Enhancing the Efficiency of Mild-Temperature Photothermal Therapy for Cancer Assisting with Various Strategies

✅ 全文

借助多种策略增强癌症温和光热疗法的效率

作者 Pei Wang; Biao‐Qi Chen; Yunyan Zhan; Lianguo Wang; Jun Luo; Jia Xu; Lilin Zhan; Zhihua Li; Yuangang Liu; Junchao Wei 期刊 Pharmaceutics 发表日期 2022 ISSN 1999-4923 DOI 10.3390/pharmaceutics14112279 类型原创研究 (Original Research)

📄 中文摘要 Chinese Abstract

中文

传统光热疗法（PTT）使用高于48°C的温度消融肿瘤细胞，但会对周围正常组织造成损伤，引发炎症反应，并因肿瘤内容物快速释放而增加转移风险。为减轻这些副作用，发展了温和温度光热疗法（MTPTT），其工作温度为42–45°C。然而，由于癌细胞的热抵抗性——主要由热休克蛋白（HSPs）和自噬介导——MTPTT的治疗效果较低。本综述探讨了通过靶向这些保护机制并将MTPTT与其他疗法联合使用来增强其疗效的策略。

📋 英文结构化总结 English Structured Summary

全文整理

Background:

Conventional photothermal therapy (PTT) uses temperatures above 48 °C to ablate tumor cells but causes damage to surrounding normal tissues, inflammatory responses, and risk of metastasis due to rapid release of tumor contents. To mitigate these side effects, mild-temperature photothermal therapy (MTPTT) was developed, operating at 42–45 °C. However, MTPTT suffers from low therapeutic efficacy due to cancer cells’ thermoresistance, primarily mediated by heat shock proteins (HSPs) and autophagy. This review explores strategies to enhance MTPTT effectiveness by targeting these protective mechanisms and combining MTPTT with other therapies.

Methods:

This is a review article summarizing recent advances in MTPTT for cancer treatment. The authors analyzed studies involving nanoplatforms designed to inhibit HSPs (e.g., using inhibitors like gambogic acid, 17-AAG, quercetin, or siRNA), disrupt nuclear function, suppress energy metabolism (via glucose oxidase or diclofenac), or modulate autophagy (using chloroquine or Beclin-1). Additionally, synergistic approaches combining MTPTT with chemotherapy, radiotherapy, photodynamic therapy, gas therapy, and immunotherapy were reviewed. No original experimental methodology was conducted; the work synthesizes findings from published literature.

Results:

Key findings show that inhibiting HSPs—particularly HSP70 and HSP90—via pharmacological agents or gene silencing significantly sensitizes cancer cells to MTPTT, enabling effective apoptosis at mild temperatures. Nanocarriers delivering HSP inhibitors (e.g., GA-loaded ICG-Mn nanoparticles or 17AAG-loaded HMONs) demonstrated enhanced tumor suppression in vitro and in vivo. siRNA targeting HSP70 or BAG3 reduced thermal resistance and increased apoptosis rates (up to 72.2%). Nuclear-targeting agents (e.g., V₂C-TAT quantum dots) improved MTPTT by damaging DNA. Energy inhibition strategies, such as glucose oxidase-mediated starvation or diclofenac-induced glucose uptake blockade, lowered ATP levels and HSP synthesis. Autophagy modulation—either inhibition (with chloroquine) or overactivation (via Beclin-1)—also enhanced MTPTT efficacy. Furthermore, combining MTPTT with other therapies yielded synergistic effects: chemotherapy improved drug release and uptake; radiotherapy benefited from MTPTT-induced tumor reoxygenation; photodynamic therapy generated more ROS under improved oxygenation; gas therapy (e.g., NO release) showed ~85% tumor inhibition when combined with MTPTT; and immunotherapy leveraged MTPTT-triggered antigen release to activate anti-tumor immunity.

Data Summary:

In vitro studies reported IC50 reductions from 100 μg/mL to 3.13 μg/mL for Qu-Fe²⁺P nanoparticles under laser irradiation. Tumor volumes decreased by 32.5% after 21 days with GOx-based starvation plus MTPTT. Apoptosis rates reached ~70% with NO-releasing nanoplatforms combined with MTPTT, doubling that of MTPTT alone. In vivo, two-thirds of tumors disappeared after 16 days with siRNA-mediated HSP70 knockdown plus MTPTT. Western blot analyses confirmed downregulation of HSP70/HSP90, reduced Glut1 expression, and decreased ATP levels (by up to 52.7%) in treated groups.

Conclusions:

MTPTT offers a safer alternative to conventional PTT by minimizing damage to healthy tissues while maintaining therapeutic potential when combined with strategies that overcome cancer cell thermoresistance. Targeting HSPs, disrupting energy metabolism, modulating autophagy, and integrating synergistic therapies significantly enhance MTPTT outcomes. Multifunctional nanoplatforms enable precise, image-guided, and stimuli-responsive treatment, demonstrating strong preclinical efficacy and potential for clinical translation.

Practical Significance:

The reviewed strategies provide a foundation for developing clinically viable MTPTT-based cancer treatments that reduce side effects, prevent recurrence, and improve patient quality of life. By leveraging nanotechnology and combination therapies, MTPTT could become a key component of precision oncology, particularly for deep or metastatic tumors where traditional PTT is limited.

📋 中文结构化总结 Chinese Structured Summary

中文

背景:

方法:

本文为综述性文章，总结了MTPTT在癌症治疗中的最新进展。作者分析了涉及纳米平台的研究，这些平台旨在抑制HSPs（例如使用抑制剂如藤黄酸、17-AAG、槲皮素或siRNA）、破坏细胞核功能、抑制能量代谢（通过葡萄糖氧化酶或双氯芬酸）或调节自噬（使用氯喹或Beclin-1）。此外，还综述了将MTPTT与化疗、放疗、光动力疗法、气体疗法和免疫疗法联合使用的协同策略。本文未进行原创性实验，而是综合了已发表文献的研究成果。

结果:

关键发现表明，通过药物或基因沉默抑制HSPs（特别是HSP70和HSP90）可显著增强癌细胞对MTPTT的敏感性，使其在温和温度下有效诱导凋亡。负载HSP抑制剂的纳米载体（如GA负载的ICG-Mn纳米颗粒或17AAG负载的HMONs）在体外和体内均显示出增强的肿瘤抑制效果。靶向HSP70或BAG3的siRNA降低了热抵抗性，并将凋亡率提高至72.2%。核靶向剂（如V₂C-TAT量子点）通过损伤DNA改善了MTPTT疗效。能量抑制策略，如葡萄糖氧化酶介导的饥饿疗法或双氯芬酸诱导的葡萄糖摄取阻断，降低了ATP水平和HSP合成。自噬调节——无论是抑制（使用氯喹）还是过度激活（通过Beclin-1）——也增强了MTPTT的疗效。此外，将MTPTT与其他疗法联合使用产生了协同效应：化疗改善了药物释放和摄取；放疗受益于MTPTT诱导的肿瘤再氧合；光动力疗法在改善的氧合条件下产生活性氧（ROS）增多；气体疗法（如一氧化氮释放）与MTPTT联合使用时肿瘤抑制率约为85%；免疫疗法则利用MTPTT触发的抗原释放激活抗肿瘤免疫。

数据总结:

体外研究表明，在激光照射下，Qu-Fe²⁺P纳米颗粒的IC50从100 μg/mL降至3.13 μg/mL。基于GOx的饥饿疗法联合MTPTT治疗21天后，肿瘤体积减少了32.5%。释放一氧化氮的纳米平台与MTPTT联合使用时，凋亡率达到约70%，是单独MTPTT的两倍。体内实验中，siRNA介导的HSP70敲低联合MTPTT治疗16天后，三分之二的肿瘤消失。Western blot分析证实，治疗组中HSP70/HSP90表达下调，Glut1表达减少，ATP水平下降高达52.7%。

结论:

MTPTT通过最小化对健康组织的损伤，同时在与克服癌细胞热抵抗性的策略联合使用时保持治疗潜力，为传统PTT提供了更安全的替代方案。靶向HSPs、破坏能量代谢、调节自噬以及整合协同疗法显著提高了MTPTT的疗效。多功能纳米平台实现了精确、影像引导和刺激响应性治疗，在临床前研究中显示出强大的疗效，并具有临床转化的潜力。

实际意义:

所综述的策略为开发临床可行的基于MTPTT的癌症治疗奠定了基础，这些策略可减少副作用、预防复发并提高患者生活质量。通过利用纳米技术和联合疗法，MTPTT有望成为精准肿瘤学的重要组成部分，尤其适用于传统PTT受限的深部或转移性肿瘤。

