目的：免疫检查点阻滞剂（Immune checkpoint blockers，ICBs）治疗是晚期肾细胞癌（Renal cell carcinoma，RCC）的一线治疗选择。然而，其有效性受到免疫抑制肿瘤微环境（Tumor microenvironment，TME）的阻碍。研究表明声动力疗法（Sonodynamic therapy，SDT）产生的肿瘤细胞片段可以启动宿主的抗肿瘤免疫。但是，SDT后缺氧肿瘤微环境及自噬激活通常导致肿瘤细胞产生耐药性。本研究的目的是探索一种声动力纳米药物，以增强肾细胞癌的免疫治疗效果。

方法：首先，为了制备负载HIF-2α抑制剂贝组替凡（Belzutifan）和声敏剂二氢卟吩e6（Chlorin e6，Ce6）的缺氧响应性纳米颗粒，我们使用4-硝基苯甲醇作为缺氧响应接头合成了4，4'-偶氮联苯甲醇。随后，通过1，2，4，5-环己烷四羧酸二酐（PMDA）、4，4'-偶氮联苯甲醇和聚乙二醇单甲醚（mPEG_5k）合成了缺氧反应性前药（P-APm）。然后，将Ce6、belzutifan与P-APm通过纳米沉淀自组装制备了缺氧响应性纳米颗粒(APm/Ce6/HIF)。此外，使用相同的方法将Ce6与P-APm自组装成纳米颗粒(APm/Ce6)。然后利用透射电子显微镜（Transmission electron microscope，TEM）、动态光散射仪（Dynamic light scattering，DLS）、高效液相色谱（High-performance liquid chromatography，HPLC）和电子自旋共振（Electron spin resonance，ESR）光谱仪等分别对APm/Ce6/HIF的理化特征以及功能进行验证。利用流式细胞仪（Flow cytometry，FCM）和激光共聚焦显微镜（Confocal laser scanning microscopy，CLSM）探究肿瘤细胞对APm/Ce6/HIF的摄取能力。紧接着，通过MTT、CCK8、克隆形成、细胞凋亡、活死染色等方法探究了APm/Ce6/HIF在细胞水平的抗肿瘤活性，并通过蛋白免疫印迹、免疫荧光及细胞共培养等方式探究了APm/Ce6/HIF的抗肿瘤和免疫激活机制。最后，在肾癌RENCA皮下肿瘤模型上探究了APm/Ce6/HIF的生物分布、抗肿瘤活性及与PD-1单抗（αPD-1）联合的抗肿瘤效果。

结果：TEM显示，APm/Ce6/HIF具有均匀的球形结构，直径约为120 nm。DLS分析证实，APm/Ce6/HIF的平均粒径为127.1 nm，APm/Ce6/HIF的电位为20.9 mV。HPLC分析证实APm/Ce6/HIF在低氧环境中可以有效降解并释放出包载的belzutifan及Ce6。此外，通过ESR光谱仪证实了APm/Ce6/HIF在超声波条件下可以产生活性氧（Reactive oxygen species，ROS）。在体外，APm/Ce6/HIF+US抑制肾癌细胞增殖。另外，APm/Ce6/HIF+US可以产生更多的ROS以杀伤肿瘤细胞，同时诱导免疫原性细胞死亡（Immunogenic cell death，ICD）以激活抗肿瘤免疫应答。在体内，APm/Ce6/HIF+US显著抑制肿瘤生长并激活抗肿瘤免疫。总而言之，这种治疗有效地将免疫微环境从“冷肿瘤”转变为“热肿瘤”，从而增强了ICBs的疗效。

结论：综上所述，我们设计了一种缺氧响应聚合物（P-APm），包载belzutifan和Ce6，通过自组装形成APm/Ce6/HIF。在体内经过静脉给药后，APm/Ce6/HIF会优先在肿瘤组织中积聚，进一步在肿瘤缺氧微环境下快速降解，释放包载的belzutifan和Ce6。释放的belzutifan通过抑制缺氧和自噬信号通路抑制肿瘤生长。此外，与大多数化疗和放疗不同，肿瘤内由Ce6引起的SDT可以显著促进ROS的产生，从而诱导强效的ICD，显著促进了树突状细胞的成熟以及细胞毒性T淋巴细胞和M1型巨噬细胞的浸润。随着先天性和适应性免疫应答的激活，免疫抑制微环境被改变，“免疫冷肿瘤”转化为“免疫热肿瘤”，与αPD-1联合治疗产生协同作用。总之，本研究为肾细胞癌治疗提供了一种靶向与免疫联合治疗的有效策略，具有巨大的临床转化潜力。

