*Review* **pH-Responsive Nanoparticles for Cancer Immunotherapy: A Brief Review**

#### **Yunfeng Yan \* and Hangwei Ding**

College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China; dhwpao@163.com

**\*** Correspondence: yfyan@zjut.edu.cn

Received: 31 July 2020; Accepted: 15 August 2020; Published: 17 August 2020

**Abstract:** Immunotherapy has recently become a promising strategy for the treatment of a wide range of cancers. However, the broad implementation of cancer immunotherapy suffers from inadequate efficacy and toxic side effects. Integrating pH-responsive nanoparticles into immunotherapy is a powerful approach to tackle these challenges because they are able to target the tumor tissues and organelles of antigen-presenting cells (APCs) which have a characteristic acidic microenvironment. The spatiotemporal control of immunotherapeutic drugs using pH-responsive nanoparticles endows cancer immunotherapy with enhanced antitumor immunity and reduced off-tumor immunity. In this review, we first discuss the cancer-immunity circle and how nanoparticles can modulate the key steps in this circle. Then, we highlight the recent advances in cancer immunotherapy with pH-responsive nanoparticles and discuss the perspective for this emerging area.

**Keywords:** nanoparticle; cancer; immunotherapy; pH-responsive; drug delivery

#### **1. Introduction**

Immunotherapy has revolutionized the cancer treatment by activating the innate and adaptive immune system against tumor cells with immune checkpoint inhibitors (ICIs), agonists, antigens, or engineered T cells. In contrast to the conventional cancer treatment modalities, e.g., chemotherapy, radiotherapy, and surgery, which directly kill cancer cells or resect tumor tissues, immunotherapy aims to restore the antitumor activity of the immune system to attack abnormal cells through natural mechanisms, allowing better potency and fewer off-target effects in the treatment of advanced malignancies [1–4]. Several notable clinical successes in cancer immunotherapy have been made over the past decade, including the FDA approval of the chimeric antigen receptor (CAR) T cell therapy and therapies with monoclonal antibodies (mAbs) targeting cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death 1 (PD-1), or its ligand (PD-L1) as the immune checkpoint inhibitors. Due to their contributions in the discovery of cancer therapy through the immune checkpoint blockade, the Nobel Prize in Physiology or Medicine 2018 was awarded to James P. Allison and Tasuku Honjo. Now, there is a large number of active clinical trials worldwide and immunotherapy has become a new pillar of cancer treatment owing to these tremendous achievements [5].

#### *1.1. Modulation of Anticancer Immunity*

The generation of endogenous immune response against tumors involves several distinct steps (Figure 1) [1,6,7]. First, tumor-associated antigens (TAAs) are released from cancer cells. The immunogenic signal could be proinflammatory cytokines or factors which are produced in oncogenesis (step 1). Then, TAAs are captured by antigen presenting cells (APCs) (e.g., dendritic cells) and presented on major histocompatibility complex (MHC) class I and class II molecules (step 2). Tumor antigen-loaded dendritic cells migrate to lymph nodes, resulting in the priming and activation of

T cells (step 3). This is a crucial step to generate T cell response against cancer-specific antigens. In the presence of immunogenic stimulators, the antigen presentation elicits effector T cells which are killers to cancer cells. Without such stimulation, dendritic cells will induce T cell deletion and the production of regulatory T cells which relate to the immunosuppression. The activated T cells subsequently leave the lymph node, traffic to tumors through the bloodstream, and infiltrate into tumor parenchyma (steps 4 and 5). Finally, effector T cells recognize cancer cells by the specific binding of T cell receptor (TCR) to the antigen on cancer cell surface (step 6), and kill target cancer cells (step 7). The death of cancer cells further promotes the release of TAAs and elicits the subsequent immune response.

**Figure 1.** The cancer-immunity cycle. Seven steps are involved in the generation of antitumor immunity, i.e., the release of tumor antigens from cancer cells, the presentation of tumor antigen to antigen-presenting cells (APCs), the priming and activation of T cells in lymph nodes, the trafficking and infiltration of T cells to tumors, the recognition and the killing of cancer cells.

Durable cancer immunotherapy requires the complete cancer-immunity cycle. However, a few factors may waken or suspend the generation and performance of antitumor immunity in cancer patients. The approaches to overcome these obstacles derive the major classes of cancer immunotherapy. The cancer antigens may not be sufficiently released from solid tumors and captured by dendritic cells for the further processing. In this respect, treatments with conventional chemotherapeutics or radiation induce the apoptosis of cancer cells that promotes the release of tumor antigens from dead cells [5]. The immunization could be also initiated with the delivery of exogenous vaccines including conventional protein or peptide antigens, nucleic acids, and dendritic cells. The tumor-associated proteins, peptides, and nucleic acids could be directly administered to cancer patients, then, processed by dendritic cells and cross-presented to T cells in vivo. Alternatively, dendritic cells are

engineered with specific antigens ex vivo and injected into patients for personalized immunization [8]. Stimulatory molecules are requisite for dendritic cell maturation, antigen presentation, and T cell activation. For example, agonists of Toll-like receptor (TLR), e.g., cytosine-phosphate-guanine oligonucleotide (CpG-ODN) for TLR9 and Imiquimod for TLR7, and stimulator of interferon genes (STING) are able to promote the maturation of APCs [9,10]. Incubation with interferon-α (IFN-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitates dendritic cell development and the expression of leukocyte antigen (HLA), B7 co-stimulatory molecules, MHC proteins, and CD40, which benefits the TAAs presentation and immunization [11]. Interleukin-2 (IL-2) is able to stimulate the expanding and activation of T cells in lymph nodes [12]. These cytokines and agonists are legitimately utilized to improve the immune activity of T cells for cancer immunotherapy. To circumvent the elaborate procedures of T cell priming and activation in vivo, T cells are collected from tumors or peripheral blood, selected, genetically engineered, and proliferated in vitro, followed by the reinfusion into the tumor-bearing patient [13]. This strategy of adoptive T cell therapy represents a major advancement of cancer immunotherapy in the past decade. In particular, T cells modified with chimeric antigen receptor (CARs) show exceptional immune activity which have been approved for clinical use to treat B cell acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma [14,15].

Abnormal angiogenesis and proliferation of cancer cells and cancer-associated fibroblasts (CAFs) contribute to the formation of solid tumors with high interstitial fluid pressure (IFP) that hinders the infiltration of all therapeutics into tumor parenchyma from blood vessels [16]. In addition to physical barriers, reduced blood flow and substance exchange further induce the hypoxia and acidity in tumor, resulting in the immunosuppressed tumor microenvironment (TME) that is a major cause of the resistance to the current cancer immunotherapy. The therapeutics for the normalization of TME, e.g., antiangiogenic and CAF-reprogramming agents, have been broadly utilized to improve the tumor perfusion and immunity for cancer immunotherapy [1]. The activity of effector T cells could be suppressed by immunosuppressive macrophages in the TME, including regulatory T cells, M2-like tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells because they are able to secrete a number of immunosuppressive factors (e.g., NO, reactive oxygen species, arginase, iterleukin-10, indoleamine 2,3-dixoygenase, and transforming growth factor-β) and to down-regulate the cytotoxicity of effector T cells [5]. Reprogramming or eliminating immunosuppressive cells has proven to be a complementary approach to augment the antitumor immunity of T cells in solid tumors. Cancer cells usually express immune checkpoint proteins on the surface, leading to the immune resistance when these proteins bind to the specific ligands on T cells. Inhibition of the immune checkpoints with anti-CTLA4, anti-PD-1, or anti-PD-L1 antibodies represents the most notable approach in the current cancer immunotherapy [2]. There are now at least six FDA-approved immune checkpoint inhibitors for the treatment of a wide range of cancers [3].

#### *1.2. Ongoing Challenges in Cancer Immunotherapy*

Despite the substantial progress in recent years, the broad implementation of cancer immunotherapy remains challenging. The response rate and magnitude of cancer patients to immunotherapies remains moderate. Only <13% of cancer patients effectively respond to the current immune checkpoint inhibitors because the expression level of checkpoint proteins varies with cancer types and patients [17]. CAR-T cell therapy shows high potency for the treatment of hematologic malignancies. However, its clinical application to solid tumors is still unfulfilled due to the compact and immunosuppressive microenvironment of solid tumors [18]. In addition to the unsatisfactory efficacy, the safety issues further limited the broader clinical use of immunotherapeutics. CAR-T therapy requires successive infusion of CAR-T cells that may cause severe side effects including cytokine-release syndrome (CRS) and CAR-T cell-related encephalopathy syndrome (CRES) [18]. The advance of immune checkpoint inhibitors (ICI) therapy is also associated with some immune-related adverse events (irAEs) including colitis, pneumonitis, hepatitis, myocarditis, and neurotoxic effects [19]. Some immune modulators are toxic and the repeated administration leads to accumulative toxicity

for the patients. In addition, the stimulating and activating circulating lymphocytes out of tumors may lead to the attack on normal tissues, causing the off-target side effects [2,19]. The ideal cancer immunotherapy should be capable of precise modulation on the strength and the site of immune response to optimize the clinical outcomes.

#### **2. Cancer Immunotherapy with Nanoparticles**

Nanoparticles, particles with a typical size of 1–100 nm in diameter, have been widely utilized in cancer treatments [2,16,20]. Nanoparticle-based delivery offers potent approaches for the spatiotemporal control of immunotherapeutic agents to reduce the adverse effects and maximize the therapeutic index of cancer immunotherapy [1–5]. First, the formulation of immunotherapeutics in nanoparticles can improve the pharmacological properties of drugs, including the solubility and stability. Of particular importance, some biologic drugs, e.g., nucleic acids and antibodies, require protection from degradation and macrophage clearance in blood after systemic administration. Second, nanoparticle platforms enable versatile modification or functionalization to modulate the pharmacokinetic profile of drugs and regulate the interaction between drugs and cells or organs. The level of antibodies or small drugs can be tuned by controlled release to extend the efficacy and avoid the systemic toxicity due to the instantaneous high concentration after systemic administration. In addition, the structure of nanoparticles can be readily designed for the active targeting and the smart response to external stimuli (e.g., light, electronic, and magnetic fields) or the biochemical changes from normal tissues to tumors (e.g., pH, redox potential, and enzymes), resulting in the enhanced tumor accumulation and reduced off-target side effects. For the highly hydrophilic and negatively charged nucleic acid drugs, nanoparticle carriers play significant roles in their cellular uptake, endosomal escape, and release in target cells that are the critical steps for the implementation of nucleic acid-based immunotherapy. Finally, nanoparticle technologies allow feasible combinations of immunotherapy with conventional chemotherapy, radiotherapy, as well as photothermal and photodynamic therapy for the normalization of immunosuppressive TME and improved immunotherapy efficacy.

#### **3. pH-Responsive Nanoparticles for Cancer Immunotherapy**

pH-responsive nanoparticles have received intensive attention in cancer immunotherapy because of the distinct acidic features of a tumor microenvironment compared with normal tissues. Deregulated glycolysis in cancer cells results in the high level of lactic acid and consequent acidic pH (6.5–6.9) in tumor tissues [21]. pH-sensitive nanoparticles afford cancer immunotherapy improved pharmacology and enhanced accumulation of immunotherapeutics in tumor tissues. In addition, the intracellular trafficking of drug nanoparticles usually undergoes early endosomes, late endosomes, and fusion with lysosomes with a decreased pH from 6.5 to 4.5 [22,23]. The formulation of biologic drugs with pH-sensitive carriers is able to response to the subtle pH change, facilitating the endosomal escape and avoiding the degradation of nucleic acids or proteins in lysosomes. It is worth noting that pH-responsive nanoparticles have been extensively used in other cancer therapies (e.g., chemotherapy) besides immunotherapy. For these topics, readers may refer to the published reviewer articles [24–26]. This review will focus on the recent advances in cancer immunotherapy using pH-responsive nanoparticles and offer perspectives on this burgeoning field.

Nanoparticles' response to pH change in two typical ways: the protonation/ionization of functional groups and the degradation or the cleavage of acid-labile bonds (Figure 2). pH-dependent protonation/ionization is frequently utilized in the design of pH-triggered delivery systems. In this strategy, various ionizable groups (e.g., amines and carboxyl acids) are incorporated into the delivery carriers, endowing drug-loaded nanoparticles with sensitivity to acid environments. pH variation alters the protonation of amines or the ionization of carboxyl groups that result in the change of the surface charge, the stability, and the interaction with cells or tissues. The pH-dependent protonation of carriers enables the "proton sponge" effect in the intracellular trafficking of nanoparticles which is crucial for the endosomal escape of biological immunotherapeutics (e.g., nucleic acids and proteins) [23]. The protonation of amines increases with the acid degree in endosomes, leading to the extensive influx of water and counterions into the late endosomes, which further results in the rapture of the endosomal membrane and the release of cargoes into the cytoplasm, therefore, avoiding the degradation of the biological drugs in lysosomes. In contrast to the physical changes in pH-responsive protonation/ionization, the increase of acidity may lead to the break of covalent bonds including amide, ester, imine, oxime, acetal, and ketal bonds or the disintegration of inorganic components, altering the physicochemical properties of drug-loaded nanoparticles. For example, acid cleavable PEG segments are incorporated into nanocarriers to shield nanoparticles from protein adsorption and aggregation under physiological conditions. When nanoparticles are circulated to the acidic tumors, the acid-labile linkers break and shielding PEGs are detached from nanoparticles, shifting the surface from a hydrophilic to a hydrophobic one or from a neutral to a positive one that improves the accumulation and retention of nanoparticles in tumor tissues [27,28]. In contrast to changes on the surface, the hydrolysis inside the nanoparticles triggers the degradation and disassembly of nanoparticles under acidic conditions, leading to the rapid release of immune cargoes and facilitating the antigen presentation in the target tissue [29,30]. Several pH-responsive inorganic components have been utilized to construct nanocarriers for cancer immunotherapy because they are highly sensitive to pH change from a physiological to a tumor environment [30,31].

**Figure 2.** The typical approaches to the pH-response of nanoparticles for cancer immunotherapy. (**a**) Protonation/ionization and (**b**) cleavage of acid-labile shells or degradation of nanoparticles at acid pH enables the significant change of surface properties or the disintegration of nanoparticles.

