet of graphite with various oxygen-containing moieties either on the basal plane or at the edges. Meanwhile, reduced graphene oxide (rGO) can be summarily described as an "intermediate" structure between graphene and GO, with variable and higher C/O elemental ratios compared with GO, but remaining residual oxygen and structural defects with reference to the pristine graphene structure. Although GO was reported a couple of centuries ago, GO and rGO nanomaterials have mainly emerged for various applications after the discovery of graphene since GO is a precursor to prepare graphene, and both present distinctive physical and chemical properties that differ from graphene. As a result, countless and rising fast illustrations of graphene derivatives for antibacterial applications are regularly reported [121,275–280].

#### 4.2.4. Lipid-Based Systems

Due to their amphiphilic nature (typically hydrophilic "head" and hydrophobic "tail"), some lipids—natural and synthetic—have been extensively studied to develop efficient biocompatible delivery systems—initially for drugs and DNA/RNA, but also for aPDT PSs—with synthetic flexibility and structural diversity. Among the prevalent examples, we can distinguish the micelles (lipid monolayers with polar units at the surface and hydrophobic core) and the liposomes (one or more concentric lipid bilayer with a hydrophilic surface and an internal aqueous compartment). Although sharing similar chemical constituents, the micelles and liposomes present significant differences to be taken into consideration depending on the intended application (nature of the target) and the nature of the PSs. Indeed, the micelles are typically smaller than liposomes (with a diameter starting from a few nanometers for the micelles, and ca. 20 nm for the liposomes), with distinct stability and permeability in biological medium and uptake pathways for the PSs into bacteria. With reference to the nature of the transported PSs, the liposomes display the additional flexibility to carry both hydrophilic PSs (in the core compartment or between the bilayers) or/and hydrophobic PSs (within the lipid bilayer), while the micelles are usually easier and cheaper to prepare [80,156]. As often critical to address for biomedical applications, the surface charge of these nano-objects can be tailored to further optimize the interactions with the bacteria, with cationic modification of liposomes identified as a promising aPDT efficiency "amplifier" [80,156,281]. In addition to recent examples such as the hypericin loaded liposomes against Gram(+) bacteria [282–286], another emerging and promising alternative includes the development of modified liposome-like derivatives labeled either as "ethosomes", "transfersomes", or "invasomes", which can be briefly described as ultra-deformable vesicular carriers with upgraded transdermal penetration and increased permeability into the skin for the PSs compared with conventional liposomes [287–290]. On the other hand, recently reported aPDT micellar systems refer to micelles loaded with various hydrophobic PSs such as curcumin, BODIPY, porphyrins, hypocrellin A, or hypericin among others [121,291–293]. Furthermore, solid lipid NPs (SLN) composed of solid biodegradable lipids have been recently highlighted as delivery systems used for actual mRNA COVID-19 vaccines [294], but they also have been reported as transporters

for curcumin for the treatment of oral mucosal infection [121]. Besides, we may also include nanoemulsions in this category of lipid-based nanovehicles since the latter conventionally involve lipids in the oily phase during the formulation process, with recent curcumin/curcuminoid nanoemulsions [121,295].

#### 4.2.5. Polymer-Based Systems

In direct correlation with the above-mentioned lipid-based systems, polymer-based nanocarriers have been positioned as a logical extension with the objective to implement a "degree of freedom" in the synthesis flexibility while expanding the panel of building blocks available in the design of aPDT nanostructures. A categorization of polymeric systems for the delivery of PSs to therapeutically relevant sites can be done using as criteria either the nature of the polymer(s) involved or the type of polymeric (nano)structures. Thus, in the following, we will use a classification primarily based on the structure of the polymeric systems with differentiation between NPs (including hydrogels, biopolymers, and aerogels), polymersomes, polymeric micelles, dendrimers, fibers, and polymeric films and layers (including hybrid systems and nanocomposites). The nanostructure of polymeric systems is implicitly highly correlated to the molecular structure, i.e., composition (nature of the polymer(s), ratios, and distribution and amount of hydrophilic and hydrophobic moieties), charge, and size, as reviewed recently by Osorno et al. [296]. The refined control of these parameters enables the design and fine-tuning of specific polymeric nanostructures spanning from long-ranged ordered lamellar sheets, tubes, and fibers to oval or spherical particles, micelles, and polymersomes [296] (Figure 2C).

#### Conjugated Polymers as PSs or Polymer-Functionalized PSs

One of the easiest approaches reported to develop polymeric carrier systems is to enhance water solubility and biocompatibility of PS molecules through the use of functionalized polymers, by introducing the PSs in a post-modification reaction to a hydrophilic and/or biocompatible polymer, or to modify the PS with a polymerizable group (e.g., acrylate) to react as a monomer for further polymerization with suitable monomers. Other apt options are conjugated polymers incorporating a backbone with alternating double and single bonds which provide photodynamic and optical properties, and therefore might act as PSs themselves [297,298]. For example, poly[(9,9-bis{60 -[N-(triethylene glycol methyl ether)-di(1H-imidazolium)-methane]hexyl}-2,7-fluorene)-co-4,7-di-2-thienyl-2,1,3 benzo-thiadiazole] tetrabromide (PFDBT-BIMEG) is a conjugated polymer which affords salt bridges and electrostatic interactions with microorganisms; these interactions enable the simultaneous detection and inhibition of microorganisms [297]. Conjugated polymers were intensively investigated for the development of multifunctional NPs in antibacterial applications because of their generally low-toxicity toward eukaryotic cells, their flexibility, and their high potential to vehicle versatile therapeutic molecules [298]. Improved PSs based on conjugated polymers or polymerized PSs with antimicrobial photodynamic properties have been amply reported in recent years [299–303]. For example, Huang et al. demonstrated the efficiency and selectivity of polyethyleneimine-Ce6 conjugated in potential aPDT/PDI applications [304].

Other polymeric systems also show antimicrobial properties or preferential target infection sites and are thus suitable for aPDT applications when combined with PSs. For example, cationic polymers, which are known to be highly hydrophilic, can be used to target cell walls and thus microbial infections. Hence, such polymers are adequate for potential aPDT applications [305,306]. Exemplary amphiphilic or cationic poly(oxanorbornene)s doped with PSs, which exhibit pronounced antimicrobial activity (99.9999% efficiency) against *E. coli* and *S. aureus* strains, have been reported [307]. By contrast, anionic polymer particles and nanocarriers with negatively charged surfaces and membranes invade the reticuloendothelial system, which leads to elongated blood circulation times [308–310]. Polymers with large fractions of functional groups, such as the biopolymers mentioned above, can be easily modified and offer many loading sites for suitable PSs. Thus, among

many suitable materials, hyperbranched and dendrimeric polymers are particularly promising candidates for PS nanocarriers.

