Research Progress of Nanomedicine-Based Mild Photothermal Therapy in Tumor

Introduction

No more words are needed to emphasize the harm brought to people by tumors, and the traditional treatments like surgery, chemotherapy, and radiotherapy are still unable to improve the prognosis of patients effectively due to various contraindications, serious adverse reactions, and other limitations.1–3 There is an urgent need for a novel therapeutic modality for Cancer. Fortunately, with the rapid development of nanotechnology in recent years, nanomedicine has gradually entered people’s vision. Photothermal therapy (PTT), as an important part of nanomedicine, is a new effective tumor treatment method that makes use of photothermal agents (PTAs) to transform light energy into heat energy to kill tumor cells under the irradiation of external light sources, especially near-infrared light (NIR).4,5 PTT has been broadly applied in tumor therapy research due to the spatiotemporal controllability, non-invasiveness, and minimal side effects compared with traditional tumor treatments.4,5 The heat produced by PTAs is the key factor of PTT and directly determines the therapeutic effect of tumor as well as some biological reactions, and PTT is divided into conventional PTT and mild PTT (mPTT) according to temperature. As for conventional PTT, high local temperature (>50°C) is used to completely ablate the tumor tissues mainly through necrosis.6 However, such excessive hyperthermia inevitably damages the surrounding normal tissues by non-specific heat diffusion, and the necrosis induced by PTT will cause severe local inflammation which may further impair normal tissues and even increase the risk of tumor metastasis.7 Moreover, it has been proved that the host antitumor immunity is also suppressed attributed to the lesion of immune antigens at an overheating tumor microenvironment (TME) during PTT,8 which is extremely unfavorable for clinical translation as the fact that the antitumor immune response plays an important role in inhibiting the metastasis and recurrence of tumors.9 Therefore, mPTT at a relatively low temperature (usually 42–45°C) has been more and more widely used in recent years to avoid the drawbacks mentioned above.

Compared to the high-temperature PTT, mPTT produces negligible side effects by inducing apoptosis rather than necrosis while treating tumors, and the non-specific heat diffusion generated by such a relatively low temperature can be inconsiderable.10 In addition, the biological effects of mPTT in favor of treatment make it more promising in tumor therapy. For instance, mPTT is able to loosen the dense structure in tumor tissues, enhance blood perfusion, and alleviate the hypoxic microenvironment, which can increase the infiltration of immune cells, improve the delivery of antitumor drugs, and boost the generation of reactive oxygen species (ROS), respectively.11–13 However, mPTT utilizes low temperature that rarely causes harm to normal tissues, but the therapeutic efficacy is largely discounted and cannot ablate tumors as effectively as harsh PTT (hPTT). For this reason, extensive researches have been conducted to investigate how to improve the efficacy of mPTT in tumor therapy, mainly consisting of three aspects: (1) improving the property of PTAs and constructing multifunctional nanosystems, (2) taking mPTT as the leading role to maximize its effect by blocking the cell defense mechanisms, and (3) regarding mPTT as a supporting role to assist other therapies to achieve synergistic antitumor curative effect.

Since mPTT is a nanomedicine-based therapeutic method, the performance of nanomaterials directly determines the application prospects of mPTT. Researches on PTAs have emerged endlessly in recent years, which can be categorized into inorganic PTAs and organic PTAs. Inorganic PTAs have the advantages of excellent stability and high photothermal conversion efficiency (PCE), mainly comprising precious metals,14,15 transition metals,16 and some carbon-based nanomaterials,17,18 while organic PTAs have the advantages of good biosafety and biodegradability, mainly including cyanine dyes,19,20 conjugated polymers and polyaniline nanomaterials.21 Whether inorganic PTAs or organic PTAs, the most basic but the most significant point is that they are supposed to possess extremely strong NIR absorption capacity and superb PCE, by reason of only with these features can lower quality NIR lasers and lower drug concentrations are employed during mPTT, as milder conditions undoubtedly improve the biosafety of therapy. In addition to developing novel PTAs with excellent PCE, researchers have issued a wide variety of multifunctional nanosystems to optimize the application of mPTT in tumor treatment. For instance, PTAs are endowed with the ability of active targeting to enhance accumulation in tumor tissues by being wrapped with biomimetic nanomaterials like cancer cell hybrid membranes.22 Furthermore, the breakthrough in nanomaterials allows mPTT to apply laser radiation in the NIR-II window (1000–1350nm, especially 1064nm) with deeper penetration, higher maximum exposure to the skin, and lower signal-to-noise ratio than initial NIR-I window (750–1000nm, especially 808nm).23,24 Last but not the least, imaging capabilities such as fluorescence imaging (FLI), Ultrasound (US), photoacoustic imaging (PAI), computed tomography (CT), and magnetic resonance imaging (MRI), also provide mPTT with favorable information for tumor diagnosis and treatment. For instance, the biodistribution of nanodrugs can be detected by imaging techniques, which identify tumors to provide diagnostic information, help determine the optimal starting irradiation time to obtain the maximum utilization of nanocomposites, and monitor the treatment process and outcomes. Each imaging modality has its own outstanding advantages. FLI has superior sensitivity and US can realize real-time monitoring of deep tissue, while PAI and MRI have excellent spatial resolution.

Although great efforts have been put into promoting mPTT by optimizing the performance of PTAs, mPTT monotherapy is still unable to fully eradicate tumors owing to the existence of a series of defense mechanisms of tumor cells. On the one hand, heat shock proteins (HSPs), a sort of molecular chaperone that involves in the process of repairing misfolded or denatured protein, are elevated to weaken the heat injury during mPTT when the tissue temperature rises to 41°C.25 On the other hand, autophagy is activated under heat pressure and able to provide nutrition and energy for cell regeneration by degrading misfolded proteins and organelles, ultimately playing a protective role to resist mPTT.26 Therefore, various strategies and multifunctional nanosystems have been meticulously designed to reduce HSPs expression or regulate the autophagy process to maximize mPTT efficacy. Besides, the combinations of mPTT with other therapies have also shown great clinical transformation value in tumor treatment due to its outstanding regulation ability and unique biological effects. Mild hyperthermia can realize controlled release and enhanced internalization of agents for combined application with chemotherapy and gene therapy, alleviate tumor hypoxia and increase ROS production for combined application with radiotherapy (RT), photodynamic therapy (PDT), and sonodynamic therapy (SDT), catalyze Fenton/Fenton-like reactions for combined application with chemodynamic therapy (CDT), decompose azo initiator to generate oxygen-independent alkyl radicals (R) for combined application with thermodynamic therapy (TDT), and convert “cold” tumors into “hot” tumors to improve immunosuppressive microenvironment for combined application with immunotherapy.