#### *3.1. Nanoparticles with pH-Responsive Protonation*/*Ionization for Cancer Immunotherapy*

pH-responsive polycations (e.g., poly(2-diethylamino ethyl methacrylate), PDEAEMA) take positive charges upon the protonation of amine groups and enable the proton sponge effect after internalization into cells (Figure 3). Therefore, they could be used to encapsulate negatively charged immunotherapeutic nucleic acids, improve their cellular uptake, and protect intrinsically unstable nucleic acids from degradation in acidic lysosomes in dendritic cells [32]. For example, poly(dimethylaminoethyl methacrylate)-*b*-(dimethylaminoethyl methacrylate-*co*-butyl methacrylate-*co*-propylacrylic acid) (P(DMAEMA)-*b*-(DMAEMA-*co*-BMA-*co*-PAA)) (Figure 3a) was utilized for the delivery of a RNA agonist of the retinoic acid gene (3pRNA) to dendritic cells. The pH-responsive polymer-nucleic acid nanoparticles reduce nuclease degradation and

improve cellular uptake and endosomal escape of 3pRNA, enhancing the immunostimulatory activity and the therapeutic efficacy of anti-PD-1 immune checkpoint blockade in a CT26 colon cancer model [33]. Similar pH-responsive polymers, poly(ethylene glycol)-*b*-poly(diisopropanol amino ethyl methacrylate-*co*-hydroxyethyl methacrylate) (PEG-*b*-P(DPA-*co*-HEA)) (Figure 3b) and 1,2-epoxytetradecane alkylated oligoethylenimine (OEI-C14) were utilized to deliver a photosensitizer (PS) and small interfering RNA against PD-L1 (siPD-L1) for combination of photodynamic therapy and RNA interference (RNAi)-based PD-L1 blockade [34]. At physiological pH, the carriers and payloads form stable nanoparticles wherein the fluorescence of photosensitizers is quenched due to the fluorescence resonance energy transfer, implying reduced dark toxicity in the blood circulation upon laser irradiation. After entering the weakly acidic endocytic vesicles in tumor cells (pH 5.0–6.0), the protonation of the tertiary amines of PDPA increases, resulting in the dissociation of nanoparticles and the release of photosensitizers into tumor cells that mediates photodynamic immunotherapy with laser irradiation. Furthermore, the photodynamic treatment induces tumor-specific reactive oxygen species (ROS) and promotes the release of antigens, stimulating the adaptive anti-tumor immunity. The combination of photodynamic immunotherapy and PD-L1 knockdown on the acid-activatable nanoplatform significantly inhibits tumor growth and metastasis in a B16-F10 melanoma xenograft tumor model.

**Figure 3.** Representative materials with pH-dependent protonation/ionization for cancer immunotherapy. (**a**) P(DMAEMA)-*b*-(DMAEMA-*co*-BMA-*co*-PAA): poly(dimethylaminoethyl methacrylate)-*b*-(dimethylaminoethyl methacrylate-*co*-butyl methacrylate-*co*-propylacrylic acid);

(**b**) PEG-*b*-P(DPA-*co*-HEA): poly(ethylene glycol)-*b*-poly(diisopropanol amino ethyl methacrylate*co*-hydroxyethyl methacrylate); (**c**) pH-responsive lipids; (**d**) PC7A: poly(ethylene glycol)-*b*-poly(2-(hexamethyleneimino)ethyl methacrylate); (**e**) MGlu-Dex: dextran derivatives having 3-methylglutarylated residues; (**f**) CECm: amphoteric methacrylamide *N*-carboxyethyl chitosan; HTCCm: methacrylamide *N*-(2-hydroxy)propyl-3-trmethylammonium chitosan chloride; (**g**) histamine and mPEG modified poly(β-amino ester)s; (**h**) PAH: PEG-histidine modified alginate; and (**i**) PEG-*b*-PAEMA-PAMAM/Pt: platinum (Pt)-prodrug conjugated and poly(ethylene glycol)-*b*-poly(2-azepane ethyl methacrylate)-modified polyamidoamine. The key pH-sensitive groups are indicated in red.

Messenger RNA (mRNA) has great potential in cancer immunotherapy [35]. A successful delivery of TAAs-encoded mRNA into DCs enhances the antigen presentation and the tumor specific immune response. In comparison with a short double-stranded RNA, a single-stranded mRNA is much longer, more flexible, and less stable. Further application of mRNA in cancer immunotherapy requires robust delivery carriers [36]. pH-responsive lipid nanoparticles (Figure 3c) are proven carriers for the cellular uptake and the endosomal escape of mRNA both in vitro and in vivo [17]. There is not a universal carrier for the delivery of any RNAs in different tumor models. In general, a proper content of pH-responsive amines and a delicate balance between hydrophilicity and hydrophobicity is needed to tackle the complicate challenge in RNA delivery. Of particular importance, amines with a p*K*a of 6.0–6.5 are crucial for the binding, cellular uptake, and the release of RNAs. A recent research shows that the alteration of the component of lipid nanoparticles changes the global apparent p*K*a and the protein corona of nanoparticles that enables the selective delivery of RNAs and proteins to target cells and tissues [37].

Ultra-pH-sensitive nanoparticles consisting of copolymers containing varying tertiary amines have been developed for the delivery of protein antigens to APCs in draining lymph nodes. Due to the robust response to the subtle pH change in organelles, the leading nanoparticle PC7A (Figure 3d) enables excellent cytosolic delivery and efficient surface presentation of tumor antigens, generating a strong cytotoxic T cell response with low systemic cytokine expression [38].

Carboxyl groups have been incorporated into pH-responsive nanoparticles for cancer immunotherapy because their pH-dependent ionization enables the change of hydrophilicity/hydrophobicity of carriers and the modulation of drug release from nanoparticles at varying pHs. For example, dextran was functionalized with carboxyl pendants and C12 alkyl side chains for the fabrication of pH-responsive liposomes for the delivery of a model antigen, ovalbumin (OVA) (Figure 3e). The modified liposomes are stable at neutral pH but destabilized at weakly acidic pH because the solubility of carboxy-bearing dextran decreases with pH, enhancing the release of OVA in the cytosol of dendritic cells. The pH-sensitive OVA-loaded liposomes demonstrate significant suppression of tumors upon subcutaneous injection to E.G7-OVA tumor-bearing mice [39].

In addition to improving the delivery efficacy at a cellular level, pH-responsive nanoparticles benefit cancer immunotherapy by enhancing the accumulation of immunotherapeutic drugs or targeting TAMs in tumor tissues through the charge change in acidic conditions. Paclitaxel (PTX) and interleukin-2 were encapsulated in nanogels composed of hydroxypropyl-β-cyclodextrin acrylate, red blood cell membrane, and two opposite charged chitosan to remodel the immunosuppressive tumor microenvironment (Figure 3f). With the pH decrease in the tumor environment, the ionization of –COOH decreases while the protonation of –NH2 increases, reversing the main driving force in nanogels from electrostatic attraction to repulsion, which further leads to the disintegration of the nanogel and the release of drugs in tumor tissues. The combinational chemotherapy and immunotherapy with the tumor microenvironment responsive nanogel significantly enhance the infiltration of immune effector cells and reduce the immunosuppressive factors in a murine melanoma model [40].

TAM is one of the key targets for the cancer immunotherapy besides tumor cells. Reversing TAMs from a tumor supportive phenotype to a tumoricidal phenotype is an effective way to remodel the immunosuppressive TME and enhance the antitumor immunity of immunotherapy. Histamine and mPEG modified poly(β-amino ester)s (Figure 3g) were prepared for the delivery of IL-12 to re-educate TAMs in TME. The drug-polymer nanoparticles swell under weak acidic conditions (e.g., pH 6.5), resulting in effective accumulation and prolonged release of IL-12 in TME that reverses the tumor-infiltrated macrophage phenotype from M2 to M1. This nanoparticle platform shows great potential in local re-education of TAMs in solid tumors with low systemic side effects in cancer immunotherapy [41]. In another report, pH-sensitive PEG-histidine modified alginate (PAH, p*K*a ~ 6.9) (Figure 3h) was developed for the delivery of a combination of CpG oligodeoxynucleotide (ODN), anti-IL-10 ODN and anti-IL-10 receptor ODN, to alter the phenotype of TAMs and stimulate their antitumor immunity. Galactosylated cationic dextran was selected for the fabrication of a ODNs nanocomplex (GDO) for TAM targeting because of high level of galactose-type lectin on TAMs. GDO forms nanoparticles with PAH via electrostatic attraction at physiological pH. After entering the acidic TME, the charge of PAH changes from negative to positive, resulting the detachment of PAH from the GDO complex and the exposure of galactose for TAM targeting. The acidic tumor microenvironment-responsive and TAM-specific approach significantly reduces the systemic side effects of cancer immunotherapy by inhibiting the upregulation of serum proinflammatory cytokines [42].

Cancer therapy can be improved by targeting the delivery of chemotherapeutics and immune modulators to both TAMs and tumor cells. BLZ-945, a small molecule inhibitor of colony stimulating factor 1 receptor (CSF-1R) of TAMs, was encapsulated in ultra-pH-sensitive cluster nanoparticles (SCNs) which was constructed from the self-assembly of platinum (Pt)-prodrug conjugated and poly(ethylene glycol)-*b*-poly(2-azepane ethyl methacrylate)-modified polyamidoamine (PEG-*b*-PAEMA-PAMAM/Pt) (Figure 3i). At neutral pH, PAEMA is hydrophobic and maintains the stable nanoparticles for prolonged blood circulation and reduced systemic toxicity of payloads. PAEMA is rapidly protonated at tumor pH and becomes hydrophilic, leading to instantaneous disintegration of SCNs into small dendrimer nanoparticles (<10 nm) for deep tumor penetration and the release of BLZ-945 for TAM depletion. Comparing with BLZ-945 or Pt-loaded nanoparticles, the spatial targeting nanoparticles demonstrate better tumor growth suppression, metastasis inhibition, and mouse survival in multiple tumor models [43].

Silica nanoparticles with a pH-responsive surface have been used as scaffolds for the controlled release of drugs or enhanced accumulation in tumor tissues [28,31]. Mesoporous silica nanoparticles with pH- and GSH-responsive molecular gates were developed for doxorubicin (DOX) delivery for the treatment of metastatic tumors. The highly integrated nanoplatform demonstrates a robust response to the simultaneously acidic and reductive tumor microenvironment, enabling a precise release of drugs in tumor tissues. The smart nanoparticles not only show good chemotherapy efficacy but also stimulate the maturation of DCs and the release of antitumor cytokines [31]. Hollow silica nanoparticles were coated with PEG and 2-propionic-3-methylmaleic anhydride (CDM)-grafted PEI, enabling prolonged blood circulation and negative-to-positive charge conversion at acidic pH. The catalase and photosensitizer-loaded hybrid nanoparticles show enhanced retention in tumor tissue, leading to greatly relieved tumor hypoxia via decomposition of tumor endogenous H2O2 and improving anti-PD-L1 checkpoint blockade therapy [28].

#### *3.2. Nanoparticles with pH-Responsive Bond Cleavage or Degradation for Cancer Immunotherapy*

In addition to the physical change induced by protonation/ionization, the break of covalent bonds triggered by pH change can lead to the physicochemical property change on the surface of nanoparticles or the disintegration of the nanoparticle that mediates the targeting or the controlled release of payloads to tumors.

OVA was grafted to alginate (ALG) via pH-sensitive Schiff base bonds and formed nanovaccines with mannose modified ALG by CaCl2 crosslinking (Figure 4a). The nanovaccines are relatively stable at pH 7.4 and release OVA remarkably in the release in the endo/lysosomes (pH 4.5–5.5) due to the detachment of OVA from delivery vehicles after cleavage of a Schiff base linkage at acidic pH. Subcutaneous administration of the nanovaccines enables efficient trafficking of the OVA-bearing nanoparticles from the injection site to the draining lymph nodes, remarkably stimulating the major cytotoxic T lymphocytes (CTL) response and inhibiting E.G7 tumor growth in C57BL/6 mice [44]. Similarly, pH-responsive hydrazone bond has been utilized for the construction of alltrans retinal-loaded nanogels which show long-term stability at physiological pH but dissociate and release antigens in acidic lysosomes in DCs (Figure 4b) [29]. In a recent study, amphiphilic charge-altering releasable transporters (CARTs) (Figure 4c) were developed for mRNA delivery to multiple lymphocytes in which cationic poly(α-amino ester)s bind mRNA at acidic pH but release mRNA after a time-dependent rearrangement of poly(α-amino ester)s to neutral small molecules (diketopiperizine) at pH 7.4, resulting in enhanced lymphocyte transfection in primary T cells and in vivo in mice. In contrast to conventional polycations, CARTs provide a new mechanism for mRNA release and great potential to avoid polycation-associated tolerability issues [45].

**Figure 4.** Representative materials with acid-labile bond cleavage or degradation for cancer immunotherapy. (**a**) ALG = OVA: ovalbumin-conjugated alginate; (**b**) GDR: galactosyl dextran-retinal conjugates; (**c**) CART D13:A11: charge-altering releasable transporter with 13 carbonate repeating units and 11 amino ester repeating units; (**d**) PEG2000-hydrazone-C18: conjugates of polyethylene glycol (molecular weight of 2000) and stearic hydrazide; (**e**) PEG-CDM-PDEA: conjugates of PEG and poly(2-(diethylamino) ethyl methacrylate with 2-propionic-3-methylmaleic anhydride linkers; (**f**) DiPt-*ASlink*-PEG2k: conjugates of PEG2000 and hexadecyl-oxaliplatin(IV) with 2-propionic-3-methylmaleic anhydride linkers; (**g**) PEG = TMC-Man: PEG and mannose doubly modified trimethyl chitosan; PAH-Cit (PC): citraconic anhydride-grafted poly (allylamine hydrochloride); (**h**) HA-ADH-DOX: conjugates of hyaluronic acid and doxorubicin with hydrazine linkers; (**i**) PLGA: poly(lactic-*co*-glycolic acid); (**j**) PLGA-NaHCO3/NH4HCO3 NP: NaHCO3 or NH4HCO3-encapsulated PLGA nanoparticle; and (**k**) MOF: Metal-organic framework. The acid-labile groups are indicated in red.

Acid liable-PEG has been frequently used in pH-responsive nanoparticles to stabilize nanoparticles in the blood circulation while enhancing the nanoparticle accumulation in acidic tumor tissues. For example, nanoparticles composed of acid liable-PEG-hydrazone-C18 (PHC) (Figure 4d), poly(lactic-*co*-glycolic acid) (PLGA), and *O*-stearoyl mannose (M-C18) enable a decreased accumulation in the mononuclear phagocyte system (MPS) owing to the PEG shielding at normal pH, and thus reduce the off-target immune activation, while they can be effectively accumulated in TAM via mannose-receptor recognition after the hydrolysis of hydrazone bonds and the detachment of PEG in acidic TME [46].