#### Dendrimers

Dendrimers, dendrimeric polymers, or dendrons are highly ordered and highly branched polymers, which may form spherical three-dimensional structures with diameters typically ranging from 1–10 nm [311]. The dimensions of dendrimers are relatively small compared with other drug delivery systems, but as they consist of individual welldefined molecules, the drug loading can be subtly established with reproducibility. The incorporation or encapsulation of drugs may be achieved by covalent binding of the PSs or drug molecules to reactive functional groups along the dendrimeric structure [312,313]. Alternatively, the PS or drug molecule may act as a scaffold from which the dendrimer is synthesized. Finally, drug or PS encapsulation may occur inside the voids of the dendrimer [311,314,315]. Thus, dendrimers offer a substrate for PSs with many reaction sites, and controllable sterical and hydrophobic properties depending on the backbone of the dendrimer. Approaches using hyperbranched polymers for stimuli-responsive release of PSs, such as porphyrins, under acidic or reductive conditions, with improved targeting of bacterial sites have been reported, such as mannose-functionalized polymers by Staegemann et al. [316,317].

#### Polymeric NPs and Nanocomposites

In many cases, polymeric nano-systems are not clearly classified, but instead typically grouped under the terminology "polymeric NPs", since the exact structure may not be fully characterized or considered less relevant with respect to the application efficiency. Polymeric NPs are commonly defined as physically or chemically crosslinked polymer networks with a size in the range of 1 to 1000 nm, but if not further defined may also include nanocapsules, such as polymersomes, micelles, chitosomes, and even highly branched polymers and dendrimers [318].

In aPDT, polymeric NPs may be either used to encapsulate PS molecules (loading with PSs), or built from inherent photoactive polymers acting as PSs [303]. Various PS molecules (e.g., RB, porphyrin derivatives, or curcumin) encapsulated in biocompatible polymeric NPs, such as polystyrene-, PEG-, polyester- (including poly-beta-amino esters), or polyacrylamide-based NPs, are widely used in aPDT/PDI approaches, as recently reported [319–321]. In addition to the role of nanovehicle, the polymeric particles may also help to address the lack of solubility in the biological medium from the PSs and/or reduce their toxicity, as reported among others by Gualdesi et al. [320,322]. For instance, polystyrene NPs with encapsulated hydrophobic TPP-NP(5,10,15,20-tetraphenylporphyrin) have been reported as nano-PS for efficient aPDI approaches towards multi-resistant bacteria [321]. In addition, biocompatible PLGA (polylactic-co-glycolic acid) NPs were employed to encapsulate curcumin, which could potentially serve as an orthodontic adhesive antimicrobial additive [322]. Alternatively, combinatory approaches such as polymeric NPs merging a polymeric PS, a photothermal polymeric agent, and poly(styrene-co-maleic anhydride) as dispersants have recently been reported for coupled aPDT/PTT nano-platforms in aqueous media [323,324]. Furthermore, Kubát et al. reported an increase in the stability of physically crosslinked polymeric NPs (polystyrene) to comparable nanocapsules (PEGpoly(ε-caprolactone) (PCL) micelles) equipped with identical PS [319].

The term polymeric NP also includes NPs obtained from natural polymers (e.g., chitosan and alginate), hydrogels, or aerogels. Hydrogels are chemically or physically crosslinked polymeric networks, which are able to absorb large amounts of water due to their pronounced hydrophilicity, but do not dissolve because of the crosslinking, whereas aerogels are formed by replacement of liquid with a gas resulting in low-density polymeric structures [325]. Recently, Kirar et al. reported PS-loaded biodegradable NPs fabricated from gelatin, a naturally occurring biopolymer, and hydrogel, for application in aPDT [326]. Moreover, also recently, some polymers such as Carbopol-forming hydrogel matrix en-

trapping PS molecules attracted attention because of their bio/muco-adhesive property, allowing a prolonged local PS delivery. Nevertheless, the viscosity of such systems can prevent the efficiency of PDT by decreasing the photostability and ROS production, thus calling for further optimizations [327,328]. Furthermore, the incorporation of hydrophobic PSs such as curcumin in polyurethane hydrogel has demonstrated to be an efficient PS release system [329].

Another suitable hydrogel for aPDT is chitosan, which is a polycationic biopolymer with good biocompatibility and antibacterial properties [330,331]. Indeed, cationic polymers, which can be antimicrobials by themselves such as chitosan and poly-Lysine, can assure a better recognition toward bacteria thus improving antimicrobial efficiency. Typically, polycationic chitosan may form nanogels or NPs in the presence of (poly)anionic molecules. Alternatively, another promising aPDT approach for the treatment of *Aggregatibacter actinomycetemcomitans* was recently reported [332,333], in which anionic PS indocyanine green was used to form PS-doped chitosan NPs. These NPs were shown to significantly reduce biofilm growth-related gene expression. Other similar approaches have also been reported, in which PS doped chitosan NPs showed enhanced cellular uptake and improved antimicrobial properties compared with free PS against different bacteria [212,334,335].

The above-mentioned approach has not only been implemented with NPs, but also in thin films and layers to create biocompatible and antimicrobial surfaces [336]. Indeed, in aPDT and especially aPDI, polymers are also used to create antimicrobial surfaces or matrices to embed PS units. Thus, combinatory approaches—using cellulose derivatives, alginates, chitosan, and other polymer-based materials as biocompatible substrates for PSs and nanocomposites to create photoactive antimicrobial surfaces—have recently been reported [337–339]. The development of such surfaces, and in particular antimicrobial membranes, is of considerable interest, especially in numerous and diverse fields in which providing hygienic and sterile surfaces is essential. As an illustration, Müller et al. reported polyethersulfone membranes doped with polycationic PS that provide antimicrobial properties for potential use as filter membranes in water purification or medicine [340]. Other recent distinctive systems for aPDI refer to self-sterilizing and photoactive antimicrobial surfaces made from (i) natural polymers such as chitosan doped with chlorophyll [336], (ii) "bioplastic" poly(lactic acid) surfaces coated with a BODIPY PS [341], or (iii) synthetic polyurethanes doped with curcumin and cationic bacterial biocides [336,342–345].