📖 英文全文 English Full Text

2103 pharmamdpi Pharmaceutics Pharmaceutics Multidisciplinary Digital Publishing Institute (MDPI) PMC9695556 9695556 9695556 36365098 10.3390/pharmaceutics14112279 Enhancing the Efficiency of Mild-Temperature Photothermal Therapy for Cancer Assisting with Various Strategies Wang Pei 1 2 3 Chen Biaoqi 4 Zhan Yunyan 1 2 3 Wang Lianguo 1 2 3 Luo Jun 1 2 3 Xu Jia 1 2 3 Zhan Lilin 1 2 3 Li Zhihua 1 2 3 Liu Yuangang 4 * Wei Junchao 1 2 3 * Nowakowska Maria Academic Editor Su Chia-Hao Academic Editor Thangudu Suresh Academic Editor 1 School of Stomatology, Nanchang University, Nanchang 330006, China 2 Jiangxi Province Key Laboratory of Oral Biomedicine, Nanchang 330006, China 3 Jiangxi Province Clinical Research Center for Oral Diseases, Nanchang 330006, China 4 Institute of Pharmaceutical Engineering, College of Chemical Engineering, Huaqiao University, Xiamen 361021, China * Correspondence: ygliu@hqu.edu.cn (Y.L.); weijunchao@ncu.edu.cn (J.W.) 24 10 2022 14 11 2279 2279 26 11 2022 © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/ ). Abstract Conventional photothermal therapy (PTT) irradiates the tumor tissues by elevating the temperature above 48 °C to exert thermal ablation, killing tumor cells. However, thermal ablation during PTT harmfully damages the surrounding normal tissues, post-treatment inflammatory responses, rapid metastasis due to the short-term mass release of tumor-cellular contents, or other side effects. To circumvent this limitation, mild-temperature photothermal therapy (MTPTT) was introduced to replace PTT as it exerts its activity at a therapeutic temperature of 42–45 °C. However, the significantly low therapeutic effect comes due to the thermoresistance of cancer cells as MTPTT figures out some of the side-effects issues. Herein, our current review suggested the mechanism and various strategies for improving the efficacy of MTPTT. Especially, heat shock proteins (HSPs) are molecular chaperones overexpressed in tumor cells and implicated in several cellular heat shock responses. Therefore, we introduced some methods to inhibit activity, reduce expression levels, and hinder the function of HSPs during MTPTT treatment. Moreover, other strategies also were emphasized, including nucleus damage, energy inhibition, and autophagy mediation. In addition, some therapies, like radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy, exhibited a significant synergistic effect to assist MTPTT. Our current review provides a basis for further studies and a new approach for the clinical application of MTPTT. Keywords: mild-temperature photothermal therapy, immunotherapy, heat shock proteins, nanoplatforms, thermal resistance status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2022 Sep 22; Accepted 2022 Oct 23; Collection date 2022 Nov. 1. Introduction Hyperthermia was used to treat breast tumors in Egypt, tracing back to 5000 B.C. Tumor tissue presents increased blood vessels, blood stasis, poor heat dissipation, high resistance, difficult heat dissipation, easy heat accumulation, and rapid temperature increase [ 1 , 2 ]. Thus hyperthermia is effective for tumor treatment [ 3 ]. Photothermal therapy (PTT) is a kind of thermal therapy whereby light energy is converted into heat energy to improve the temperature of lesions to achieve a therapeutic effect [ 4 , 5 ]. Exogenous photothermal agents (PAs) are not necessary for PTT but can improve the efficiency and efficacy of therapy [ 6 ]. PTT is widely applied for the treatment of various types of tumors by promoting apoptosis or necrosis of tumor cells at high temperatures [ 4 , 7 , 8 ]. PTT relying on the introduction of an exogenous laser can achieve high accuracy, high efficiency, mild toxicity, and non-invasive treatment compared with traditional chemotherapy, radiotherapy, and surgery [ 5 , 9 , 10 ]. In addition, the laser can be used as a “light-trigger switch” to achieve remote drug control release (light stimulation response) [ 11 , 12 ]. In contrast, the heat can destroy the lysosome to help the drug-loaded to escape from the lysosome. Nowadays, a division between the concentration of preclinical and clinical PTT research is obvious, with preclinical studies focused on new PAs, whereas clinical studies concentrated on the exploitation of integrated laser devices [ 6 ]. The difference may reflect the fact that the effectiveness of PTT can easily be demonstrated in preclinical research, enabling the preparation and application of a wide variety of novel nanomaterials. Nevertheless, PAs hold potential in clinical transformation on account of better selectivity for the target tissue, enabling the utilization of lower-power lasers and simplifying device design. Previous studies have made significant efforts to optimize PAs by modulating the shape, size, and surface chemistry of nanoparticles [ 7 , 13 , 14 ]. Moreover, the rapid development of nanotechnology has increased advances in PTT through the development of multi-functional nanoparticles [ 15 ]. For instance, plasmonic nanoparticles, like gold nanoparticles, and platinum nanoparticles, are chosen as PAs in many reports [ 16 , 17 ]. In addition, synergistic therapy with PTT improves the therapeutic effect of PTT against tumors [ 18 ]. PTT directly kills tumor cells or enhances other therapies by promoting drug delivery, stimulating release, mediating tumor microenvironment (TME), eliciting tumor-specific antigen release, or modulating other biologically related responses [ 19 , 20 , 21 , 22 , 23 , 24 , 25 ]. However, the clinic application of PTT has been hindered to some extent by several limitations. For instance, it is challenging to completely kill tumor cells using PTT, thus augmenting the risk of tumor recurrence and metastasis owing to limited tissue penetration of the laser (NIR-I widow laser 1~2 cm, NIR-II widow laser > 2 cm) [ 26 ]. Therefore, to achieve a high treatment temperature, researchers often increase the laser power or dosage of PAs. However, the American National Standards Institute (ANSI) has established standard tolerance threshold values for the clinically safe use of PTT on the skin [ 27 ]. The 808 nm laser power threshold ranges from 330 to 350 mW cm −2 with an exposure time of 10–1000 s. Moreover, PTT inevitably damages normal tissue around the tumor site and leads to in vivo toxicity and side effects [ 28 ]. Furthermore, several cell contents and some residual tumor cells caused by thermal ablation may cause a series of side effects, including inflammation, tumor metastasis, harm to normal tissues, and tumor recurrence [ 29 ]. To circumvent these limitations, mild-temperature photothermal therapy (MTPTT), with a temperature range from 42 °C to 45 °C [ 3 , 30 ], was introduced to reduce the temperature used, thus alleviating the side effects. In addition, MTPTT does not significantly affect the quality of life of the patient owing to the milder temperature used. However, MTPTT is associated with poor therapeutic effects. Therefore, studies have been exploring methods to achieve better therapeutic efficacy using nanocarriers under MTPTT. Though heat shock protein (HSPs) inhibitors or other compounds can be encapsulated into the nanoplatforms, the antitumor efficacy and safety still need more comprehensive and in-deep studies. Nevertheless, it is a significant integrative treatment and exhibits great potential in future clinical applications [ 31 ]. The current review comprehensively summarizes the recent advances and functions of novel nanosystems comprising MTPTT for the treatment of tumors ( Figure 1 ). The review explores (1) the mechanism of action of MTPTT, (2) diverse approaches of MTPTT, (3) the combination of MTPTT with other therapeutic modalities, (4) challenges and future development of MTPTT to provide a basis for improving the efficacy of MTPTT. The studies explored the role of heat shock response (HSR) in the efficacy of MTPTT and how they can be used to improve the efficacy of MTPTT by designing drug delivery nanosystems. Finally, the current crucial challenges faced in the MTPTT field are explored, and some considerable future research directions are proposed to improve the existing strategies and to lay a basis for developing new strategies to improve the effectiveness of MTPTT. Figure 1 Scheme illustrating the use of MTPTT for cancer treatment via various strategies. 2. The Mechanism of MTPTT MTPTT effectiveness in cancer treatment does not depend on precise devices or special methods to control the mild temperature but on methods for maintaining treatment efficacy at mild temperature. MTPTT therapeutic effect is attributed to damage to the self-protective mechanism of tumor cells and preventing serious damage from heat stress. Studies report that MTPTT exerts its activity through two self-protective mechanisms, including heat shock reaction and autophagy [ 32 , 33 ]. In conventional PTT (>48 °C), thermal ablation induces severe and irreversible denaturation of proteins, DNA damage, and denaturation, and destroys the effective defense of the self-protective mechanism. Notably, the self-protective mechanism has a significant effect on the repair of unfolded proteins in MTPTT (<45 °C). Therefore, inhibiting the pathway of the self-protective mechanism is the most effective way to achieve the high efficacy of MTPTT. Studies report that HSR and autophagy are key targets for mediating self-protective mechanisms during MTPTT ( Figure 2 ). Figure 2 ( A ) Schematic of the process of heat shock reaction after hyperthermia and the blocking function of heat shock reaction via siRNA, nuclear damage, HSPs inhibitors, and energy inhibition. ( B ) Illustrating the physiological functions of HSPs: assists protein folding into its native form in MTPTT. Hyperthermia above 41 °C causes protein denaturation and temporary cell inactivation, which may last for several hours [ 34 , 35 ]. As a result, upregulation of expression of HSPs is induced by HSR, thus effectively preventing aggregation of other proteins. HSR is a cellular defense mechanism present in all organisms and plays a role in preventing damage from hyperthermia or other adverse stress conditions. HSR limits the therapeutic efficacy of MTPTT through its cytoprotective and antiapoptotic effects [ 36 ]. Moreover, HSPs can interact with apoptosis signaling pathway proteins to inhibit the occurrence of apoptosis, thus reducing the therapeutic effect of hyperthermia [ 37 , 38 ]. In addition, tumor cells overexpress HSPs compared with normal cells, which makes them less sensitive to heat treatment and enables them to remain active at high temperatures [ 39 , 40 ]. Tumor cells mainly regulate the expression of HSPs by activating heat shock transcription factors (HSFs) [ 41 ]. Previous studies have explored four HSFs, including HSF1, HSF2, HSF3, and HSF4. Notably, HSF1 is the main transcription factor that mediates HSR. HSF1 is a highly expressed protein in various tumor cells and is related to tumor progression and poor prognosis. The main mechanism of action of HSF1 is by enhancing phosphorylation of its own 326 site serine, thus upregulating expression of HSP70 and HSP27 and ultimately promoting malignant proliferation and apoptosis resistance [ 42 , 43 ]. Expression levels of HSPs are low, and only 1–2% of the total protein exists under normal physiological conditions [ 44 ]. HSF1 is activated and bound to the promoter region of the downstream HSPs gene to promote the expression of HSPs after stimulation by high temperatures, excessive reactive oxygen species (ROS), or inflammation. HSP70 is mainly the first expressed protein as a result of HSR in many HSP families [ 45 , 46 ]. B-cell lymphoma-2 (Bcl-2) associated athanogene 3 (BAG3) is the chaperone protein of HSP70 and can bind to the ATPase domain of HSP70 through the bag domain to modulate HSP70 function [ 47 , 48 ]. In addition, the BAG3-HSP70 complex can bind to Bcl-2 and protect it from degradation, thus inhibiting the apoptosis pathway or inhibiting tumor cell apoptosis induced by hyperthermia therapy and chemotherapy [ 49 , 50 , 51 ]. Therefore, inhibition of HSR can reduce the thermoresistance of tumor cells to increase the effectiveness of sensitizing PTT. Several studies have explored the inhibition of HSR by gene-mediated silencing technology (small interfering RNA or short hairpin RNA, siRNA, or shRN and A), studies are developing heat-sensitive drugs. The efficacy of MTPTT is mainly achieved by blockingHSR, and is mainly through two aspects, including (1) reducing the synthesis of HSPs from HSR [ 52 ], and (2) inhibiting the activity of HSPs [ 53 ]. The current research mainly focuses on the mechanism of HSPs in improving the efficacy of PTT. The efficacy of PTT can be improved through the following three ways: use of HSPs inhibitors, silencing HSPs gene by siRNA and reducing ATP synthesis. Therefore, it is important to combine HSPs inhibitors (or siRNA, ATP inhibitors) with PAs in the nanosystem, thus improving the sensitivity of tumor cells to heat [ 54 ]. Besides, autophagy as a cellular self-protective mechanism rapidly activates cancer cells to maintain energy production and offer recycled materials in response to hyperthermia stress. Autophagy-related tolerance also acts a crucial role in thermal resistance [ 55 ]. There are three types of autophagy identified according to different routes in which substrates eventually enter into the lysosomal lumen: microautophagy, chaperone-mediated autophagy, and macroautophagy ( Figure 3 ). Damaged and denatured proteins and organelles are engulfed by autophagosomes, then degraded in the lysosome to provide energy, and macromolecular precursors, and can be recycled to sustain cellular metabolism [ 56 , 57 , 58 ]. Therefore, intercepting the autophagy pathway can improve the efficacy of MTPTT. Autophagy can be blocked by inhibiting (1) formation of autophagosome (3-methyladenine, wortmannin) [ 56 ], (2) fusion of autophagosome and lysosome (hydroxychloroquine, chloroquine, vinblastine) [ 59 ], and (3) degradation of autolysosome (pepstatin A) [ 60 ]. On the contrary, excessive autophagy does not protect cells but destroys homeostatic functions and induces autophagy-mediated cell death (ACD), known as type II programmed cell death [ 61 ]. The excessive autophagy activity far exceeds the degradation capacity of the autolysosome, resulting in the formation of micron vacuoles and degradation blockage [ 62 ]. When autophagy fails to stop effectively or is overstimulated, the autophagic activities cannot recycle the cancer cellular components and accelerate ATP depletion, which ultimately leads to cell death and further enhance the therapeutic efficacy of MTPTT. Therein, excessive autophagy is induced via cutting off the inhibition pathway of autophagy or using autophagy inducers, including carbamazepine, C2-ceramide, rapamycin, and xestospongin B/C [ 63 ]. Figure 3 Schematic of the process of macroautophagy after hyperthermia and the various strategies to inhibit or induce autophagy. 3. Various Approaches to Improve the Efficacy of MTPTT Thermal ablation (above 48 °C) directly induces necrosis in tumor cells, whereas the surrounding normal tissues are damaged by heat diffusion [ 34 ]. This implies that PTT has a high therapeutic effect. However, it is characterized by adverse effects. Reduction in the temperature reduces the efficacy of PTT the owing to thermoresistance of tumor cells [ 3 ]. Therefore, developing strategies for overcoming thermoresistance is important to promote the efficacy of MTPTT. The next section explores the mechanism of thermal tolerance and summarizes approaches for constructing multifunctional nanosystems to improve the efficacy of MTPTT. 3.1. Heat Shock Proteins Inhibitors HSPs are mainly classified as HSP27 (~27 kDa), HSP40 (~40 kDa), HSP60 (~60 kDa), HSP70 (~70 kDa), HSP90 (~90 kDa) and HSP110 (~110 kDa) based on their molecular weight [ 44 ]. HSP70 and HSP90 play important roles in HSR [ 39 , 64 , 65 , 66 ]. HSPs have some similarities in structure and function. All HSPs classes comprise three domains ( Figure 4 A), including the N-terminal domain, intermediate domain, and C-terminal domain [ 42 , 44 ]. The N-terminal domain is the binding site of ATP. Proline residues in the ATPase domain can induce conformational changes and cause hydrolysis, thus inducing the activity of HSPs [ 43 ]. The intermediate domain is the binding site of a guest protein and chaperone protein and is the active region of HSPs [ 45 ]. The C-terminal domain is a binding site for chaperone protein and is responsible for substrate binding and refolding (the dimerization of HSPs), resulting in “blocked” conformation to protect the guest protein [ 67 ]. Figure 4 Several examples showing HSP70 and HSP90 inhibitors incorporated nanoplatforms that efficiently achieve MTPTT of the tumor. ( A ) Schematic representation of the structure and function of HSP70 and HSP90 [ 43 , 67 ]. Copyright © 2022 and 2016, Elsevier. ( B ) A scheme to illustrate a one-step synthesis of one-dimensional nanoscale coordination polymers and to overcome thermal resistance by inhibiting HSP90 [ 68 ]. Copyright © 2022, John Wiley and Sons. ( C ) Schematic illustration of the construction of the ICG-17AAG@HMONs-Gem-PEG nanoplatforms for fluorescence/photoacoustic imaging-guided MTPTT/chemotherapy [ 69 ]. Copyright © 2022, American Chemical Society. ( D ) Schematic representation of the one-pot synthesis of a family of poly(vinylpyrrolidone) protected metal ion-quercetin (Qu) coordination nanodrugs, intrinsically integrating precise diagnosis, excellent MTPTT efficacy, ROS elimination, and anti-inflammatory action, dynamic disassembly, and renal clearance ability into a single nanoparticle [ 70 ]. Copyright © 2022, Elsevier. HSPs inhibitors ( Table 1 ) can specifically bind to the intermediate domain of HSPs, preventing binding of the guest protein and thus losing the ability to protect cells during HSR [ 71 ]. Gambogic acid (GA) is a natural prenylated xanthone moiety isolated from Garcinia hanburyi , and it presents various biological activities, such as anticancer, anti-inflammatory, antioxidant, and antibacterial activities. In addition, GA plays an important role by binding to the N-terminal ATP-binding domain of HSP90 without competing with, ATP thus inhibiting the catalysis of ATP hydrolysis [ 72 , 73 ]. GA has used an inhibitor of HSP90 due to this function and is combined with PAs to improve MTPTT efficacy in cancer treatment. Smart nanosystems are designed to achieve rapid release in tumor tissues or cells, thus improving the efficacy of drugs targeting HSPs. Yang et al. [ 68 ] designed poly (ethylene glycol) (PEG)-modified one-dimensional indocyanine Green (ICG)-Mn nanomaterials loaded with GA for MTPTT ( Figure 4 B). The one-dimensional ICG-Mn nanomaterial has the advantages of a high loading rate and pH stimulation response. In the acidic microenvironment of the tumor, the structure dissociates and rapidly releases GA. The cell survival rate of the GA-loaded nanoparticles group in vitro was significantly less compared with that of other groups at ~43 °C. In addition, the Western blot test showed that GA downregulated the expression of HSP90. GA-loaded nanocarriers can induce effective apoptosis of tumor cells under relatively mild temperatures by inhibiting HSP90 rather than thermal ablation (above 50 °C). It helps minimize the non-specific thermal effects on normal organs and improves the efficacy of PTT treatment of large or deep tumors. Table 1 HSP inhibitors for MTPTT. The Species of HSPs Agents PAs Reference HSP70 2-phenylethynesulfonamide (PES) PEG-PAu@SiO2-SNO [ 80 ] quercetin Qu-Fe II P nanoparticles [ 70 ]

B780/Qu NPs [ 78 ] HSP90 17-AAG ICG-17AAG@HMONs-Gem-PEG [ 69 ]

DOX-17AAG@B-PEG-cRGD [ 81 ] Gambogic acid NCPs/GA [ 68 ]