In this review, we extensively summarize the latest advances in the therapeutic applications of mPTT in cancer, mainly including two sections: (1) taking mPTT as a leading role to maximize its effect by blocking the cell defense mechanisms, and (2) regarding mPTT as a supporting role to assist other therapies to achieve synergistic antitumor curative effect (Figure 1). Meanwhile, we also summarize the special characteristics and imaging capabilities of nanoplatforms applied in various therapies. At last, we put forward the bottlenecks and challenges in the current research path of mPTT, and at the same time, possible solutions and research directions in future are proposed correspondingly.

Figure 1 Schematic illustration of mPTT playing a leading role or a supporting role in tumor therapy.

mPTT Plays a Leading Role in Tumor Therapy

Unlike hPTT at an overheating temperature, which rudely degrades all the protective proteins produced by tumors under heat stress, mPTT cannot bypass the stress protector mechanisms at a relatively mild temperature, and therefore the efficacy is largely diminished. Among defensive mechanisms, HSPs and autophagy play an important role in keeping tumors from heat injury. Primarily, heat-induced misfolding and dysfunctional proteins are prevented from aggregating by activated HSPs, and subsequently, HSPs can restore the function of impaired proteins, then repair the thermal damage.27,28 In addition to HSPs, autophagy also plays a critical role in protecting cells from stress and various kinds of tumor treatments. Autophagy, as a highly conserved catabolic process and an important mechanism for maintaining intracellular homeostasis, exists in most cells, and autophagy mainly includes three steps: autophagosome formation, fusion with lysosome, and autolysosome degradation.29,30 Once the progression of autophagy proceeds smoothly, the degradation of autolysosome which engulfed damaged organelles and dysfunctional proteins provides nutrition and energy for cell regeneration,31 resulting in the survival of cancer cells and contributing to the inefficiency of a broad range of tumor treatment methods. As a result of HSPs and autophagy, cancer cells are able to escape heat injury and be highly resistant to therapies. To this end, studies on potentiating mPTT by inhibiting HSPs and manipulating autophagic process have been widely reported, including initial drug treatment and various novel strategies in the latest years. Of note, whether it is the inhibition of HSPs or the regulation of autophagy process, mPTT is always the key factor to kill tumor cells, so we call it the leading role in this part.

mPTT Plays a Leading Role Through HSPs Inhibition

Based on their molecular mass, HSPs are usually classified into small HSPs (HSPB, molecular mass < 40 kDa), HSP40 (DNAJ), HSP60 (HSPD), HSP70 (HSPA), HSP90 (HSPC), and HSP110 (HSPH).32,33 It has been demonstrated that HSPs with different molecular mass not only carry out many of the protein chaperone functions independently, but also collaborate on other protein structure-modifying functions. HSP70 involves in the early stage of folding and is related to a wide range of protein regions, while HSP90 recognizes specific conformations, and HSP60 promotes the folding, unfolding, and degradation of mitochondrial proteins.34,35 These three types of HSPs are currently the most extensively studied among all HSPs due to their thermal resistance and antitumor apoptotic properties. Recently, heat resistance has been overcome by a variety of HSPs inhibition strategies based on the rapid development of nanomedicine, mainly including HSPs inhibitors-mediated HSPs inhibition, ATP-mediated HSPs inhibition, gas-mediated HSPs inhibition, and gene editing-mediated HSPs inhibition (Table 1).

Table 1 mPTT Plays a Leading Role Through HSPs Inhibition

HSPs Inhibitors-Mediated HSPs Inhibition

Up to today, researchers have exploited a variety of inhibitors to directly target different types of HSPs. Among them, VO2+, which can be transformed from vanadium oxide under a mildly acidic condition (pH 6.5–6.9), is able to inhibit the expression of HSP60.63 For example, Zhao et al ingeniously developed a PEG-modified virus-like silicon oxide nanoplatform (SiOX/CeO2/VOX, SCV-PEG) loading CeO2 and VOX simultaneously to achieve VO2+- mediated HSP60 inhibition for enhancing mPTT (Figure 2a).36 Virus-like structures could load more drugs and transfer them into cells due to a large specific surface area, which facilitated the cell adhesion and phagocytosis of nanodrugs in tumors,64 and SiOX was able to maintain the local temperature at 45°C under a 1064nm laser excitation in the NIR-II window. In addition, VO2+ converted from VOX significantly inhibited the expression of HSP60 at a mild acidic TME. Accordingly, immunofluorescence staining assay results showed that the expression level of HSP60 in SCV-PEG+NIR group was obviously lowered than any other group. At the same time, SCV-PEG could also serve as a peroxidase(POD) due to the existence of CeO2 capable of reacting with hydrogen peroxide (H2O2) to generate hydroxyl radical (·OH). Synergistic with nanozyme catalytic therapy, SCV-PEG+NIR exhibited the highest tumor-killing efficiency in vitro and the smallest tumor volume in vivo, which resulted from the down-regulation of HSP60 that magnified the curative effect of mPTT and the production of toxic free radicals. Similarly, a novel VO2-ICG nanocomposite fabricated by Wang et al also realized the degradation of VO2-ICG under weak acidic TME and downregulated the expression of HSP60 to enhance photothermal tumor ablation.37 Moreover, indocyanine green (ICG), a US Food and Drug Administration (FDA)-approved small molecular chromophor, not only endowed the nanocomposite with the ability of photothermal conversion but also the capacity of FLI to achieve imaging-guided enhanced mPTT.65