The detachment of the PEG shell also enables the exposure of positively charged groups or negative-to-positive charge conversion on the nanoparticle surface [27,47]. The PEG block was conjugated with poly(2-(diethylamino) ethyl methacrylate (PDEA) using 2-propionic-3-methylmaleic anhydride (CDM) and formed mix micelles with PEI-PDEA for the delivery of siRNA against PD-L1 and a mitochondrion-targeting photosensitizer (Figure 4e). The detachment of the PEG corona at acidic pH endows nanoparticles with significant size reduction and surface charge increase in TME, facilitating the penetration of nanoparticles to tumors and improving the antitumor immune response in vivo. Using the same pH-sensitive linker, the PEG block was attached to a lipid with two tails (Figure 4f). The nanoparticle is negatively charged at neutral pH due to the partial ionization of carboxyl groups in the side chain. After entering acidic tumor tissue, the acid-labile linker was cleaved, resulting in the detachment of the PEG shell with negative carboxyl groups and the charge conversion from negative to positive. The smart nanoparticle shows enhanced tumor accumulation and deep penetration, consequently enabling efficient delivery of a combination of immunoregulators to suppress tumor growth and metastasis in mice [47].

Moreover, the cleavage of PEG induces simultaneous exposure of targeting ligands and positively charged groups that benefit cancer immunotherapy with enhanced accumulation and specific targeting to M2-TAMs or cancer cells in tumor tissues. For example, nanoparticles composed of PEG and mannose doubly modified trimethyl chitosan and citraconic anhydride-grafted poly (allylamine hydrochloride) (PC) have been developed for the delivery of siRNAs against the vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) to breast cancer cells and M2-TAMs (Figure 4g). The PEG shell is able to mask mannose to reduce the uptake by resident macrophages in the reticuloendothelial system and improve the blood circulation time. When nanoparticles enter acidic tumor tissues, the benzamide bond between PEG and the mannose-modified trimethyl chitosan is cleaved, which results in the detachment of PEG and the exposure of mannose and cationic amines, enabling effective accumulation in tumor tissues and uptake by cancer cells and macrophages. In the more acidic late endosomes or lysosomes (pH 4.5–5.5), the side chains of PC are hydrolyzed, leading to the charge reversal from negative to positive and promoting the endosomal/lysosomal escape of siRNAs. The dual pH-responsive nanoparticle could be a robust platform to reverse TME from pro-oncogenic to anti-tumoral and suppress the tumor growth and metastasis [48].

A combination of an acid-cleavable bond and pH-dependent ionization has been utilized to construct a sequential pH-responsive delivery system for the co-delivery of a TLR7/8 agonist R848 and chemotherapeutic doxorubicin (DOX) (Figure 4h). Hyaluronic acid-DOX (HA-DOX) conjugates were prepared by coupling using acid-cleavable hydrazone bonds. R848 was bound with poly(L-histidine) (PHIS) and the PHIS/R848 nanocomplex were further coated with HA-DOX to form HA-DOX/PHIS/R848 nanoparticles. When pH decreases from neutral to acidic, the mixed nanoparticles undergo two distinguished changes. The ionization of PHIS at pH 6.5 leads to the hydrophobic-to-hydrophilic transition and the release of the encapsulated R848 in to TME. The cleavage of the hydrazone bond around pH 5.5 triggers the release of the covalently bound DOX to the cytosol of cancer cells, enabling the synergistic effects of immunotherapy and chemotherapy against breast cancer in 4T1 tumor-bearing mice [49].

In contrast to the cleavage of pH-responsive linkers, the break of acid-liable bonds in the polymer backbone induces the degradation of polymers and the disintegration of nanoparticles that facilitates the release of payloads into an acidic compartment of APCs or tumor tissues. For example, biodegradable PLGA-based nanoparticles have been used for the delivery of protein antigens (Figure 4i) (e.g., gp100, OVA) and JSI-124 (a small molecule inhibitor of activator of transcription-3, STAT3) to protect the immunoregulators and achieve the sustained release of drugs to DCs [50,51]. Biodegradable nanoparticles could be prepared by using acid degradable primary amine monomer and cross-linker in which anti-DEC-205 mABs were encapsulated for cytotoxic T lymphocyte activation [52].

The incorporation of pH-responsive inorganic components into nanoparticles is a robust approach to promote the release of antigens in APCs because they can generate CO2 and/or NH3 in acidic conditions which leads to the rupture of antigen-loaded nanoparticles (Figure 4j). In comparison with the degradation of polymers, the disintegration is more readily available for nano-sized inorganic components in mild acidic conditions. For example, ammonium bicarbonate (NH4HCO3) was encapsulated in PLGA nanoparticles and could react

with hydrogen ions in endosomes and lysosomes that produce NH3 and CO2, mediating the dissociation of nanoparticles and the rapid release of encapsulated OVA to cytoplasm [30]. Similarly, PLGA-sodium bicarbonate (NaHCO3) hybrid nanoparticles have been utilized to deliver an agonist 522 to stimulate the maturation of DCs and the secretion of pro-inflammatory cytokines. The incorporation of bicarbonate salts into PLGA nanoparticles enables 33-fold higher loading of the hydrophobic agonist and the rapid rapture of nanoparticles at acidic pH, resulting in an increased expression of co-stimulatory molecules and improved antigen presentation [53].

Metal-organic frameworks (MOFs) have acid-labile metal–ligand bonds and are therefore able to respond to the acidic environment endo/lysosome (Figure 4k). OVA was incorporated into the frameworks during the synthesis of MOF using Eu and guanine monophosphate (GMP). The OVA-loaded MOFs were further coated with CpG as an endosomal-acting oligonucleotide adjuvant. The MOF-based nanocarriers show high loading of antigens. Moreover, the coordination of Eu and GMP is interrupted under pH 5.0 which results in the rapid degradation of MOF and facilitates the endosomal escape and cytosol release of antigens [54].

#### **4. Conclusions and Perspectives**

pH-responsive nanoparticles show tremendous potential in cancer immunotherapy because they are capable of targeting the acidic microenvironment of tumor tissues and organelles in cells, therefore, reducing the off-targeting toxic side effects and improving therapeutic efficacy. Current pH-responsive nanoparticles for cancer immunotherapy are constructed via distinct mechanisms involving the protonation/ionization of pH-sensitive groups or the break of acid-labile covalent bonds. The key components of pH-responsive nanoparticles and their delivery payloads as well as the related disease models in this review are summarized in Table 1. The protonation/ionization varies with pH, leading to the change of surface charges or the hydrophilicity/hydrophobicity balance of nanoparticles, consequently promoting the accumulation of nanoparticles and release of payloads in target sides. The acid-cleavable bonds have been incorporated into the backbone of polymers or the linker of PEG and the backbone in pH-responsive nanoparticles. The pH-sensitive linkers are stable at physiological pH, affording nanoparticles' great serum stability upon protection by PEG corona while their cleavage at acidic pH leads to the detachment of the PEG corona and the subsequent exposure of positively charged or targeting groups on nanoparticles, resulting in the enhanced accumulation of nanoparticles in tumors or target cells. The degradation of polymer backbone at acidic pH results in the dissociation of nanoparticles, promoting the release of immunotherapeutic drugs into target sides. Incorporating pH-responsive inorganic components into nanoparticles is a robust approach to the construction of pH-sensitive nanoparticles for cancer immunotherapy because they are able to rapidly respond to the acidic environment and trigger the instant disintegration of nanoparticles at low pH.


**Table 1.** Selected pH-responsive nanoparticle systems for cancer immunotherapy.


**Table 1.** *Cont.*

The convergence of pH-responsive nanotechnology and immunotherapy provides a promising strategy for improving the unsatisfactory efficacy and reducing the off-target side effects in cancer treatments. Despite the substantial advances in animal models, challenges remain in the clinical translation of cancer immunotherapy with pH-responsive nanoparticles. The therapeutic efficacy should be further evaluated in clinical relevant tumor models. Moreover, clinically translatable materials should be developed for the construction of pH-responsive nanoparticles to meet the strict requirements in clinical use. With the rapid progresses in material chemistry, immunology, and nanotechnology, pH-responsive nanoparticles are expected to exert a significant role in cancer immunotherapy in the near future.

**Author Contributions:** Y.Y. and H.D. drafted and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** We gratefully acknowledge the financial support from the National Natural Science Foundation of China (U1932164) and the Zhejiang Provincial Natural Science Foundation (LY19B040004).

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

#### **References**


© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Nanoparticle Systems for Cancer Phototherapy: An Overview**

**Thais P. Pivetta 1,2,†, Caroline E. A. Botteon 3,†, Paulo A. Ribeiro 2, Priscyla D. Marcato <sup>3</sup> and Maria Raposo 2,\***


**Abstract:** Photodynamic therapy (PDT) and photothermal therapy (PTT) are photo-mediated treatments with different mechanisms of action that can be addressed for cancer treatment. Both phototherapies are highly successful and barely or non-invasive types of treatment that have gained attention in the past few years. The death of cancer cells because of the application of these therapies is caused by the formation of reactive oxygen species, that leads to oxidative stress for the case of photodynamic therapy and the generation of heat for the case of photothermal therapies. The advancement of nanotechnology allowed significant benefit to these therapies using nanoparticles, allowing both tuning of the process and an increase of effectiveness. The encapsulation of drugs, development of the most different organic and inorganic nanoparticles as well as the possibility of surfaces' functionalization are some strategies used to combine phototherapy and nanotechnology, with the aim of an effective treatment with minimal side effects. This article presents an overview on the use of nanostructures in association with phototherapy, in the view of cancer treatment.

**Keywords:** nanoparticles; phototherapy; cancer; photodynamic therapy; photothermal therapy

#### **1. Introduction**

Cancer is a leading cause of death worldwide, with an estimated 19.3 million new cases and nearly 10 million deaths caused by cancer in 2020 [1]. During the 20th century, there was an undeniable technological development aiming to enhance the treatment of cancer, mainly regarding to the discovery of chemotherapy. Nowadays, chemotherapy is one of the pillars for cancer treatment, along with surgery and radiotherapy [2,3]. However, it is known that chemotherapy and radiotherapy have severe side effects to the patient, mainly due to the non-specificity of the treatment [4]. Within this context, phototherapy has gained attention as an alternative treatment with reduced side effects [4].

Photodynamic therapy (PDT) and photothermal therapy (PTT) are photo-mediated therapies with different damage mechanisms that consist in the generation of reactive oxygen species (ROS) and heat, respectively [4,5]. These effects result in the cells' death, thereby, with a potential application for treatment of several types of cancer [6]. PDT requires the application of photosensitizer drugs (PS) that will be triggered by radiation.

These drugs generally present poor solubility in physiological conditions, which can impair therapy's success [7]. For this purpose, it is necessary to find appropriate nanoparticulate systems that can deliver these drugs to the cancer cells. Currently, there is not a unique definition that is accepted internationally, however nanomaterials are often described in the scale of 1–1000 nm [8]. Nanotechnology emerged in order to enhance problems related to drugs' solubility and provide a targeted treatment, enabling to reduce drugs' dosage and

**Citation:** Pivetta, T.P.; Botteon, C.E.A.; Ribeiro, P.A.; Marcato, P.D.; Raposo, M. Nanoparticle Systems for Cancer Phototherapy: An Overview. *Nanomaterials* **2021**, *11*, 3132. https:// doi.org/10.3390/nano11113132

Academic Editor: James Chow

Received: 26 October 2021 Accepted: 16 November 2021 Published: 20 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/).

also minimize several side effects in patients [9]. Additionally, through nanotechnology research, there are several types of nanoparticles, particularly metallic nanoparticles such as gold nanoparticles, that can generate heat upon exposition to light, which can be useful for PTT [10] as it induces hyperthermia in the tumor environment, consequently leading to cancer cells' death [11]. PTT is a non-invasive and selective technique which can potentially suppress many kinds of tumors [12]. Cancer treatment with the PTT approach offers many advantages, such as sensitization of hypoxic regions, reinforcement of the immune system, releasing of thermo-sensitive substances and increasing susceptibility of cancer cells to chemotherapeutic agents [13]. The combination of PDT and PTT is also possible through the use of a sensitizing agent able to produce ROS and hyperthermia [5].

NPs for phototherapy have been extensively investigated and reported in the literature [14,15], and in this work, new issues concerning NP systems' design, in view of cancer treatment under photodynamic and photothermal therapies, will be addressed. The referred new issues are intended to exemplify recent approaches related to nanoparticle conditions, such as the targeting of drugs in the tumor site and problems and/or new achievements related to the phototherapy. The overall situation and trend of research in both therapies using nanoparticles is clearly demonstrated in Figure 1, which shows, in the last ten years, both number of publications and number of citations listed in the Web of Science platform using "Photodynamic Therapy AND Nanoparticles" and "Photothermal Therapy AND Nanoparticles" as search topics, where both number of publications and of citations are increasing strongly in recent years.

**Figure 1.** *Cont*.

**Figure 1.** Updated number of publications and number of citations in the last ten years listed in the Web of Science platform using as search topics: (**a**) "Photodynamic Therapy AND Nanoparticles" and (**b**) "Photothermal Therapy AND Nanoparticles" (November 2021).

#### **2. Photodynamic Therapy**

#### *2.1. A Brief Introduction*

PDT has been used for centuries, mainly to treat skin disorders, with most treatments involving the intake of extracts of some types of plants followed by exposition to the sun [16]. The main discovery took place in Germany in 1900, where Oscar Raab and Hermann von Tappeiner were investigating the behavior of protozoan *Paramecium* spp. in the presence of the dye acridine orange. They verified that the protozoan died after the exposure to the sunlight coming from an adjacent window. This discovery was important later for the successful achievements on the human skin carcinoma treatment, and by 1904, it was found that the presence of oxygen was important for the treatment, originating the name photodynamic [17]. Currently, PDT is a highly successful and barely or non-invasive type of treatment for several skin disorders, such as psoriasis and cancers [18]. There are three important elements to perform PDT, which are a photosensitizer drug, the light source, and the presence of oxygen. The interaction of these elements results in reactive oxygen species (ROS), which play a key role in the treatment [19]. Upon a specific light wavelength, the photosensitizers (PS) can absorb a photon, which will lead to a conversion from the single basic state to the single excited state, as shown in Figure 2. From there, it can make an intersystem, crossing to a metastable triplet state, which in turn can take two possible paths known as PDT type I or type II. In type I, the activated photosensitizer can trigger a series of reactions with biomolecules generating radicals that interact with oxygen molecules, creating ROS. On the other hand, in PDT type II, the PS by itself can transfer energy directly to oxygen, resulting in ROS molecules [20,21]. Due to their high oxidizing power, ROS molecules have cytotoxic effects, however, due to the short lifetime, the effect of ROS on cell damage will occur around the created species [22].