HAS/dc-IR825/GA [ 74 ] Notably, insufficient tumor tissue accumulation and excessive liver retention availably limit the curative effect and biocompatibility of plenty of nanomedicines. Wu et al. reported smart theranostic nanocarriers consisting of GA as an HSP90 inhibitor, dc-IR825 as a fluorescence imaging probe and photothermal agents, and biocompatible human serum albumin [ 74 ]. The nanocarriers showed the synergy of chemotherapy and MTPTT, thus improving the efficacy of cancer treatment. In the nanocarriers, cytosolic translocation of GA can be promoted through ROS-mediated mitochondrial disruption under near-infrared (NIR) laser irradiation, further blocking the overexpression of HSP90. Hence, the nanocarriers can kill cancer cells under MTPTT, thus improving effectiveness in cancer treatment. 17-allylamino-17-demethoxy-geldanamycin (17-AAG) is an HSP90 inhibitor derived from the geldanamycin antibiotic and can cause the apoptosis of tumor cells [ 75 , 76 ]. Moreover, 17-AAG can effectively inhibit several cell signals transduction pathways, such as decreasing cellular levels of serine/threonine kinase 38 (STK38)/nuclear Dbf2-related 1 (NDR1) and the activity of STK38 kinase [ 77 ]. Wu et al. prepared hollow mesoporous organosilica nanocapsules (HMONs) to provide a versatile nanoplatform for imaging-guided MTPTT/chemotherapy, thus achieving high theragnostic efficacy ( Figure 4 C) [ 69 ]. 17AAG and ICG were loaded onto HMONs and then simultaneously released when the gemcitabine (Gem) gatekeeper was specifically open because of hydrolysis of the acetal bonds at acid TME. Then, 17AAG induces downregulation of HSP90 and thus reverses the thermoresistance of tumor cells to achieve the aim of MTPTT. This nanoplatform with, exhibiting the synergistic effect of MTPTT/chemotherapy, is the potential for precise cancer theranostics. Quercetin (Qu) is a natural polyphenol-rich in hydroxyl groups widely distributed in vegetables, fruit peels, seeds, beverages, and Chinese herbs. It exhibits excellent antioxidant, anticancer, prevention, and treatment of cardiovascular and cerebrovascular diseases. Moreover, Qu inhibits HSP70 expression by regulating HSF transcriptional activity [ 78 , 79 ]. Yang et al. designed novel Qu-Fe Ⅱ P nanoparticles using quercetin, an HSP70 inhibitor ( Figure 4 D), as the framework [ 70 ]. High temperatures can induce inflammation and damage normal cells around tumor tissue. Therefore, quercetin plays an important role in clearing ROS and presents anti-inflammatory activity. The IC50 of Qu-Fe Ⅱ P on MCF-7 cells was 100 μg/mL in vitro. However, the IC50 of Qu-Fe Ⅱ P under laser irradiation was 3.13 μg/mL. After 20 days of treatment in vivo, the Qu-Fe Ⅱ P + laser irradiation group showed a good tumor inhibition effect under MTPTT, and three-quarters of the tumors disappeared completely. Western blot analysis of tumor tissue showed that expression level HSP70 was lower compared with other proteins, thus significantly reducing the thermal tolerance of tumor cells and improving MTPTT efficacy. 3.2. siRNA RNA interference (RNAi) technology has great potential in the biomedical field. It provides a novel approach for the design and development of new drugs owing to its high efficiency, high specificity, and mild toxicity. Rational design, precise chemical modifications, and nanocarriers provide available opportunities to overcome the limitations of siRNA, such as rapid degradation, poor cellular uptake, and off-target effects. siRNA is an effective vector for RNA interference which inhibits the expression of HSPs or BAG3 by abrogating the expression of specific genes, making cancer cells more vulnerable to PTT effects, thus providing a strategy for inhibition of HSR [ 54 ]. However, the approach is characterized by limitations such as low serum stability and elimination in vivo during siRNA delivery to target cells. Therefore, its effectiveness can be improved by using nanosystems to deliver siRNA. Ding et al. [ 34 ] used siRNA as a crosslinking agent to construct DNA-grafted polycaprolactone (DNA-g-PCL) nanoparticles ( Figure 5 A) and then packaged them using polydopamine (PDA) and modified them using PEG (PP-NG-siHSP70). The gene silencing activity of PP-NG-siHSP70 with laser irradiation group presented the lowest expression of HSP70 mRNA and the highest expression of caspase-3 mRNA in agreement with the western blot results. The cellular apoptosis rate for the PP-NG-siHSP70 laser irradiation group in vitro was 72.2%, which indicated that the nanoparticle-induced effective target gene knockdown and apoptosis under mild conditions rather than cell necrosis. Notably, two-thirds of tumors in the PP-NG-siHSP70 laser irradiation group disappeared after 16 days of treatment. Wang et al. [ 82 ] prepared a gold nanorod (GNRs) platform loaded with BAG3 siRNA with gene silencing ability to improve the efficacy of MTPTT ( Figure 5 B). The findings using oral squamous cell carcinoma in vitro and in vivo showed that the nanorod improved the sensitivity of tumor cells to PTT and increased apoptosis after through downregulation expression of BAG3. The relative volume of GNRs-siRNA in mice treated with siRNA decreased by 18.4% after laser treatment. Figure 5 Specific means of siRNA and nucleus damage in favor of MTPTT. ( A ) Schematic illustration of the synthesis of PDA-coated nucleic acid nanogel and the mechanism of siRNA-mediated MTPTT induced by PEG-PDA-Nanogel [ 34 ]. Copyright © 2022, Elsevier. ( B ) Schematic illustration of the design of GNRs-siRNA in the improved MTPTT platform [ 82 ]. Copyright © 2022, Elsevier. ( C ) Schematic diagram of the cancer cell membrane and nucleus organelle dual-target V 2 C-TAT@Ex-RGD nanoagents for multimodal imaging-guided MTPTT in the NIR-II biowindow [ 83 ]. Copyright © 2022, American Chemical Society. ( D ) Illustration of DNAzyme-based nanosponges for highly efficient PTT [ 84 ]. Copyright © 2022, The Author(s). 3.3. Nucleus Damage PAs target the nucleus, resulting in the destruction of the structure of genetic material in the nucleus, thus improving the efficacy of MTPTT. TAT is a cell-penetrating peptide that exhibits its function by targeting the nucleus. Thus, it can be used to target the nucleus by modification of ultra-small nanoparticles or quantum dots [ 85 ]. Therefore, the design of novel nuclear targeting PAs with efficient photothermal conversion properties and high intranuclear accumulation is significantly desired for MTPTT. Cao et al. [ 83 ] prepared small-size vanadium carbide (V 2 C)-TAT quantum dots that can accumulate nucleus and destroy genetic material, thus enhancing the effect of MTPTT ( Figure 5 C). The V 2 C-TAT quantum dots were coated by exosomes modified with RGD (V 2 C-TAT@Ex-RGD), which has a long-life cycle, high biocompatibility, and good tumor-targeting ability. V 2 C-TAT@Ex-RGD has a significant therapeutic effect in vivo owing to long blood circulation time, strong targeting ability of tumor cells, and less tumor accumulation under irradiation with a 1064 nm laser with a power density of 0.96 W cm −2 . Therefore, the V 2 C-TAT@Ex-RGD can achieve nuclear targeting for guiding by multimodal imaging at mild temperature, which shows a good prospect for biomedical, and clinical application. In addition to the cell-penetrating peptide-mediated nuclear target, ultrasmall nanoparticles are more likely to enter the nucleus through the nuclear pore (~40 nm size) and nuclear pore complex [ 86 ]. Liu et al. [ 87 ] prepared special ultrasmall chitosan-coated ruthenium oxide nanoparticles (CS-RuO 2 NPs) with a nuclear target for MTPTT application in cancer in the near-infrared window and synthesized RuO 2 NPs using a simple one-pot method ( Figure 5 D). Analysis of the nuclear power source with different sizes and surface charges showed that only the nuclear power source with ultrasmall size (2 nm) and positive charge could help effectively enter the nucleus destroying DNA and protein. The CS-RuO 2 NPs revealed strong absorption in the NIR-II window with great photothermal conversion efficiency. In addition, they are ideal materials for PAs and photoacoustic imaging (PAI). 3.4. Energy Inhibition HSPs are ATP-dependent proteins and are synthesized in large quantities under the presence of ATP [ 45 , 47 , 67 ]. Therefore, HSPs levels can be reduced by limiting the availability of energy. The main pathways for obtaining energy in tumor cells are glutamine metabolism, glycolysis, and autophagy and not oxidative phosphorylation of normal cells [ 88 , 89 ]. Therefore, these pathways can be regulated to limit the production of ATP in tumor cells. Starvation therapy aims to inhibit tumor cells’ access to or utilization of nutrients so that they can “starve to death” due to lack of energy [ 90 ]. Zhou et al. [ 29 ] used the catalysis of glucose oxidase (GOx) to oxidize glucose to gluconic acid and H 2 O 2 , thus limiting the utilization of glucose by tumor cells ( Figure 6 A). Mesoporous hollow PB NPs loaded glucose oxidase was designed based on this mechanism for a combination of starvation therapy and MTPTT for tumor treatment. In addition, PB NPs were used to catalyze H 2 O 2 to improve the hypoxia level in tumor tissue. Starvation therapy limits the supply of ATP and inhibits the synthesis of HSPs, thus reducing the heat tolerance of cancer cells. The findings showed that the intracellular oxygen concentration decreased from 5.1 mg/mL to 0.04 mg/mL under the catalysis of GOx for 10 min, indicating that GOx catalyzed glucose oxidation. The tumor volume decreased by 32.5% after 21 days of treatment using synergistic therapy. Figure 6 Strategies that enhance the therapeutic effect of MTPTT through inhibiting energy or metabolism. ( A ) Illustration of GOx-induced starvation for enhanced MTPTT in a hypoxic TME via the PHPBNs-mediated tumor reoxygenation [ 29 ]. Copyright © 2022, American Chemical Society. ( B ) Schematic illustration of thermosensitive liposomes encapsulating GOx, ICG, and GA for synergistic starvation therapy, EEPT, and enhanced MTPTT of tumors [ 91 ]. Copyright © 2022 John Wiley and Sons. ( C ) Schematic illustration of GNR/HA-DC for selectively sensitizing tumor cells to MTPTT by interfering with the anaerobic glycolysis metabolism [ 92 ]. Copyright © 2022, American Chemical Society. In another attempt to address the toxic side effects of the high dose of HSP inhibitor, Gao et al. demonstrated a thermosensitive GOx/indocyanine green/gambogic acid (GA) liposomes (GOIGLs) for enhancing the efficiency of MTPTT via synergistic inhibition of HSPs from GA and GOx which induced glucose consumption via catalyzing glucose into gluconic acid ( Figure 6 B), together with enzyme-improved phototherapy effect [ 91 ]. In addition, H 2 O 2 , as the product of the oxidation of glucose, can be converted into hydroxyl radical (·OH) under light irradiation, which effectively eliminates cancer cells to realize enzyme-enhanced phototherapy (EEPT). From the results of cancer cells and tumor-bearing mice experiments, the significant antitumor efficacy of “GOIGLs + Laser + Light” demonstrated that GOx-mediated tumor starvation and phototherapy improved the therapeutic efficiency of MTPTT. Compared with conventional PTT, MTPTT not only can achieve an effective antitumor therapy at a relatively low temperature (below 45 °C) but also reduce thermal damage to the normal tissues from the safety evaluation results. Consuming a large amount of glucose is one effective way to burn energy, hindering the efficiency of HSPs. In addition, glucose uptake is also a vital target for inhibiting the metabolic pathway of cancer cells. Chen et al. [ 92 ] used diclofenac to inhibit the activity of glucose transporters (Gluts) ( Figure 6 C), thus limiting the uptake of glucose by tumor cells. It achieved the purpose of MTPTT by downregulating the synthesis of HSP70 and HSP90 after reducing the anaerobic glycolysis of tumor cells, thus reducing the level of ATP. Western blot analysis presented that the amount of Glut1 protein decreased significantly after HeLa cells and MCF-7 cells were cultured with GNR/HA-DC for 12, 24, and 48 h. GNR/HA-DC caused a significant reduction in glucose uptake by cancer cells, which inhibited cell functions, including anaerobic glycolysis. ATP decreased by 52.7% and 35%, respectively, after 48 h of culture. 3.5. Autophagy Mediation Autophagy, as a dynamic cellular pathway, degrades and recovers damaged or aged proteins and organelles. Dysfunctional autophagy is related to cancer, microbial infection, neurodegeneration, and aging, indicating that autophagy plays a key role in these diseases [ 61 ]. Several studies report that drug inhibition of autophagy or gene knockout of autophagy-related genes (ATG) can increase the sensitivity of cancer cells to a variety of drugs [ 93 , 94 ]. Chloroquine (CQ) inhibits autophagy and enhances the anticancer activity of histone deacetylase inhibitors against chronic myeloid leukemia [ 95 ]. Moreover, inhibition of autophagy can enhance the anticancer effect of bevacizumab against hepatocellular carcinoma. Besides, CQ is also applied for the treatment of malaria and autoimmune diseases [ 96 ]. Several studies have explored the modulation of autophagy for the development of drugs for cancer treatment. The heat transformed by the photothermal effect in PTT activates autophagy by damaging cytoplasmic components. Therefore, inhibition of autophagy can improve the therapeutic effect of MTPTT. CQ inhibits the degradation of autophagy by inhibiting lysosomal activity. Zhou et al. used CQ to inhibit the autophagy of tumor cells, thus improving the efficacy of MTPTT ( Figure 7 A) [ 97 ]. The findings showed that the tumor volume of the PDA-PEG/CQ group was about 30 mm 3 , and the temperature of laser irradiation was controlled at ~42 °C. Figure 7 Two examples illustrate that autophagy sensitizes the photothermal killing of cancer cells. ( A ) Schematic of autophagy inhibition sensitizes photothermal killing of cancer cells. Western blot of LC3-I and LC3-II in HeLa cells treated with CQ and 3-MA as autophagy inhibitors, respectively [ 97 ]. Copyright © 2022, Elsevier. ( B ) The illustration depicts beclin 1-induced autophagy sensitizing photothermal killing of cancer cells. Western blot of LC3-I/LC3-II conversion and P62 in MDA-MB-231 cells treated with beclin 1, PP, PPB, and PPBR [ 98 ]. Copyright © 2022, Elsevier. Moderate autophagy helps cells survive in an adverse environment; however, excessive autophagy leads to cell death. Unlike apoptosis, autophagy is characterized by the forming of a large number of autophagosomes wrapped in cytoplasm and organelles. Beclin1 coupled polymer nanoparticles promote autophagy activity in tumor cells and further inhibit the growth of the tumor. Beclin1 induced autophagy abrogates the homeostasis function of autophagy, activates the autophagy cell death pathway, and improves the therapeutic effect of PTT. Zhou et al. [ 98 ] prepared multifunctional nanoparticles for tumor targeting and for improving PTT efficacy through autophagy induction ( Figure 7 B). The nanoparticles comprised PDA nanoparticles and Beclin 1-derived peptide, PEG, and cyclic Arg-Gly-ASP peptide (PPBR). PPBR improved the autophagy activity of cancer cells and significantly promoted the killing efficiency of PTT. Findings from animal experiments showed that PPBR could upregulate autophagy of tumor cells, and the combination therapy inhibited tumor growth more effectively compared with single therapy in a breast tumor model. 4. The Synergistic-Therapy Strategies Nowadays, many preclinic and clinic researchers have demonstrated that many monotherapies show low efficiency in curing tumors, high recurrence rate, severe toxic side effects like hematologic toxicity, abnormal liver function, and high toxicity to normal cells, and immune system disorder. The limited penetration depth of light lowers the efficacy of PTT in inhibiting tumor growth outside the radiation range [ 99 ]. Monotherapies are inefficient in abrogating tumor growth, including PTT monotherapy. Although PTT has a high therapeutic effect, its limitations may contribute to the incomplete elimination of tumor cells, ultimately leading to tumor recurrence and metastasis. The combination of MTPTT with other treatments can improve therapeutic efficacy. In addition to providing a supplement, the combination of different therapeutic approaches results in a synergistic treatment effect. 4.1. Chemotherapy MTPTT improves the therapeutic effect of chemotherapy through several mechanisms, including (1) increasing toxicity of some drugs, (2) increasing uptake of nanoparticles by tumor cells, (3) stimulating the rapid release of drugs from nanoparticles, and (4) increasing sensitivity of multidrug-resistant cells to chemotherapy. In addition, chemotherapy can kill the metastatic tumor cells, whereas MTPTT does not eliminate metastatic cells. Therefore the combination of MTPTT and chemotherapy exhibits a good synergistic effect on tumor treatment [ 13 , 100 ]. Fu et al. [ 81 ] designed novel multifunctional boron-based nanoplatforms combining chemotherapy and MTPTT. The boron-based nanoplatforms targeted αvβ3 integrin overexpressed in tumor cells through functionalization with cyclo (Arg-Gly-Asp) (cRGD) peptide ( Figure 8 A). DOX (603 mg g −1 ) and 17AAG (417 mg g −1 ) were loaded with boron nanosheets. The DOX-17AAG@B-PEG-cRGD systems exhibited controlled, and NIR induced DOX and 17AAG release. DOX-17AAG@B-PEG-cRGD systems significantly enhanced the cellular uptake of cancer cells compared with healthy cells. The presence of 17AAG in a combination of MTPTT and DOX chemotherapy improves anticancer activity. These multifunctional nanoplatforms are promising candidate platforms for tumor therapy. Wu et al. [ 69 ] designed HMONs loaded with ICG and 17AAG, and the antitumor drug gemcitabine was modified through a pH-sensitive acetal covalent bond to block the pore. Furthermore, NH 2 -PEG can be introduced through the benzamide bond, which improves the cycling performance of the nanoparticles. ICG-17AAG@HMONs-Gem-PEG nanoparticles exhibit pH-responsive molecular release and glutathione (GSH) dependent degradation in TME. ICG and 17AAG can be released on demand owing to hydrolysis of the acetal bond under weak acidity (<6.0). Subsequently, 17AAG regulates HSP90, thus abrogating the heat tolerance of tumor cells. In addition, it can effectively induce cancer cell apoptosis under relatively mild-temperature PTT. Gemcitabine acts as a gatekeeper and can be released from nanocapsules as a chemical for cancer chemotherapy. The near-infrared fluorescence and PAI of nanocapsules present high contrast owing to the strong near-infrared absorption of ICG, which helps in targeted treatment. 4.2. Radiotherapy MTPTT alone does not completely eliminate deep tumors owing to its limitations. RT can damage DNA and cause cancer cell death without depth limitation [ 101 ]. However, the degree of cell damage is significantly affected by the level of intracellular oxygen ions induced by ionizing radiation. Therefore, the hypoxia TME significantly limits the effectiveness of RT [ 2 ]. Notably, MTPTT induced hyperthermia can accelerate tumor blood flow and improves TME oxygen status, thus increasing the sensitivity of tumor cells to RT [ 102 , 103 ]. Therefore, the combination of RT and PTT is a promising strategy for tumor eradication, which shows advantages such as improving treatment efficacy and reducing side effects. In addition, hyperthermia can effectively inhibit the repair of nonlethal X-ray injury, resulting in a significant synergistic effect of MTPTT/RT. Song et al. [ 104 ] developed Bi 2 Se 3 hollow nanocubes (HNCs) modified with hyaluronic acid loaded with GA (HNC-S-S-HA/GA) ( Figure 8 B). Downregulation of HSPs mediated by GA reduces the resistance of cancer cells to heat stress. HNC-S-S-HA/GA effectively induces apoptosis of cancer cells and has a significant ablation effect on cancer cells. The heat generated by light and heat increases blood flow resulting in the delivery of more oxygen into cancer cells, thus alleviating the hypoxic TME. HNC-mediated enhanced RT showed an improved cancer cell-killing effect under X-ray irradiation. Novel HNC-S-S-HA/GA nanocubes have several unique advantages such as (1) high stability, drug loading capacity, and absorption coefficient, (2) HNC-S-S-HA/GAS has enhanced permeability and retention effect/CD44 mediated bimodal tumor targeting and GSH sensitive drug release, further reducing toxicity to normal cells, (3) MTPTT can inhibit proliferation of surrounding tissues, (4) inhibition rate of MTPTT combined with RT is significantly higher compared with that of MTPTT or RT alone. Novel HNC-S-S-HA/GA-based TME responsive nanodrugs show potential for use in mild-temperature MTPTT/RT guided by multispectral optoacoustic tomography (MSOT)/computed tomography (CT) imaging, with high anti-tumor efficiency and minimal invasion in normal tissues. 