**1. Introduction**

Cancer remains one of the leading causes of death worldwide. Due to its complex nature, multiple metabolic pathways and ability to resist numerous drugs, so far, selective elimination of cancer cells without influencing healthy tissues has not yet been achieved [1,2].

**Citation:** Stavroulaki, D.; Kyroglou, I.; Skourtis, D.; Athanasiou, V.; Thimi, P.; Sofianopoulou, S.; Kazaryan, D.; Fragouli, P.G.; Labrianidou, A.; Dimas, K.; et al. Influence of the Topology of Poly(L-Cysteine) on the Self-Assembly, Encapsulation and Release Profile of Doxorubicin on Dual-Responsive Hybrid Polypeptides. *Pharmaceutics* **2023**, *15*, 790. https://doi.org/10.3390/ pharmaceutics15030790

Academic Editor: Ana Isabel Fernandes

Received: 20 January 2023 Revised: 16 February 2023 Accepted: 23 February 2023 Published: 27 February 2023

**Copyright:** © 2023 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/).

Most of the anticancer drugs used are poorly water soluble, leading to poor absorption and low bioavailability. Therefore, it is necessary to design and synthesize smart drug delivery systems which can transport therapeutic agents in a timely and spatially controlled manner. This need can be satisfied by using nanotechnology for the synthesis of nanostructured materials as drug carriers, the most common being micelles [3–5], liposomes [6], polymersomes, magnetic NPs and mesoporous silica NPs as well as hydrogels [7–9]. Among the materials used, polymers play a pivotal role due to their high functionalization. Drugs can be encapsulated or chemically conjugated to polymers, lowering their toxicity and increasing their solubility as well as their circulation time in the blood and preventing renal clearance for more efficient accumulation in the solid tumor either through the EPR effect or an active targeting mechanism [10].

Ideally, smart drug delivery systems to target cancer cells should have the ability to bypass numerous biological obstacles such as vasculature and non-vasculature barriers and tumor microenvironment as well as intracellular barriers [11]. The tumor microenvironment is dynamic and is characterized by acidity, hypoxia and ischemia. Usually, the pH value of a tumor tissue is 6.3–6.8, while higher concentrations of various biological substances are detected in cancer cells such as GSH and matrix metalloproteinase 2 (MMP2) as well as active oxidative species (ROS) [12,13]. The blood serum has a pH value of 7.4, while the endosomal and extracellular pH of the cancer tissues exhibits a pH range from 6.3–6.8, and that of the lysosomal compartments of the cell can be between pH 5.5–5.0 [14,15]. Therefore, pH-responsive systems can induce controlled drug release and penetrate deeper into cancer cells, due to pH variations between the intracellular organelles of the cell and the extracellular matrix. In the case of polypeptides bearing an amine or carboxylic acid in their side chains, pH changes activate the protonation/deprotonation mechanism, leading to the disassembly of their conformation and the desired triggered release of their cargo.

Therefore, the NPs that will be used to treat cancer should exhibit the following main characteristics: good biocompatibility and minimal cytotoxicity without systemic side effects, highly selective accumulation in pathological tissues, accurate stimulus responses to result in selective release of the cargo and long-term stability in blood circulation as well as minimal cargo loss before arrival at the target. In addition, drug delivery systems should be inert and stable in the aggressive environment of the blood compartment. We envisioned synthetic NPs that fulfill the above properties and can change their structure when they reach the pathological environment, releasing the cargo in a controlled way, leading to efficient and highly selective elimination of cancer cells.

PHis is a unique polypeptide with the ability to respond to physiological pH variations as its imidazole ring with a pKa around 6.5 can be protonated and deprotonated within the physiological values, altering the hydrophilicity of the polypeptide [16]. Bilalis et al. [17] have described the synthesis of PHis-grafted mesoporous silica NPs (MSNs) which can efficiently encapsulate the anticancer drug DOX, and can release it in a pH-controlled manner. Our group has also referred to the synthesis of linear and 3-miktoarm star hybrid polypeptides based on PHis, poly(L-glutamic acid), poly(L-lysine) and PEO. These novel structures could load the anticancer drug Everolimus and release it in response to pH changes [18].

