抗肿瘤药物高效纳米递送系统的细胞及亚细胞转运机制

The current project aims at exploration of mechanisms involved in the cellular and subcellular transfer of nanostructured delivery systems for improving the effectiveness of antitumor drugs. During recent years there has been much interest in the use of nanoparticles for delivery of antitumor drugs in animals and humans. To this end it is necessary to increase our understanding of how these nanoparticles are taken up and transported within the cells. In the present project, the multifunctional polymeric nanoparticles whose size and surface charge could be simultaneously controlled were prepared and used as model nanocarriers, techniques in cell biology and biochemistry were employed to explore the cellular and subcellular transfer of nanocarriers which had been insufficiently investigated. The investigation of endocytic mechanisms was performed by combining the use of different inhibitors, mutated proteins and siRNAs because most methods described for elucidating the endocytic mechanisms involved were not specific for any of the endocytic mechanisms.The endocytic pathways favorable in cellular entry of nanocarriers that could pass biological barriers would be elucidated. The differential transport efficiency mediated by various endocytic pathways in different cell lines，cell states and cell cycles would be exhibited, and the respective intracellular traffick which was related to different endocytic pathways among subcellular compartments inclouding lysosome，mitochondria，nucleus and cytoplasm would be revealed. Besides, the cellular export of internalized antitumor drugs delivered by nanocarriers was also investigated. These mechanisms above could be used to ascertain the impact of surface properties and functionalities on cellular and subcellular transfer of drug-loaded nanocarriers.The antitumor drug nanocarriers which designed on the base of the resulted mechanisms were subsequent studies in vivo in the terms of antitumor efficacy, drug distribution in tumor and tumor cell for further verification of the mechanisms. These results could serve as guidelines in the rational design of antitumor drug nanostructured delivery systems with maximized therapeutic efficacy.

本项目致力于研究纳米给药系统高效递送抗肿瘤药物的细胞及亚细胞转运机制。选择可同时调控粒径和表面电荷并可进行多功能表面修饰的聚合物纳米粒为模型纳米载体，结合细胞生物学及生物化学等手段，阐明可克服细胞水平上生物学屏障的纳米递送系统的细胞摄取机制，获得胞饮、网格蛋白、小窝蛋白及网格蛋白和小窝蛋白非依赖性介导的内吞在不同肿瘤细胞、不同细胞状态与不同细胞周期的转运效率；揭示不同内吞机制下纳米粒在细胞内溶酶体、细胞质、线粒体和细胞核等亚细胞隔室的转运路径及靶向规律；研究肿瘤细胞对经纳米载体递送入胞的胞内药物的外排；全面解析纳米粒表面性质及功能修饰对载药纳米粒的细胞及亚细胞转运的影响规律；根据机理研究结果合理设计抗肿瘤药物纳米递送系统，进行体内抗肿瘤试验及肿瘤组织与细胞药物分布研究，对细胞及亚细胞转运机制进行进一步体内验证。为基于合理设计的抗肿瘤药物高效纳米传递系统构建提供理论和方法指导。

阐明抗肿瘤药物高效纳米递送系统的细胞及亚细胞转运机制可指导其合理设计。项目制备了羧甲基壳聚糖纳米粒（CMCNPs）模型纳米粒，研究其主要性质对细胞黏附和内吞、细胞摄取及机制、胞内转运、亚细胞分布的影响和机制，以紫杉醇（PTX）为模型抗肿瘤药物，考察纳米粒理化性质对载药纳米粒的体外释药、细胞增殖抑制和亚细胞分布及其体内肿瘤组织分布和抗肿瘤功效的影响。结果表明，200 nm 纳米粒主要经网格蛋白和小窝蛋白介导的内吞入胞，1000 nm 纳米粒主要经网格蛋白介导的内吞入胞，800 nm 纳米粒主要经非网格蛋白、非小窝蛋白介导的内吞入胞；三种纳米粒均可转运至溶酶体、线粒体、内质网和高尔基体，200 nm 和1000 nm纳米粒的转运路径为细胞膜（网格蛋白）→溶酶体→胞质，800 nm纳米粒的转运路径为细胞膜（内吞囊泡）→高尔基体→内质网→胞质；H-22荷瘤小鼠尾静脉注射200、800和1000 nm PTX-CMCNPs，PTX肿瘤分布显著提高，抑瘤率分别为89.1%、75.1%和74.1%。以TAT肽修饰介孔硅纳米粒（TAT-MSN）经静电作用依次吸附聚丙烯胺盐酸盐-柠康酸酐共聚物（PAH-Cit）和半乳糖修饰的巯基化壳聚糖季铵盐（GTC），制备多层结构纳米粒（MLNs），同时包载阿霉素（DOX）和血管内皮生长因子（VEGF）siRNA（siVEGF），结果表明，MLNs的药物释放速率具pH敏感性，pH 5.0时DOX和siRNA的释放速率均显著高于pH 7.4和pH 6.5；在QGY-7703肝癌细胞体外细胞模型上，MLNs组DOX和siRNA的细胞摄取量分别为未修饰半乳糖纳米粒组的1.6倍和2.0倍，且受氯丙嗪和网格蛋白重链-1（CHC-1）siRNA抑制，入胞后MLNs与转铁蛋白大量共定位，表明经半乳糖受体介导的网格蛋白途径入胞； CLSM观察结果表明MLNs可从内涵体/溶酶体快速逃逸至细胞质；核质分离试验结果表明TAT肽可促进纳米粒的入核转运；QGY-7703肝癌裸鼠模型静脉注射后，MLNs可高效抑制肿瘤生长，沉默肿瘤组织VEGF基因表达。上述研究可为基于合理设计的抗肿瘤药物高效纳米传递系统构建提供理论和方法指导。