4.3. Photodynamic Therapy PDT is a therapeutic approach mainly using photosensitizers (PSs) to transfer energy to oxygen to form highly active singlet oxygen ( 1 O 2 ) under laser irradiation, selectively destroying cancer cells by oxidating the proteins, nucleic acids, and lipids (Type II mechanism) [ 31 , 105 , 106 ]. In addition, PSs produce ROS, such as a hydroxyl radical or a superoxide anion, which is the Type I mechanism [ 107 ]. Due to its advantages of being non-invasive, high safety, spatiotemporal control, and broad spectrum, PDT has attracted much attention and is considered a potential tumor treatment. However, one of the biggest bottlenecks in Type II PDT is hypoxic TME, which severely decreases the 1 O 2 yield. MTPTT significantly improves tumor oxygen supply by increasing the blood flow, which is a benefit to enhancing the singlet oxygen generation efficiency. Moreover, PDT interferes with tumor physiology and microenvironment and enhances the thermal sensitivity of tumor cells. The combination of MTPTT and PDT exhibits several advantages in improving the effectiveness of tumor therapy [ 108 ]. Hence, a variety of nanosystems have been developed for MTPTT/PDT synergistic therapy [ 109 , 110 ]. Notably, GQDs-based nanocomposites for PDT can act not only as PSs but also nanoplatforms for delivering PSs [ 111 ]. In addition, some inorganic nanomaterials also become PSs and delivery systems. However, most of these systems require complex integration or assembly components to achieve an “integrated” function [ 112 , 113 ]. Studies are currently exploring the use of organic metal frameworks (MOFs) in PDT/PTT and multimode imaging to reduce additional integration of other components. PSs can be directly used as a component (linkers) of MOFs to achieve non-invasive PDT of tumors [ 114 ]. However, the production of ROS limits therapeutic effect, mainly because the single PDT treatment does not present an effective anti-cancer effect [ 115 , 116 ]. Therefore, there is a need to develop multifunctional MOFs to improve diagnostic accuracy and treatment efficacy. Zhang et al. [ 117 ] developed new multifunctional zirconium-ferriporphyrin MOF (Zr-FeP-MOF) nanoshuttles ( Figure 8 C). HSP70 inhibitor siRNA was modified using PEG and loaded with siRNA to prepare the siRNA/Zr-FeP-MOF therapeutic platform. Notably, the siRNA/Zr-FeP-MOF can catalyze endogenous hydrogen peroxide (H 2 O 2 ) and O 2 to generate abundant hydroxyl radical (·OH) and 1 O 2 under NIR laser. siRNA/Zr-FeP-MOF nanoswitches have a photothermal effect on MTPTT owing to the introduction of siRNA. It indicated that PDT combined with MTPTT could significantly inhibit cancer growth in vitro and in vivo. 4.4. Gas Therapy Organisms have gaseous signaling molecules that act as messengers in the process of specific binding with multivalent transition metals like hydrogen sulfide (H 2 S), nitric oxide (NO), and carbon monoxide (CO) [ 118 ], and thus have a variety of physiological functions in almost all human systems, such as the nervous system, cardiovascular system, and immune system. The gas signaling molecules play an important role in normal human physiological processes and are implicated in the regulation of pathological processes. A high concentration of NO, CO and H 2 S in the blood can cause poisoning; however, in a relatively mild concentration range, they have significant anti-cancer activity [ 119 ]. For instance, NO mainly reacts with the superoxide anion to form reactive nitrogen species (RNS), such as peroxynitrite, that react with DNA to induce a variety of DNA damage [ 120 ]. The efficacy of gas therapy is highly correlated with gas concentration. Therefore, the controlled release of therapeutic gas in the lesion is very critical in gas therapy [ 121 , 122 ]. Laser is the most convenient and effective exogenous stimulus, therefore, light-controlled drug release has been explored in several studies [ 123 , 124 ]. The penetration depth of ultraviolet and visible light significantly limits the application of light-responsive gas release in vivo and can easily cause phototoxicity. On the contrary, NIR light has better tissue penetration depth and milder phototoxicity. Therefore, NIR laser-responsive gas release has a broader application prospect. Laser irradiation ensures remote control of gas therapy, and the thermal effect of PTT can promote the release of the gas [ 125 , 126 , 127 ]. For instance, Gao et al. utilized PAs to combine with a photo-triggered NO generator (thiolated transferrin), thus promoting the release of NO under the irradiation of 808 nm near-infrared light [ 128 ]. However, the application of NO in biomedicine is limited by several aspects, such as high activity, poor selectivity, and short half-life (less than 3s). Therefore, selective delivery of NO based on nanoplatforms to the lesion area should be explored to ensure maximum utilization of NO in anti-cancer therapies. The thermal effect of MTPTT promotes a spatiotemporally controlled release of NO. Yao et al. [ 80 ] designed gold nanorods coated with mesoporous silica, linked with S-nitrosothiols, and loaded them with 2-phenylethynesulfonamide (PES) as an inhibitor of HSP70, and surface modification using PEG ( Figure 8 D). The cumulative concentration of NO reached 14.6 μM after 10 min of irradiation with 1 W/cm 2 808 nm laser, and the solution temperature was 40.8 °C. The apoptosis rate of the combination therapy comprising gas therapy and MTPTT was approximately 70%, which was two-fold that of MTPTT alone. The tumor inhibition rate of the combination of gas therapy and MTPTT was approximately 85% in vivo. Figure 8 A variety of examples reveal the superiority of the synergistic therapy MTPTT with other therapies. ( A ) A schematic illustration of the preparation of DOX-17AAG@B-PEG-cRGD nanosheets and the synthetic approach of MTPTT with chemotherapy [ 81 ]. Copyright © 2022, Royal Society of Chemistry. ( B ) Illustration of synthesis procedure of HNC-s-s-HA/GA and the combination of MTPTT with radiotherapy [ 104 ]. Copyright © 2022, John Wiley and Sons. ( C ) Schematic illustration of siRNA/Zr-FeP MOF nanoshuttles for multimode imaging diagnosis and combination of MTPTT and PDT for cancer treatment [ 116 ]. Copyright © 2022, John Wiley and Sons. ( D ) Schematic illustration of the preparation of PEG-PAu@SiO 2 -SNO nanocomposites and the process of mild heat-enhanced gas therapy under NIR irradiation in MCF-7 cells [ 80 ]. Copyright © 2022, American Chemistry Science. 4.5. Immunotherapy Cancer immunotherapy is the most promising and epochal treatment that boosts anti-cancer immunity or eliminates immune suppression to achieve immune cell-mediated tumor clearance and improve the survival of cancer patients [ 129 , 130 , 131 ]. Normally, the immune system recognizes and kills abnormal tumor cells, but tumor cells can trigger a large of strategies to avoid recognition and elimination by the immune system through a process known as “immune escape” [ 132 ]. Cancer immunotherapy is a treatment to eradicate tumors by restarting and maintaining the cancer-immunity cycle [ 133 ]. The cancer-immunity cycle is divided into the following seven links: (1) the release of antigens from the dead cancer cells, (2) antigen presentation, (3) priming or activating T cells, (4) T-cell migration to tumors, (5) tumor tissues infiltrating T-cells, (6) T cells recognize tumor cells, (7) eliminating cancer cells. One of the barriers in any of these links can result in failure of the anti-tumor—immune cycle and immune escape. However, immunotherapy is not effective for all tumor types, owing to the toxicity of high immunohorizons and low objective rate [ 134 ]. Tumor recurrence is a severe defect caused by PTT due to residual tumor cells caused by uneven heating or failure to completely remove tumor cells due to the limitation of the laser penetrating depth [ 28 ]. In addition, PTT is an effective local tumor therapy. However, it is not effective for disseminated tumors. Recent studies report that high temperatures can promote the release of anticancer substrates from dead tumor cells, leading to immune activation [ 135 , 136 , 137 ]. Previous studies report that hyperthermia can trigger an immune response by activating immune cells such as CD8 + T cells, natural killer (NK) cells, and dendritic cells (DCs), promoting the release of tumor cells exons, and upregulating expression of inflammatory cytokines and HSPs [ 138 ]. PTT-induced hyperthermia can result in apoptosis or necrosis of tumor cells and release tumor-associated antigens. The tumor-associated antigens can be received and presented by antigen-presenting cells (APCs), thus activating immune cells, and inducing an anti-cancer immune response. Moreover, a combination of PTT and immune adjuvant can be used for the development of in situ autologous cancer vaccine [ 139 , 140 ]. Cancer vaccine is efficient immunotherapy that can effectively activate the immune system to kill tumor cells by injecting tumor-associated antigens into tumor patients so as to achieve the purpose of tumor control and treatment [ 141 , 142 , 143 ]. Tumor-infiltrating dendritic cells usually present an immature and immunosuppressive phenotype and are unable to mediate the immunosuppressive response of tumors fully. Immunotherapy adjuvants with CpG oligonucleotides as Toll-like receptor (TLR) agonists can activate tumor-infiltrating dendritic cells to enhance vaccine-specific immunity [ 144 , 145 ]. Nevertheless, the shortcoming of CpG oligonucleotide-based immunotherapy is usually counteracted by immunosuppressive TME [ 146 , 147 , 148 , 149 ]. For modulating the microenvironment toward immune activation, Li et al. prepared a photothermal CpG nanotherapeutics (PCN) ( Figure 9 A) to induce an immunofavorable TME by casting heat (43 °C) after laser irradiation in the tumor site [ 150 ]. The apoptosis results showed that an MTPTT from laser illumination could mediate tumor cell apoptosis and necrosis. The antigen released from tumor cells and CpG-activated macrophages and DCs to promote activation/maturation demonstrated from the results of the increased serum IL-6 level, the upregulated expression of BMDC maturation markers, CCL8 and Clec4e. As a results, MTPTT was successfully proven to improve the innate and adaptive immune response. Figure 9 MTPTT assists in distinct types of immunotherapies. ( A ) The schematic of the disassembly process of PCN and an immunofavorable TME was established via a fever-like immune response induced by the photothermal effect of PCN [ 150 ]. Copyright © 2022, The Author(s). ( B ) Schematic structure of GO-FA-SNX-2112 and its application for MTPTT of the tumor to induce overactivation of autophagy; stimulated autophagy not only causes tumor cells to die directly but also makes them be captured by immunity because of the decrease in PDL1 receptor expression; residual surviving tumor cells are also gradually killed by restored immune cells, to achieve efficient inhibition of tumor growth [ 32 ]. Copyright © 2022, American Chemical Society. ( C ) Schematic illustration of the symbiotic mild photothermal-assisted immunotherapy via a combined all-in-one and all-in-control strategy [ 159 ]. Copyright © 2022, The Author(s). HSP90 inhibition improves tumor immunotherapy by upregulating the expression of the interferon response gene. HSP90 is highly correlated with autophagy and protein kinase B (AKT). However, severe side effects and tumor recurrence limits traditional treatment. Thus, it is challenging to obtain a satisfactory survival rate. Treatment can be more effective and milder by targeting the specific region or functions of cancer. “Automatic treatment” widely represents the “double-edged sword” phenomenon. On the one hand, autophagy plays a significant role in cancer eradication due to providing nutrition and limiting T cell-mediated cytotoxicity. On the other hand, the accumulation of autophagosomes can exert anti-tumor activity by inducing is associated with ACD. Therefore, drug-induced or inhibition of autophagy can kill tumor cells. Tumor cells produce a stress protein (HSPs) when exposed to laser irradiation to protect themselves through the normal function of HSPs from the invasion of MTPTT. On the contrary, autophagy can lead to cancer cell apoptosis when HSP90 is downregulated, which plays a role in several key ways to maintain the dynamic stability of the cell environment. This implies that the function of autophagy may be reversed in the absence of HSP90 when facing a high-energy environment. The application of MTPTT to regulate autophagy is a novel approach to tumor therapy. Overactivation of autophagy induced by MTPTT and regulation of HSP90 inhibitors play key roles in the efficacy of MTPTT [ 33 ]. Deng et al. designed graphene oxide (GO) loaded with SNX-2112 and folic acid (FA) for MTPTT ( Figure 9 B) [ 32 ]. Changes in HSPs levels are related to the activity of AKT because HSP90 is an early protein in the AKT signaling pathway. During autophagy, HSP90 inhibits AKT and inactivates AKT under stress conditions. Therefore, the pathways associated with vital signs were evaluated by Western blot analyses. The results showed that expression of level p-AKT and autophagy-related genes were significantly different in the GO-folic acid- SNX-2112 group and expression of HSP90 was inhibited by SNX-2112. These findings indicated that autophagy is activated and the AKT pathway is inhibited by MTPTT. In addition, the expression of programmed death-ligand 1 (PDL1), which is implicated in tumor immune function, was significantly downregulated compared with the level of the control group and GO-folic acid group. These findings indicate a relationship and crosstalk among autophagy, p-AKT, and PDLL. Immune checkpoint therapy that aims to regulate the activity of T cells through inhibition or stimulation of signals from the immunosuppressive TME is the main treatment of immunotherapy [ 151 , 152 ]. However, recent research presented that many patients presented with “non-immunogenic” tumors, also known as “cold” tumors, characterized by a lack of tumor-infiltrating lymphocytes [ 153 , 154 ]. Therefore, it is a grand challenge for immunotherapy how to convert “cold” tumors to “hot” tumors. There are five strategies to switch cold tumors to hot tumors [ 155 ]: (1) improve the tumor inflammation, (2) neutralize the immunosuppressive factors in the TME, (3) target the tumor blood vessels and stroma, (4) target tumor cell signaling pathways, (5) improve the longevity and function of anti-tumor immune T cells. The combined therapy with various therapeutic treatments has enhanced the immune checkpoint therapy efficacy [ 156 , 157 ]. Moreover, the mild temperature is favorable to immunological responses in TME [ 115 , 158 ]. Huang et al. loaded a photothermal agent (IR820) and an anti-programmed death-ligand 1 antibody (aPD-L1) into a lipid mixture for combining the immune checkpoint blockade antibodies with MTPTT ( Figure 9 C) [ 159 ]. After measuring the immune response of immune cells in lymph nodes, spleens, and tumors of 4T1 and B16F10 mice, the results showed that the treatment-induced cell differentiation of naive T cells to CD8 + T cells. Therefore, the precise control of MTPTT temperature is critical in sensitizing the immunosuppressive tumors for converting cold tumors to hot tumor as well as potentiating immune checkpoint therapy. 5. Summary and Perspectives Over the past decades, PTT of tumors has drawn extensive attention, and studies demonstrate broad prospects for small, unresectable tumors or for patients who are poor surgical candidates. High-temperature thermal ablation by agent-free PTT or contrast-enhanced PTT significantly affects healthy tissues, induces undesirable inflammation, and largely impairs immune antigens and immune cells related to antitumor immunity. Therefore, MTPTT was introduced to circumvent the side effects associated with PTT at a temperature below 45 °C. Studies report that a combination of MTPTT with HSPs inhibitors or other various agents significantly improves the efficacy of mild hyperthermia as these agents alleviate thermal resistance. In addition, well-designed and multifunctional nanoplatforms can improve tumor specificity of photosensitizers improving tumor targeting, selectivity, activation, or image guidance, and/or through combination with other therapies. Nanosystems can achieve the high therapeutic efficiency of MTPTT by restricting the function or reducing the expression of HSPs. In the current review, the mechanism and recent strategies in multifunctional nanoformulations for enhanced MTPTT were summarized. The use of HSPs inhibitors, integrating siRNA, targeting the nucleus, blocking energy inhibition, and impacting autophagy can alleviate the thermal resistance of tumor cells and protect normal cells from MTPTT effects. In addition, synergistic therapy can combine the advantages and offset the disadvantages of individual therapies, thus improving therapeutic outcomes. The current review explored the recent developments in combining MTPTT with other therapies, including chemotherapy, radiotherapy, PDT, gas therapy, and immunotherapy. Notably, MTPTT can modulate and rebuild the immunosuppressive tumor environment from “cold” to “hot,” thus preventing tumor recurrence and metastasis. In summary, MTPTT has a wide clinical prospects and further advances in biomedicine compared with PTT. Although previous studies have reported several multifunctional nanoagents and their positive outcomes, MTPTT is still in a nascent stage. The clinical application of MTPTT is limited by the poor penetration depth of laser light, accumulation at the target locations, and poor biocompatibility with nanomaterials in vivo. Further studies should explore NIR-Ⅱ PAs should be explored for orthotopic tumors. In addition, the use of nanoparticles is limited by undesirable immune reactions and rapid clearance from the body by the reticuloendothelial systems. Biomimetic and bioinspired coating can be used to overcome physical barriers, such as the use of cell membrane cloak nanomaterials. Broad particle size distribution of nanoparticles can result in high risks when used in humans. Biodegradable and naturally derived PAs can be used in MTPTT. Moreover, new technology or therapy modalities such as chiral nanoparticles, nanoenzymes, nanorobots, chemodynamic therapy, ultrasound therapy, and microwave therapy can be combined with MPPTT to improve therapeutic efficacies for MTPTT-based cancer treatment. Acknowledgments We sincerely acknowledge the assistance from Fei Tong and Jun Guo. Author Contributions Conceptualization, P.W., B.C., Y.L., J.W.; writing—original draft preparation, P.W., Y.Z., L.W.; writing—review and editing, J.L., J.X., L.Z., Z.L.; visualization, P.W.; supervision, Z.L.; funding acquisition, P.W., J.X., L.Z., Z.L., Y.L., J.W. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Funding Statement This research was funded by National Natural Science Foundation of China grant number [52163016, 32171337, 82060198], Jiangxi Province Key Research and Development Program grant number [20203BBGL73157], Natural Science Foundation of Jiangxi Province of China grant number [20192BAB205055, 20212BAB206072], and the Technological Plan of Health Commission of Jiangxi Province of China grant number [202130519]. Footnotes Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Hildebrandt B., Wust P., Ahlers O., Dieing A., Sreenivasa G., Kerner T., Felix R., Riess H. The cellular and molecular basis of hyperthermia. Crit. Rev. Oncol./Hematol. 2002;43:33–56. doi: 10.1016/S1040-8428(01)00179-2. 2. Vaupel P. Tumor microenvironmental physiology and its implications for radiation oncology. 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# 借助多种策略增强癌症轻度温热光热治疗的效率

