近红外光和尺寸联合调控肿瘤渗透增强的多级载体构建及siRNA递送

Developing efficient vectors to continuously conquer the barriers of blood, tumor and cells is important for successful tumor gene therapy. Stimuli-responsive vectors have paved a road for overcoming blood and cell barriers. However, tumor barrier still seriously depresses the gene delivery efficiency because of the poor tumor penetration of the vectors. This project will focus on constructing multiple stimuli-responsive gene vector characterized with NIR and size synergistically regulating tumor penetration. Firstly, the dual stimuli-responsive sub gene vector will be synthesized by the strategy of in situ self-assembly and core cross-linking. Then the sub gene vector and Glu-PEG will be loaded on the gold nanorod though layer-by-layer self-assembly method and stimuli-responsive bonds. Thus the multiple stimuli-responsive gene vector will be constructed. The stimuli-responsive properties and gene delivery performances of the vector will be investigated at different delivery stages. The improvement of the vector’s tumor penetration by the synergetic regulation of NIR and size will be identified. Furthermore, the efficient gene delivery performances of the vector benefiting from long blood circulation, effective tumor penetration, targeted cell endocytosis, rapid intracellular gene release will be evaluated. The successful implementation of this project will provide scientific guidance for the design of efficient gene vectors to overcome multi-barriers in tumor gene therapy.

发展连续克服血液屏障、肿瘤屏障、细胞屏障的高效基因载体，是肿瘤基因治疗成功的关键。环境响应基因载体为克服血液和细胞屏障提供了解决途径，但是肿瘤屏障导致的渗透障碍仍严重制约了基因递送的效率。本项目拟构建近红外光（NIR）和尺寸联合调控的多级环境响应基因载体，通过载体的NIR光热效应和尺寸响应，实现肿瘤渗透增强的基因高效递送。首先采用接枝诱导原位自组装和核交联策略可控构建胞内双重环境响应次级载体，然后利用肿瘤环境响应化学键，通过分子识别的逐层自组装，将次级载体与糖基修饰聚乙二醇有序装载在金纳米棒母体上，构筑NIR和尺寸响应的多级基因载体。研究载体在基因递送不同阶段的环境响应特征和基因递送性能，明确NIR和尺寸联合调控的肿瘤渗透增强途径，揭示载体兼顾血液稳定循环、肿瘤有效渗透、靶向胞吞与胞内基因快速释放的基因高效递送机制。本项目将为连续克服多级屏障基因载体的设计和肿瘤基因治疗提供理论依据。

发展连续克服血液屏障、肿瘤屏障、细胞屏障的高效基因载体，是肿瘤基因治疗成功的关键。环境响应基因载体为克服血液和细胞屏障提供了解决途径，但是肿瘤屏障导致的渗透障碍仍严重制约了基因递送的效率。本项目构建了近红外光（NIR）和尺寸联合调控的多级环境响应基因载体，通过载体的NIR光热效应和肿瘤微环境诱导尺寸响应，实现肿瘤渗透增强的基因高效递送和免疫治疗。首先，采用介孔硅包覆金纳米棒（GNR），经苯硼酸表面修饰后，顺序装载siRNA（siPD-L1）、富含二硫键的壳聚糖衍生物(烯丙基甘露糖修饰壳聚糖，AMLC）和N-异丙基丙烯酰胺（NIPAM），并在孔道内原位自由基聚合AMLC和NIPAM,形成低临界溶解温度42 oC的温敏多糖凝胶，然后，装载吲哚菁绿（ICG），制备多刺激响应簇状纳米载体。该载体在内源性唾液酸和pHe及外源性NIR刺激下，硼酸酯键分解、光热响应收缩，实现载体的尺寸响应；同时，光热响应导致肿瘤间质液压降低40%，引起肿瘤细胞凋亡，改善了肿瘤渗透性，从而实现siRNA及ICG的肿瘤深度递送。一方面，温敏凝胶被肿瘤相关巨噬细胞（TAMs）靶向吞噬进入细胞后，在谷胱甘肽作用下，温敏凝胶二硫键断裂，释放ICG进入细胞内部。并且，这种响应引起谷胱甘肽耗尽，协同GNR光热效应的O2扩散改善，使ROS产率提高了4.1倍。增强的ROS介导TAMs从M2表型重编程为M1表型，解除了肿瘤免疫抑制微环境，并促进T细胞增殖和肿瘤浸润。另一方面，siRNA作用于肿瘤细胞，抑制了其表面PD-L1的表达，减弱了肿瘤细胞对T细胞的免疫耐受。二者协同作用，促进了肿瘤免疫治疗，实现肿瘤抑制。本项目的研究表明，含光敏剂的簇状多刺激响应纳米载体设计策略有效增强了siRNA肿瘤深度递送和免疫治疗，有望激发肿瘤免疫治疗的进一步研究与应用。

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