Figure 2 (a) Schematic depicting the synthesis of virus-like SCV-PEG and its application in VO2+-mediated HSP60 inhibition for enhancing mPTT. Reprinted with permission from Zhao R, Zhang R, Feng L, et al. Constructing virus-like SiO/CeO/VO nanozymes for 1064 nm light-triggered mild-temperature photothermal therapy and nanozyme catalytic therapy. Nanoscale. 2022;14(2):361–372.36 Copyright 2022 Royal Society of Chemistry. (b) Schematic illustrating the temperature-mediated precise management of HSPs expression, where 47°C with the least HSP70 expression was applied for mPTT. Reprinted from Colloids Surf B Biointerfaces, 217, Xia Y, Li C, Cao J, et al. Liposome-templated gold nanoparticles for precisely temperature-controlled photothermal therapy based on heat shock protein expression. 112686, Copyright 2022, with permission from Elsevier.39 (c) Schematic showing the synthesis of [email protected] and its application for multimodal imaging-guided and multimodal-targeted mPTT. Reprinted from J Control Release, 347, Shu X, Chen Y, Yan P, et al. Biomimetic nanoparticles for effective mild temperature photothermal therapy and multimodal imaging. 270–281, Copyright 2022, with permission from Elsevier.55 (d) Synthesis of the nanoplatform and its application for multimodal combination therapy. Reprinted with permission from Cao J, Qiao B, Luo Y, et al. A multimodal imaging-guided nanoreactor for cooperative combination of tumor starvation and multiple mechanism-enhanced mild temperature phototherapy. Biomater Sci. 2020;8(23):6561–6578.53 Copyright 2020 Royal Society of Chemistry.

Besides HSP60 inhibitors, HSP70 inhibitors are also wildly employed in mPTT for accomplishing satisfactory tumor treatment outcomes. Although researchers have demonstrated that HSPs expressions are decided by temperature, where the maximum expression content is reached at 43 °C for HSP70 and 45 °C for HSP90 in tumor cells, the correlation between HSPs expression and PTT temperature in vivo remains unclear.66 In order to realize the precise management of HSPs expression, Xia et al made use of liposome-templated gold nanoparticles which had low-temperature fluctuation (smaller than 1°C) to probe the expression of HSP70 and HSP90 versus temperature (42,45,47,50,55°C) in 4T1 tumor-bearing Bal/b mice (Figure 2b).39 Quantitative real time PCR analysis showed that the maximum expression of HSP70 and HSP90 were at 42°C and 47°C(3.9-fold and 2.3-fold compared to PBS group), similar to the in vitro results of Mantso’s work,66 while the minimal expression of HSP70 and HSP90 were both almost at 55°C(0.5-fold compared to PBS group). Interestingly, it found that the expression of HSP70 was more susceptible to temperature changes than HSP90, so the inhibition of HSP70 might be more beneficial to tumor treatment. Despite the lowest HSP70 expression and the best therapeutic efficacy at 55°C, it led to severe tumor necrosis and inflammation. Thus, the second lowest HSP70 expression, 47°C, was selected as the optimal temperature to combine 2-Phenylethynesulfonamide (PES, an HSP70 inhibitor) for enhancing mPTT, since lower expression of HSP70 meant less heat resistance and lower concentration of HSP inhibitors. Undoubtedly, the results showed that the introduction of PES greatly elevated the therapeutic outcome at a mild temperature. Such an accurate temperature-controlled mPTT based on the expression of HSPs strategy opened a new field of vision for tumor ablation.

Compared to HSP60 and HSP70 inhibitors, there is a greater variety of HSP90 inhibitors applied to mPTT. Among them, gambogic acid (GA) has almost occupied the largest proportion of HSP90 inhibitors in recent years’ researches. For instance, Sun et al designed a nanoagent (COF-GA) for enhanced PTT against cancer at low temperature.25 Covalent organic frameworks (COFs), a novel type of porous materials composing of molecules linked by covalent bonds, had shown great potential in bioMedical field because of their long-range ordered structure with large and regular pores and components of only light atoms (such as C, H, O, N and B, etc.), which endowed them with high drug loading capacity and excellent biocompatibility.67–70 The fabricated COF-GA nanoagent exerted a low-temperature (about 44°C) PTT on 4T1 tumor-bearing Balb/C mice upon 635nm NIR laser irradiation. After two weeks post treatment, the relative tumor volumes were obviously inhibited while neither the body weights of treated mice nor the serum biochemical measurements (ALT, AST, CRER, URER) significantly changed during the observation period, demonstrating excellent antitumor efficacy and negligible nonspecific thermal damage of this enhanced mPTT strategy and broadening our vision towards the different applications of COFs in biomedicine field. Apart from GA, other types of HSP90 inhibitors applied in mPTT had also achieved satisfactory cancer treatment outcomes. Zhang et al synthesized a mitochondria-targeted photosensitizer consisting of IR780 iodide and BIIB021 (an HSP90 inhibitor).46 After selectively aggregating on mitochondria, the prepared PEG-IR780-BIIB021 released BIIB021 under 808nm laser irradiation to increase tumor heat sensitivity, which could reduce the mitochondrial membrane potential and induce the release of key intrinsic apoptotic factors (Cyt-C, Caspase-9, Bcl-2, and Bax) to activate the mitochondrial apoptotic pathway. In another example, Jiang group loaded phenolic epigallocatechin 3-gallate (EGCG, an HSP90 inhibitor) into the flower-like NiS2 coated-NaLuF4:Nd nanoparticle (Lu:[email protected]2).50 This novel therapeutic agent (Lu:[email protected]2-EGCG) achieved remarkable antitumor efficacy under the dual-mode imaging guidance of short-wave infrared light imaging and MRI. In addition, 17-allylamino-geldanamycin (17-AAG) and its second-generation derivative 17-dimethylamino-ethylamino-17-demethoxydeldanamycin (17-DMAG) had been proved to inhibit HSP90 expression for enhancing mPTT.48,49