Alternatively, other combinatory approaches—in which polymeric nanocomposites embedding inorganic nanomaterials with relevant complementary features are conceived have garnered attention in recent years. Examples include fullerenes or silver NPs that are incorporated as PSs into polymeric matrices [346], phthalocyanine-silver nanoprism conjugates [347], or mesoporous silica NPs loaded into polymer membranes [348]. Additionally, the embedding of zinc-based PS—such as Zn (II) porphyrin [349]—into polymer matrices to create antimicrobial polymers and polymeric surfaces for aPDI and possibly aPDT has also been reported, with pronounced antimicrobial activity against several bacteria strains and viruses [350,351]. Furthermore, protein-based approaches were developed as reported by Ambrósio et al. [352] and Silva et al. [353] using BSA (bovine serum albumin) NPs or BSA conjugated to PSs to improve solubility and biocompatibility.

Lastly, stimuli-responsive polymeric NPs are generally known to be sensitive to an internal or external stimulus (e.g., pH, temperature, light) and so of utmost interest for the controlled release of PSs for PDT and aPDT, whereas the stimuli-responsiveness depends on the structure and properties of the used polymer such as the assembly of polymer chains and linkages [354]. For example, Dolanský et al. reported light- and temperature-triggered ROS and NO release from polystyrene NPs for combinatory aPDT/PTT approaches [355]. Unlike micelles or polymersomes, crosslinked NPs have been reported to be thermodynamically stable, while stimulus-responsive behavior such as pH-responsive release of PSs has been more often achieved using self-assembled micellar or vesicular structures [319,356–358].

#### Polymersomes

Polymersomes are polymeric vesicles that resemble liposomes, which were previously described in Section 4.2.4 dedicated to lipid-based systems. Polymersomes are formed from amphiphilic copolymers, which self-assemble in aqueous media, resulting in capsules. The lumen of the polymersomes is filled with an aqueous medium and the wall comprises a hydrophobic interior with a hydrophilic corona on both inner and outer interfaces. Polymersomes typically form when the weight fraction of the hydrophilic parts (e.g., PEG in a PEG-b-poly(lactic acid) (PLA) block copolymers) comprises up to 20–40% of the polymer. They also form at comparatively large water fractions in the solvent shift method [359–361]. At higher weight fractions, the system tends to form micellar structures [296,362,363]. The hydrophilic and hydrophobic compartments inside the polymersomes enable the uptake and encapsulation of both hydrophobic and hydrophilic molecules [364–367], making polymersomes a relevant candidate for the development of advanced nanocarriers for PSs [368]. In aPDT and similar approaches, efficient delivery of the PSs to the targeted tissue is essential, not only to minimize toxic side effects and overcome low solubility in body fluids, but also to enable elongated circulation in the blood stream and prevent dimerization and quenching of the PSs. Li et al. reported that PSs encapsulated in polymeric nanocarriers exhibit an increased singlet oxygen <sup>1</sup>O<sup>2</sup> quantum yield compared with nonencapsulated PSs [308]; by contrast, non-encapsulated PSs may tend to aggregate and lose efficiency [308,369,370]. Additionally, most polymersomes offer certain advantages compared with the established liposome-based systems such as enhanced biocompatibility, lower immune response, controlled membrane properties, stimuli-responsive drug release, biodegradability, and higher stability, with those mainly resulting from the individual design of polymers and polymersomes for the anticipated applications [296,371].

The encapsulation of PSs into specifically designed nanosized polymersomes with stimulus-triggered release of PSs has been reported in recent years, including temperature, pH, and light-induced release of the PSs [296,372,373]. For instance, Lanzilotto et al. recently reported a system used for aPDT consisting of polymersomes of the tri-blockcopolymer poly(2-methyl-2-oxazoline)-block-poly-(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA34–PDMS6–PMOXA34) encapsulating water-soluble porphyrin derivatives [372].

#### Polymeric Micelles

In contrast to polymersomes or liposomes, micelles are particles that contain a hydrophobic core. Typical micelles range between 10 to 100 nm in size. More specifically, polymeric micelles are formed of amphiphilic polymers, with a higher ratio of hydrophilic parts in the case of block copolymers compared with polymersomes [296,362,363]. They also form at comparatively small water fractions in the solvent shift method [359–361]. Due to their structures, micelles are implicitly used to encapsulate hydrophobic drugs or agents, such as PSs, to convey the latter to the target (e.g., cancer cells or microbial infections) while overcoming their low solubility in aqueous media [374]. Additionally, encapsulation may reduce the toxicity of the PSs [375,376]. When compared with polymersomes, one disadvantage is the lack of flexibility to encapsulate or transport both hydrophilic and hydrophobic molecules. On the other hand, as reported in the review by Kashef et al. [377], polymeric micelles are easier to produce, hence they are more cost-efficient than liposomes and potentially polymersomes, while providing similar applicable features [378,379]. Among examples, an aPDT system of polymeric micelles, fabricated from methoxy-PEG and PCL and loaded with the hydrophobic PS curcumin and ketoconazole for the PDI of fungal biofilms, has been reported with an increased water solubility and controlled release of the PSs on display [380]. Additionally, Caruso et al. recently reported the synthesis of thermodynamically stable PEG-PLA micelles for efficient aPDI of *S. aureus*, suggesting that this delivery system is promising in aPDI applications, which also reduces toxicity compared with pure PSs [291]. Moreover, a significant advantage of polymeric micelles compared with lipid-based micelles resides notably in the adjustability of properties by

individual design of the polymer and membrane surface with respect to the selected application. Hence, the polymeric micelles can be equipped with target ligands and/or their morphology can be tuned to increase the cellular uptake of the micelle, PS, or other therapeutic agents in a specific tissue, such as in tumors [381]. Like polymersomes, polymeric micelles can be tuned to release drugs or collapse by application of external stimuli, such as light, temperature or pH [357,358].

#### Niosomes

Niosomes are closely related to liposomes. They are vesicular structures consisting of non-ionic surfactants, including polymers, and lipids such as cholesterol. The addition of lipids to the non-ionic surfactants leads to increased rigidity of the membrane and the vesicular structure. Niosomes range from 10 to 3000 nm in size, and also may include multi-layered systems typically consisting of more than one bilayer. Niosomes offer enhanced stability and biocompatibility compared with vesicular structures based on ionic surfactants [288]. Due to the bilayer structure and adjustability of the selected polymers for the applications of choice, they exhibit similar properties and offer similar advantages as polymersomes, and can be used for encapsulation of a variety of hydrophobic and hydrophilic molecules [288,358]. They may also be equipped with targeting ligands, such as folic acid that enhances uptake into cancer cells for PDT of cervical cancer [382]. A niosome-based system, using MB as encapsulated PS, has been reported for aPDT treatment of hidradenitis suppurativa [383].