Redox responsive nano-structures, whose action is determined by the microenvironment of cancer tissue, also induce controlled drug release [19,20]. The redox reaction depends on the concentration of active substances in the cell organelles. For example, the concentration of GSH is four times higher in cancerous tumors compared to its concentration in healthy tissues. Different concentrations of GSH were also found in intracellular (~2–10 mM) and extracellular (~2–10 μM) fractions. In particular, intracellular compartments comprising the cytoplasm, mitochondria, and nucleus contain higher concentrations of GSH compared to extracellular fluids [21–23]. It is well known that disulfide bonds are sensitive to GSH, as GSH can cause the rapid cleavage of disulfide bonds, leading to cytosolic delivery of anticancer agents [24–27].

Wang et al. [28] managed to synthesize redox-responsive SCL micelles based on poly(ethylene glycol)-*b*-poly(L-cysteine)-*b*-poly(L-phenylalanine) triblock copolymers, which could load DOX. A sustained release profile of DOX was observed from these NPs where the PCys was in a block form. Moreover, Wu et al. [29,30] reported the synthesis of DOX-loaded and gold-embedded micelles based on poly(L-cysteine) which exhibit synergistic chemo- and photothermal therapy of cancer cells. In both works, PCys was in a block form and a similar sustained release of the drug was observed. In a previous publication by our group [31], we presented the synthesis of disulfide crosslinked polypeptide nanogels consisting of PHis and PCys, which show satisfying pH, redox and thermo-responsiveness to the external stimuli. In that work, the disulfide bonds were randomly distributed along the polymeric chains. We have shown that the macromolecular architecture and topology of the blocks can play a critical role on the self-assembly of amphiphilic polymers [32–36]. Although the topology of cysteine along the polypeptide chain can play a critical role at the release profile of the drug, to our knowledge, there is no publication so far which studies this parameter on the structure and drug release profile of NPs.

Herein, we present the synthesis of three series of hybrid polypeptide copolymers composed of PEO, PCys and PHis. PEO was always the first block in all these series. At the first series, PCys was the second block, while PHis was the last block. At the second series, PHis was the second block while PCys was the last. Finally, at the third series, PHis and PCys were randomly distributed along the chain. Two different compositions of PHis and PCys were used, keeping the total number of the monomeric units of PHis and PCys the same, while the PEO block was the same for all hybrid polypeptides. It was found that the empty hybrid terpolymers can self-assemble into micellar nanostructures and exhibit pH and redox responsiveness. Moreover, they can effectively load the anticancer drug DOX in differently structured NPs compared to the empty one and can release in a controlled manner, in response to pH and redox variations. The release profiles depended on the structure of the NPs. In order to elucidate the influence of PCys topology at the release profile of DOX, the release was studied in media with various concentrations of GSH as well as different pH values. Finally, in vitro studies of the efficacy of the NPs in breast cancer cell lines prove that the DOX-loaded NPs could be potentially used for cancer treatment. In order to elucidate the impact of the PCys topology on encapsulation efficiency and release profiles, the results were compared to the NPs obtained under similar conditions with similar polymers such as PEO-*b*-PHis or PEO-*b*-Poly(sarcosine)-*b*-PCys (PEO-PSAR-PCys).