ATP-Mediated HSPs Inhibition

Since the expression, activation, and synthesis of HSPs primarily rely on intracellular adenosine triphosphate (ATP) levels, preventing intracellular ATP synthesis may assist to combat HSPs-dependent tumor heat tolerance and enhance the effectiveness of PTT at low temperature.71,72 According to the Warburg effect, ATP produced by tumor cells occurs mainly through the glycolytic pathway rather than the oxidative phosphorylation pathway, even in the presence of sufficient oxygen, thus interfering with the process of glycolysis and reducing the level of glucose within the tumor are effective strategies to reduce ATP production.73,74 Inspired by this, Shu et al loaded lonidamine (LN, a glycolysis inhibitor) and DL-menthol (DLM) into hollow mesoporous Prussian blue nanoparticles (HPB NPs) (Figure 2c).55 DLM had the property of controllable solid-liquid-gas (SLG) triphasic change because of its boiling point of 212°C and melting point of 32–36°C, which made it not only a blocker agent of LN to avoid its early leakage, but also a US imaging agent.75,76 After 793nm laser irradiation, the heat produced by HPB NPs was able to induce DLM vaporization and boost LN release, which could be observed by US imaging in real time. Moreover, the coating of 4T1 cancer cell membrane (CCM) endowed nanoparticles with the ability of homologous targeting and better biocompatibility. In conclusion, under the guidance of PAI and US imaging, [email protected] NPs could specifically target tumors and decrease the level of intracellular ATP, thereby simultaneously reducing the expression of HSP70 and HSP90, so as to achieve enhanced mPTT.

Similarly, glucose oxidase (GOx), which can convert glucose into gluconic acid and H2O2, has been broadly applied in mPTT for tumors due to its property capable of reducing the level of glucose in tumor, which subsequently decreases the production of ATP and downregulates the expression of HSPs.77–79 For instance, Cao et al elaborated a nanoreactor (Fe-PDAP/GOx/ICG) that simultaneously loaded GOx and ICG into Fe-doped polydiaminopyridine (Fe-PDAP) (Figure 2d).53 As a nanozyme with intrinsic catalase-like activity, Fe-PDAP could not only convert H2O2 generated from GOx-mediated glucose decomposition and naturally possessed H2O2 within tumors to oxygen, but also consumed glutathione (GSH, ROS scavenger) via metal reduction reaction after releasing Fe3+ to sensitize the ICG-mediated PDT. Meanwhile, the ICG-mediated mPTT could also be promoted attributed to the reduction of glucose and ATP as well as the decreased expression of HSPs. Moreover, Fe-PDAP/GOx/ICG nanoreactor had shown superior performance in imaging to simultaneously achieve FLI/PAI/MRI.

In addition to glucose-regulation-mediated downregulation of ATP, mitochondria, known as the factory of ATP production, plays an important role in ATP-dependent HSPs production, therefore interfering with the function of mitochondrial can also be used as a strategy to downregulate HSPs. Qi et al reported for the first time that biomimetic mesoporous Fe-SAzyme could realize mitochondrial damage-mediated mPTT (Figure 3a).56 Although single-atom nanozyme (SAzyme) based on carbon material had emerged remarkable potential in cancer therapy and antibacterial therapy due to their distributed metallic architecture, which enabled SAzyme to maximize the usage of metal catalytic sites, the hard surface modification of carbon-based materials made it difficult for SAzyme to achieve tumor targeting.80–83 For this reason, platelet membrane (PM) was coated onto the surface of Fe-SAzyme to attain tumor-targeting ability attributable to the tendency of PM to vascular injury areas and abnormal vascular structures in tumor tissue.84,85 After specific aggregation to tumor tissue, PM-coated Fe-SAzyme (PMS) with POD activity could catalyze H2O2 to produce·OH, causing mitochondrial damage and down regulating HSPs, thereby enhancing mPTT under NIR-II excitation (Figure 3b and 3c). In another example of SAzyme, Chang et al added glutathione oxidase (GSHOx) activity on Pd-SAzyme with POD activity to induce ferroptosis that could produce a large number of lipid peroxides (LPO) to cleave HSPs, firstly achieving ferroptosis-boosted mPTT based on SAzyme.15 These findings provided new insights into the current application of single-atom nanozyme in mPTT.

Figure 3 (a) Schematic showing the synthesis of PMS and its working mechanisms in mPTT. (b) DCF fluorescence and JC-1 fluorescence intensity after the indicated treatments. (c) Western blot analysis of HSP90 after treatments. (a–c) Reprinted with permission from Qi P, Zhang J, Bao Z, et al. Nanozyme for mitochondrial damage-mediated mild-temperature photothermal therapy. ACS Appl Mater Interfaces. 2022;14(17):19081–19090.56 Copyright 2022 American Chemical Society. (d) Schematic illustration of the synthesis of mPEG (CO). (e) Schematic depicting the nanobomb consisting of mPEG (CO) and PBPTV. (f) Schematic illustration of the working mechanisms of nanobomb. (g) The UV spectrum shift exhibits the release of CO into the aqueous nanobomb solution measured by reduced hemoglobin. (d–g) Reprinted with permission from Ma G, Liu Z, Zhu C, et al. Responsive NIR-II AIE nanobomb for carbon monoxide boosting low-temperature photothermal therapy. Angew Chem Int Ed Engl. 2022;61(36):e202207213. © 2022 Wiley-VCH GmbH.62

Gas-Mediated HSPs Inhibition

In the latest research, gas-mediated HSPs downregulation which employs therapeutic auxiliary gases (such as CO, H2, NO, etc.) or their precursor medications to inhibit the expression of HSPs has received a lot of interest in low-temperature PTT. For instance, Ma et al established a nanobomb ([email protected](CO)) based on the self-assembly of NIR-II AIE polymer PBPTV and carbon monoxide (CO) storage polymer mPEG, which could be destroyed by the excessive H2O2 in TME to release CO and suppressed the overexpression of HSPs (Figure 3d–f).62 In order to assess the CO release ability of the nanobomb, they used reduced hemoglobin (Hb) for measuring CO content that could be calculated in the light of the absorption peak value of reduced Hb and HbCO (Figure 3g).86 In a simulated environment of TME, the nanobomb released CO vigorously at the speed of 1494/s, while barely released in the absence of H2O2. Western blot analysis proved that the expression of HSP70 was significantly inhibited by CO under a low temperature (43°C). With the guidance of FLI, this nanobomb exhibited excellent tumor inhibition effect both in vivo and in vitro.