However, PDT has a limited application that can depend on several factors to achieve a successful treatment. A special mention should be given to the light source. This is an important variable to take into consideration because different light wavelengths have different penetration depths in tissues. For example, ultraviolet (UV) light is known to cause several damages in biomolecules, such as the DNA presents low penetration compared to longer light wavelengths [23–27]. Near-infrared (NIR) light, on the other hand, is capable of higher penetration depths, with the capability of generation of local heat even with low energy input. NIR is also safer than UV, which can cause sunburns, inflammation, and even skin cancer [23,28].

**Figure 2.** Jablonski diagram representation and the photodynamic therapy mechanism of action.

Another factor that can impair the efficacy of PDT is the hypoxic tumor microenvironment. To overcome this challenge, some strategies involved the elaboration of nanoparticles with molecules such as catalase, that can react and generate oxygen, or hemoglobin and perfluorocarbon, that serve as an oxygen carrier. Therefore, the inclusion of these molecules in nanoparticles is able to improve the PDT efficacy [29].

The photosensitizer drugs themselves are another variable that can interfere with the PDT. For example, some PS can present poor solubility under physiologic conditions and impair the correct distribution of the drug to the target tissue, which of course will interfere with the therapy's success. To circumvent such a drawback, the use of nanoparticulate systems is addressed, enhancing the drug's solubility and the cellular uptake, and consequently the PDT efficacy [7,30]. Many types of nanoparticles for PDT have been attempted for different types of cancers, and some aspects of nanoparticle systems for PDT will be discussed in this section.

#### *2.2. Nanoparticles with Application for PDT*

As mentioned before, in phototherapy, the delivery of PS molecules to the target tissue is a relevant issue, as it is in all cases of drug delivery systems. The NP systems to be used should be suitable to release the active components over a defined period of time with control over the nanoparticle size. The raw materials employed, and their biodegradability, is also important to consider for nanoparticles' preparation. The most common NPs used in PDT are not only organic-based but also inorganic, such as silica and magnetic NPs. In the next sections, some of the best achievements with the use of nanocarriers will be presented.

#### 2.2.1. Organic Nanoparticles

Organic nanoparticles are the most used systems to encapsulate molecules which can be used in PDT. There are several categories based on different materials and respective organization. Figure 3 is a representation that summarizes the most common categories of organic nanoparticles, and in Table 1, there is a brief description with examples of nanoparticulate systems that are cited in this review.

**Figure 3.** Different organic nanoparticles that can be used for photodynamic therapy: (**A**) solid lipid nanoparticles, (**B**) liposomes, (**C**) micelles, (**D**) nano-emulsions, (**E**) polymeric nanoparticles, (**F**) cyclodextrins and (**G**) protein nanoparticles.

#### Solid Lipid Nanoparticles

Solid lipid nanoparticles (SLNs) were developed in the 1990s, and ever since, these particles have become the perfect model of safe nanoparticles with an occlusive effect that can also increase the drug permeation in the skin [31]. Generally, these NPs are composed of a surfactant layer, with a lipidic nucleus (Figure 3A, and can be prepared by the Müller and Lucks method based on high-pressure homogenization (1996) or by the microemulsion technique developed by Gasco (1993) [32,33].

Either way, SLNs' production requires lipids that are solid at body temperature, such as some triglycerides or glycerides mixtures. Due to their composition, SLNs are well-tolerated, are biodegradable and can easily be produced on a large scale and at low cost [31,34,35]. However, SLNs present some disadvantages, such as the limited encapsulation efficiency and the possibility of drug release during the storage time. In order to overcome these problems, a second generation of SLNs was developed [34], the so-called nanostructured lipid carriers (NLCs). They consist of SLNs with a less ordered solid matrix based on a mixture of lipids. There are three types of NLCs: imperfect, amorphous and multiple [34]. The imperfect NLCs are composed by a blend of solid lipids with different chain lengths as well as the lipid saturation degree, characteristics that lead to the creation of an imperfect solid matrix. The amorphous type is produced from special solid and liquid lipids, creating a solid particle that does not crystalize. The combination of solid lipids with higher amounts of liquid lipids results in the multiple type, in which there is the creation of oil nano-compartments inside the solid matrix [34,36].

Nanostructured lipid carriers with a photosensitizer precursor (5-aminolevulinic acid) were developed by Qidwai and collaborators [37], aiming for use in basal-cell carcinoma treatment. In their study, the nanoparticles exhibited a sustained release profile, higher retention of the drug in the skin layers and enhanced toxicity. Similarly, solid lipid nanoparticles were used to encapsulate curcumin, a natural product with potential in phototherapy application. Curcumin nanoparticles were revealed to enhance drug uptake into the lung cancerous cells and were able to produce ROS under light exposition, thus presenting potential for phototherapy [38].

Most of the studies employing SLNs and NLCs are intended for skin delivery. For example, Goto et al. [39] developed solid lipid nanoparticles containing aluminum chloride phthalocyanine for melanoma treatment. The developed system showed great stability and the measurements of forced stability indicated that the system would be stable for 12 months. In vitro studies showed no toxicity under dark conditions but, when submitted to a light source, the toxicity was seen dependent on the radiation dose. Almeida et al. [40]

also encapsulated phthalocyanine in lipid nanoparticle formulations and demonstrated an enhancement of the drug penetration in the skin, when compared to the control group. Interestingly, NLC formulation with higher amounts of the liquid lipid oleic acid showed greater retainment (89.5%) in the deeper skin layers when compared to the NLC with less oleic acid and the solid lipid nanoparticle. In vitro studies carried out on melanoma cells revealed that that the free drug did not lead to cell toxicity under light conditions, probably due to poor accumulation in the cells but, on the other hand, drugs encapsulated in NLC showed a significant reduction in cell viability starting from 0.1 μg/mL. Therefore, the composition of solid lipid nanoparticles is a relevant parameter that can directly result in a higher effect in therapeutics.

#### Liposomes

Liposomes are formed by auto-organization of phospholipids in bilayers that, in an aqueous medium, tend to fold on themselves, creating vesicles (Figure 3B) [41]. Due to the lipid's amphiphilic nature, hydrophilic and hydrophobic drugs can be stored in different compartments of liposomes [42]. These vesicles are usually employed as a model in the study of cell membranes, considering the similarity between them, however, liposomes can also be applied to drug delivery [43,44]. The lipid composition provides great biocompatibility, biodegradability and additionally, does not present toxicity [42,45]. The functionalization of these particles with polyethylene glycol (PEG) can lead to the creation of stealth liposomes, that are able to evade from the immune system and increase their blood circulation [46,47]. Other types of ligands can be used in the functionalization, such as antibodies, which in turn manage a robust targeted drug delivery [48]. Due to the system's versatility, liposomes are great candidates for photodynamic therapy application.

Foscan® is a commercial photosensitizer formulation already approved in Europe for neck and head cancers' application. The active drug of Foscan®, known as temoporfin, also originated Foslip® and Fospeg®, which are liposomes formulations [49]. The temoporfin encapsulation in the lipid carriers presents a similar phototoxicity as Foscan® with significantly lower toxicity. Fospeg®, a derivative from Foslip® and distinguished by a PEGylation, is able to provide enhanced pharmacokinetics with longer circulation in the blood [50,51]. Studies in HeLa spheroids showed that the drug delivery via liposomes is a way to decrease the drug's toxicity in the absence of light, increase the cellular internalization and, consequently, PDT effectiveness [49]. Foslip® and Fospeg® are just examples of formulations developed that are currently approved, however many other liposomal systems containing photosensitizers can be explored targeted to different tumor types.

To overcome issues related with low encapsulation efficiency, drug expulsion and quenching caused by molecules' aggregation, Cai et al. [52] incorporated fluorogens with singular aggregation-induced emission characteristics (AIEgens) in the lipid, creating a conjugate. Liposomes produced from these conjugates (AIEsomes) were able to show a superior ROS production compared to conventional liposomal systems containing photosensitizer molecules. In vitro studies carried out under dark conditions proved that both AIEsomes and conventional liposomes were toxic for a breast cancer cell line, however when irradiated with white light, AIEsomes exhibited more toxicity compared to conventional liposomes. Afterwards, in vivo studies revealed AIEsomes' ability to target and image in the tumor site, factors intrinsically related to their accumulation mainly in tumors. Furthermore, irradiation of animals after injection of AIEsomes was able to suppress tumor growth and induce necrosis in the tissue, which did not happen to other experimental groups, revealing the potential of liposomes prepared with AIEgen-lipid conjugates for targeted PDT.

A similar technique was employed by Kim et al. [53] with liposomes prepared from lipid conjugated with pheophorbide A, which were used as photosensitizers aiming for photo-induced immunotherapy in cholangiocarcinoma. Regardless of whether the technique used to exploit photosensitizer incorporation in liposomes consists in a PS-lipid conjugation or encapsulation, these systems have been studied for PDT in several types

of cancer, such as gastric, breast, ovarian, liver, skin and others [45,54–57]. Liposomes' features provide an extensive range of new possibilities to create therapeutic carriers that can improve PDT.

#### Micelles

Similar to the previous description of liposomes formation, micelles (Figure 3C) are also formed by the self-organization of amphiphilic molecules, and the resultant particle is different from the vesicles because of the different packing parameters [58,59]. The concentration of amphiphilic molecules must reach values above the designated critical micellar concentration (CMC) to form stable micelles, with a confined hydrophobic interior isolated from the aqueous medium. Polymers can also be materials used for micelles' preparation if the polymers present hydrophobic and hydrophilic segments. Therefore, the choice of the amphiphilic molecule that will be used is important due to different CMC values [60].

Aiming at a dual action of chemo- and photo-therapy in melanoma, Zhang et al. [61] investigated the preparation of micelles from block copolymer for the co-delivery of the classical anticancer agents Doxorubicin and pheophorbide A. These compounds were incorporated in the polymer chain, and the prepared micelles were successfully internalized into melanoma cells with ROS formation induced by light observed in vitro and in vivo. Micelles showed high inhibition of tumor growth, almost twice that of micelles without irradiation treatment, and significantly higher than treatment with only Doxorubicin.

To obtain a target system for ovarian cancer and metastatic melanoma cells, Lamch et al. [62] developed micelles with a di-block copolymer mPEG45-PLLA70 conjugated with folic acid for the encapsulation of the photosensitizer zinc (II) phthalocyanine. Wang et al. [63], in turn, used hyaluronic acid functionalization in micelles containing protoporphyrin IX to target cells with overexpression of CD44 receptors. The in vitro application of these micelles in monolayers and spheroids of human lung adenocarcinoma cells suggested that the enhanced cytotoxicity was due to higher internalization, and the effect of the interaction between the ligand hyaluronic acid and the receptor. Therefore, these studies suggest that micelles' functionalization can be an approach to enhance photodynamic therapy using this kind of nanostructure.

#### Nano-Emulsions

A nano-emulsion is a mixture of oil and surfactant in aqueous phase, which demands energy to form small droplets of 20–200 nm (Figure 3D) [64]. Nano-emulsions can be employed as a strategy to enhance the bioavailability of several lipophilic drugs. For example, studies by Machado et al. [65] on formulations of nano-emulsions containing curcumin, a natural product, as a photosensitizer drug revealed that curcumin-nano-emulsion was highly phototoxic to breast cancer cells and produced high levels of ROS. Mongue-Fuentes et al. [66] also used natural raw materials for the development of nano-emulsions for PDT. In their work, acai oil was used for the nano-emulsion preparation, which, combined with light irradiation, resulted in 85% death of melanoma cells, results which were also confirmed by animal studies in mice, with a decrease of tumor volume.

#### Polymeric NPs

On the nanotechnology timeline, polymer-based nanoparticles were firstly reported in 1976 [67]. Since then, the great interest in these NPs resulted in the development of several methods to produce polymeric nanoparticles or PNPs (representation of 1-nanospheres and 2-nanocapsules in Figure 3E), such as nanoprecipitation and solvent evaporation. The solvent evaporation method is an example of a two-step procedure where an emulsion is created, homogenized or sonicated, and then an evaporation step is required to remove the organic solvent in which the polymer was dissolved. On the other hand, nanoprecipitation is a one-step procedure where the polymer and drug are dissolved in a solvent miscible in water and dripped in an aqueous solution containing stabilizer. In both methods, organic solvents are employed, and although toxic solvents such as chloroform are no longer used, ether and acetone are currently used for the preparation of nanoparticles. In these cases, evaporation and purification methods are required to remove solvent residues from the dispersion [68–70].

Eltahan and collaborators developed polymeric nanoparticles co-loaded with NVP-BEZ235 and Chlorin-e6 (Ce6), named NVP/Ce6@NPs [71]. Ce6 was the selected photosensitizer and NVP-BEZ235 was used due to its ability to inhibit the PI3K/AKT/mTOR pathway that is related to tumor progression and proliferation and inhibit the repair of DNA damage in tumor cells. This sophisticated system plus irradiation was able to generate ROS by the Singlet Oxygen Sensor Green method, followed by tests in the triple-negative breast cancer cell line, and by flow cytometry, the authors discovered that treatment with NVP/Ce6@NPs and irradiation presented a fluorescence approximately 5 times greater compared to the control and nanoparticles without Ce6. These achievements showed the effect of a biochemical and PDT combination to treat a severe type of cancer.

Polymeric nanoparticles can be used to enhance the solubility of drugs as well as to provide drug's stability and sustained release [72]. PNPs were used to encapsulate the photosensitizer zinc phthalocyanine, and as result, the phototoxicity showed a 500 times increase compared to the free drug in a lung cancer cell line [73]. Polymers' functionalization is another strategy able to achieve multifunctional nanoparticles [74]. The addition of some type of ligand such as an antibody to the nanoparticle surface allows it to bind specifically to sites where there is an overexpression of the receptor (Figure 4). Transferrin receptors, for example, are overexpressed in breast cancer. Regarding this, Jadia and collaborators [75] functionalized polymers with a peptide (hTf) that is able to bind to transferrin receptor and prepared nanoparticles containing the drug benzoporphyrin monoacid. As expected, functionalized nanoparticles exhibited specificity to the cell line in this study and enhanced the phototoxicity compared to non-functionalized nanoparticles. This successful strategy led to the synthesis of polymers containing different ligands, resulting in nanoparticles with different biological activities such as bioimaging and photodynamic therapy [74].