#### Polymeric Fibers

Another approach to obtain PS carrier systems relies on polymeric or polymer-hybrid fibers with diameters in micro and nano range [384–386]. The fibers can either be loaded after formation with various PSs, such as cationic yellow or RB [387] incorporated into wool/acrylic blended fabrics to obtain antimicrobial properties, or they can be electrospun from PS containing polymer solution to form polymeric fibers loaded with PSs [388]. Since many polymers (such as nylon, cellulose acetate, or polyacrylonitrile) are used in the textile industry, those materials may offer a novel approach to create substrates or textiles with self-sterilizing and antimicrobial properties in the presence of visible light [386,389]. Furthermore, combinatory approaches using various NPs, such as magnetite NPs, to form polymeric fiber-based nanocomposites have shown some efficiency for antimicrobial photodynamic chemotherapy [390,391].

To conclude this Section 4.2, the list of above-mentioned aPDT nanosystems is non exhaustive and aims at providing an overview of the diversity and richness in the composition of aPDT nanomaterials. The next section focusing on "combinatorial strategies" partly overlaps with the description of aPDT systems, which further complicates the classification process. Moreover, a distinction must be settled between strict "mixtures" of components and "chemical combinations" of components in the development of aPDT treatments with possible synergistic behaviors.

#### **5. Focus on Combinatory aPDT Approaches**

Given its intrinsic characteristics, PDT is highly amenable to versatile combinations with other drugs, treatments, or modalities in view to potentiate therapeutic effects, which include enhanced efficacy, limitation of side effects, and reduction in the risk of resistance emergence. Proof-of-concept for various combinatory aPDT are thus being increasingly recorded, exploiting the additive/synergistic effects arising from single or multiple therapeutic species acting via different mechanistic pathways. The following part aims to review recent works following such strategies (Table 2).


**Table 2.** Examples of recent studies that combined aPDT with other antimicrobial actives or treatments.

**Table 2.** *Cont.*




ALA, alanine; MB, methylene blue; RB, rose bengal; Ce6, chlorin e6; ICG, indocyanine green; SPION, superparamagnetic iron oxide NP. 1 , hematoporphyrin monomethyl ether enclosed into yolk-structured up-conversion core and covalently linked RB on SiO2 shell; 2 , photothermal agent poly(diketopyrrolopyrrole-thienothiophene) (PDPPTT) and the photosensitizer poly(2-methoxy-5-((2-ethylhexyl)oxy)-pphenylenevinylene) (MEH-PPV) in the presence of poly(styrene-co-maleic anhydride).

### *5.1. "Basic" aPDT Combinations*

#### 5.1.1. Combination of Several PSs

The simplest combinatory aPDT approach probably consists in combining two or more PSs in a single treatment. Thus, besides aPDT making use of a single PS, aPDT may rely on the simultaneous use of several PSs in an attempt to obtain additive or synergistic antimicrobial effects. The PSs combined may exhibit different photophysical characters showing complementarities. For example, carboxypterin-based aPDT upon sunlight irradiation demonstrates a significant planktonic bacterial load reduction [419]. However, eradication of biofilm formation needs a PS concentration 500 times higher than assays performed with planktonic forms. When combined with MB, Tosato et al. showed that reasonable concentrations of both PSs exert synergistic effect on both biofilm and planktonic MDR bacteria [411]. In other studies, alternative dual-PSs aPDT systems have shown even better antibacterial and antibiofilm properties [35,412].

#### 5.1.2. Addition of Inorganic Salts

The combination of PSs with inorganic salts can modulate the PDT effectiveness, potentiating or inhibiting the antimicrobial activity through the production of additional reactive species or quenching <sup>1</sup>O<sup>2</sup> [420]. As an example, azide sodium can modulate aPDT effectiveness by promoting or inhibiting the binding of bacteria with PSs, depending on lipophilicity of the latter. Indeed, azide sodium is a <sup>1</sup>O<sup>2</sup> quencher, but can also produce highly reactive azide radicals, via electron transfer from PS at the excited state. This has been reported with many phenothiazinium dyes and also fullerenes [93,421,422]. Other salts can amplify the bacterial killing mediated by MB-PDT such as potassium iodide (KI), a very versatile salt, involved in the generation of short-lived reactive iodine radicals (I•/I<sup>2</sup> •−) [423,424]; similar findings have been obtained when using KI with cationic BOD-IPY derivatives [425] or porphyrin–Schiff base conjugates bearing basic amino groups [426]. Potassium thiocyanate and potassium selenocyanate could also form reactive species, such as the sulfur trioxide radical anion and selenocyanogen (SeCN)2, respectively [427]. In addition, interactions between PSs and target microorganisms could be improved thanks to inorganic salts. For example, calcium and magnesium cations can modulate the electrostatic interaction between PSs and bacterial membranes [428]. It is worth noting here that most of the above-mentioned studies were conducted under in vitro settings. While inorganic salts can be useful to enhance in vitro or ex vivo aPDT effects, the use of such additives in animals or human beings would need to be carefully examined, considering the dose to be used and potential concomitant side effects.

#### *5.2. Combinations of aPDT with Other Antimicrobial Drugs or Antimicrobial Therapies*

Various classes of antimicrobial drugs or other antimicrobial therapies, which action does not depend on light, have been considered with regard to their aPDT compatibility.

#### 5.2.1. Antibiotics

Antibiotherapy is an obvious complementary therapy to aPDT, which may allow obtaining stronger antimicrobial effects and/or restore antibiotic susceptibility. Combinations of PSs with antibiotics have been investigated in numerous studies addressing various infectious diseases, such as skin and mucosal infections as recently reviewed [429]. Aminosides are the most common antibiotics used in combination with a PS. For instance, kanamycin, tobramycin, and gentamicin combined respectively to RB, porphyrin, and 5-Alanine have been reported, showing potent effects against bacteria of clinical interest, notably by improving biofilm clearance and reducing microbial loads [392,430–433]. In addition, combinations with other antibiotic classes, such as nitroimidazoles (e.g., metronidazole) or glycopeptides (e.g., vancomycine), also showed some effect against biofilms of *F. nucleatum* and *P. gingivalis* [393], as well as *S. aureus* [434]. Further, PSs can also be useful to combine with antibiotics in order to restore the susceptibility of bacteria to the latter, notably to last-resort antibiotics. As a recent example, Feng et al. reported the photodynamic inactivation of bacterial carbapenemases, both restoring bacterial susceptibility to carbapenems and enhancing the effectiveness of these antibiotics [396]. However, all combinations of PSs and antibiotics may not be effective, since in some cases antibiotics can have an antagonistic effect on PS activity [435]. Finally, it is noteworthy that some antibiotics can act themselves as PSs; Jiang et al. indeed reported light-excited antibiotics for potentiating bacterial killing via ROS generation [436].