Equally, H2 has been used to reduce the expression of HSPs to optimize mPTT. Different from the previous example, Ge group took advantage of the hot electron transfer property to produce H2 in TME to damage mitochondria instead of carrying gas from the outside, thus reducing the expression of HSPs (Figure 4a).61 The developed gold nanorod/titanium dioxide lollipop-like nanoparticle(L-TiO2-GNR) could generate abundant hot electrons on the GNRs under irradiation at 808nm. On the one hand, some of the electrons generated on the GNRs were able to raise local temperature to 48°C to produce mPTT effect on tumors (Figure 4b). On the other hand, the rest electrons could yield ·OH, 1O2, and H2 by irreversibly injecting into the conduction band of TiO2 through the Schottky junction, thus inducing PDT effect and the simultaneous production of H2 was capable of reducing the expression of HSPs (Figure 4b). Further, the introduction of Nile blue derivative ENBS and PEG enabled nanoparticles to have FLI ability and higher biocompatibility. Therefore, this [email protected]2-GNR nanoparticle which had synergistic PDT, mPTT, and H2 therapy only with the support of NIR irradiation opened a new window for gas-enhanced mPTT. Besides CO and H2, NO also played a role in gas-mediated HSPs degradation. A mitochondria-targeting CuS-PEI/NO-TPP nanoplatform that combined mPTT and NO gas treatment, two distinct therapeutic modalities, in a single nanomaterial to synergistically improve cancer therapy was described by Wu et al60 Under 1064 nm laser irradiation, NO released from nanomaterial could interfere with the function of mitochondria to decrease ATP production, causing the suppression of HSP90 and enhancing mPTT.

Figure 4 (a) Schematic showing the construction of [email protected]2-GNR nanoparticle and its application in synergistic PDT, mPTT, and H2 therapy. (b) The mechanism of ROS generation and photothermal effect by [email protected]2-GNR nanoparticle upon NIR irradiation. (a and b) Reprinted with permission from Ge H, Du J, Long S, et al. Near-Infrared Light Triggered H Generation for Enhanced Photothermal/Photodynamic Therapy against Hypoxic Tumor. Adv Healthc Mater. 2022;11(3):e2101449. © 2021 Wiley-VCH GmbH.61 (c) Schematic illustrating the synthesis of PP-NG and its working mechanisms in combination therapy. (d) Western blot analysis of HSP70 and Caspase-3 after the indicated treatments. (c and d) Reprinted from Biomaterials, 245, Ding F, Gao X, Huang X, et al. Polydopamine-coated nucleic acid nanogel for siRNA-mediated low-temperature photothermal therapy. 119976, Copyright 2022, with permission from Elsevier.57 (e) The synthesis of Cas9/sgHSP90α and its application for gene editing and mPTT combination therapy. Reprinted with permission from Li X, Pan Y, Chen C, et al. Hypoxia-responsive gene editing to reduce tumor thermal tolerance for mild-photothermal therapy. Angew Chem Int Ed Engl. 2021;60(39):21200–21204. © 2021 Wiley-VCH GmbH.58

Gene Editing-Mediated HSPs Inhibition

Gene editing-mediated HSPs down-regulation by silencing HSPs at the genomic level which mainly includes small interfering RNA (siRNA) and the clustered regularly interspaced short palindromic repeats-associated protein 9 system (CRISPR-Cas9) has become an indispensable part of mPTT. However, one of the biggest challenges is that the exposed siRNA will be degraded by various biological molecules such as nuclease, which increases the difficulty of its application. For this purpose, Ding et al elaborately designed a PEGylated PDA-coated siRNA-bearing nucleic acid nanogel (PEG-PDA-NG) with three shields (Figure 4c).57 Under the protection of three-layered barriers comprising nucleic acid nanogel, PDA, and PEG, siRNA could be free from the degradation of lysosomes and effectively silenced HSP70. Under 808nm laser irradiation, the secondary barrier PDA also functioned as a PTA to realize mPTT. The Western blot results showed that the expression of HSP70 in PEG-PDA-NG+NIR group was significantly reduced compared with the rest groups, along with a remarkable increase level in caspase-3 (a representative protein of apoptosis) (Figure 4d). This work gave a classic example of the realization of gene-mediated mPTT while also providing an easily scalable and applicable nano-delivery platform that could be applied as a vehicle for other combination therapies.

In the recent development of gene editing technologies, CRISPR-Cas9 composing of Cas9 nuclease and single-guide RNA (sgRNA) undoubtedly occupies the most important position and has been increasingly applied in cancer research because of its high specificity, easy operation, well efficiency, and simultaneous silencing of multiple targets.87 Li et al constructed a hypoxia-responsive nanoplatform by connecting CRISPR-Cas9 system with Au nanorods through azobenzene-4, 4’dicarboxylic acid (P-AZO, a hypoxia-responsive azobenzene linker) to achieve Cas9-mediated mPTT (Figure 4e).58 Under normal partial pressure of oxygen, CRISPR-Cas9 system was covalently cross-linked to Au nanorods through P-AZO, but by reaching the tumor area, a hypoxic microenvironment, the N-N double bond of P-AZO would be cleaved, causing the release of Cas9/sgRNA ribonucleoprotein mixture (Cas9 ribonucleoprotein, Cas9 RNP) and specifically knocking down Hsp90α (a subunit of Hsp90). Similarly, Chen et al took another approach to regulate the release of Cas9 RNP.59 According to the principle of base complementary pairing, Cas9 RNP was assembled with Thiol-modified DNA (DNA-SH) fragments which were conjugated to CuS NPs. Under 808nm light irradiation, heat introduced by CuS could break the double chain and promote the release of Cas9 RNP to knock out Hsp90α. Both studies, although utilizing different modes of regulation, were successful in achieving Cas9-mediated mPTT by specifically knocking down Hsp90α.