### **2. Experiment**

### *2.1. Materials and Methods*

### 2.1.1. Materials

Ethyl acetate (>99.9%, Carlo Erba, Val de Reuil, France) was fractionally distilled over phosphorous pentoxide. Tetrahydrofuran (THF) (>99.9%, Carlo Erba) was purified over Na-K alloy, using standard high vacuum techniques. The purification of *N*,*N*-Dimethylformamide (DMF) (99.9%, Alfa Aesar, Waltham, MA, USA anhydrous, amine free) was performed by short-path fractional distillation under high vacuum in a custom-made apparatus, and the middle fraction was used. The final product was stored in a vacuum flask at 3 ◦C. Benzene (99%, thiophen-free grade, Sigma Aldrich, Saint Louis, MO, USA) was treated with calcium hydride and was allowed to be stirred overnight for moisture removal. It was then distilled under high vacuum and stored in a different flask containing *n*-BuLi. Diethyl ether (>99.5%), Dichloromethane (99.8%) and Chloroform (>99.8%) were purchased from Fluka, Charlotte, NC, USA. *n*-Hexane (>95%) was obtained from Carlo Erba, Val de Reuil, France. Methyl Sulfoxide (DMSO) (99.8+%, for peptide synthesis) was supplied from Acros Organics, Waltham, MA, USA. BOC-His(*Trt*)-OH (>99%) was acquired from Christof Senn Laboratories AG, Dielsdorf, Switzerland. Sarcosine (98%) was purchased from Alfa Aesar. *S*-tert-Butylmercapto-L-Cysteine (99%) was provided by Sigma Aldrich. Methoxypolyethylene glycol amine (*m*PEO-NH2) with average *M*<sup>n</sup> = 10,000 g mol−<sup>1</sup> was

obtained from Sigma Aldrich. Triethylamine (Et3N) (99.83%, Fluka) was dried over calcium hydride for one day and then distilled and stored under vacuum over sodium. Triphosgene (99%), Thionyl chloride (99.5+%), Hydrogen peroxide (ACS reagent, 30 wt.%, solution in water, non-stabilized) and DL-1,4-Dithiothreitol (99%) were provided by Acros Organics, Waltham, MA, USA. (R)-(+)-Limonene (97%) was purchased from Alfa Aesar. Trifluoroacetic acid (TFA) (>99%) was obtained from Fluka. Triisopropylsilane (98%) was supplied from Sigma Aldrich. L-Glutathione (98%, reduced form) was obtained from FluoroChem, Hadfield, UK. Sodium Chloride (99.9%) was purchased by Penta chemicals, Prague, Check Republic. Sodium Hydroxide pearls (99.4%) was acquired by Lachner, Neratovice, Check Republic. Hydrochloric Acid 1 mol L−<sup>1</sup> and Acetic Acid glacial (99.8%) were obtained from Chem-Lab, Zedelgem, Belgium. Tris base ultrapure (99.9%) was purchased from Duchefa Biochemie, RV Haarlem, Netherlands. Sodium Phosphate Monobasic (98–100.5%) was supplied from Riedel-de Haen, Charlotte, NC, USA. Doxorubicin Hydrochloride (>99%) was obtained from Selleckchem, Planegg, Germany. Distilled water was further purified by a Milli-Q Direct Water purification system (18.2 MΩ·cm, Merck Millipore, Darmstadt, Germany).

### 2.1.2. NMR Spectroscopy

1H-NMR measurements were carried out on a 400 MHz Bruker Avance Neo instrument, Billerica, MA, USA. A mixture of deuterium oxide (D2O) and deuterium chloride (DCl 1%) was used as the solvent for the polymers, while deuterated chloroform (CDCl3) was employed as the solvent for NCAs, at room temperature.

### 2.1.3. FT-IR Spectroscopy

Fourier transform infrared (FT-IR) spectroscopy measurements were conducted using a Perkin Elmer Spectrum One instrument (Waltham, MA, USA), in KBr pellets at room temperature, in the 450–4000 cm−<sup>1</sup> range.