## 摘要

传统光热治疗（PTT）通过将肿瘤组织温度升高至48°C以上来实现热消融，从而杀伤肿瘤细胞。然而，PTT过程中的热消融会损伤周围正常组织，引发治疗后炎症反应，并因肿瘤细胞内容物在短期内大量释放而导致快速转移或其他副作用。为克服这一局限性，轻度温热光热治疗（MTPTT）被引入以替代PTT，其治疗温度范围为42–45°C。然而，由于癌细胞的热耐受性，MTPTT的治疗效果显著降低，尽管其解决了一些副作用问题。本文综述了MTPTT的作用机制及提高其疗效的各种策略。热休克蛋白（HSPs）作为分子伴侣在肿瘤细胞中过表达，参与多种细胞热应激反应。因此，本文介绍了在MTPTT治疗过程中抑制HSPs活性、降低其表达水平及阻碍其功能的方法。此外，本文还重点介绍了其他策略，包括细胞核损伤、能量抑制和自噬介导。同时，放疗、化疗、光动力治疗和免疫治疗等疗法与MTPTT联合应用可产生显著的协同效应。本综述为进一步研究提供了基础，并为MTPTT的临床应用提供了新思路。

**关键词：** 轻度温热光热治疗；免疫治疗；热休克蛋白；纳米平台；热耐受状态

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## 1. 引言

早在公元前5000年的埃及，热疗便被用于治疗乳腺肿瘤。肿瘤组织具有血管增多、血液淤滞、散热不良、阻力高、散热困难、易蓄热及温度快速升高等特点[1,2]。因此，热疗对肿瘤治疗具有显著效果[3]。光热治疗（PTT）是一种将光能转化为热能以提高病灶温度从而实现治疗效果的热疗方法[4,5]。外源性光热转换剂（PAs）虽非PTT所必需，但可提高治疗的效率和疗效[6]。PTT通过促进肿瘤细胞在高温下发生凋亡或坏死，被广泛应用于多种类型肿瘤的治疗[4,7,8]。与传统化疗、放疗和手术相比，PTT借助外源激光可实现高精度、高效率、低毒性和无创治疗[5,9,10]。此外，激光可作为"光触发开关"实现远程药物控释（光刺激响应）[11,12]。同时，热量可破坏溶酶体，帮助载药体系从溶酶体中逃逸。

目前，临床前与临床PTT研究之间存在明显分野：临床前研究聚焦于新型PAs的开发，而临床研究则集中于集成激光设备的应用[6]。这种差异可能反映了PTT在临床前研究中易于展示其效果，从而促进了多种新型纳米材料的制备和应用。然而，PAs因其对靶组织具有更好的选择性、可使用更低功率的激光并简化设备设计，在临床转化方面具有潜力。先前研究通过调控纳米颗粒的形状、尺寸和表面化学性质对PAs进行了大量优化[7,13,14]。此外，纳米技术的快速发展通过开发多功能纳米颗粒推动了PTT的进步[15]。例如，金纳米颗粒和铂纳米颗粒等贵金属纳米颗粒常被选作PAs[16,17]。此外，与PTT的协同治疗提高了PTT对肿瘤的治疗效果[18]。PTT通过促进药物递送、刺激释放、介导肿瘤微环境（TME）、引发肿瘤特异性抗原释放或调节其他生物学相关反应，直接杀伤肿瘤细胞或增强其他疗法[19–25]。

然而，PTT的临床应用在一定程度上受到若干局限性的制约。例如，使用PTT完全杀伤肿瘤细胞具有挑战性，由于激光的组织穿透深度有限（NIR-I窗口激光1~2 cm，NIR-II窗口激光>2 cm），增加了肿瘤复发和转移的风险[26]。因此，为实现较高的治疗温度，研究人员往往提高激光功率或PAs的剂量。然而，美国国家标准学会（ANSI）已制定了PTT在皮肤上临床使用的安全耐受阈值标准[27]。808 nm激光功率阈值范围为330~350 mW cm⁻²，曝光时间为10~1000 s。此外，PTT不可避免地损伤肿瘤部位周围的正常组织，导致体内毒性和副作用[28]。此外，热消融引起的多种细胞内容物及残留肿瘤细胞可能引发一系列副作用，包括炎症、肿瘤转移、正常组织损伤和肿瘤复发[29]。

为克服这些局限性，MTPTT（温度范围为42°C至45°C）[3,30]被引入以降低使用温度，从而减轻副作用。此外，由于使用较温和的温度，MTPTT对患者生活质量的影响不显著。然而，MTPTT的治疗效果较差。因此，研究一直在探索在MTPTT条件下利用纳米载体实现更好治疗效果的方法。虽然热休克蛋白（HSPs）抑制剂或其他化合物可被包封到纳米平台中，但其抗肿瘤疗效和安全性仍需更全面和深入的研究。尽管如此，MTPTT是一种重要的综合治疗方法，在未来临床应用中展现出巨大潜力[31]。

本综述全面总结了用于肿瘤治疗的新型纳米系统（包含MTPTT）的最新进展和功能（图1）。本综述探讨了（1）MTPTT的作用机制，（2）MTPTT的多种方法，（3）MTPTT与其他治疗方式的联合应用，（4）MTPTT的挑战及未来发展，以提高MTPTT的疗效。研究探讨了热应激反应（HSR）在MTPTT疗效中的作用，以及如何通过设计药物递送纳米系统来改善MTPTT的疗效。最后，本文探讨了MTPTT领域当前面临的关键挑战，并提出了若干重要的未来研究方向，以改进现有策略并为开发新策略以提高MTPTT的有效性奠定基础。