**Figure 4.** Representation of examples of functionalization to NPs with PEG for stealth NP, with fluorophores for imaging. Functionalization with ligands (e.g., antibody, peptide, carbohydrate and others) can show an advantage in abnormal cells with receptor's overexpression to enhance uptake by the cells mediated by a receptor endocytosis.

Polyethylene glycol (PEG) has gained attention due to its stealth behavior [72]. PEG has shown promising application due to several properties, namely, inertness in biological systems combined with the non-activation of immune components and low adsorption of biomolecules, such as proteins providing a prolonged circulation in the blood [30,76]. The importance of PEG in PDT was investigated by Yang and collaborators [77] using Ce6 as a photosensitizer, a PDT light source based on a 660 nm laser and synthetized polymers with different densities. It was demonstrated that the drug was detected in the circulation for a prolonged time and a higher amount of Ce6 was detected with high-density PEG nanoparticles. On the other hand, the PDT effectiveness was dependent on the cellular internalization, which is maximized when low-density PEG nanoparticles are applied [77]. Therefore, these achievements debate the need of a parameter's balance in the design of the nanoparticles to achieve an effective therapy.

Studies developed by Luo et al. [78] focused on the development of polymeric nanoparticles with co-encapsulation of Doxorubicin and a photosensitizer. To avoid the known toxicity of Doxorubicin, the strategy used was to link DOX to the polymer, a link that can be cleaved by ROS, and thereby the activation of the nanoparticle is ROS-dependent. They encapsulated the catalase enzyme to act on the intracellular H2O2 to produce more O2 and functionalized particles with a peptide IF7 to target the tumor. This versatile and complex system (IF7-ROSPCNP) was shown to be an effective nanoparticle with accurate tumor targeting, that was able to inhibit tumor growth and prolong survival time when submitted to laser irradiation (Figure 5A–D). Mice treated with ROSPCNP and IF7-ROSPCNP, but not irradiated, were also submitted to histopathological studies, which showed that other tissues were no different from the control group, which suggests that the nanoparticles were safe (Figure 5F).

**Figure 5.** Evaluation of animal studies treated with several samples, such as free DOX, ROSPCNP and IF7-ROSPCNP, in the presence or absence of laser irradiation. (**A**,**B**) Evolution of tumor volume, (**C**) relative inhibition rate of tumor (IRT), (**D**) survival of the animals along the days of the experiment, (**E**) evolution of body weight and (**F**) histopathological analysis of heart, liver, spleen, lung and kidney of animals treated with different approaches (reproduced from Reference [78] with permission from Elsevier. Copyright 2019. Nanomedicine: Nanotechnology, Biology and Medicine).

Deng and collaborators [79] developed systems with tetrakis(4-carboxyphenyl)porphyrin as a photosensitizer, where the drug Doxorubicin was encapsulated forming π-π interactions with PNP to enhance the drug loading. These researchers obtained high drug loading (17.9%) and encapsulation efficiency (89.3%) associated with π-π interactions, as proven by the fluorescence method. Furthermore, in vivo studies showed that the PNPs developed were able to inhibit the growth of breast tumor in Balb/c mice when exposed to laser irradiation. The studies discussed in this topic were a few examples among many reports of photodynamic therapy exploiting PNPs in several types of cancer, such as in cervical adenocarcinoma, glioblastoma, highly aggressive breast cancer and hepatocellular carcinoma, showing the versatility of combining PNPs and PDT for cancer treatment [72,79–81].

#### Cyclodextrins

Cyclodextrins (CD) are biodegradable and biocompatible structures composed by oligosaccharides of D(+)-glucose that are able to form nanosized particles by self-organization in aqueous medium [82,83]. As shown in Figure 3F, CD present a conic shape where the hydrophobic cavity provides a way for the solubilization and delivery of hydrophobic drugs [84,85]. The conjugation of the photosensitizer (phthalocyanine) and cyclodextrin was a strategy employed to increase the PS solubility. Assays performed in human bladder cancer cells demonstrated that those conjugates, with higher solubility in water, were more phototoxic to the cells [86]. A similar strategy was adopted by Semeraro and collaborators with a cyclodextrin-chlorophyll *α* conjugate, with a potential photo-induced toxicity in human colorectal adenocarcinoma cells reiterating the versatility of CD-PS complexation for PDT applications [87].

#### Protein Nanoparticles

Proteins are polymeric-type macromolecules formed by repeated amino acid monomers. Due to their biodegradability and low toxicity, proteins gained attention as drug delivery systems, as represented in Figure 3G [88,89]. Recently, Ye and Chi [90] published a review about the recent progress in drug and protein encapsulation. This includes a revision on the different encapsulation techniques, namely, emulsion evaporation, self-emulsifying drug delivery system as well as supercritical fluid, and proposed a novel method using foam that can be quite interesting in the encapsulation. Many types of proteins have been explored for the formation of protein-based nanoparticles, such as albumin.

Nanoparticle albumin-bound (NAb™) technology was developed to produce albumin nanoparticles. The success of these NPs has already generated a commercial formulation containing paclitaxel, Abraxane®, which presented advantages mainly with respect to tumor targeting and drugs' toxicity decrease [91,92]. In order to be applied to PDT, the association of protein nanoparticles with photosensitizers such as chlorin e6 was investigated by Phuong and collaborators using NAb™ technology [92]. The treatment with the nanoparticles and submission to 660 nm light radiation resulted in a significantly higher toxicity in breast cancer cells and in vivo tumor suppression of 7 times less than the control group, revealing a promising application of protein nanoparticles in PDT.


*Nanomaterials* **2021**, *11*, 3132

99


**Table 1.** *Cont*.

#### 2.2.2. Carbon-Based Nanomaterials

Nanotubes, fullerenes and graphenes are among the several carbon-based nanomaterials that became widely explored for medical purposes, mainly due to the π-π interactions in their chemical structure and the ability to produce ROS, as a result of acting as a photosensitizer in PDT [93–95].

The potential of graphitic carbon nitride nanoparticles in PDT using visible light was analyzed by Heo et al. [95] using cervical cancer cells. Their study showed that the PDT allied with nanoparticles selectively killed more cancer cells than the normal cell lines. Other light sources in the NIR region are also found in the literature to carry out PDT with carbon nanoparticles derived from glucose, which resulted in an efficient ROS production [93]. The surface modification technique can also be employed to bind specifically to receptors that are overexpressed in some cancer cells types, as investigated by Xie and collaborators [96]. In their studies, hollow carbon nanospheres with Doxorubicin presented peptide and hyaluronic acid moieties in the surface to enhance the uptake and damage by dual targeting in a lung cancer cell line.

Carbon dots are carbon-based nanomaterials that can be applied for bioimaging, drug delivery and can also be used for PDT [97]. He et al. [98] designed diketopyrrole-based fluorescent carbon dots and the in vitro and in vivo studies showed that they were able to inhibit the tumor growth when irradiated.

#### 2.2.3. Silica Nanoparticles

Silica nanoparticles (SNPs) present several advantages that can be useful for the design of nanoparticles for PDT, such as the easy production, possibility of functionalization and to obtain particles with a controlled size [99]. An efficient anti-tumor effect was achieved by Liu et al. [100] when exploring the complex combination of a photosensitizer (rose Bengal), carbon dots and the drug Doxorubicin in mesoporous silica nanoparticles. In their studies, the developed nanoparticle had high drug loading capacity and the problems related to carbon dots and PS aggregation were prevented. This system was also able to produce a higher amount of singlet oxygen compared to PS rose Bengal, and the combination with Doxorubicin provided a synergy between chemotherapy and phototherapy that resulted in a 90% decrease of cell viability.

The high surface area of silica nanoparticles is another advantage as it allows its modification and functionalization, as demonstrated by the work of Lin and collaborators [101], who developed silica nanoparticles with the PS chlorin e6 encapsulated and a gene plasmid at the surface. Through a photo-induced cleavage of coumarin and detachment of the polycation PDMAEMA, with which the cytocidal gene presented an interaction, the nanoparticles could provide the release of the gene, activation of the PS and therefore a synergistic effect of the gene and phototherapy.

Bretin et al. [102] studied the anticancer potential of the photosensitizer 5-(4 hydroxyphenyl)-10,15,20-triphenylporphyrin (TPPOH) and developed silica nanoparticles coated with the conjugate xylan-TPPOH for photodynamic therapy of cancer. In the xenograft tumor model of colorectal cancer, they studied the biodistribution using Cy5.5-labeled free TPPOH and TPPOH-X SNPs. The fluorescence signal was observed at 24 h post-injection, and as shown in Figure 6A, it was a strong signal for TPPOH-X SNPs, while it showed a minimal accumulation for free TPPOH administration. An ex vivo fluorescence imaging of tumors and organs showed that liver and kidney presented higher intensity compared to the others, but the fluorescence of tumors treated with TPPOH-X SNPs had a superior intensity compared to the other organs when compared to the free TPPOH (Figure 6B). It was also confirmed by a quantitative analysis of fluorescence (Figure 6C).

**Figure 6.** Evaluation of Cy5.5-labeled free TPPOH and TPPOH-X SNPs biodistribution by fluorescence imaging at 24 h post-injection. (**A**) In vivo fluorescence imaging of HT-29 tumor-bearing mice, (**B**) ex vivo fluorescence of tumors and organs, (**C**) fluorescence analysis of tumor and organs. Data are shown as mean ± SEM (*n* = 3). \* *p* < 0.05 and NS: not significant (adapted from Reference [102] with permission from MDPI. Copyright 2019, Cancers).

#### 2.2.4. Magnetic Nanoparticles

Due to their magnetics properties, magnetic nanoparticles can be used in therapy based on the application of an external magnetic field to a targeted tissue. Besides this, it is also possible to attach molecules to it, thus working as a carrier [103].

For example, a delivery system prepared with iron oxide magnetic nanoparticles was employed for the targeted delivery of the anticancer Doxorubicin and PDT therapy using a hematoporphyrin. The synergistic effect of PDT with the anticancer drug was shown to provide an effective inhibition of breast cancer in vivo [104]. Recently, Zhang et al. [105] used nanomotors with iron oxide nanoparticles for the delivery of zinc phthalocyanine, and due to the magnetic properties of the iron nanoparticles, the NPs can be targeted to the desired tumor tissue These nanomotors generate O2 by catalyzing endogenous H2O2 for the creation of O2 as power to create the nanomotor's displacement. The system allowed an extended distribution of the photosensitizer as well as ROS generation. Additionally, the generation of O2 also supplied an efficient PDT process.

#### 2.2.5. Hybrid Nanoparticles

The hybrid NPs consist in a combination of two or more types of NPs to achieve a unique multifunctional structure [106]. Hybrid NPs composed by the combination of polymers and lipids is a quite common topic found in the literature over the past few years that can also be applied to PDT, as investigated by Pramual and collaborators [107]. In their study, the polymer-lipid-PEG nanoparticles were used for the encapsulation of a PS molecule that exhibited enhanced ROS production and phototoxicity in thyroid cancer cells.

#### **3. Photothermal Therapy**

#### *3.1. A Brief Overview*

Photothermal therapy (PTT) is a therapeutic strategy using a near-infrared (NIR) laser/light to heat the tumor region and induce cancer cells' death [108] (Figure 7). Other radiation sources able to generate hyperthermia include visible light, microwaves, radiofrequency and ultrasound waves [109]. PTT has many advantages when compared with conventional therapeutic approaches, including minimal invasiveness and high specificity [110]. In general, PTT approaches explore two mechanisms: The first one involves the exposition of the tumor site to high temperatures (superior to 45 ◦C) for a few minutes, leading to cellular death by thermal ablation. This approach usually results in stasis in tumor vessels and hemorrhage, which prevent the combination with other treatments. The second one refers to the mild hyperthermia and involves the increasing and setting of temperatures between 42 and 43 ◦C, prompting cellular damage and enhancing permeability of tumor vessels, which can be used to improve nanoparticles' uptake by tumors [111,112]. Tumor tissues are more hypoxic and acidic than normal tissues [109]. It is believed that these characteristics make them more susceptible to temperature, thus allowing PTT to selectively destroy cancer cells and protect healthy ones around the tumor [113]. Therefore, since the cancer cells are responsive to this temperature range, this procedure allows the union with synergistic therapies.

**Figure 7.** Mechanism of cell death induced by a photothermal agent in the presence of NIR light.

PTT displays promising therapeutic efficacy in the treatment of primary tumors or metastasis, in such a way that it has been studied in animal models with various types of cancer, including bone, lung or lymph metastasis [110]. The photothermal effect can also be enhanced using organic dyes or photothermal nano-agents, including metallic nanoparticles, nanocarbons, metal oxide nanomaterials and organic nanostructures [113,114].

A synergistic way to improve cancer treatment is its combination with current available therapies, such as chemotherapy, immunotherapy and radiotherapy [109]. The combination of hyperthermia therapy and chemotherapy is commonly explored through hydrophobic interactions, in which nanostructures loading antitumor drugs, such as Doxorubicin and paclitaxel, demonstrated anticancer effects. Moreover, imaging-guided PTT is another improvement to minimize adverse effects and to provide better patient outcomes [115,116], making it possible to plan a therapeutic strategy before and during treatment.

In the following sections, attention will be given to the recent developments in nanotechnology for photothermal applications of cancer.

#### *3.2. Nanoparticles with Application in PTT* 3.2.1. Metallic Nanoparticles Gold Nanoparticles

Gold nanoparticles (AuNPs) have attracted great interest as photothermal agents for cancer therapy, as they demonstrate efficient local heating after light irradiation [115]. The photothermal conversion phenomenon in AuNPs is based on the collective oscillations of free electrons at AuNPs surface (Surface Plasmon Resonance, SPR) in the presence of electromagnetic radiation (Figure 8). Due to electron excitation and relaxation, this single physicochemical property supplies high localized heating around AuNPs, resulting in destruction of cancer tissues [113,116].