Objective: Immune checkpoint blockers (ICBs) therapy is the first-line treatment option for advanced renal cell carcinoma (RCC). However, its effectiveness is hampered by the immunosuppressive tumor microenvironment (TME). Studies have shown that fragments of tumor cells produced by sonodynamic therapy (SDT) can initiate anti-tumor immunity in the host. However, the hypoxia tumor microenvironment and upregulated autophagy after SDT often lead to drug resistance in tumor cells. This study aimed to explore a sonodynamic nanomedicine to enhance the immunotherapeutic effects of renal cell carcinoma cancer.

Methods: To prepare hypoxia-responsive nanoparticles loaded with a HIF-2α inhibitor (belzutifan), and the ultrasonic sensitizer (Chlorin e6, Ce6) for further enhancing their synergistic effect, herein, we synthesized 4,4’-azobisbenzenemethanol using 4-nitrobenzyl alcohol as a hypoxia-responsive linker, which was confirmed by 1H-NMR. Subsequently, to obtain hypoxia-responsive prodrugs (P-APm), the polymer was synthesized via 1,2,4,5-cyclohexanetetracarboxylic dianhydride (PMDA), 4,4’-azobisbenzenemethanol and polyethylene glycol monomethyl ether (mPEG_5k). Then, the hypoxia-responsive nanoparticles (APm/Ce6/HIF) were prepared by encapsulating Ce6 and belzutifan simultaneously with P-APm through self-assembly. Additionally, nanoparticles (APm/Ce6) encapsulated with Ce6 only were prepared using the same method. Then the morphology, particle size and zeta potential of APm/Ce6/HIF were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), high-performance liquid chromatography (HPLC), electron spin resonance (ESR) spectrometer, respectively. Flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) were used to investigate the uptake of APm/Ce6/HIF by cancer cells. Then, the anti-cancer activity of APm/Ce6/HIF at the cellular level was investigated by MTT, CCK8, clony formation, apoptosis, and Live/Dead staining, and the anti-tumor and immune activation mechanism of APm/Ce6/HIF was investigated by western blotting, immunofluorescence and cell co-culture. Finally, the Biodistribution and anti-tumor effect of APm/Ce6/HIF combined with PD-1 monoclonal antibody (αPD-1) were investigated in RENCA tumor-bearing mice.

Results: TEM revealed that APm/Ce6/HIF have a uniform, spherical shape with an approximate diameter of 120 nm. DLS analysis confirmed that the average particle size of APm/Ce6/HIF was 127.1 nm, and the zeta potential of APm/Ce6/HIF was -20.9 mV. We observed the release of Ce6 and belzutifan from APm/Ce6/HIF, as confirmed by HPLC analysis in the hypoxic environment. Furthermore, the ability of APm/Ce6/HIF to generate reactive oxygen species (ROS) under ultrasonic conditions was confirmed by ESR spectrometer. In vitro, APm/Ce6/HIF+US inhibited the proliferation of renal carcinoma cells. APm/Ce6/HIF+US can further lead to the production of ROS to kill tumor cells, while inducing immunogenic cell death (ICD) to activate the anti-cancer immune response. In vivo, APm/Ce6/HIF+US significantly inhibits tumor growth and activates anti-tumor immunity. Moreover, the presence of APm/Ce6/HIF+US altered the immunosuppressive TME and transformed the “immune-cold” tumor into an “immune-hot” tumor, leading to improved efficacy antitumor immunotherapy.

Conclusion: In summary, we designed a hypoxia-responsive polymer (P-APm) loaded with belzutifan and Ce6, forming APm/Ce6/HIF through self-assembly. APm/Ce6/HIF, when injected systemically, preferentially accumulate in tumor tissues. In the hypoxic TME, APm/Ce6/HIF rapidly degrade, releasing belzutifan and Ce6. The released belzutifan effectively suppresses tumor growth by targeting hypoxia and autophagy signaling pathways. Furthermore, the Ce6 enhances SDT, significantly increasing the generation of ROS and triggering ICD. This process reprograms the “immune-cold” tumor into “immune-hot” tumor by promoting DCs maturation and tumor infiltration of cytotoxic T cells and M1 macrophages, leading to a synergistic effect when combined with αPD-1 therapy. Overall, our study presents a promising targeted therapy combined with immunotherapy strategy for the treatment of RCC, demonstrating significant clinical application potential.