程序响应型纳米粒用于逆转免疫抑制微环境及协同肿瘤化疗的研究

The immunosuppressive tumor microenvironment is the main pathological barrier for chemotherapy and immunotherapy. It is critically important to design novel therapeutic strategies to prime the tumor immune microenvironment for superior immune response and outcomes. In consideration of the dominance of tumor-associated macrophages (TAMs) in the immunosuppressive tumor microenvironment, here we report a novel multi-stage responsive nanoparticle to reverse the immunosuppressive tumor microenvironment by targeting the TAMs, and maximize the efficacy of immunotherapy by combination the immunogenic cell death (ICD) effect of chemotherapy and TAM priming. The nanoparticle can stay “OFF” during circulation, and release the ICD triggering module in the acidic tumor microenvironment. The TAM-targeting module, PEG-b-iPDPA-R848, remains particle state with 30 nm in diameter and positive charge, which is tend to be endocytosed by macrophages. Subsequently, the TLR7/8 agonist R848 in the hydrophobic core is exposed in the acidic endosomes to re-educate the immunosuppressive M2-type TAM into immuno-supportive M1-type, leading to tumor immune microenvironment priming. Meanwhile, PEG-b-PEPA-hydrazone-DOX is released as doxorubicin prodrug with 5 nm in diameter, which prefers to penetrate into deep tumor tissues and release free DOX after cleavage in the acidic endosomes, killing most tumor cells and inducing ICD effect. Finally, the combination of immune response of M1-type TAM and ICD effect results in synergistic immunotherapy to maximize tumor eradication effect. By integrating tumor acid response, immune microenvironment modulation and chemotherapy, our nanotechnology may provide a powerful strategy for the combination of chemotherapy and immunotherapy in cancer therapy.

免疫抑制微环境是导致癌症化疗耐药和免疫治疗失效的重要因素。因此，如何有效调控肿瘤免疫微环境，打破肿瘤免疫的生物学屏障，对于肿瘤治疗至关重要。本项目提出一种程序性靶向肿瘤相关巨噬细胞（TAM）逆转免疫抑制微环境，并联合化疗实现肿瘤免疫最大化的新型智能响应纳米递送系统。它在血液循环中保持“OFF”状态，在肿瘤酸性微环境中发生一级解散。TAM靶向模块保持30nm的粒子且带正电，易于被TAM摄取，并通过特异性靶向酸性内涵体发生二级解散，激活TLR7/8诱导免疫抑制的M2型TAM转变为免疫增强的M1型，从而逆转肿瘤免疫抑制微环境；释放的肿瘤靶向模块则形成5nm的DOX前药，穿透致密的组织间隙，进入肿瘤细胞并在内涵体酸性作用下释放DOX，发挥化疗及ICD效应。最后，M1型TAM与ICD协同发挥免疫治疗作用。本项目为纳米药物在肿瘤化疗与免疫治疗的联合应用提供了新思路，具有重要科学意义和潜在临床应用价值。

药物在亚细胞水平的分布对生物学效应和疗效都具有重要作用。纳米药物在细胞内不同分布情况同样也能调控细胞的命运。基于pH超敏感技术，我们在国际上率先建立了一系列不同pH转变点（pHt 5~7）的纳米药物，能够对细胞的早期内吞体（EE）-晚期内吞体(LE)-溶酶体（Ly）的内吞体成熟通路进行多达10个阶段的划分，每个阶段pH差异仅0.2个单位，从而将纳米药物精准靶向递送至特定阶段的内吞细胞器。我们在国际上首次发现了细胞内吞成熟过程能够时空操控纳米药物的效应机制及疗效，并系统阐明了早期内吞体靶向的活性氧通过特异性激活内吞体膜上的磷脂酶C信号通路和下游的Caspase3/GSDME信号通路，高效诱导肿瘤细胞焦亡；而靶向递送至晚期内吞体和溶酶体阶段的活性氧则通过经典的溶酶体膜渗透性增加，引起细胞杀伤效率较低的凋亡模式。这种通过内吞体成熟过程调控细胞死亡模式和疗效的策略在十多种肿瘤细胞上都具有良好的普适性。相对于晚期内吞体和溶酶体靶向的活性氧，早期内吞体靶向的活性氧在体外和体内的肿瘤治疗效果分别能提升40倍和20倍。利用该发现，我们在多种皮下/原位肿瘤模型上实现了安全高效的肿瘤治疗。. 基于细胞焦亡纳米调控技术，我们进一步构建了pH/ROS程序响应型纳米药物。一方面，通过诱导肿瘤细胞焦亡，产生高效肿瘤免疫原性死亡效应，释放肿瘤抗原，ATP和HMGB1等具有免疫佐剂效应的模式信号分子，以及钙网蛋白等“eat-me”信号，激活抗原提呈、免疫应答和肿瘤免疫效应。另一方面，通过光动力产生的单线态氧释放ROS敏感的免疫佐剂TLR激动剂IMDQ，调控肿瘤免疫微环境，实现了安全高效的光-免疫治疗。

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