#### 5.2.2. Antifungals

The combination of PSs with antifungals is a powerful approach to thwart growing antifungal resistance, especially to fluconazole, which is commonly observed in *C. albicans* strains [437]. Quiroga et al. showed that sublethal photoinactivation mediated by a tetracationic tentacle porphyrin allowed to reduce the MIC of fluconazole in *C. albicans* [438]. Nystatin, a common antifungal used to prevent and treat candidiasis, was combined with photodithazinebased PS and red light in *C. albicans*-infected mice. The combined therapy reduced the fungal viability and decreased the oral lesions and the inflammatory reaction [54]. Moreover, other antifungals (e.g., terbinafine, itraconazole and voriconazole) combined to ALA reported promising results. These combinations could be alternative methods for the treatment of refractory and complex cases of chromoblastomycosis [401,439].

#### 5.2.3. Other Antimicrobial Compounds

Many other antimicrobial compounds may be used in combination with PSs. Antimicrobial peptides (AMPs) are oligopeptides (commonly consisting of 10–50 amino acids) with high affinity for bacterial cells thanks to an overall positive charge. Their antimicrobial spectrum can be modulated through variation of their amino acids sequence. AMPs encompass a large set of natural compounds that may be used in aPDT. For example, de Freitas et al. reported that sub-lethal doses of PSs (either Ce6 or MB) and aurein peptides (either aurein 1.2 monomer or aurein 1.2 C-terminal dimer) were able to prevent biofilm development by *E. faecalis* [398]. In another study, a membrane-anchoring PS, named TBD-anchor, demonstrated both bacterial membrane-anchoring abilities and ROS production [440]. In a similar way to peptide therapy, LPS-binding proteins were used to improve the contact of PSs with the cytoplasmic membrane of bacteria. For instance, the antibacterial efficacy of a complex consisting of a combination of RB with the lectin concanavalin A (ConA) was demonstrated in a planktonic culture of *E. coli*; ConA-RB conjugates increased membrane damages and enhanced the RB efficacy up to 117-fold [399]. In addition, coupling pump efflux inhibitors, such as CCCP EPI (carbonyl cyanide mchlorophenylhydrazone), to PSs was also investigated. This highlighted the interest to combine PSs with molecules acting on targets susceptible to induce resistance modulations [40]. Other antimicrobial molecules may be good candidates to be used in aPDT strategies. For example, quinine used in combination with antimicrobial blue light was shown efficient to photo-inactivate MDR *P. aeruginosa* and *A. baumannii* [400]. Some cationic molecules, such as cationic lipids, can have a good affinity for bacterial cell membrane and a good antibacterial activity [126,441]. Thus, PS-amphiphiles conjugates would also deserve to be investigated for aPDT applications.

#### 5.2.4. Viral NPs and Phagotherapy

In recent years, viral NPs (VNPs) deriving from phages, animal, or plant viruses have been proposed as biological vehicles for delivery of PSs. Such carriers can exhibit a series of advantageous properties including natural targeting, easy manufacture and good safety profile [442]. Compared with nanomaterials used as PS carriers, VNPs are natural proteinbased NPs that may display higher biocompatibility and tissue specificity. In addition, VNPs can be tuned through genetic and synthetic engineering with appropriate biological and chemical modifications (e.g., surface decoration). Alternatively, the combination of aPDT with so-called phagotherapy may be useful for various reasons, following different strategies. One study suggests that, following a PDT treatment, ROS damages can cause

quorum sensing and virulence pathway alterations rendering micro-organisms more susceptible to other therapies. Among these, phagotherapy is known to be modulated by multiple virulence factors [443]. Another approach can consist of conjugating phages with PSs, in order to improve interaction/delivery of the latter into target microorganisms. For example, Dong et al. showed that a phage carrying the chlorophyll-based PS pheophorbide efficiently induced apoptosis in *C. albicans*, thus demonstrating the potential of phototherapeutic nanostructures for fungal inactivation [409]. However, such an approach has to be prudently considered regarding the occurrence of phage resistance already reported in numerous investigations [444].

#### *5.3. Combinations of aPDT with Other Light-Based Treatments*

Multiple modalities entirely controlled by light stimuli may be combined with aPDT in multifunctional antimicrobial treatments. The following part reports some very recent studies illustrating multiple light-based antimicrobial strategies combined to operate independently, with potential additive or synergistic effects and without reciprocal interferences. This may be obtained with a single PS displaying such multiple properties and/or through combination of PSs with other light-activatable compounds exhibiting complementary properties.

#### 5.3.1. aPDT and Photothermal Therapy

Photothermal therapy (PTT) is a local treatment modality relying on the property of a PS to absorb energy and convert it into heat upon stimulation with an electromagnetic radiation, such as radiofrequency, microwaves, near-infrared irradiation, or visible light. The localized hyperthermia can lead to various damages resulting in microbial inactivation in the treatment area. Following this principle, ruthenium NPs have been used for PDT/PTT dual-modal phototherapeutic killing of pathogenic bacteria [414]. Moreover, GO demonstrated antibacterial effect against *E. coli* and *S. aureus* as a result of both PDT and PTT effects following irradiation with ultra-low doses (65 mW/cm<sup>2</sup> ) of 630 nm light [415]. Furthermore, combination of sonodynamic, photodynamic, and photothermal therapies with an external controllable source recently reported against breast cancer [445] may also show promising applications for treating bacterial infection. Mai et al. reported a FDA-approved sinoporphyrin sodium (DVDMS) for photo- and sono-dynamic therapy in cancer cells and photoinactivation of *S. aureus* strains, in in vitro and in vivo models. However, no bacterial sonoinactivation by DVDMS was obtained [446].