**图1** 通过多种策略利用MTPTT进行癌症治疗的示意图。

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## 2. MTPTT的作用机制

MTPTT在癌症治疗中的有效性不依赖于精确的设备或特殊方法来控制温和温度，而依赖于在温和温度下维持治疗效果的方法。MTPTT的治疗效果归因于破坏肿瘤细胞的自保护机制，防止热应激造成严重损伤。研究表明，MTPTT通过两种自保护机制发挥作用，包括热休克反应和自噬[32,33]。在传统PTT（>48°C）中，热消融引起蛋白质严重不可逆变性、DNA损伤和变性，并破坏自保护机制的有效防御。值得注意的是，自保护机制在MTPTT（<45°C）中对未折叠蛋白的修复具有显著作用。因此，抑制自保护机制的通路是实现MTPTT高效性的最有效途径。研究表明，HSR和自噬是介导MTPTT过程中自保护机制的关键靶点（图2）。

**图2** （A）热疗后热休克反应过程及通过siRNA、细胞核损伤、HSPs抑制剂和能量抑制阻断热休克反应的示意图。（B）HSPs的生理功能示意图：在MTPTT中协助蛋白质折叠成天然构象。

高于41°C的热疗引起蛋白质变性和暂时性细胞失活，可持续数小时[34,35]。因此，HSR诱导HSPs表达上调，有效防止其他蛋白质聚集。HSR是存在于所有生物体中的细胞防御机制，在防止热疗或其他不良应激条件造成的损伤中发挥作用。HSR通过其细胞保护和抗凋亡效应限制MTPTT的治疗效果[36]。此外，HSPs可与凋亡信号通路蛋白相互作用以抑制凋亡的发生，从而降低热疗的治疗效果[37,38]。此外，与正常细胞相比，肿瘤细胞过表达HSPs，使其对热处理的敏感性降低，并能在高温下保持活性[39,40]。

肿瘤细胞主要通过激活热休克转录因子（HSFs）来调节HSPs的表达[41]。先前研究探索了四种HSF，包括HSF1、HSF2、HSF3和HSF4。值得注意的是，HSF1是介导HSR的主要转录因子。HSF1在多种肿瘤细胞中高表达，与肿瘤进展和不良预后相关。HSF1的主要作用机制是通过增强其326位丝氨酸的磷酸化，上调HSP70和HSP27的表达，最终促进恶性增殖和抗凋亡[42,43]。

在正常生理条件下，HSPs的表达水平较低，仅占总蛋白的1–2%[44]。在高温、过量活性氧（ROS）或炎症刺激后，HSF1被激活并结合到下游HSPs基因的启动子区域，促进HSPs的表达。HSP70是HSR后许多HSP家族中首先表达的蛋白[45,46]。B细胞淋巴瘤-2（Bcl-2）相关抗凋亡基因3（BAG3）是HSP70的伴侣蛋白，可通过BAG结构域结合到HSP70的ATP酶结构域以调节HSP70功能[47,48]。此外，BAG3-HSP70复合物可结合Bcl-2并防止其降解，从而抑制凋亡通路或抑制热疗和化疗诱导的肿瘤细胞凋亡[49–51]。因此，抑制HSR可降低肿瘤细胞的热耐受性，提高PTT的增敏效果。

多项研究探索了通过基因介导的沉默技术（小干扰RNA或短发夹RNA，siRNA或shRNA）抑制HSR，同时研究也在开发热敏药物。MTPTT的疗效主要通过阻断HSR实现，主要包括两个方面：（1）减少HSR产生的HSPs合成[52]，以及（2）抑制HSPs的活性[53]。当前研究主要聚焦于HSPs在提高PTT疗效中的机制。PTT的疗效可通过以下三种途径提高：使用HSPs抑制剂、通过siRNA沉默HSPs基因以及减少ATP合成。因此，将HSPs抑制剂（或siRNA、ATP抑制剂）与PAs在纳米系统中联合使用，以提高肿瘤细胞对热的敏感性，这一点非常重要[54]。

此外，自噬作为一种细胞自保护机制，在热应激下被迅速激活，以维持能量生产并提供回收材料。自噬相关耐受性在热耐受性中也发挥关键作用[55]。根据底物最终进入溶酶体腔的不同途径，自噬分为三种类型：微自噬、伴侣介导的自噬和巨自噬（图3）。受损和变性的蛋白质及细胞器被自噬体吞噬，随后在溶酶体中降解以提供能量和大分子前体，并可被回收以维持细胞代谢[56–58]。因此，阻断自噬通路可提高MTPTT的疗效。

自噬可通过抑制以下环节被阻断：（1）自噬体形成（3-甲基腺嘌呤、渥曼青霉素）[56]，（2）自噬体与溶酶体融合（羟氯喹、氯喹、长春花碱）[59]，以及（3）自噬溶酶体降解（胃蛋白酶A）[60]。相反，过度自噬不仅不能保护细胞，反而破坏稳态功能并诱导自噬介导的细胞死亡（ACD），即II型程序性细胞死亡[61]。过度自噬活性远超自噬溶酶体的降解能力，导致微空泡形成和降解障碍[62]。当自噬不能有效停止或被过度刺激时，自噬活动无法回收癌细胞组分并加速ATP耗竭，最终导致细胞死亡并进一步增强MTPTT的治疗效果。因此，可通过切断自噬抑制通路或使用自噬诱导剂（包括卡马西平、C2-神经酰胺、雷帕霉素和xestospongin B/C）来诱导过度自噬[63]。

**图3** 热疗后巨自噬过程及抑制或诱导自噬的各种策略示意图。

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## 3. 提高MTPTT疗效的多种方法

热消融（高于48°C）直接诱导肿瘤细胞坏死，而周围正常组织则因热扩散而受损[34]。这意味着PTT具有较高的治疗效果，但伴随不良反应。由于肿瘤细胞的热耐受性，降低温度会降低PTT的疗效[3]。因此，开发克服热耐受性的策略对于促进MTPTT的疗效至关重要。以下部分探讨了热耐受机制，并总结了构建多功能纳米系统以提高MTPTT疗效的方法。

### 3.1. 热休克蛋白抑制剂

HSPs主要根据其分子量分为HSP27（~27 kDa）、HSP40（~40 kDa）、HSP60（~60 kDa）、HSP70（~70 kDa）、HSP90（~90 kDa）和HSP110（~110 kDa）[44]。HSP70和HSP90在HSR中发挥重要作用[39,64–66]。HSPs在结构和功能上具有一些相似性。所有HSPs类别均包含三个结构域（图4A），包括N端结构域、中间结构域和C端结构域[42,44]。N端结构域是ATP的结合位点。ATP酶结构域中的脯氨酸残基可诱导构象变化并引起水解，从而诱导HSPs的活性[43]。中间结构域是客蛋白和伴侣蛋白的结合位点，是HSPs的活性区域[45]。C端结构域是伴侣蛋白的结合位点，负责底物结合和重折叠（HSPs的二聚化），形成"封闭"构象以保护客蛋白[67]。

**图4** 若干实例展示了整合HSP70和HSP90抑制剂的纳米平台，有效实现肿瘤的MTPTT。（A）HSP70和HSP90结构与功能的示意图[43,67]。版权所有 © 2022和2016，Elsevier。（B）一维纳米级配位聚合物一步合成及通过抑制HSP90克服热耐受性的示意图[68]。版权所有 © 2022，John Wiley and Sons。（C）用于荧光/光声成像引导的MTPTT/化疗的ICG-17AAG@HMONs-Gem-PEG纳米平台的构建示意图[69]。版权所有 © 2022，American Chemical Society。（D）聚乙烯基吡咯烷酮保护的金属离子-槲皮素（Qu）配位纳米药物家族的一锅合成示意图，将精确诊断、优异的MTPTT疗效、ROS清除和抗炎作用、动态解组装和肾清除能力固有地整合到单一纳米颗粒中[70]。版权所有 © 2022，Elsevier。

HSPs抑制剂（表1）可特异性结合HSPs的中间结构域，阻止客蛋白结合，从而在HSR过程中失去保护细胞的能力[71]。藤黄酸（GA）是一种从藤黄树中分离的天然异戊烯化呫吨酮类化合物，具有多种生物活性，如抗癌、抗炎、抗氧化和抗菌活性。此外，GA通过与HSP90的N端ATP结合域结合发挥重要作用，不与ATP竞争，从而抑制ATP水解的催化[72,73]。由于这一功能，GA被用作HSP90抑制剂，并与PAs联合使用以提高MTPTT在癌症治疗中的疗效。

智能纳米系统被设计为在肿瘤组织或细胞中快速释放，从而提高靶向HSPs药物的疗效。Yang等[68]设计了聚乙二醇（PEG）修饰的一维吲哚菁绿（ICG）-锰纳米材料，负载GA用于MTPTT（图4B）。一维ICG-锰纳米材料具有高载药率和pH刺激响应的优点。在肿瘤的酸性微环境中，结构解离并快速释放GA。在约43°C下，负载GA的纳米颗粒组的体外细胞存活率显著低于其他组。此外，Western blot实验显示GA下调了HSP90的表达。负载GA的纳米载体可通过抑制HSP90在相对温和的温度下诱导肿瘤细胞有效凋亡，而非热消融（高于50°C）。这有助于最大限度地减少对正常器官的非特异性热效应，并提高PTT治疗大型或深部肿瘤的疗效。

**表1** 用于MTPTT的HSP抑制剂。

| HSPs种类 | 药物 | PAs | 参考文献 | |---|---|---|---| | HSP70 | 2-苯乙炔磺酰胺（PES） | PEG-PAu@SiO₂-SNO | [80] | | | 槲皮素 | Qu-FeⅡP纳米颗粒 | [70] | | | | B780/Qu NPs | [78] | | HSP90 | 17-AAG | ICG-17AAG@HMONs-Gem-PEG | [69] | | | | DOX-17AAG@B-PEG-cRGD | [81] | | | 藤黄酸 | NCPs/GA | [68] | | | | HAS/dc-IR825/GA | [74] |

值得注意的是，肿瘤组织蓄积不足和肝脏过度滞留有效限制了许多纳米药物的疗效和生物相容性。Wu等报道了由GA（HSP90抑制剂）、dc-IR825（荧光成像探针和光热转换剂）和人血清白蛋白（生物相容性材料）组成的智能诊疗纳米载体[74]。该纳米载体展示了化疗与MTPTT的协同作用，从而提高了癌症治疗的疗效。在纳米载体中，GA的胞质转位可通过近红外（NIR）激光照射下线粒体ROS介导的破坏来促进，进一步阻断HSP90的过表达。因此，该纳米载体可在MTPTT条件下杀伤癌细胞，从而提高癌症治疗的有效性。

17-烯丙基氨基-17-去甲氧基格尔德霉素（17-AAG）是一种源自格尔德霉素抗生素的HSP90抑制剂，可引起肿瘤细胞凋亡[75,76]。此外，17-AAG可有效抑制多种细胞信号转导通路，如降低丝氨酸/苏氨酸激酶38（STK38）/核Dbf2相关1（NDR1）的细胞水平及STK38激酶活性[77]。Wu等制备了中空介孔有机硅纳米胶囊（HMONs），为成像引导的MTPTT/化疗提供了多功能纳米平台，实现了高诊疗疗效（图4C）[69]。17-AAG和ICG被负载到HMONs上，当吉西他滨（Gem）门控分子在酸性TME中因缩醛键水解而特异性打开时，二者同时释放。随后，17-AAG诱导HSP90下调，从而逆转肿瘤细胞的热耐受性，实现MTPTT的目标。该纳米平台展示了MTPTT/化疗的协同效应，在精确癌症诊疗方面具有潜力。

槲皮素（Qu）是一种富含羟基的天然多酚，广泛分布于蔬菜、果皮、种子、饮料和中草药中。它具有优异的抗氧化、抗癌及心脑血管疾病预防和治疗作用。此外，Qu通过调节HSF转录活性抑制HSP70表达[78,79]。Yang等以槲皮素（HSP70抑制剂）为框架设计了新型Qu-FeⅡP纳米颗粒（图4D）[70]。高温可引发炎症并损伤肿瘤组织周围的正常细胞。因此，槲皮素在清除ROS和发挥抗炎活性方面发挥重要作用。Qu-FeⅡP对MCF-7细胞的体外IC₅₀为100 μg/mL。然而，在激光照射下，Qu-FeⅡP的IC₅₀为3.13 μg/mL。经过20天的体内治疗，Qu-FeⅡP +激光照射组在MTPTT下显示出良好的肿瘤抑制效果，四分之三的肿瘤完全消失。肿瘤组织的Western blot分析显示，HSP70的表达水平低于其他蛋白，从而显著降低了肿瘤细胞的热耐受性并提高了MTPTT的疗效。

### 3.2. siRNA

RNA干扰（RNAi）技术在生物医学领域具有巨大潜力。由于其高效率、高特异性和低毒性，它为新型药物的设计和开发提供了新途径。合理的精确化学修饰和纳米载体为克服siRNA的局限性（如快速降解、细胞摄取差和脱靶效应）提供了可行机会。siRNA是RNA干扰的有效载体，通过沉默特定基因的表达来抑制HSPs或BAG3，使癌细胞更容易受到PTT效应的影响，从而为抑制HSR提供了策略[54]。然而，该方法存在血清稳定性低和在递送至靶细胞过程中被体内清除等局限性。因此，可通过使用纳米系统递送siRNA来提高其有效性。

Ding等[34]以siRNA作为交联剂构建了DNA接枝聚己内酯（DNA-g-PCL）纳米颗粒（图5A），随后用聚多巴胺（PDA）进行包覆并用PEG进行修饰（PP-NG-siHSP70）。PP-NG-siHSP70 +激光照射组的基因沉默活性显示HSP70 mRNA表达最低，caspase-3 mRNA表达最高，与Western blot结果一致。PP-NG-siHSP70 +激光照射组的体外细胞凋亡率为72.2%，表明该纳米颗粒在温和条件下诱导了有效的靶基因敲低和细胞凋亡，而非细胞坏死。值得注意的是，PP-NG-siHSP70 +激光照射组中三分之二的肿瘤在治疗16天后消失。

Wang等[82]制备了负载BAG3 siRNA的金纳米棒（GNRs）平台，该基因沉默能力可提高MTPTT的疗效（图5B）。口腔鳞癌的体内外研究结果表明，该纳米棒通过下调BAG3表达提高了肿瘤细胞对PTT的敏感性并增加了凋亡。经激光处理后，siRNA处理的小鼠中GNRs-siRNA的相对体积减少了18.4%。