**Figure 8.** Surface Plasmon Resonance of gold nanoparticles.

It is known that the SPR band of noble metal nanoparticles is much stronger than other metals [117]. Changing AuNPs sizes and shapes, the range of the SPR wavelength of AuNPs is shifted from the visible to the near-infrared (NIR) region, and optical properties can be readily tuned. One of the most interesting parts of the diminished nanoparticles' diameter is due to the fact that decreasing the size (<5 nm) allows them to be excreted by urine, improving their clearance from the body [118].

Moreover, AuNPs size affects the cellular uptake and influences the photothermal conversion efficiency. According to Mie's theory, smaller nanoparticles show superior heat conversion compared to the larger ones. It was reported that 20 nm gold nanospheres exhibited 97–103% of conversion efficiency [119]. Saw et al. [120] studied the use of four sizes of cystine/citric acid-coated confeito-like gold nanoparticles (confeito-AuNPs) (30, 60, 80 and 100 nm) (Figure 9A) in the photothermal treatment of breast cancer cells. The authors observed that the smallest sizes (30 and 60 nm) of confeito-AuNPs showed higher cellular uptake into MDA-MB-231 cells, compared to larger sizes of AuNPs (Figure 9B). This same size range has been reported in the literature [121]. In vitro studies showed that smaller sizes reached the better PTT cytotoxicity activity against cancer cells (Figure 9C). This result is due to the high surface area in relation to the total mass of NPs, which is observed in smaller nanoparticles.

Sun et al. [115] employed gold nanoparticle-coated Pluronic-b-poly(L-lysine) nanoparticles (Pluronic-PLL@AuNPs) for the delivery of paclitaxel (PTX) in PTT of solid tumors. The nanoparticles showed efficient photothermal heating capabilities after exposure to an 808 nm NIR laser irradiation and a synergistic effect of chemo-photothermal treatment. The temperature of the PTX-loaded Pluronic-PLL@Au NP-injected tumors increased to 34 ◦C, which was adequate to eliminate tumors in vivo.

**Figure 9.** (**A**) Characterization of confeito-AuNPs at (i) 30 nm, (ii) 60 nm, (iii) 80 nm and (iv) 100 nm, by (a) FESEM images and (b) TEM images. (**B**) Evaluation of the cellular uptake of confeito-AuNPs into MDA-MB-231 cells. \* *p* < 0.05 (ANOVA). (**C**) In vitro photothermal treatment: MDA-MB-231 cell viability after laser treatment (2 W/cm2, 1 min of irradiation) with confeito-AuNPs. \* *p* < 0.05 (ANOVA) (adapted from Reference [120] with permission from Elsevier. Copyright 2018, Colloids and Surfaces B: Bio-interfaces).

Gold nanoparticles are readily synthesized and allow easy surface modification. Binding a specific ligand on AuNPs surface promotes their targeting to the disease areas and their interactions with cells, such as cancer cells. This procedure increases the effectiveness of the treatment and decreases possible toxic effects in healthy areas of the body [122].

One of the strategies of passive targeting to the tumor site is based on the enhanced permeation and retention mechanism that takes place when gold nanoparticles are intravenously administered. However, the AuNPs presence in the blood can arouse attention of the mononuclear phagocytic system (MPS), leading to the rapid elimination of the nanoparticles from the body [123].

The functionalization with polyethylene glycol (PEG) is one of the most effective strategies to optimize the hydrophilic surface and to improve the blood circulation time of nanoparticles [124]. Wang et al. [125] developed PEGylated hollow gold nanoparticles (mPEG@HGNPs) for combined X-ray radiotherapy and photothermal therapy in cancer cells. The in vitro results using the combination of the 808 nm NIR laser and X-ray radiation demonstrated a synergistic antitumor effect with cell viability decreased to 61% and 65% for HGNPs and mPEG@HGNPs, respectively. The nanoparticle cytotoxicity was decreased after PEGylation, due to less mPEG@HGNPs internalized into the cells. Despite that, the targeting enhanced to the tumor site by the mPEG@HGNPs was confirmed using CT imaging in xenografted breast tumor models, due to the EPR effect.

Cheng et al. [126] reported photolabile AuNPs covalently cross-linkable with a diazirine (DA) terminal group of PEG ligand on the AuNPs surface. The 20.5 nm diazirinedecorated AuNPs (dAuNPs) were obtained after laser excitation at 405 nm. The photothermal therapy in tumor-bearing BALB/c mice was investigated by monitoring the average tumor size in different mice groups. The mice groups that were treated with dAuNPs + λ405 nm and dAuNPs + NIR showed weak tumor inhibition, while the group treated with dAuNPs exhibited a high tumor volume decrease upon 808 nm irradiation (0.75 W cm<sup>−</sup>2). The tumor region reached 26.7 ◦C after 10 min of light exposure. The PTT efficacy was further confirmed through tissue analysis, which showed extensive necrosis in dAuNPs + λ405 nm + NIR group.

The extracellular environment of solid tumors has an acidic pH. pH-sensitive AuNPs with potential application in PTT have been reported in the literature [127]. Natural peptides can be used as tumor-targeting agents. Barram et al. [122] used glutathione (GSH), soluble in water, as a coating for AuNPs. GSH is a pH-sensitive polymer, with its isoelectric point (IEP) close to the pH of the cancer cells (~6). Consequently, GSH-AuNPs become responsive to the tumor's acidic environment, improving its targeting to the desired location. In vitro photothermal therapy was applied to rhabdomyosarcoma (RD) cancer cells, using two types of low-power laser (visible green light (532 nm) and infrared light (NIR) (800 nm)). The study observed cell death values of tumor cells for both types of lasers, and these values were proportional to the longer periods of radiation exposure and, even more so, to the highest concentrations of GSH-AuNPs.

From the molecular point of view, several studies on the effect of nanoparticles on DNA molecules and DNA bases have been performed. These studies clearly demonstrate the damage effect of the gold nanoparticles. Recently, Marques et al. [128] analyzed the decomposition of halogenated nucleobases by Surface Plasmon Resonance excitation of gold nanoparticles. In fact, the halogenated uracil derivatives can be of great interest for cancer therapy [129,130] and the authors demonstrated that the presence of irradiated gold nanoparticles decomposes the ring structure of uracil and its halogenated derivatives with similar efficiency. This decomposition is associated with the fragmentation of the pyrimidine ring, for 5-bromouracil, with cleavage of the carbon-halogen bond, whereas for 5-uorouracil, this reaction channel was inhibited. Locally released halogen atoms can react with molecular groups within DNA, and hence this result indicates a specific mechanism by which doping with 5-bromouracil can enhance DNA damage in the proximity of laserirradiated gold nanoparticles.

#### Gold Nanorods

Gold nanorods (AuNRs) are one of the many tools employed in cancer photothermal therapy, due to their high capability to transform near-infrared light into heat. The investigation of their aspect ratio allows to adjust a particular SPR band in the NIR [131], consequently reducing damage in normal tissues as these ones have minimal NIR energy absorption. Despite their ability as PTT agents, AuNRs are considered to be toxic to cells, because of the stabilizers, e.g., hexadecyl-trimethylammonium bromide (CTAB), used in their synthesis [132]. Several approaches have been used to minimize the toxicity of AuNRs, such as the binding of polymers to increase their biocompatibility. Kirui et al. [133] improved biocompatibility of AuNRs using poly(acrylic acid) (PAA) for coating of nanoparticles. Liu et al. [134] reduced the toxicity of PEG-AuNRs using multidentate PEG (AuNT-PTP Gm950).

PTT induced by NIR is known to improve chemotherapeutic efficacy by triggering drug release or increasing the cancer cells' sensitivity to chemotherapeutics [135]. Hauck et al. [136] revealed that the heat produced by gold nanorods together with the chemotherapeutic drug cisplatin killed 78% more cancer cells than cisplatin alone. Combination therapy can reduce toxicity associated with chemotherapeutics through reducing the effective drug dosage. Duan et al. [137] developed gold nanorods coated with chitosan (CS) derivatives as a carrier of Doxorubicin (DOX) to combine chemical and photothermal effects. In vitro studies demonstrated that these nanoparticles showed low cytotoxicity and potential against cancer cells. Wang et al. [138] developed gold nanorods coated with polydopamine (PDA) and loaded with thiolated poly(ethylene glycol) tumor-homing peptides (NGR and TAT), as a carrier of Doxorubicin. NGR/TAT-DOX-PDA@GNRs allowed a pH-triggered controlled release of DOX and a synergistic effect with the combination of chemo-photothermal therapy.

Moreover, the efficacy of a targeted synergistic photothermal chemotherapy of breast cancer using gold nanorods (GNRs) functionalized with hyaluronic acid (HA) and folate (FA) to deliver DOX was demonstrated by Xu et al. [139]. The therapeutic system showed a long blood circulation time and high tumor site accumulation. In vitro photothermal chemotherapy was evaluated. Cell viability of MCF-7 cells treated with GNRs-HA-FA-DOX + NIR was reduced to 31%. The authors also investigated the synergistic effect of PTT chemotherapy in vivo and GNRs-HA-FA-DOX exhibited an excellent antitumor effect against MCF-7 breast tumors in nude mice. After 5 min of light exposure, the temperature of MCF-7 breast tumors in nude mice treated with GNRs-HA-FA-DOX reached 67.5 ◦C (1.5 W/cm2), leading to irreversible tumor cell death.

In metastasis, cancer cells migrate and invade the surrounding tissues, and therefore collective cell migration is directly related to cancer aggressiveness. This process involves interactions between neighboring cells through the cell junctions and contraction motions of the cytoskeleton filaments [140]. Studying the migration and invasion of cancer cells, Wu et al. [141] developed AuNRs functionalized with PEG and Arg-Gly-Asp (RGD) peptides. These authors found morphological changes of many cytoskeletal and cell junction proteins after PTT treatment, suggesting that interactions between integrin-targeted AuNRs and cells could trigger inhibition of cancer collective migration.

It should also be reported that an effective combined therapy of paclitaxel-loaded gold nanorods against head, neck and lung cancer cells was developed by Ren et al. [142]. Paclitaxel was loaded into a hydrophobic pocket of the polymeric monolayer on the surface of NIR-absorbing AuNRs, which allowed the efficiently direct cellular release of the hydrophobic drug via a cell membrane mimicking two-phase solution. It was demonstrated that the PTT approach with this developed nanocomplex led to total eradication of tumor cells at a dosage of 0.5 nm of nanomaterials with low-intensity (0.55 W/cm2) NIR light.

Stimuli-responsive materials have attracted attention due to their capability to control the timed release of the entrapped drugs. Near-infrared light (NIR)-responsive polymers have been used for triggered drug delivery in specific tissues [143]. Hribar et al. [144] reported a NIR light-sensitive polymer−nanorod composite for controlled drug release, in the range of body temperature. As the glass transition temperature is near to the physiological temperature, it can be used to control and improve the release of a molecule. The researchers applied this heating system to trigger release of the Doxorubicin from the nano-system. After NIR light exposure, Doxorubicin-encapsulated microspheres were able to decrease 90% of the activity of T6-17 cells.

#### Gold Hybrid Nanoparticles

Although the research on hybrid nanoparticles to improve the diagnosis and cancer treatment has attracted attention due to its potential use in medicine, its safe application in therapy still remains limited [145,146]. In recent years, it has been reported that the development of iron-gold nanocomplexes are used for the combined PTT with magnetic resonance imaging (MRI). The Au shell composes the light-responsiveness portion, while the iron core can be used to improve the ratio of water molecules' transverse relaxation, leading

to strong MRI signals. Additionally, the magnetic center allows the nanocomplex to be directed to the tumor site by means of a magnetic field [147,148].

Dong et al. [149] developed gold-nano-shelled magnetic hybrid nanoparticles functionalized with anti-human epidermal growth factor receptor 2 (Her2) antibodies (Her2-GPH NPs) for multi-modal imaging and cancer treatment. The nanoparticles were produced by loading gold nano-shells with poly (lactic-co-glycolic acid) (PLGA) attached to perfluorooctyl bromide (PFOB) and superparamagnetic iron oxide nanoparticles, and then binding the antibody. Her2-GPH NPs showed high ability as a contrast agent for both ultrasound (US) and magnetic resonance (MR) imaging. The in vitro cytotoxicity studies demonstrated that Her2-GPH NPs specifically promoted Her2-positive human breast cancer SKBR3 cells' death after NIR exposure. Abed et al. [148] directed Iron (III) oxide–gold (Fe2O3@Au) coreshell nanoparticles to the tumor site through a magnetic field in Balb/c mice bearing a CT26 colorectal tumor model after intravenous administration of the nanoplatform. The in vivo antitumor studies showed the complete tumor growth eradication after magnetic targeting and subsequent NIR eradication.

The toxicity of Au and magnetic nanocomplexes is still concerning. These nanoparticles can lead, among others, to DNA damage, production of free radicals and modification in cell signaling. Additionally, the toxicity can be caused by nanoparticles' aggregation in biological fluids. Using biocompatible and water-soluble polymers as a coating makes it possible to improve the colloidal stability and decrease nanoparticles' aggregation, thus diminishing the cytotoxicity. Abedin et al. [150] improved the colloidal stability of Au–Fe3O4 NPs in aqueous media using poly-l-lysine (PLL) polymer as a surface coating. Additionally, PLL-Au-Fe3O4 NPs demonstrated cytocompatibility and NIR light absorption ability.

Mesoporous silica nanoparticles (MSNPs) are highly versatile drug carriers due to their biocompatibility and high surface area, consequences of their well-defined internal mesopore structure, varying from 2 to 10 nm in diameter and with large pore volume. Depending on surface charge and nanoparticle size, the characteristics such as nanoparticle cytotoxicity and cellular uptake can change [151]. Yang et al. [152] designed a system composed of ultra-small gold nanoparticles attached to mesoporous silica nanoparticles (MSN) through Au-S bonds. The in vitro studies showed the fast release of DOX upon NIR light irradiation and synergistic cytotoxic effect against A549 cells.

Gold nanoparticles lose their ability to convert light into heat under repetitive NIR laser irradiation, including gold nanorods that can change their shape and extinction after NIR exposure. Cheng et al. [153] projected gold/mesoporous silica hybrid nanoparticles (GoMe) for lung cancer detection and treatment. This hybrid system has a good photothermal ability and stability, and maintains its capacity of photothermal conversion after repetitive NIR exposures. In addition, 64Cu-labeled GoMe was used to detect lung tumors in vivo through PET imaging, demonstrating to be a potential theranostic system for cancer therapy.