#### 5.3.2. aPDT and NO Phototherapy

Combination of aPDT with NO phototherapy is gaining increasing interest for antimicrobial applications [447]. For instance, light-responsive dual NO and <sup>1</sup>O<sup>2</sup> releasing materials showed phototoxicities against *E. coli* [355,417]. More recently, Parisi et al. developed a molecular hybrid based on a BODIPY light-harvesting antenna producing simultaneously NO and <sup>1</sup>O<sup>2</sup> upon single photon excitation with green light for anticancer applications; according to the authors, this system may also act as an effective PS and NO photodonor antibacterial agent [448].

#### 5.3.3. aPDT and Low Laser Therapy

Photobiomodulation (PBM), also called low-level laser therapy, is a non-destructive process that may alleviate pain and inflammation or promote tissue healing and regeneration. The use of this method coupled to aPDT is a very recent approach. A concomitant use of aPDT and PBM was reported as an adjunct treatment for palatal ulcer [449]. In a clinic-laboratory study, aPDT and PBM showed similar improvement in gingival inflammatory and microbiological parameters compared with conventional treatment [450]. More recently, some benefits of this combined therapy were reported such as the modulation of inflammatory state, pain relief, and acceleration of tissue repair of patients contracting *herpes simplex labialis* virus or orofacial lesions in patients suffering from COVID-19 [451–453].

## *5.4. Coupling of aPDT with Other Physical Treatments*

### 5.4.1. aPDT and Sonodynamic Therapy

Sonodynamic therapy (SDT), combining so-called sonosensitizers (SS) and ultrasounds (US) is a relatively new approach for treating microbial infections [454,455]. The ultrasonic waves have the property to induce a cavitation phenomenon thus enhancing the efficacy of combined antimicrobial treatments. The rationale for combining PDT with SDT relies on specific advantages of the latter, notably, a deeper propagation of US into the tissue than light; therefore, PDT/SDT may be used to treat deeper lesions in vivo, alleviating the limitations of light propagation and delivery presented by aPDT [405,456]. An approach combining PDT and SDT, called sonophotodynamic therapy (SPDT), has been reported to improve microbial inactivation compared with individual aPDT or SDT [457]. Because of the complicated system of SPDT, its mechanisms have not been clearly revealed yet. Some studies have demonstrated that sonoporation mechanism induced by US improves the transfer of large molecules into the bacteria by forming transient pores. Moreover, US waves could potentiate the microorganisms dispersion in the medium resulting in (i) a better biodisponibility of therapeutic agent and light diffusion and (ii) a reduction of microbial aggregation and networks, such as biofilm [457,458]. The mention of a new class of PS characterized by the dual ability to be activated by both US and light, for SPDT application, has been questioned. Indeed, Harris et al. recently suggested that this specific PS/SS class could be useful for antimicrobial application – beside previously reported anticancer application – with initial investigation using chlorins as dual PS/SS agent [459]. Since this study by Harris et al., a few dual-activated PS/SS have been described that would warrant further investigations [460,461].

#### 5.4.2. aPDT and Electrochemotherapy

Electrochemotherapy, also called pulsed electromagnetic fields (PEMFs) or electropermeabilization, is a method consisting in applying an electrical field to cells in order to enhance their permeability to therapeutic molecules (often chemical drugs or DNA). Combination of PDT with electrochemotherapy has been used many times to treat cancer diseases [14,462,463]. One study showed that, compared with aPDT used alone, hypericin combined with electrochemotherapy allows to achieve more than 2 to 3 log<sup>10</sup> CFU reduction in *E. coli* and *S. aureus*, respectively [407]. To our knowledge, no other study combining electrochemotherapy with aPDT was recorded since then. However, combination of aPDT and a cell-permeabilization technique with a controllable toxicity degree may be highly relevant since most PSs can act in extracellular medium without having a specific target. Accordingly, electrochemotherapy may allow boosting aPDT activity by promoting PSs internalization into target microorganisms.

#### *5.5. aPDT and Other Antimicrobial-Related Therapies*

Immunotherapeutic effects may be obtained as a result of PDT itself or due to other treatments used in combination. For instance, Schiff base complexes with differential immune-stimulatory and immune-modulatory activities were reported efficient to eliminate both Gram(+) and Gram(−) bacteria. Furthermore, upon photoactivation, these complexes blocked the production of the inflammatory cytokine TNFα, thereby allowing to treat at once bacterial infections associated with damaging inflammation [403]. One recent study proposed the first application of antimicrobial photoimmunotherapy (PIT) by developing a PS-antibody complex, selective to the HIV antigen anchored to the infected cell membranes [404]. Such an approach supports the therapeutic applicability of PDT against antimicrobial infections, especially those mediated by intracellular pathogens. In addition, photodynamic therapy using PSs at sub-lethal concentrations may exhibit interesting properties for inflammatory and infectious conditions [464]. It was shown effective to alter immune cell function and alleviate immune-mediated disease, to hasten the process of wound healing, and to enhance antibacterial immunity. PDT thus appears as

a promising therapeutic modality in infectious and chronic inflammatory diseases such as inflammatory bowel disease and arthritis.

#### **6. Other aPDT Perspectives: New Strategies to Efficiently Target Bacteria**

Irrespective of the biomedical applications, achieving a precise targeting is crucial to guarantee both efficiency and specificity. For anticancer PDT, many targeting studies have been done, notably for evaluating PSs covalently attached to molecules having affinity for neoplasia or ligands for receptors expressed on tumors. By this way, PSs may be chosen considering primarily their ability to achieve high PDT effects rather than depending on their intrinsic targeting properties. Following the same rationale, aPDTbased combinatory systems can be developed, benefiting from earlier studies performed in multimodal oncology [465].

#### *6.1. Aggregation-Induced Emission (AIE) Luminogens*

AIE luminogens exhibit, in the aggregated state, nonradiative decay and show bright fluorescence due to the restriction of intramolecular motions [9]. Recently, their interests for antimicrobial applications have been reported, showing the possibility to simultaneously perform detection and image-guided elimination of bacteria for theranostics applications [466]. In comparison with classical PSs, AIE luminogens in an aggregated state do not exhibit self-quenched fluorescence and ROS production is better. For instance, Gao et al. reported a tetraphenylethylene-based discrete organoplatinum(II) metallacycle electrostatically assembled with a peptide-decorated virus coat protein. This assembly showed strong membrane-intercalating ability, especially in Gram(−) bacteria, and behaved as a potent AIE-PS upon light irradiation [467].