In general, various approaches of inhibiting HSPs mentioned above to sensitize mPTT all achieved favorable efficacy. We believe that there is no best but only the most suitable strategy to down regulate the expression of HSPs, which mainly benefits from the rapid development of nanotechnology which allows the drawbacks of different therapies to be addressed one by one. For instance, nanoplatform can realize non-targeted drugs to reach tumor tissue specifically, make gas-mediated therapy possible, and prevent nucleic acid from being degraded by biomolecules. Therefore, it is undoubtedly the wisest to select a strategy for HSPs inhibition based on the unique properties of the nanoplatform.

mPTT Plays a Leading Role Through Autophagy Regulation

Autophagy, a highly conserved catabolic process and an important mechanism for maintaining intracellular homeostasis, has been argued as a double-edged sword in tumorigenesis.29,30 Autophagy can lead to the survival of tumor cells and the inefficiency of a wide range of tumor therapeutic approaches by alleviating cellular stress, but autophagy, in turn, can limit tumorigenesis through multiple pathways.31,88 The fact is, in recent years of researches on tumor therapy, autophagy mostly plays a protective role (Table 2). The inhibition of autophagy has therefore emerged as an effective approach to enhance mPTT.

Table 2 mPTT Plays a Leading Role Through Autophagy Regulation

All kinds of novel nanoplatforms have been designed to block the process of autophagy to potentiate mPTT, where the targeted delivery of autophagy inhibitors is initial and the most typical method. Among them, chloroquine (CQ), which can inhibit the fusion of autophagosome with lysosome to block autophagic progression, is widely used to enhance mPTT. Shi et al applied poly(lactic-co-glycolic acid) (PLGA) as a carrier to load IR820 (PTA), ZnCdSe/ZnS quantum dots (QDs, FLI agent), and CQ to afford a multifunctional nanoplatform.26 Under the mild acidic TME, the developed PLGA/IR820/QDs/CQ nanoparticles (PIFC NPs) would be biodegraded and released CQ that could greatly reduce degradation of autolysosome and effectively prevent damaged cells from repairing. With 808nm laser irradiation, the PIFC NPs showed enhanced tumor-killing effect both in vitro and in vivo under a relatively low temperature(45°C). At the same time, the PIFC NPs achieved excellent real-time FLI capability attributed to the introduction of inorganic fluorescent emitter QDs, which had stronger fluorescence intensity and higher light stability compared with organic fluorescent dyes.95

As research continues to deepen, methods to inhibit autophagy are being gradually excavated. Nanodiamonds (NDs), known as a kind of nanocarrier possessing numerous advantages like large specific surface area, have been reported to serve as an autophagy inhibitor by blocking the NUPR1-mediated autophagy process.96–98 NUPR1 (nuclear protein 1), a transcriptional coregulator, is aberrantly expressed in tumor cells and dramatically induced upon stress conditions.99 Since NUPR1 involves in regulating the late stage of autolysosome processing, it may downregulate autophagic flux and reduce degradation of autolysosome by depleting NUPR1.99 Encouraged by the unique performance of NDs, Cui et al formulated a multifunctional NDs-based nanoplatform consisting of ICG, apoptozole (APZ, an HSP70 inhibitor), and doxorubicin (DOX) through a layer-by-layer self-assembly approach (Figure 5a).92 This nanoplatform achieved robust antitumor efficacy by using two strategies to simultaneously increase the sensitivity of tumor cells to the combination therapy of mPTT/chemotherapy. Firstly, APZ enhanced mPTT by reducing the expression of HSP70. Secondly, NDs-mediated inhibition of autophagy further increased the efficacy of mPTT while reducing the resistance of tumor cells to chemotherapy agent. This work proved that autophagy could be blocked not only by specific autophagy inhibitors, but also by unique nanocarriers, thus realizing enhanced mPTT.

Figure 5 (a) Schematic showing the synthesis of NDs-based nanoplatform and its application for multimodal imaging-guided combination therapy. Reprinted with permission from Cui X, Liang Z, Lu J, et al. A multifunctional nanodiamond-based nanoplatform for the enhanced mild-temperature photothermal/chemo combination therapy of triple negative breast cancer via an autophagy regulation strategy. Nanoscale. 2021;13(31):13375–13389.92 Copyright 2021 Royal Society of Chemistry. (b) The synthesis of BNN-Bi2S3 NPs and their working mechanisms for enhanced mPTT. (c) Western blot analysis of p62 and LC3-I/LC3-II was examined to evaluate autophagic flux after the indicated treatments. (d) Flow cytometry was examined to determine the percentages of apoptosis with different treatments. (b–d) Reproduced from Zhang X, Du J, Guo Z et al Efficient Near Infrared Light Triggered Nitric Oxide Release Nanocomposites for Sensitizing Mild Photothermal Therapy. Adv Sci (Weinh). 2019;6(3):1801122. © 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.93

Apart from specific autophagy inhibitors and nanocarriers, gas like NO has also been demonstrated to inhibit the progression of autophagy and optimize mPTT. Zhang et al synthesized a heat-triggered NO controlled release nanocomposites based on bismuth sulfide (Bi2S3) nanoparticles loading hydrophobic NO donor, N, N′-Di-sec-butylN,N′-dinitroso-1,4-phenylenediamine (BNN) (Figure 5b).93 Compared with Bi2S3 NPs, BNN-Bi2S3 NPs with NO assistance showed a stronger tumor inhibition effect under mPTT. Besides the partial toxic effect of NO itself, the author further explored the in-depth molecular mechanism to explain this synergistic effect. Hence, autophagy inducer rapamycin (Rapa) and inhibitor 3-methyladenine (3-MA) were used to analogize the role of NO in autophagy regulation under a mild temperature. The results demonstrated that Rapa could relieve tumor cell death, whereas 3-MA, like NO, could promote tumor cell death during the mPTT. Subsequently, Western blot and flow cytometry further validated that NO could inhibit the progression of autophagy to promote tumor cell apoptosis (Figure 5c and d). This study for the first time reported NO-sensitized mPTT and provided a new strategy for autophagy inhibition.