**图5** 有利于MTPTT的siRNA和细胞核损伤的具体方式。（A）PDA包覆核酸纳米凝胶的合成及PEG-PDA-纳米凝胶介导的MTPTT机制示意图[34]。版权所有 © 2022，Elsevier。（B）GNRs-siRNA在改进的MTPTT平台中的设计示意图[82]。版权所有 © 2022，Elsevier。（C）癌细胞膜和细胞核细胞器双靶向V₂C-TAT@Ex-RGD纳米试剂用于NIR-II生物窗口中多模态成像引导的MTPTT示意图[83]。版权所有 © 2022，American Chemical Society。（D）基于DNAzyme的纳米海绵用于高效PTT的示意图[84]。版权所有 © 2022，The Author(s)。

### 3.3. 细胞核损伤

PAs靶向细胞核可破坏细胞核中遗传物质的结构，从而提高MTPTT的疗效。TAT是一种细胞穿透肽，通过靶向细胞核发挥功能。因此，它可用于通过修饰超小纳米颗粒或量子点来实现细胞核靶向[85]。因此，设计具有高效光热转换性能和高度核内蓄积的新型核靶向PAs对于MTPTT具有重要意义。

Cao等[83]制备了可蓄积于细胞核并破坏遗传物质的小尺寸碳化钒（V₂C）-TAT量子点，从而增强MTPTT的效果（图5C）。V₂C-TAT量子点被RGD修饰的外泌体包覆（V₂C-TAT@Ex-RGD），具有长循环周期、高生物相容性和良好的肿瘤靶向能力。V₂C-TAT@Ex-RGD在体内具有显著的治疗效果，因为其在1064 nm激光（功率密度0.96 W cm⁻²）照射下具有长血液循环时间、强肿瘤细胞靶向能力和较少的肿瘤蓄积。因此，V₂C-TAT@Ex-RGD可在温和温度下实现多模态成像引导的核靶向，在生物医学和临床应用方面展现出良好前景。

除细胞穿透肽介导的核靶向外，超小纳米颗粒更容易通过核孔（~40 nm大小）和核孔复合体进入细胞核[86]。Liu等[87]制备了具有核靶向功能的特殊超小壳聚糖包覆氧化钌纳米颗粒（CS-RuO₂ NPs），用于近红外窗口中癌症的MTPTT应用，并采用简单的一锅法合成了RuO₂ NPs（图5D）。对不同尺寸和表面电荷的核电源的分析表明，只有超小尺寸（2 nm）和带正电荷的核电源可有效进入细胞核，破坏DNA和蛋白质。CS-RuO₂ NPs在NIR-II窗口中显示出强吸收和优异的光热转换效率。此外，它们是PAs和光声成像（PAI）的理想材料。

### 3.4. 能量抑制

HSPs是ATP依赖性蛋白，在ATP存在下大量合成[45,47,67]。因此，可通过限制能量供应来降低HSPs水平。肿瘤细胞获取能量的主要途径是谷氨酰胺代谢、糖酵解和自噬，而非正常细胞的氧化磷酸化[88,89]。因此，可通过调节这些通路来限制肿瘤细胞中ATP的产生。

饥饿疗法旨在抑制肿瘤细胞获取或利用营养，使其因缺乏能量而"饿死"[90]。Zhou等[29]利用葡萄糖氧化酶（GOx）的催化作用将葡萄糖氧化为葡萄糖酸和H₂O₂，从而限制肿瘤细胞对葡萄糖的利用（图6A）。基于该机制设计了负载葡萄糖氧化酶的中空介孔普鲁士蓝（PB）纳米颗粒，用于饥饿疗法与MTPTT联合治疗肿瘤。此外，PB NPs被用于催化H₂O₂以改善肿瘤组织的缺氧水平。饥饿疗法限制ATP供应并抑制HSPs合成，从而降低癌细胞的热耐受性。结果表明，在GOx催化10分钟后，细胞内氧浓度从5.1 mg/mL降至0.04 mg/mL，表明GOx催化了葡萄糖氧化。使用协同疗法治疗21天后，肿瘤体积减少了32.5%。

**图6** 通过抑制能量或代谢增强MTPTT治疗效果的策略。（A）GOx诱导的饥饿在低氧TME中通过PHPBNs介导的肿瘤复氧增强MTPTT的示意图[29]。版权所有 © 2022，American Chemical Society。（B）封装GOx、ICG和GA的热敏脂质体用于协同饥饿疗法、EEPT和增强肿瘤MTPTT的示意图[91]。版权所有 © 2022，John Wiley and Sons。（C）GNR/HA-DC通过干扰厌氧糖酵解代谢选择性增敏肿瘤细胞MTPTT的示意图[92]。版权所有 © 2022，American Chemical Society。

在另一项旨在解决高剂量HSP抑制剂毒副作用的尝试中，Gao等展示了热敏GOx/吲哚菁绿/藤黄酸（GA）脂质体（GOIGLs），通过GA和GOx（通过将葡萄糖催化为葡萄糖酸诱导葡萄糖消耗）对HSPs的协同抑制来提高MTPTT的效率（图6B），同时结合酶增强光疗效应[91]此外，葡萄糖氧化的产物H₂O₂可在光照射下转化为羟基自由基（·OH），有效消除癌细胞，实现酶增强光疗（EEPT）。癌细胞和荷瘤小鼠实验结果表明，"GOIGLs +激光+光"的显著抗肿瘤疗效表明GOx介导的肿瘤饥饿和光疗提高了MTPTT的治疗效率。与传统PTT相比，MTPTT不仅可在相对低温（低于45°C）下实现有效的抗肿瘤治疗，而且从安全性评估结果来看可减少对正常组织的热损伤。

消耗大量葡萄糖是燃烧能量、阻碍HSPs效率的有效途径。此外，葡萄糖摄取也是抑制癌细胞代谢通路的重要靶点。Chen等[92]使用双氯芬酸抑制葡萄糖转运蛋白（Gluts）的活性（图6C），从而限制肿瘤细胞对葡萄糖的摄取。通过减少肿瘤细胞的厌氧糖酵解后下调HSP70和HSP90的合成，降低ATP水平，实现了MTPTT的目的。Western blot分析显示，HeLa细胞和MCF-7细胞与GNR/HA-DC共培养12、24和48小时后，Glut1蛋白量显著减少。GNR/HA-DC导致癌细胞葡萄糖摄取显著减少，抑制了包括厌氧糖酵解在内的细胞功能。培养48小时后，ATP分别减少了52.7%和35%。

### 3.5. 自噬介导

自噬作为一种动态细胞通路，降解和回收受损或老化的蛋白质及细胞器。自噬功能障碍与癌症、微生物感染、神经退行性疾病和衰老相关，表明自噬在这些疾病中发挥关键作用[61]。多项研究表明，药物抑制自噬或自噬相关基因（ATG）的基因敲除可增加癌细胞对多种药物的敏感性[93,94]。氯喹（CQ）抑制自噬并增强组蛋白去乙酰化酶抑制剂对慢性髓系白血病的抗癌活性[95]。此外，抑制自噬可增强贝伐单抗对肝细胞癌的抗癌作用。此外，CQ也用于治疗疟疾和自身免疫性疾病[96]。多项研究探索了调节自噬以开发癌症治疗药物。

PTT中光热效应转化的热量通过损伤细胞质组分激活自噬。因此，抑制自噬可提高MTPTT的治疗效果。CQ通过抑制溶酶体活性来抑制自噬降解。Zhou等使用CQ抑制肿瘤细胞自噬，从而提高MTPTT的疗效（图7A）[97]。结果表明，PDA-PEG/CQ组的肿瘤体积约为30 mm³，激光照射温度控制在约42°C。

**图7** 两个实例说明自噬增敏癌细胞的光热杀伤。（A）自噬抑制增敏癌细胞光热杀伤的示意图。用CQ和3-MA（分别为自噬抑制剂）处理的HeLa细胞中LC3-I和LC3-II的Western blot[97]。版权所有 © 2022，Elsevier。（B）Beclin 1诱导的自噬增敏癌细胞光热杀伤的示意图。用Beclin 1、PP、PPB和PPBR处理的MDA-MB-231细胞中LC3-I/LC3-II转化和P62的Western blot[98]。版权所有 © 2022，Elsevier。

适度自噬帮助细胞在不利环境中存活；然而，过度自噬导致细胞死亡。与凋亡不同，自噬的特征是形成大量包裹细胞质和细胞器的自噬体。Beclin1偶联的聚合物纳米颗粒促进肿瘤细胞的自噬活性，进一步抑制肿瘤生长。Beclin1诱导的自噬破坏了自噬的稳态功能，激活了自噬细胞死亡通路，提高了PTT的治疗效果。Zhou等[98]制备了多功能纳米颗粒，用于肿瘤靶向和通过自噬诱导提高PTT疗效（图7B）。该纳米颗粒由PDA纳米颗粒、Beclin 1衍生肽、PEG和环状Arg-Gly-Asp肽（PPBR）组成。PPBR提高了癌细胞的自噬活性，显著促进了PTT的杀伤效率。动物实验结果表明，PPBR可上调肿瘤细胞的自噬，在乳腺肿瘤模型中，联合治疗比单一治疗更有效地抑制了肿瘤生长。

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## 4. 协同治疗策略

如今，许多临床前和临床研究表明，许多单一疗法在治愈肿瘤方面效率较低，复发率高，毒副作用严重，如血液毒性、肝功能异常和对正常细胞的高毒性，以及免疫系统紊乱。光的穿透深度有限降低了PTT在抑制辐射范围外肿瘤生长方面的疗效[99]。单一疗法在抑制肿瘤生长方面效率低下，包括PTT单一疗法。尽管PTT具有较高的治疗效果，但其局限性可能导致肿瘤细胞消除不完全，最终导致肿瘤复发和转移。MTPTT与其他治疗方法的联合应用可提高治疗效果。除了提供补充外，不同治疗方法的综合应用产生协同治疗效果。

### 4.1. 化疗

MTPTT通过多种机制提高化疗的治疗效果，包括（1）增加某些药物的毒性，（2）增加肿瘤细胞对纳米颗粒的摄取，（3）刺激药物从纳米颗粒中快速释放，以及（4）增加多药耐药细胞对化疗的敏感性。此外，化疗可杀伤转移性肿瘤细胞，而MTPTT不能消除转移细胞。因此，MTPTT与化疗的联合应用在肿瘤治疗中表现出良好的协同效应[13,100]。

Fu等[81]设计了结合化疗和MTPTT的新型多功能硼基纳米平台。硼基纳米平台通过环（Arg-Gly-Asp）（cRGD）肽的功能化靶向肿瘤细胞中过表达的αvβ3整合素（图8A）。阿霉素（DOX，603 mg g⁻¹）和17-AAG（417 mg g⁻¹）与硼纳米片共同负载。DOX-17AAG@B-PEG-cRGD系统表现出可控的NIR诱导DOX和17-AAG释放。DOX-17AAG@B-PEG-cRGD系统显著增强了癌细胞相对于健康细胞的细胞摄取。17-AAG在MTPTT和DOX化疗联合中的存在提高了抗癌活性。这些多功能纳米平台是有前景的肿瘤治疗候选平台。

Wu等[69]设计了负载ICG和17-AAG的HMONs，抗肿瘤药物吉西他滨通过pH敏感的缩醛共价键修饰以封闭孔道。此外，NH₂-PEG可通过苯甲酰胺键引入，提高了纳米颗粒的循环性能。ICG-17AAG@HMONs-Gem-PEG纳米颗粒在TME中表现出pH响应性分子释放和谷胱甘肽（GSH）依赖性降解。由于弱酸性（<6.0）下缩醛键的水解，ICG和17-AAG可按需释放。随后，17-AAG调节HSP90，从而消除肿瘤细胞的热耐受性。此外，它可在相对温和的温度PTT下有效诱导癌细胞凋亡。吉西他滨作为门控分子，可从纳米胶囊中释放用于癌症化疗。纳米胶囊的近红外荧光和PAI由于ICG的强近红外吸收而呈现高对比度，有助于靶向治疗。

### 4.2. 放疗

由于局限性，MTPTT单独使用不能完全消除深部肿瘤。放疗（RT）可损伤DNA并导致癌细胞死亡，且无深度限制[101]。然而，细胞损伤程度受电离辐射诱导的细胞内氧离子水平的显著影响。因此，低氧TME显著限制了RT的有效性[2]。值得注意的是，MTPTT诱导的热疗可加速肿瘤血流并改善TME氧状态，从而提高肿瘤细胞对RT的敏感性[102,103]。因此，RT与PTT的联合应用是一种有前景的肿瘤根除策略，具有提高治疗效果和减少副作用等优势。此外，热疗可有效抑制非致死性X射线损伤的修复，产生显著的MTPTT/RT协同效应。

Song等[104]开发了透明质酸修饰的Bi₂Se₃空心纳米立方体（HNCs），负载GA（HNC-S-S-HA/GA）（图8B）。GA介导的HSPs下调降低了癌细胞对应激的耐受性。HNC-S-S-HA/GA有效诱导癌细胞凋亡，对癌细胞具有显著的消融效果。光热产生的热量增加血流，导致更多氧气输送到癌细胞，从而缓解低氧TME。HNC介导的增强RT在X射线照射下显示出改善的癌细胞杀伤效果。新型HNC-S-S-HA/GA纳米立方体具有若干独特优势，如（1）高稳定性、载药量和吸收系数，（2）HNC-S-S-HA/GA具有增强渗透和滞留效应/CD44介导的双模态肿瘤靶向和GSH敏感药物释放，进一步降低对正常细胞的毒性，（3）MTPTT可抑制周围组织的增殖，（4）MTPTT联合RT的抑制率显著高于MTPTT或RT单独使用。基于新型HNC-S-S-HA/GA的TME响应性纳米药物在多光谱光声断层扫描（MSOT）/计算机断层扫描（CT）成像引导的轻度温热MTPTT/RT中展现出应用潜力，对正常组织具有高抗肿瘤效率和最小侵入性。