#### Silver Nanoparticles

Silver nanoparticles (AgNPs) are multifunctional materials which have been used for many applications, such as biosensors, electronic compounds, antimicrobials and medicines [154]. Their general use is due to singular characteristics such as size and shape being controllable, easily modified surface and optical and electrical properties [155]. Additionally, their antibacterial activities are widely known [156].

AgNPs can be produced through various physical and chemical methods [157]. Spherical AgNPs are frequently synthesized using the Turkevich method [158] with citrate as a reducing and stabilizing agent or with NaBH4 as a reducing agent [159]. In recent years, many researchers are using biological methods to produce AgNPs. These techniques utilize plants, fungi, algae and other organic sources to synthesize nanoparticles with great stability [160].

Application of AgNPs in the biomedical field is still limited due to the concern of their intrinsic toxicity. Interactions of AgNPs with the human body are not yet wellunderstood [161]. Modifying its surface with biodegradable molecules and polymers or

incorporating these nanoparticles into hybrid systems are some of the ways that many researchers have found to increase the biocompatibility of AgNPs. Kim and coworkers [162] developed bovine serum albumin (BSA)-coated silver NPs (BSA-silver NPs) by a single-step reduction process.

Similar to gold nanoparticles, SPR of silver nanoparticles can be tuned to the infrared region by altering their size and shape [163]. Boca et al. [164] designed chitosan-coated silver nanotriangles (Chit-AgNTs) for hyperthermia of human non-small lung cancer cells (NCI-H460) using a 800 nm laser. Wu et al. [165] engineered a nanoplatform for fluorescent probe and label-free imaging of cell surface glycans composed of DNA/silver nanoclusters (DNA/AgNCs) via hybridization chain reaction (HCR). The nanoparticles showed a great ability to convert light to heat, reaching 53.6 ◦C after irradiation with the 808 nm laser at 1 W cm−<sup>2</sup> for 10 min. The confocal results demonstrated the applicability of the DNA/AgNCs for labeling glycans on the surface of tumor cells. Moreover, in vivo experiments showed that DNA/AgNCs were able to ablate and inhibit tumor growth under the laser exposure.

PEGylated bovine serum albumin (BSA)-coated silver core/shell nanoparticles loaded with ICG ("PEG-BSA-AgNP/ICG") were synthesized by Park et al. [166]. These nanoparticles were tested for anticancer activity in B16F10 cells after light exposure. The cytotoxicity results revealed a cell viability of 6% when temperature reached at 50 ◦C. PEG-BSA-AgNP/ICG also displayed a long plasma half-life, which led to the higher accumulation in the tumor. At 4 h post-administration of PEG-BSA-AgNP/ICG in a B16F10 nude mice model, the tumor temperature reached 49.6 ◦C with a laser power of 0.95 W. Furthermore, among the treatment groups, the "PEG-BSA-AgNP/ICG + PTT group" was the only one that exhibited significant inhibition in tumor growth.

#### 3.2.2. Carbon-Based Nanomaterials

#### Carbon Nanotubes

Carbon nanotubes (CNTs) are cylindrical structures constructed from a sheet of graphene [167]. These NPs are allotropic forms of carbon, with diameter in the nanometric dimension and various millimeters in length [168]. CTNs are classified into two types, according to the number of layers in their structure: single-walled carbon nanotubes (SWCNTs), which consist of a single graphene sheet, and multiwalled carbon nanotubes (MWCNTs), consisting of several sheets forming concentric cylinders [169].

CNTs have a wide range of properties that make them unique nanomaterials, such as excellent electrical, thermal and optical conduction, mechanical strength [170] and high surface areas, which can be easily functionalized [171]. Indeed, CNTs are usually modified with molecules that help to enhance their biocompatibility or enable specific functions [172]. Attachments to PEG is one of the major types of CNTs' functionalization to improve biocompatibility, water solubility and stability [173]. Sobhani et al. [174] successfully attached PEG onto the CNTs's surface.

CNTs have a wide NIR absorption which is dependent on the size and shape of these nanomaterials [175]. Exposing CNTs to NIR light releases vibrational energy in the form of heat, and could be used for cancer cell ablation [176]. The application of CNTs in PTT for the treatment of various kinds of human cancer xenografts in animal models has been investigated in the literature and has been demonstrated to be effective [177].

Li and collaborators [178] designed an interesting system for curcumin (Cur) delivery composed of functionalized single-walled carbon nanotubes by phosphatidylcholine and polyvinylpyrrolidone (SWCNT-Cur). Results of the cellular uptake study showed that SWCNT-Cur effectively improved the delivery of Cur into cells within 4 h. Compared with native Cur, the formulation developed obtained an uptake amount 6-fold higher. Additionally, biodistribution studies demonstrated that SWCNT-Cur could enhance curcumin blood concentration up to 18-fold. Lastly, this system was evaluated for its ability of photothermal ablation in an in vivo model. Among all the groups tested (saline + laser, Cur + laser, SWCNT + laser, SWCNT-Cur + laser), the SWCNT-Cur and laser (808 nm) groups showed the most significant suppression on tumor weight and volume, indicating the synergistic anticancer effect of Cur and PTT.

Waghray et al. [179] synthesized MWCNTs coated with phospholipid-poly(ethylene glycol) and conjugated with an anti-P-glycoprotein (Pgp) antibody, to enhance Pgp-specific cellular uptake. Pgp is an ATP-binding transporter, expressed on tumor cell membranes, and it is related to cancer drug resistance. The phototoxicity of Pab-MWCNTs was investigated in 3T3 and 3T3-MDR1 cells and in a tumor spheroid model (NCI/ADR-RES cells). The nanostructures demonstrated not only Pgp-specific endocytosis by 3T3-MDR1, but they also exhibited dose-dependent phototoxicity only in 3T3-MDR1 cells. Moreover, NCI/ADR-RES spheroids treated with Pab-MWCNTs showed the highest cell death after NIR laser irradiation when compared with control groups.

Zhang and colleagues [180] engineered MWNTs/gemcitabine/lentinan (MWNTs-Ge-Le) to overcome Gemcitabine's clinical application problems related to short plasma half-life and low cellular uptake. It was observed that the MWNTs-Ge-Le conjugated with rhodamine were internalized by MCF-7 cells about 3 h after incubation. Additionally, encouraged by the results in vitro, the authors evaluated the synergistic antitumor effect of MWNTs-Ge-Le on tumor-bearing mice. MWNTs-Ge-Le nanoparticles have reached the tumor site through the EPR effect. After 3 min of NIR irradiation, the temperature of the tumor surface increased to approximately 42.6 ◦C, while the PBS group only reached about 36.6 ◦C. Moreover, it was observed that the size of the tumor significantly decreased, confirming the high synergetic effect of chemotherapy and PTT.

Zhao et al. [181] developed SWCNTs and MWCNTs coated with peptide lipid (PL) and sucrose laurate (SL) (denoted as SCNT-PS and MCNT-PS, respectively), which were conjugated with siRNA (anti-survivin siRNA) for synergistic PTT and gene therapy (GT). The engineered CNTs exhibited excellent temperature-sensitivity and biocompatibility. The effective cellular internalization was confirmed after they observed nanoparticles' presence in the cytosol of HeLa cells. The in vitro cytotoxicity after 808 nm laser irradiation was evaluated treating cells with 30 μg/mL of SCNT-PS or MCNT-PS. The results showed that 76.2% ± 4.4% and 75.3% ± 3.5% of the cells were led to death by combined therapy in the SCNT-PS and MCNT-PS, respectively. The PT efficacy of CNTs was also evaluated in vivo. After tumor irradiation (1 W/cm2 for 5 min), the local temperature reached 42–45 ◦C. Furthermore, they investigated whether the SCNT-PS/siRNA and MCNT-PS/siRNA complexes could be able to downregulate survivin. The data obtained were promising, indicating that cells that received treatment with SCNT-PS + P + G and MCNT-PS + P + G, followed by PTT, had about a 60% decrease of survivin expression in comparison with GT alone (SCNT-PS + G and MCNT-PS + G).

Besides PTT generating anticancer immune responses, evidence suggests that it could also induce an effect called the abscopal effect [182]. This effect refers to the immune response generated when the primary tumor site is irradiated, which can lead to the regression of metastatic cancer in distant sites that were not irradiated [183]. Nevertheless, some tumors are capable of creating inhibitory binders, which connect to inhibitory coreceptors (immune checkpoints) expressed on tumor immune cells [184]. This activity induces negative regulatory pathways leading to loss of immunological control, allowing tumor growth progression and decreasing immune response to various therapies [185]. New immunotherapeutic approaches are focusing on blocking immune checkpoints in order to recover the suppressed immune response [186]. Among the immune checkpoints, the cytotoxic T-lymphocyte antigen 4 (CTLA-4) is an inhibitory receptor expressed by regulatory and conventional T cells, which suppresses T cell activation via cell intrinsic and extrinsic pathways. Ipilimumab, an antibody against the inhibitory co-receptor CTLA-4, is one of the main targets of immunotherapy [187,188].

CNT-mediated photothermal therapy in combination with checkpoint inhibitors can be used to maximize the abscopal effects of PTT. Li et al. [189] designed SWNT functionalized with glycated chitosan (GC), an immunoadjuvant, for specific treatment of an aggressive 4T1 murine breast cancer model, upon 1064 nm laser irradiation. Putting together

SWNT-GC-laser therapy with anti-CTLA-4, they have achieved synergistic immunomodulatory effects, inducing antitumor immune response and an increase of the survival time of the treated mice group (up to 58 days).

Recently, McKernan and his group [190] presented a delivery nano-system to treat metastatic breast cancer composed of SWCNTs that integrates PT therapy and checkpoint inhibitor immuno-stimulation with anti-CTLA-4. The SWCNTs were functionalized with the protein annexin A5 (ANXA5), which has great affinity to the anionic phospholipid phosphatidylserine expressed on endothelial cells of the tumor vasculature and on tumor cell membranes. The authors noted that PTT with SWCNT-ANXA5 alone was able to destroy primary EMT6 tumors, reaching a temperature of 54 ◦C at the site, but failed to eliminate the metastasis. On the other hand, the combination of photothermal therapy with SWCNT-ANXA5 and anti-CTLA-4 improved overall survival, leading to 55% of the treated mice surviving at 100 days post-injection. Moreover, in animals who received this combined therapy, increases in the numbers of helper T cells CD4+ and cytotoxic T cells CD8+ were observed, indicating an increase in the immune response.

#### Hollow Carbon Nanospheres

Hollow carbon nanospheres (HCNs) are mesoporous nanomaterials with high pore volume and surface area [191]. Due the carbon chains, a great amount of a hydrophobic drug can be loaded into their structure, making them a potent drug carrier [192]. Similar to carbon nanotubes, HCNs have a great ability to convert NIR light into heat, which can be used to modulate the drug release at the tumor site [193].

Wang et al. [194] produced biocompatible HCNs for loading and release of paclitaxel (PTX) and PT therapy. The nanoparticles have demonstrated excellent photostability and ability to effectively release the loaded PTX. Additionally, in vitro experiments showed great thermal ablation of HCT116 cells using 50 μg/mL of HCNs and a 3 W/cm2 laser power density for 180 s.

Xu and his group [195] produced a hollow carbon nanosphere capped with olyethylene glycol-graft-polyethylenimine (HPP) as a photothermal agent. Optical properties were investigated using a 1064 nm laser and power density of 0.6 W/cm2. After 7 min of laser exposure of the nanoparticle's dispersion, an increased temperature in the range of 17 to 44 ◦C was observed, indicating an excellent heat conversion efficiency. The photothermal therapeutic effect in vitro (4T1 cells) and in vivo (Balb/c mice inoculated subcutaneously with 4T1 cells) was also evaluated. The percentage of cell death for in vitro experiment varied from 40% up to 95%, using the HPP concentrations of 10, 20, 40, 80 and 160 μg/mL. 4T1 tumor-bearing mice treated with only 40 μg/mL were irradiated. After 14 days, tumors were measured, showing a significant decrease of volume.

#### 3.2.3. Metal Oxide Nanoparticles

#### Iron Oxide Nanoparticles

Magnetic nanoparticles are mostly formed using magnetite (Fe3O4), maghemite (γ-Fe2O3) or a combination of both [196]. Due to their intrinsic magnetic properties (superparamagnetism), these nano-systems have emerged as potent contrast agents (CAs) for magnetic resonance imaging (MRI) and biomedical purposes [197].

In the field of cancer therapy, magnetic nanoparticles can be specific delivery drugs by application of alternating magnetic fields to targeting tumor sites and eliminating them using localized moderate heating [198].

Cabana and coworkers [199] compared the application of photothermal (PT) therapy using magnetic multicore nanoflowers versus magnetic hyperthermia (MHT) of magnetic nanospheres. The NPs' performance in MHT and PT was carried out in water and in cancer cells. They found that nanoflowers are heaters that are more effective for both modalities. In the cellular environment, PT showed excellent results at low doses, while MHT was lost for all NPs. Additionally, magnetite nanoflowers demonstrated the highest cellular uptake and the best antitumor activity after the laser exposure (0.3 W/cm2).

Liu and collaborators [200] synthesized the polyethylene glycol-coated ultrasmall superparamagnetic iron oxide nanoparticle-coupled sialyl Lewis X (USPIO-PEG-sLex) with excellent photothermal conversion properties. The nanoparticles were applied for MRI and PTT in human nasopharyngeal carcinoma (NPC) xenografts on a mouse model. After 808 nm laser exposure, the cytotoxicity results showed a reduction of viability of NPC 5-8F cells at reasonable concentrations of USPIO-PEG-sLex nanoparticles. Moreover, the NPs were able to inhibit xenografts' tumor progression after in vivo post-injection.

Iron oxide NPs exhibit excellent photothermal conversion efficiency, good chemical stability and low toxicity in the biological environment [201]. The in vivo application of iron oxide NPs has been approved by the US Food and Drug Administration (FDA) [202]. However, their use in many clinical approaches is limited due to the low tumor delivery efficiency of the NPs [203].

Aiming to enhance the tumor delivery of iron oxide NPs, Wang and his group [203] developed an Ac-Arg-Val-ArgArg-Cys(StBu)-Lys-CBT probe coupled with monodispersed carboxyl-decorated SPIO NPs to form SPIO@1NPs. When SPIO@1NPs entered tumor cells overexpressing furin, a reaction chain developed, resulting in SPIO NPs' aggregates by cross-linking. The self-aggregation of NPs improved their retention in the tumor site, leading to better T2 imaging results and PTT of cancer cells more effective at low doses.