In contrast to the above examples, activation of autophagy has been similarly exploited to augment mPTT for tumor therapy. For example, Zhou et al constructed a multifunctional nanoparticle (PPBR) with autophagy activation characteristic, which consisted of PEGylated PDA modified by Arg-Gly-Asp (RGD) peptides and beclin-1 peptide.94 The modification of RGD peptides which could specifically bind overexpressed αvβ3 integrin receptor on the tumor cell surface enabled PPBR to selectively aggregate in tumor, and the modification of beclin-1 peptide could activate the beclin-1-involved the pathway of autophagic cell death.100 As a result, the efficacy of mPTT was significantly improved with the assistance of beclin-1-induced autophagy.

Collectively, since autophagy acts as a double-edged sword in the progression of tumorigenesis, the priority is to identify the relationship between autophagy and mPTT, although it mainly plays a protective role. The examples mentioned above demonstrated that both inhibition of protective autophagy and promotion of pro-death autophagy were effective in potentiating mPTT.

mPTT Plays a Supporting Role in Tumor Therapy

Although inhibiting the expression of HSPs and regulating the progression of autophagy can significantly increase the efficacy of single-mode mPTT by reducing the resistance of tumor cells to stress, it still has some limitations. Researchers have been plagued by the conundrum of uneven distribution of heat inside tumors, and the limited penetration ability of NIR fails to effectively cover deep tumor tissues. Residual tumor cells located at the edge and deep part of the tumor tissues inevitably result in tumor recurrence and metastasis. Furthermore, although the enhanced mPTT has exhibited powerful therapeutic effect on local lesions, it has little efficacy on distant tumors and cannot reduce the risk of tumor metastasis either. There is a desperate need to seek out suitable strategies to deal with the above problems. Fortunately, with the continuous efforts of researchers, the above-mentioned issues have been addressed by the combination therapy of mPTT with other therapies, including chemotherapy, gene therapy, radical-based therapy, and immunotherapy. Of note, besides the weak tumor-killing effect, mPTT mainly plays an adjuvant role to enhance the efficacy of the combination therapy when it is synergistic with other therapies, so we call it a supporting role in this part, and the synergistic mechanisms will be discussed in the corresponding section.

mPTT Plays a Supporting Role in Synergizing with Chemotherapy

Chemotherapy, as one of the three traditional therapies for cancer, has a pivotal position in clinical application, however, its efficacy is still unsatisfactory due to serious systemic side effects and drug resistance. Thanks to the rapid development of nanotechnology, mPTT based on nanomedicine has raised the application of chemotherapy in tumors to a new level, mainly from the following aspects. (1) Most chemotherapeutics suffer from several disadvantages such as low solubility, low bioavailability, and non-targeting ability, causing severe side effects.101–103 Compared with free chemotherapeutic agents, nanocarrier delivery systems can achieve a longer half-life and lower systemic toxicity.104 Meanwhile, precision medicine is made possible under the auspices of passive targeting by enhanced permeability and retention (EPR) effect as well as active targeting by artificial modification. (2) Owing to the presence of distorted tumor vessels and dense extracellular matrix (ECM), nanocarriers are usually located at the edge of the tumor after reaching, which terribly compromises the therapeutic efficacy.10 It has been well recognized that mild hyperthermia can destroy the dense ECM, improve the blood fluid of tumor tissue and the permeability of tumor blood vessels, and increase the permeability and fluidity of tumor cell membranes to assist the intracellular uptake of drugs.105,106 At the same time, the chemical bonds or physical interactions between chemotherapeutic agents and nanocarriers will be disrupted by mild hyperthermia thus achieving spatiotemporally controllable on-demand release.107,108 (3) Mild hyperthermia can also reverse drug resistance of tumor cells by regulating drug resistance-related markers like P-gp.109 Therefore, both the nanocarriers on which mPTT depends and the biological effects exerted by mPTT itself circumvent the problems faced by mono chemotherapy and increase its efficacy (Table 3).

Table 3 mPTT Plays a Supporting Role in Synergizing with Chemotherapy

mPTT Facilitates Drug Delivery, Release, and Uptake

As mentioned above, mPTT based on nanomedicine can accomplish the targeted delivery of chemotherapeutic drugs, spatiotemporally controllable on-demand release, and improve drug uptake in tumor cells, solving the shortcomings faced by single-mode chemotherapy and boosting its efficacy. Accordingly, in the following section, we will illustrate two aspects including the targeted delivery of chemotherapeutics and the role of mPTT after reaching the tumor.