### 4.3. 光动力治疗

光动力治疗（PDT）是一种主要利用光敏剂（PSs）在激光照射下将能量转移给氧气形成高活性单线态氧（¹O₂）的治疗方法，通过氧化蛋白质、核酸和脂质选择性破坏癌细胞（II型机制）[31,105,106]。此外，PSs产生活性氧，如羟基自由基或超氧阴离子，即I型机制[107]。由于其无创性、高安全性、时空控制和广谱性等优点，PDT受到广泛关注，被认为是一种有潜力的肿瘤治疗方法。然而，II型PDT的最大瓶颈之一是低氧TME，其严重降低了¹O₂产率。MTPTT通过增加血流显著改善肿瘤氧供应，有利于增强单线态氧生成效率。此外，PDT干扰肿瘤生理和微环境，增强肿瘤细胞的热敏感性。MTPTT与PDT的联合应用在提高肿瘤治疗效果方面具有若干优势[108]。因此，多种纳米系统被开发用于MTPTT/PDT协同治疗[109,110]。

值得注意的是，基于石墨烯量子点（GQDs）的纳米复合材料不仅可作为PSs，还可作为递送PSs的纳米平台[111]。此外，一些无机纳米材料也成为PSs和递送系统。然而，大多数这些系统需要复杂的集成或组装组件来实现"一体化"功能[112,113]。研究目前正在探索有机金属框架（MOFs）在PDT/PTT和多模式成像中的应用，以减少额外组件的集成。PSs可直接用作MOFs的组分（连接体），实现肿瘤的无创PDT[114]。然而，ROS的产生限制了治疗效果，主要是因为单一PDT治疗未呈现有效的抗癌效果[115,116]。因此，需要开发多功能MOFs以提高诊断准确性和治疗效果。

Zhang等[117]开发了新型多功能锆-铁卟啉MOF（Zr-FeP-MOF）纳米穿梭体（图8C）。HSP70抑制剂siRNA经PEG修饰并负载siRNA，制备siRNA/Zr-FeP-MOF治疗平台。值得注意的是，siRNA/Zr-FeP-MOF可在NIR激光下催化内源性过氧化氢（H₂O₂）和O₂生成丰富的羟基自由基（·OH）和¹O₂。siRNA/Zr-FeP-MOF纳米开关因siRNA的引入而对MTPTT具有光热效应。结果表明，PDT联合MTPTT可在体内外显著抑制癌症生长。

### 4.4. 气体治疗

生物体具有气态信号分子，作为信使在特异性结合多价过渡金属的过程中发挥作用，如硫化氢（H₂S）、一氧化氮（NO）和一氧化碳（CO）[118]，因此在几乎所有人体系统中具有多种生理功能，如神经系统、心血管系统和免疫系统。气态信号分子在正常人体生理过程中发挥重要作用，并参与病理过程的调节。血液中高浓度的NO、CO和H₂S可引起中毒；然而，在相对温和的浓度范围内，它们具有显著的抗癌活性[119]。例如，NO主要与超氧阴离子反应形成活性氮物种（RNS），如过氧亚硝酸盐，与DNA反应诱导多种DNA损伤[120]。

气体治疗的疗效与气体浓度高度相关。因此，在治疗性气体在病变部位的可控释放在气体治疗中非常关键[121,122]。激光是最方便有效的外源刺激，因此，光控药物释放已在多项研究中得到探索[123,124]。紫外光和可见光的穿透深度显著限制了光响应性气体释放的体内应用，且易引起光毒性。相反，NIR光具有更好的组织穿透深度和更温和的光毒性。因此，NIR激光响应性气体释放具有更广阔的应用前景。激光照射确保气体治疗的远程控制，PTT的热效应可促进气体释放[125–127]。例如，Gao等利用PAs与光触发NO发生器（硫醇化转铁蛋白）联合，促进在808 nm近红外光照射下NO的释放[128]。然而，NO在生物医学中的应用受到若干方面的限制，如高活性、选择性差和半衰期短（小于3秒）。因此，应探索基于纳米平台向病变区域选择性递送NO，以确保NO在抗癌治疗中的最大利用。MTPTT的热效应促进NO的时空可控释放。

Yao等[80]设计了介孔二氧化硅包覆的金纳米棒，连接S-亚硝基硫醇，并负载2-苯乙炔磺酰胺（PES）作为HSP70抑制剂，表面用PEG修饰（图8D）。在1 W/cm² 808 nm激光照射10分钟后，NO的累积浓度达到14.6 μM，溶液温度为40.8°C。气体治疗与MTPTT联合治疗的凋亡率约为70%，是MTPTT单独治疗的两倍。气体治疗与MTPTT联合治疗的体内肿瘤抑制率约为85%。

**图8** 多种实例揭示了MTPTT与其他疗法协同治疗的优越性。（A）DOX-17AAG@B-PEG-cRGD纳米片的制备及MTPTT与化疗联合的合成方法示意图[81]。版权所有 © 2022，Royal Society of Chemistry。（B）HNC-s-s-HA/GA的合成程序及MTPTT与放疗联合的示意图[104]。版权所有 © 2022，John Wiley and Sons。（C）siRNA/Zr-FeP MOF纳米穿梭体用于多模态成像诊断和MTPTT与PDT联合治疗癌症的示意图[116]。版权所有 © 2022，John Wiley and Sons。（D）PEG-PAu@SiO₂-SNO纳米复合材料的制备及MCF-7细胞在NIR照射下轻度热增强气体治疗过程的示意图[80]。版权所有 © 2022，American Chemistry Science。

### 4.5. 免疫治疗

癌症免疫治疗是最有前景和划时代的治疗方法，通过增强抗癌免疫或消除免疫抑制来实现免疫细胞介导的肿瘤清除并提高癌症患者的生存率[129–131]。正常情况下，免疫系统识别并杀伤异常肿瘤细胞，但肿瘤细胞可通过被称为"免疫逃逸"的过程触发大量策略来避免被免疫系统识别和消除[132]。癌症免疫治疗是一种通过重新启动和维持癌症-免疫循环来根除肿瘤的治疗方法[133]。

癌症-免疫循环分为以下七个环节：（1）死亡癌细胞释放抗原，（2）抗原呈递，（3）T细胞启动或激活，（4）T细胞向肿瘤迁移，（5）肿瘤组织T细胞浸润，（6）T细胞识别肿瘤细胞，（7）消除癌细胞。任何一个环节的障碍都可能导致抗肿瘤免疫循环失败和免疫逃逸。然而，免疫治疗并非对所有肿瘤类型都有效，因为高免疫原性毒性和低客观缓解率[134]。

由于加热不均匀或激光穿透深度限制未能完全消除肿瘤细胞，PTT因残留肿瘤细胞而导致肿瘤复发，这是一个严重的缺陷[28]。此外，PTT是一种有效的局部肿瘤治疗方法，但对播散性肿瘤无效。近期研究表明，高温可促进死亡肿瘤细胞释放抗癌底物，导致免疫激活[135–137]。先前研究表明，热疗可通过激活免疫细胞（如CD8⁺ T细胞、自然杀伤（NK）细胞和树突状细胞（DCs））、促进肿瘤细胞外泌体释放以及上调炎症细胞因子和HSPs的表达来触发免疫反应[138]。PTT诱导的热疗可导致肿瘤细胞凋亡或坏死并释放肿瘤相关抗原。肿瘤相关抗原可被抗原呈递细胞（APCs）接收和呈递，从而激活免疫细胞并诱导抗癌免疫反应。此外，PTT与免疫佐剂联合可用于开发原位自体癌症疫苗[139,140]。

癌症疫苗是一种有效的免疫治疗方法，通过向肿瘤患者注射肿瘤相关抗原，有效激活免疫系统杀伤肿瘤细胞，从而实现肿瘤控制和治疗的目的[141–143]。肿瘤浸润性树突状细胞通常呈现未成熟和免疫抑制表型，不能完全介导肿瘤的免疫抑制反应。以CpG寡核苷酸作为Toll样受体（TLR）激动剂的免疫治疗佐剂可激活肿瘤浸润性树突状细胞以增强疫苗特异性免疫[144,145]。然而，基于CpG寡核苷酸的免疫治疗的缺点通常被免疫抑制性TME所抵消[146–149]。

为调节微环境向免疫激活方向发展，Li等制备了光热CpG纳米药物（PCN）（图9A），通过在肿瘤部位激光照射后施加热量（43°C）诱导免疫有利性TME[150]。凋亡结果表明，激光照射的MTPTT可介导肿瘤细胞凋亡和坏死。肿瘤细胞释放的抗原和CpG激活的巨噬细胞和DCs促进激活/成熟，这从血清IL-6水平升高、BMDC成熟标志物CCL8和Clec4e上调表达的结果中得到证明。因此，MTPTT被成功证明可改善先天性和适应性免疫反应。

**图9** MTPTT辅助不同类型的免疫治疗。（A）PCN的解组装过程示意图，通过PCN的光热效应诱导的发热样免疫反应建立免疫有利性TME[150]。版权所有 © 2022，The Author(s)。（B）GO-FA-SNX-2112的结构示意图及其在肿瘤MTPTT中诱导过度激活自噬的应用；刺激的自噬不仅直接导致肿瘤细胞死亡，而且由于PDL1受体表达降低使其被免疫捕获；残留的存活肿瘤细胞也逐渐被恢复的免疫细胞杀伤，实现对肿瘤生长的有效抑制[32]。版权所有 © 2022，American Chemical Society。（C）通过联合"一体化"和"全控制"策略实现共生轻度光热辅助免疫治疗的示意图[159]。版权所有 © 2022，The Author(s)。

HSP90抑制通过上调干扰素反应基因的表达改善肿瘤免疫治疗。HSP90与自噬和蛋白激酶B（AKT）高度相关。然而，严重的副作用和肿瘤复发限制了传统治疗。因此，获得令人满意的生存率具有挑战性。通过靶向癌症的特定区域或功能，治疗可以更温和、更有效。"自动治疗"广泛代表"双刃剑"现象。一方面，自噬由于提供营养和限制T细胞介导的细胞毒性而在癌症根除中发挥重要作用。另一方面，自噬体的积累可通过诱导ACD发挥抗肿瘤活性。因此，药物诱导或抑制自噬可杀伤肿瘤细胞。

当癌细胞暴露于激光照射时，会产生应激蛋白（HSPs），通过HSPs的正常功能保护自身免受MTPTT的侵袭。相反，当HSP90下调时，自噬可导致癌细胞凋亡，HSPs在维持细胞环境动态稳定方面以若干关键方式发挥作用。这意味着在高能环境中，当HSP90缺失时，自噬的功能可能被逆转。应用MTPTT调节自噬是一种新型的肿瘤治疗方法。MTPTT诱导的过度自噬激活和HSP90抑制剂的调节在MTPTT的疗效中发挥关键作用[33]。

Deng等[32]设计了负载SNX-2112和叶酸（FA）的氧化石墨烯（GO）用于MTPTT（图9B）。HSPs水平的变化与AKT活性相关，因为HSP90是AKT信号通路中的早期蛋白。在自噬过程中，HSP90抑制AKT并在应激条件下使AKT失活。因此，通过Western blot分析评估了与生命体征相关的通路。结果表明，GO-叶酸-SNX-2112组中p-AKT和自噬相关基因的表达水平存在显著差异，且SNX-2112抑制了HSP90的表达。这些发现表明MTPTT激活了自噬并抑制了AKT通路。此外，与对照组和GO-叶酸组相比，参与肿瘤免疫功能的程序性死亡配体1（PDL1）的表达显著下调。这些发现表明自噬、p-AKT和PDL1之间存在关联和串扰。

免疫检查点治疗旨在通过抑制或刺激来自免疫抑制性TME的信号来调节T细胞的活性，是免疫治疗的主要治疗方法[151,152]。然而，近期研究表明，许多患者表现为"非免疫原性"肿瘤，也称为"冷"肿瘤，其特征是缺乏肿瘤浸润性淋巴细胞[153,154]。因此，如何将"冷"肿瘤转化为"热"肿瘤是免疫治疗面临的重大挑战。

将冷肿瘤转化为热肿瘤有五种策略[155]：（1）改善肿瘤炎症，（2）中和TME中的免疫抑制因子，（3）靶向肿瘤血管和基质，（4）靶向肿瘤细胞信号通路，（5）改善抗肿瘤免疫T细胞的长寿和功能。与各种治疗方法的联合治疗增强了免疫检查点治疗的疗效[156,157]。此外，温和的温度有利于TME中的免疫反应[115,158]。

Huang等[159]将光热转换剂（IR820）和抗程序性死亡配体1抗体（aPD-L1）负载到脂质混合物中，用于将免疫检查点阻断抗体与MTPTT联合（图9C）。在测量4T1和B16F10小鼠的淋巴结、脾脏和肿瘤中免疫细胞的免疫反应后，结果表明治疗诱导了初始T细胞向CD8⁺ T细胞的分化。因此，MTPTT温度的精确控制对于增敏免疫抑制性肿瘤以将冷肿瘤转化为热肿瘤以及增强免疫检查点治疗至关重要。

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## 5. 总结与展望

在过去几十年中，肿瘤的PTT引起了广泛关注，研究表明其在小型、不可切除肿瘤或手术候选条件差的患者中具有广阔前景。无造影剂PTT或造影剂增强PTT的高温热消融显著影响健康组织，诱发不良炎症，并严重损害与抗肿瘤免疫相关的免疫抗原和免疫细胞。因此，MTPTT被引入以规避低于45°C的PTT相关副作用。研究表明，MTPTT与HSPs抑制剂或其他多种药物的联合应用显著提高了轻度热疗的疗效，因为这些药物减轻了热耐受性。此外，精心设计的多功能纳米平台可提高光敏剂的肿瘤特异性，改善肿瘤靶向、选择性、活化或成像引导，和/或与其他疗法联合应用。

纳米系统可通过限制HSPs的功能或降低其表达来实现MTPTT的高治疗效率。在本综述中，总结了用于增强MTPTT的多功能纳米制剂的机制及最新策略。使用HSPs抑制剂、整合siRNA、靶向细胞核、阻断能量抑制和影响自噬可减轻肿瘤细胞的热耐受性并保护正常细胞免受MTPTT效应的影响。此外，协同治疗可综合各疗法优势并弥补其不足，从而改善治疗结果。本综述探讨了MTPTT与其他疗法联合应用的最新进展，包括化疗、放疗、PDT、气体治疗和免疫治疗。值得注意的是，MTPTT可调节和重建免疫抑制性肿瘤环境，从"冷"转变为"热"，从而防止肿瘤复发和转移。

总之，与PTT相比，MTPTT在生物医学领域具有广阔的临床前景和进一步的发展空间。尽管先前研究已报道了多种多功能纳米试剂及其积极成果，但MTPTT仍处于起步阶段。MTPTT的临床应用受到激光光穿透深度差、靶部位蓄积不足和纳米材料在体