## *6.2. Photochemical Internalization (PCI)*

PCI may be used to enhance cell internalization of diverse macromolecules. It consists of PDT-induced disruption of endocytic vesicles and lysosomes improving the release of their payloads into the cytoplasm of target cells. Although most PCI applications relate to cancer treatments, PCI could be extended to treat intracellular infections by delivering antimicrobials into infected cells [468]. For instance, Zhang et al. reported PCI as an antibiotic delivery strategy allowing to enhance cytoplasmic release of Gentamicin, to counter intracellular staphylococcal infection in eukaryotic cells and in zebrafish embryos [469].

#### *6.3. Genetically-Encoded PSs*

Internalization of PSs inside target microorganisms could be facilitated thanks to their conjugation with adjuvants, as mentioned before. Alternatively, it could be possible to use genetically-encoded ROS-generating proteins (RGPs), also called genetically-encoded PSs. Such an approach represents a powerful way to "completely localize" PSs inside target microorganisms for highly specific antimicrobial phototoxicity. Furthermore, in situ production of RGPs allows to enhance interaction with intracellular targets and better control the biodistribution of PSs, while limiting side-effects for the host tissues and environments [470]. To date, two groups of RGPs have been reported; those that belong to the green fluorescent protein (GFP) family and form their chromophores auto catalytically, and those that use external ubiquitous co-factors (flavins) as chromophores [471]. For example, Endres et al. compared eleven light-oxygen-voltage-based flavin binding fluorescent proteins and showed that most were potent PSs for light-controlled killing of bacteria [472].

### *6.4. pH-Sensitive aPDT*

Some studies have reported smart photoactive systems consisting in PSs assembled in nanoconjugates with acid-cleavable linkers. For instance, Staegemann et al. described porphyrins conjugated with acid-labile benzacetal linkers and demonstrated the cleavage of the active PS agents from the polymer carrier in the acidic bacterial environment [316]. In addition, photoacids may be useful to design pH-sensitive aPDT systems. Upon light

irradiation, such agents promote the spatial and temporal control of proton-release processes and could provide a way to convert photoenergy into other types of energy [473]. Thus, proof-of-concept was reported for the use of reversible photo-switchable chemicals as antimicrobials inducing MDR bacteria photoinactivation mediated by the acidification of intercellular environment [474]. To our knowledge, no studies have yet reported the potential of photoacids in combination with aPDT systems. However, some pH-sensitive PSs can induce remarkable variations of antimicrobial photoinactivation levels under different environmental pH [475]. These observations suggest the potential of photoacids as PDT potentiators for enhanced antimicrobial applications.

#### *6.5. DNA Origami as PS Carriers*

The quite recent development of DNA origami based on well-established DNA nanotechnology can serve as an excellent scaffold for the functionalization with different kinds of molecules and could be a powerful tool, as described by Yang at al., to study in a real-time conditions the assemble/disassemble of photo-controllable nanostructures [476]. Oligonucleotides organized as DNA origami could thus be used as PS-carrying nanostructures featuring numerous and dense intercalation sites. In addition, the tightly packed double helices can avoid the degradation by DNA hydrolases in the cellular environment. For instance, Zhuang et al. reported the uptake in tumor cells of a PS-loaded DNA origami nanostructure where it generated free radicals, releasing PSs due to DNA photocleavage, and induced cell apoptosis [477]. To our knowledge, such an approach has not yet been investigated for antimicrobial purposes.

#### **7. Discussion**

Antimicrobial PDT has the potential to fight against a wide spectrum of infective agents, including those resistant to conventional antimicrobials, under non-clinical and clinical settings. Rather than replacement, aPDT may be a complementary approach to reduce the use of current, especially last resort, antimicrobials. This review aims to give a non-exhaustive overview of the diversity and richness of synthetic, natural, or hybrid single PSs and aPDT nanosystems that were recently reported, with respect to their specific advantages, limitations, and possible evolutions. It is noteworthy that many systems and strategies primarily developed for anticancer PDT have been or could be applied—*per se* or following adaptations—to antimicrobial applications.

Beyond the "chemical space" that can be explored with individual PSs, versatile combinations with other compounds can allow the design of multimodular/multimodal systems. Along this line, the various PSs available may be considered as "basic ingredients". Apart from offering alternative possibilities for overcoming the most common limitations of PSs (i.e., solubility, delivery, and specificity), reasons for implementing PSs in complex systems can be related to (i) the ability to target several types of pathogens at once (extended antimicrobial spectrum), (ii) synergistic antimicrobial effects, (iii) reduction in the dose of each combined component, (iv) beneficial effects in severe poly-pathogenic infections, and (v) reduction in the risk of resistance emergence.

In addition to the many possible variations concerning PSs, optimizations can also consider other critical parameters in PDT, namely light irradiation and oxygenation. The latter was considered for a long time as an indispensable component. However, control of the oxygen level in the aPDT system is questionable. Indeed, additional oxygen-independent phototoxic mechanisms have been reported, for example with psoralens, which can produce more effective aPDT without oxygen [21]. Furthermore, recent studies suggest that various strategies could be used to reduce or bypass the limitations of oxygen and light supplies (read below). All combined, optimizations targeting not only the PSs, but also light irradiation and oxygen supply, could allow to evolve toward integrative, highly sophisticated, antimicrobial photodynamic therapy.

Light irradiation and its various modalities have been reviewed in depth by different authors [12,478–480]. Typically, the irradiation in PDT occurs in the UV (200–400 nm) or

in the visible light (400–700 nm) with a power of ≤ 100 mW [479]. However, the low light-penetration depth (around mm) and possible occurrence of tissue photodamage limit the applicability of this spectral range for PDT. This can be circumvented by application of a near-infrared (NIR) irradiation (750–1100 nm), particularly via a two-photon excitation, which is emerging for PDT applications [223,481]. Being a third-order nonlinear optic phenomenon and corresponding to the simultaneous absorption of two photons with half the resonant energy, it allows deeper penetration in biological tissues (around 2 cm), lower scattering losses, and a three-dimensional spatial resolution [482]. Light sources are also constantly improving. Laser is an exceptional source of radiation, capable of producing extremely fine spectral bands, intense, coherent electromagnetic fields ranging from NIR to UV. In comparison, LEDs feature other advantages, notably the possibility to be arranged in many ways, in large quantity, for irradiating wide areas while inducing negligible heating [478,479].