Surface modification of synthetic nanomaterials with biomimetic cell membranes is a smart strategy to make it harmless and invisible to the immune system [204]. Meng et al. [205] employed vesicles formed from macrophage membranes reconstructed to obtain a biomimetic system for cancer phototherapy. These vesicles coated onto magnetic iron oxide nanoparticles (Fe3O4 NPs) resulting in Fe3O4@MM NPs exhibited good biocompatibility and light-to-heat conversion efficiency. Cancer targeting of Fe3O4@MM NPs was confirmed by cellular uptake in MCF-7 cells. The authors also found that the NPs were able to evade from immune cells, and this activity could be related to the presence of cell membrane components on the nanoparticles' surface. The Fe3O4@MM NPs were targeted to the tumor site with application of an external magnetic field, in a breast cancer mouse model. The tumor volume was measured after the laser irradiation, reaching a high tumor regression over time.

Researchers have been employing phytochemical compounds together with magnetic nanoparticles in order to achieve nanomaterials for phototherapy and drug delivery systems [206]. Kharey et al. [207] obtained 15 nm eugenate (4-allyl-2-methoxyphenolate) capped iron oxide nanoparticles (E-capped IONPs) through green synthesis using a medicinal plant, Pimenta dioica. These NPs showed good biocompatibility in Human cervical cancer (HeLa) and Human embryonic kidney 293 (HEK 293) cell lines, and excellent efficacy of hyperthermia generation upon laser irradiation at NIR wavelength.

A delivery nano-system composed of R837-loaded polyphenols coating ICG-loaded magnetic nanoparticles (MIRDs) was constructed for spatio-temporal PTT/immunotherapy synergism in cancer. This system inhibited tumor growth, metastasis and recurrence, which resulted in potent anticancer therapeutic effects with few side effects [208].

Silica-coated Fe3O4 magnetic nanoparticles loaded with curcumin (NC) were synthesized by Ashkbar and colleagues [209] for hyperthermia and singlet oxygen production improvement for in vitro and in vivo experiments. Curcumin (CUR) belongs to the polyphenol class of natural compounds, known for its photosensitizing properties and antitumor activities [210]. The PDT was assessed using diode lasers at 450 nm and PTT was achieved by an 808 nm laser. After injection in a breast tumor mouse model, the results showed that CUR + PDT achieved a tumor volume reduction of about 58%, in comparison with the untreated group, while the NC + PDT + PTT group exhibited more than an 80% reduction compared with other treatment groups. The authors found that the NC + PDT + PTT treatment strategy could interrupt the tumor growth until day ten. This result was related to the synergistic effect achieved by hyperthermia plus ROS generation in the tumor site [209].

Manganese Oxide Nanoparticles

In recent years, manganese oxide nanoparticles (MONs) have emerged as contrast and photothermal agents due to their low toxicity and good T1-weighted contrast signals, constituting a promising alternative to the traditional PTT agents [211].

Xiang and colleagues [212] developed biocompatible and pH-sensitive MnO-loaded carbonaceous nanospheres (MnO@CNSs) for simultaneous PTT and MRI. The mimetic pH-responsive release of Mn2+ in the biological environments (pH 7.4, 6.5 and 5.0) was measured. They observed that MnO@CNSs were stable in neutral solution (pH 7.4), while in acidic pH, the nanoparticles quickly released Mn2+ ions (pH 6.5 and 5). These data were confirmed in in vivo experiments, which demonstrated that MRT1 signal values were higher in the acid region of the tumors. The MnO@CNSs PTT effect was investigated under irradiation by an 808 nm laser (2 W/cm2) for 10 min. The results showed an elevated efficiency of the MnO@CNSs for in vivo tumor ablation, making this system a potent nanotheranostic tool.

#### Molybdenum Oxide Nanoparticles

Molybdenum oxide nanostructures are reported to display good biocompatibility and biodegradability, making them a safe platform for cancer therapy. MoO3 nanoparticles have excellent absorption in the NIR region, and can also generate singlet oxygen under NIR light exposure, which enables their use for both photodynamic and photothermal therapy [213].

Chen et al. [214] synthesized molybdenum oxide (MoOx) nanosheets using the singlepot hydrothermal method and functionalized them with pluronic F127 (MoOX @ F127) to obtain a biocompatible nano-system with pH-dependent degradable properties for chemotherapy and photothermal therapy. It was observed that MoOx @ F127 showed reasonable stability at pH 5.4 and rapid degradation at pH 7.4, indicating that intact nanoparticles could reach the tumor site through the EPR effect. The ability of MoOx @ F127/DOX to kill tumor cells was investigated in MCF-7 cells after 5 min of 808 nm laser irradiation. Cytotoxicity assessment showed that almost 60% of cells died after treatment. Furthermore, in vivo experiments showed that mice injected with MoOx @ F127/DOX had a tumor temperature greater than 50 ◦C, suggesting high hyperthermic efficiency of the nanoparticles.

#### Zinc Oxide Nanoparticles

The element zinc has diverse medical applications [215]. Zinc oxide (ZnO) shows high chemical stability, low toxicity, optical, electrical and anticancer properties, becoming a potential alternative for PTT [216]. Production of intracellular reactive oxygen species (ROS) is one of the cytotoxic mechanisms of ZnO NPs [217]. Kim et al. [218] applied hybrid nanoparticles composed of ZnO and berberine (BER) for the chemo-photothermal therapy of lung cancer. The in vitro results revealed an effective antiproliferation activity against A549 (human lung adenocarcinoma) cells without severe toxicity signals observed in rats' blood tests.

Liu et al. [219] designed a core-shell nanoplatform based on a zinc oxide (ZnO) core and a polydopamine (PDA) shell to combine chemotherapy with Doxorubicin (DOX), gene DNAzyme (DZ) and photothermal therapy. The nanoparticles showed good photothermal conversion and stability after application of the 808 nm laser for 500 s. Additionally, confocal microscopy demonstrated that ZnO@PDA-DOX/DZ could be internalized by cells and consequently could deliver DZ to stimulate gene-silencing activity. Moreover, tumorbearing mice treated with ZnO@PDA-DOX/DZ exhibited an effective NP accumulation in the tumor site. The tumor tissue achieved a temperature of up to 47.3 ◦C, leading to death of the cancer cells and inhibition of the tumor growth. Lastly, the authors measured the levels of survivin in the tumor tissue by Western blotting. The results found low levels of survivin, suggesting the triggering of DZ for in vivo gene silencing.

#### 3.2.4. Transition Metal Dichalcogenide Nanomaterials

Molybdenum disulfide (MoS2) nanoparticles display several characteristics that make them excellent photothermal agents for cancer therapy, such as biocompatibility, wide surface plasmon resonance, good light-to-heat conversion efficiency and low cost [220]. In 2014, Liu and collaborators [221] pioneered using PEG-functionalized MoS2 nanosheets as drug carriers for therapy of cancer. Two-dimensional MoS2-PEG nanosheets have achieved excellent synergistic anti-tumor effects in in vivo studies, after intravenous administration of MoS2-PEG/DOX.

Xie and coworkers [222] synthesized egg yolk phospholipid-modified molybdenum disulfide (MoS2) as a PTT agent and drug delivery system for MCF-7 cells' treatment. The lipid layers on the surface of layered MoS2 nanosheets were modified to improve the NPs' stability and the accumulation of the nanocarrier in mice tumors. Additionally, Doxorubicin (DOX) was conjugated with MoS2-lipid nanocomposites for synergistic chemotherapy. Ding et al. [223] produced well-dispersed L-cysteine-modified MoS2 (MoS2- Cys) nanospheres measuring 422 nm in size. MoS2-Cys exhibits biocompatible and good photothermal conversion efficiency (35%) upon 808 nm laser irradiation. The in vitro PTT activity of MoS2-Cys nanospheres in S180 mouse ascites tumor cells displayed high cytotoxicity, with the IC50 value of 2.985 μg/mL. In vivo experiment data demonstrated a remarkable decrease of the tumor volume of the mice treated with MoS2-Cys nanospheres coupled with NIR irradiation.

Qian et al. [224] developed titanium disulfide (TiS2) nanosheets functionalized with polyethylene glycol (PEG), obtaining a great PTT agent for in vivo tumor ablation. Balb/c mice bearing 4T1 tumors were treated with TiS2-PEG, and after 24 h, exposed to an 808 nm laser at 0.8 W cm−<sup>2</sup> for 5 min. The researchers found that tumors in the mice were completely ablated. Moreover, TiS2-PEG nanosheets were tested as a contrast agent in photoacoustic imaging. Strong photoacoustic signals were observed around the mice tumor after injection of TiS2-PEG, indicating the efficient accumulation of these nanoparticles at the targeted site.

Cao et al. [225] produced TiS2 nanosheets using a human serum albumin (HSA) assisted exfoliation method, and later, modification with PEGylated folic acid (FA). TiS2-HSA-FA showed photothermal conversion efficiency of about 58.9% after NIR laser irradiation. In vitro and in vivo experiments demonstrated TiS2-HSA-FA to have a high biocompatibility and specificity for targeting tumors. In vivo synergistic PTT/radiotherapy (RT) evaluation was assessed in a CT26 tumor xenograft model, under 5 min laser irradiation (808 nm, 0.8 W/cm2). Researchers found that the highest tumor growth inhibition effect was achieved by TiS2-HSA-FA + NIR+RT, suggesting the combined therapy effect.

#### 3.2.5. Other Nanoparticles

Over the years, many kinds of inorganic and organic materials have been employed to build an effective PTT system. Graphene quantum dots (GQDs) have excellent photothermal conversion efficiency, incomparable morphology and ease of functionalization [226]. Fang et al. [227] fabricated graphene quantum dots (GQDs) as a pH-sensitive delivery system for chemotherapeutic drugs inside cancer cells. After their cellular uptake, the nanocarriers released Doxorubicin (DOX) upon laser irradiation and upon acidification of the intracellular environment. Studies in vitro and in vivo demonstrated the targeting of HA-functionalized carriers to the CD44 receptor overexpressing human cervical carcinoma HeLa cells and inhibition of tumor growth.

Phase change material (PCM) is a type of storage material that stores and releases energy in the form of heat [228]. An example of this kind of substance, fatty acid, has been studied in the thermal response to release drugs [229]. Yuan and coworkers [230] fabricated CuS-DOX-MBA@PCM nanoparticles by a nanoprecipitation method. The system was composed of copper sulfide (CuS) and DOX, encapsulated with stearic acid and lauric acid. Due to drug release in physiological conditions, this nanocarrier was used as a photothermal and imaging-guided agent. In vivo results exhibited improved inhibition of tumor growth related to the synergistic effect of 808 nm laser irradiation and antitumor therapy with DOX.

The potential anticancer activity of selenium nanoparticles has already been described in the literature [231]. Fang et al. [232] designed a combination of chemo- and PT-therapy based on SeNPs to carry both ICG and Doxorubicin (DOX). Additionally, they conjugated two peptides (RC-12 and PG-6) to SeNPs using chitosan (CS) as the linker. These peptides acted as specific tumor-targeting ligands, which helped to improve the cellular uptake of SeNPs-DOX-ICG-RP. The photothermal effect of NPs was confirmed by the raise of temperature to 78.2 ◦C after NIR irradiation for 100 s (3W cm−2). In vitro experiments demonstrated that SeNPs-DOX-ICG-RP generated ROS in HepG2 cells and promoted an efficient anticancer activity. Mohammadi et al. [233] engineered nanostructures of selenium-polyethylene glycol-curcumin (Se-PEG-Cur) for PTT and sonodynamic therapy (SDT). The nanoparticles showed great photothermal conversion efficiency (16.7%) and ability to trigger ROS production in C540 (B16/F10) cancer cells. The percentage of viable cells after irradiation of the 808 nm laser decreased to 33.9%, while ultrasound waves could reduce viability to 22.9%.

#### **4. Final Remarks**

This review shows that nanoparticles are being extensively investigated for phototherapies nowadays. Regardless of the type of nanoparticle, there a few characteristics, shown in Figure 10, that can summarize the current state of this technology for medical application. The main advantages include the minimally invasive method of therapy, the minimization of side effects and the possibility to target and enhance accumulation of drugs in the tumor. Therefore, it is possible to achieve a targeted therapy with a reduction of drug dosage and greater drug stability. In conclusion, nanoparticle systems are multifaceted structures that are under extensive investigation to create alternatives for conventional therapies of cancer in combination with phototherapy. There are still parameters such as the hypoxic tumor microenvironment that can be an obstacle for PDT, and for phototherapy in general, the limited penetration depth of the light can hinder the use of these systems in cancer therapy. Finally, scale-up and clinical studies are indeed the main challenges in the next few years, however, the incredible diversity of nanoparticles as well as their multiple qualities allied to phototherapy are a promising combination that can result in a more effective and safer treatment for the patients.

**Figure 10.** Strengths, weaknesses, opportunities and threats (SWOT) analysis of nanoparticles for Phototherapy.

**Author Contributions:** Conceptualization, T.P.P., C.E.A.B. and M.R.; methodology, T.P.P., C.E.A.B. and M.R.; investigation, T.P.P., C.E.A.B. and M.R., writing—original draft preparation, T.P.P., C.E.A.B. and M.R.; writing—review and editing, T.P.P., C.E.A.B., M.R., P.A.R. and P.D.M.; visualization, T.P.P. and C.E.A.B.; supervision, T.P.P., C.E.A.B., M.R., P.A.R. and P.D.M.; project administration, M.R., P.A.R. and P.D.M.; funding acquisition, M.R., P.A.R. and P.D.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Fundação para a Ciência e a Tecnologia (FCT-MCTES), Radiation Biology and Biophysics Doctoral Training Programme (RaBBiT, PD/00193/2012), the Applied Molecular Biosciences Unit—UCIBIO (UIDB/04378/2020), the CEFITEC Unit (UIDB/00068/2020), UIDB/04559/2020 (LIBPhys) and UIDP/04559/2020 (LIBPhys) and the scholarship grant number PD/BD/142829/2018, to T.P. Pivetta from the RaBBiT Doctoral Training Programme. C.E.A. Botteon and P.D. Marcato acknowledge the funding from the São Paulo State Research Support Foundation (FAPESP) (grants #2018/13465-5 and #2017/04138-8), the Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq, grants #465687/2014-8).

**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 cited author.

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

#### **References**