### 2.1.4. Size Exclusion Chromatography

Size exclusion chromatography (SEC) was employed to determine the *M*<sup>n</sup> and Ð*M* = *M*w/*M*n values. The analysis was performed using two different SEC sets. The first one was composed of a Waters Breeze instrument (Milford, MA, USA) equipped with a 2410 differential refractometer and a Precision PD 2020 two angles (15◦, 90◦) light scattering detector. The carrier solvent was a 0.10% TFA (*v*/*v*) solution of water/acetonitrile (80/20 *v*/*v*) at a flow rate of 0.8 mL min−<sup>1</sup> at 35 ◦C. Three linear Waters hydrogel columns were used as a stationary phase. The second system was composed of a Waters 600 high-performance liquid chromatographic pump, Waters Ultrastyragel columns (HT-2, HT-4, HT-5E and HT-6E), a Waters 410 differential refractometer and a Precision PD 2020 two angles (15◦, 90◦) light scattering detector at 60 ◦C. A 0.1 M LiBr in DMF solution was used as an eluent at a rate of 1 mL min<sup>−</sup>1.

### 2.1.5. Circular Dichroism

Circular Dichroism measurements were conducted via a JASCO J–815 model in a 0.1 cm cell. The aqueous solutions of polymers had a concentration of 10−<sup>5</sup> g mL−<sup>1</sup> and the desired pH was adjusted with the addition of droplets either of 0.01 N HCl, or 0.01 N NaOH. The temperature was stabilized to 25 ◦C with the use of a dedicated digital thermostat. The nitrogen flow was adjusted to 6.0 L min<sup>−</sup>1.

### 2.1.6. UV Spectroscopy

UV spectroscopy was carried out using a Perkin Elmer Lamda 650 spectrometer, (Waltham, MA, USA) in the range of 250–800 nm, at room temperature, with cells requiring 3 mL. A Waters Diode-Array 690 detector (Milford, MA, USA) was used for the calibration and on-line determination of the DOX drug loading efficiency at λ = 485 nm.

### 2.1.7. Dynamic Light Scattering

DLS measurements were conducted with a Brookhaven Instruments BI-200SM Research Goniometer system (Holtsville, NY, USA) operating at λ = 640 nm and with 40 mW laser power. Correlation functions were analyzed by the cumulant method and the Contin software. The correlation function was measured at 90◦, at 25 ◦C. All measurements were performed in either an isotonic PBS or Tris buffer (10 mM, 150 mM NaCl) at pH = 7.4, PBS buffer (10 mM, 150 mM NaCl) at pH = 6.5, and an isotonic acetate buffer (10 mM, 150 mM NaCl) at pH = 5.0. The concentration range measured was between 2 × <sup>10</sup><sup>−</sup>3–1 × <sup>10</sup>−<sup>5</sup> g mL<sup>−</sup>1.

### 2.1.8. Static Light Scattering

SLS measurements were carried out on an ALV/CGS-3 Compact Goniometer System (ALV GmbH, Langen, Germany), equipped with an ALV-5000/EPP multi-tau digital correlator with 288 channels and an ALV/LSE-5003 light scattering electronics unit for stepper motor drive and limit switch control. A JDS Uniphase 22 mW He-Ne laser was used as the light source. The instrument was connected to a Polyscience model 9102 bath for temperature control, allowing measurements at variable temperature.

### 2.1.9. Electrophoretic Mobility

The electrophoretic mobility measurements of the empty and drug-loaded nanoparticle dispersions were conducted using a Brookhaven Instruments Nanobrook Omni system (Holtsville, NY, USA) operating at λ = 640 nm and with 40 mW power, operating in PALLS mode. All the measurements were performed in isotonic Tris buffer (10 mM, 150 mM NaCl) at pH = 7.4 at 37 ◦C and were the average of at least three runs.

### 2.1.10. Transmission Electron Microscopy

Transmission electron microscopy images were obtained using a Jeol 2100 TEM, operated at 200 kV and fitted with a Gatan Ultrascan 1000 camera (Pleasanton, CA, USA). Samples for TEM analysis were prepared via drop-casting a few milliliters of sample dispersions after ultrasonication onto holey carbon grids, allowing the solvent to evaporate and leaving the sample to rest for 24 h at ambient temperature.