Targeted delivery of chemotherapeutic drugs is one of the most effective methods to reduce systemic adverse effects. In addition to passive targeting by the EPR effect, researchers have dedicated themselves to mining for overexpressed biomolecules on tumor cells to achieve active targeting, such as hyaluronic acid (HA) targeting CD44 and RGD targeting integrin αυβ3.132,133 Although the dual targeting strategy including active targeting and passive targeting has further improved the delivery efficiency of chemotherapy drugs, it mainly focuses on the treatment of solid tumor, leaving metastasis unresolved, and we know that it is metastasis instead of the tumor itself that causes more than 90% cancer-related deaths in clinical.134,135 Efforts are needed to find a strategy targeting tumor metastasis. Studies have shown that platelets can participate in all steps of the metastasis cascade to promote tumor metastasis.136 When circulating tumor cells (CTCs) enter the blood circulation, they will activate platelets to upregulate P-selectin and bind to them via CD44.137 With the help of platelet “coat”, the combined platelet-cancer cell complex can escape the surveillance of the immune system, promote the adhesion and exudation of cancer cells to the endothelium, and support the survival of CTCs in distant organs.138–140 Encouraged by the metastasis-targeting property of platelets, Zhao et al realized the increased paclitaxel (PTX) aggregation both in the primary and metastatic foci through the “platelet bridge” induced by mild hyperthermia (Figure 6a).115 They fabricated PSN (CDAEWVDVS)-modified human serum albumin (HSA) nanoparticles (PSN-HSA-PTX-IR780) loading IR780 (PTA) and PTX. In the presence of PSN, PSN-HSA-PTX-IR780 could target overexpressed P-selectin on activated platelets. After reaching the solid tumor via the EPR effect, the mild hyperthermia generated by IR780 under 808nm laser irradiation injured tumor tissue and subsequently led to the platelet recruitment and activation, which could guide more circulating nanoparticles into the tumor by recognizing P-selectin overexpressed on activated platelets in a positive feedback manner. Meanwhile, the intermolecular disulfide bonds were disrupted by GSH in tumor thereby releasing PTX, and the results showed that the amount of PTX released from PSN-HSA-PTX-IR780 was 2.86-fold higher than that from HSA-PTX-IR780 at 45°C (Figure 6b). Moreover, since activated platelets also involved in tumor metastasis, PSN-modified nanoparticles were able to complete the targeting and inhibition of metastasis via a ‘platelet bridge’. Taken together, with the assistance of mPTT, this work achieved significant efficacy in the treatment of primary and metastatic lesions by regarding platelets as a ‘bridge’ to lead the target delivery of chemotherapeutic drugs, providing new insights into the targeted delivery of drugs and the treatment of tumor metastasis.

Figure 6 (a) The working mechanisms of PSN-HSA-PTX-IR780: after aggregation in solid tumor via EPR effect, NPs could generate mild heat to induce tissue injury and then increased infiltration of platelets, which guided more circulating NPs into the tumor. (b) Quantification of intratumoral PTX concentration after treatments. (a and b) Reprinted with permission from Zhao W, Li T, Long Y, et al. Self-promoted albumin-based nanoparticles for combination therapy against metastatic breast cancer via a hyperthermia-induced “platelet bridge”. ACS Appl Mater Interfaces. 2021;13(22):25701–25714.115 Copyright 2021 American Chemical Society. (c) Schematic showing the synthesis of DOX-loaded MXene-DNA hydrogel and its application in photothermal-chemo synergistic treatment. Reprinted with permission from He PP, Du X, Cheng Y, et al. Thermal-responsive MXene-DNA hydrogel for near-infrared light triggered localized photothermal-chemo synergistic cancer therapy. Small. 2022;18(40):e2200263. © 2022 Wiley-VCH GmbH.112 (d) Schematic illustration of a tumor-selective heating approach induced by AuNP-5-FU complex. (e) Biodistribution of Au after intratumoral injection. (d and e) Reprinted with permission from Mulens-Arias V, Nicolás-Boluda A, Pinto A, et al. Tumor-selective immune-active mild hyperthermia associated with chemotherapy in colon peritoneal metastasis by photoactivation of fluorouracil-gold nanoparticle complexes. ACS Nano. 2021;15(2):3330–3348.123 Copyright 2021 American Chemical Society.

Upon the targeted arrival of the nanocomplexes at the tumor tissues, mPTT can regulate the controllable on-demand release of chemotherapeutic agents and increase drug internalization into tumor cells according to the mechanisms discussed before. For example, He et al established a thermo-responsive DNA hydrogel-based nanoplatform integrating both DOX and Ti3C2TX-based MXene, a PTA with excellent photothermal conversion efficiency (Figure 6c).112 Upon 808nm light irradiation, the mild heat produced by Ti3C2TX-based MXene could break the DNA duplex crosslinking structures in the hydrogel matrix and lead to the localized release of DOX. Intriguingly, when the NIR irradiation was removed, the DNA duplex structures and the hydrogel matrix could be reformed with adaptive shapes and the recombination of free DOX could be achieved, thereby attaining on-demand release of DOX via the reversible gel-to-solution transition of the DNA hydrogel. With the enhanced drug uptake triggered by mPTT, this nanoplatform exhibited extraordinary antitumor efficacy with negligible side effects. In another example, Mulens-Arias et al combined 5-fluorouracil(5-FU) and spherical citrate-coated gold nanoparticles (AuNPs) to form a AuNP-5-FU complex to address the shortcomings of hyperthermic intraperitoneal chemotherapy (HIPEC).123 HIPEC was mainly used to treat the residual disease after cytoreductive surgery (CRS) by circulating heated sterile solution containing a chemotherapeutic drug throughout the peritoneal cavity with the aid of a machine.141 Nevertheless, with prolonged intraperitoneal heating (most recommended at 41°C for 90 minutes), HIPEC posed many side effects such as digestive fistula or peritonitis, seriously affecting the life quality of patients.142 Luckily, the defects of HIPEC were subtly resolved by Mulens-Arias et al through a tumor-selective heating approach (Figure 6d). To begin with, they evaluated the ability of AuNP-5-FU to aggregate in tumors. The biodistribution of AuNP-5-FU was analyzed 24h after intraperitoneal injection, the time of light exposure, and quantitative analysis revealed that the vast majority of AuNP-5-FU accumulated in the tumor region, accompanied by a small fraction accumulated in spleen, liver, intestine, and kidneys (Figure 6e). After irradiation, the mild heat (<43 °C) induced 5-FU release and promoted its internalization by tumor cells. What’s more, this synergistic therapy of mPTT combined with chemotherapy achieved reprogramming of the immune microenvironment, polarizing M2 macrophages to M1 macrophages and activating a “cold” immune TME into a “hot” one. In a word, this research, from th

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