Considering that light-emitted sources can become a brake to any PDT applications, several promising options have been envisaged quite recently. Notably, Blum et al. have identified a series of "self-exiting way" that allows to abolish the need of light to achieve efficient PDT [483]. Among them, chemiluminescence was extensively investigated by using luminol or luciferase energy transfer to induce a chemiexcitation of PS as antibacterial therapeutics [484]. In addition, other methods may be based on Cherenkov radiation, which occurs when an emitted charged particle, such as an electron, moves with high speed through a dielectric medium, such as water. Thus, the polarization of electrons in the medium produces electromagnetic waves in the visible wavelengths that could activate PDT reaction. Another way to induce Cherenkov radiation is to use radioactive isotopes with high beta emissions (e.g., <sup>18</sup>F, <sup>64</sup>Cu, or <sup>68</sup>Ga) as an electronic excitation source [483]. The relevance of such approaches for the design of a self-induced aPDT system remains to be evaluated. In addition, another external source of excitation, such as microwaves, could activate photoactive molecules such as Fe3O<sup>4</sup> when they are complexed with carbon nanotubes. This was recently evaluated by Qiao et al. for treating MRSAinfected osteomyelitis [48]. Furthermore, X-ray as an activated source can also facilitate the activation of the PDT system by transferring energy harvested from X-ray irradiation to the PS used [485]. Kamanli et al. compared pulse and superpulse radiation modes, showing that the latter is more effective to produce <sup>1</sup>O<sup>2</sup> and *S. aureus* eradication than the former [486].

Oxygen is also a critical limiting factor that determines PDT efficiency, especially in poorly oxygenated environments. However, it is noteworthy that PDT can be achieved in cancers that typically feature a low oxygenation rate. To alleviate the possible limitations of oxygen supply in PDT systems, several strategies have also been recently considered. One is based on catalase grafting to achieve oxygen self-sufficient NPs in order to convert H2O<sup>2</sup> into available dissolved oxygen in the tumor environment. The abundant ROS in tumors compared with normal tissues provide a coherent substrate for catalase and thus allows an improvement of PDT activity [487]. Some multifunctional nanomaterials, called nanozymes, can also be used in combination with PSs to achieve a catalase-like activity supplying an oxygen source for the PS functioning [488,489]. In addition, it was also reported that noble metal NPs – such as Ru, Pt, and Au NPs – exhibit catalase-like nanozyme activities [490]. The use of catalases or nanozymes may have the crucial role of oxygen helpers in aPDT for treating deep infections.

The possibilities to design combinatorial aPDT strategies seem unlimited. Those presented in this review partly overlap with the description of aPDT systems, showing the complexity of any classification process. Awareness and caution may be raised about some sort of "paradox" or "dilemma"; indeed, while the seemingly boundless collection of chemical options and modular tools to develop nanoscale aPDT therapeutics implicitly defines extensive design flexibility, it staggeringly complicates and bewilders at the same time the optimization process aiming to compare, rationalize, and identify the "best" option for each application. Along this line, data concerning structure-activity relationships are

usually missing, with not many studies yet dedicated to this matter [11,42,91,491]. Similar to the CLSI guidelines defined for antibiotics, uniform research methodologies would be useful to assay aPDT systems under well-defined standards and guidelines, considering notably (i) illumination settings, (ii) positive controls [492], (iii) microorganisms and cell lines relevant for a given application, and (iv) assessment of antimicrobial efficacy; this would guarantee better-conducted preclinical and clinical trials of aPDT systems used as mono or combinatory therapy. Furthermore, a public database compiling the efficacy/side effects of various systems would also be useful, facilitating meta-analyses for delineating (quantitative) structure/activity relationships and computational simulations [493].

Finally, in view of translation to clinical practice, a series of precautions and potential limitations must also be considered, especially when dealing with combinatory strategies. Beside possible reciprocal interferences (antagonisms) between combined partners, safety and specificity parameters must also be carefully examined. One main advantage of aPDT is the possibility to control the production of ROS thanks to the use of nontoxic PSs triggered with inducers (specific light and free oxygen) and/or enhancers. The vast majority of PSs are considered safe and dark cytotoxic side effects toward non-target eukaryotic cells are rarely reported. However, in many cases, more studies are needed for examining—beyond potential side-effects—other parameters such as bioavailability, biodispersion, persistence in host cells and body, and elimination pathways. With regard to combinatory strategies, it is noteworthy that studies conducted to date were mostly based on in vitro evaluation or using animal models bearing well-defined sites of infection (Table 2). Thus, much more data in preclinical and clinical settings are required to support the actual potential of such strategies. Moreover, considering that many microbial infections are systemic, the use of modalities such as sonodynamic therapy or electrochemotherapy is at present not realistically feasible. Therefore, in spite of significant progress and real promise, further important work/innovations are needed to effectively broaden the range of infectious conditions that could be treated via aPDT approaches. Lastly, cost effectiveness of multiplexed aPDT therapies over monomodal conventional antimicrobial agents is another crucial point to be considered in view of clinical applications. Potent, but too expensive solutions may fail to be used in practice, especially in under-developed countries where conventional antimicrobials will continue to be used, allowing microorganisms to increase in resistance.

#### **8. Concluding Remarks**

In conclusion, aPDT is a versatile approach that tends to evolve from quite a simple method to reach much higher degrees of complexity, with several expected advantages, but also possible drawbacks or undesirable effects. Among the latter, any direct/indirect impact on AMR should be more thoroughly considered. It is noteworthy that aPDT clinical trials conducted to date evaluated quite simple PSs. Any increase in the complexity of therapeutic systems would lead to an increase in difficulty before being able to reach clinical applications. The development of effective and safe aPDT treatments requires expertise in many fields of research, including biology (microbiology, cell biology, biochemistry, pharmacology), chemistry, physics (optical physics), and engineering. This is even more the case with combinatory strategies involving different modalities as reviewed in this article. Translation to practical applications also implies strong collaborations with the different sectors of health care and pharmaceutical companies. In these conditions, aPDT and its many therapeutic combinations could become a frontline routine treatment to fight against microorganisms possibly responsible for the next healthcare crises [61].

**Funding:** This work was supported by grants from ANR/BMBF (TARGET-THERAPY; PIs: Tony Le Gall and Holger Schönherr; ANR grant number: ANR-20-AMRB-0009, RPV21103NNA; BMBF Förderkennzeichen: 16GW0342). We also thank the "Association de Transfusion Sanguine et de Biogénétique Gaétan Saleün" (France) and the "Conseil Régional de Bretagne" (France) for their financial support. Raphaëlle Youf is recipient of a PhD fellowship from the French "Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation" (Paris, France).

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**



## **References**


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