可高效递送siRNA至巨噬细胞的聚合物纳米粒的胞内转运分子机制

The current project is a successive application based on our previous funding project (No: 81072595). In the former project, mannose-modified trimethyl chitosan-cysteine (MTC) conjugate nanoparticles encapsulating siRNA were developed via ionic gelation with tripolyphosphate (TPP) and performed as highly-efficient vehicles for the delivery of siRNA to macrophages. Oral administration of MTC/TPP nanoparticles at the siRNA dose of 50 μg/kg led to robust in vivo anti-inflammation efficacies, which were intimately correlated with their specific intracellular trafficking involving both virus-like macropinocytosis and caveolin-mediated endocytosis. In the current project, to clarify the molecular mechanism for the intracellular trafficking of MTC/TPP nanoparticles in macrophages and provide new strategies at a molecular level to improve siRNA delivery, multiple techniques have been adopted to systematically elucidate the endocytic pathway, intracellular localization, and exocytic route of MTC/TPP nanoparticles and screen the key signaling pathway factors that affect their internalization. In vitro and in vivo studies are integrated to reveal the essential protein regulators of the intracellular trafficking of MTC/TPP nanoparticles in macrophages and clarify the interaction between macropinocytosis and caveolin-dependent endocytosis, as well as their individual contribution to the delivery of siRNA. The above information could serve as a molecular guideline in the rational design of highly-efficient and clinically-practical polymeric vectors for siRNA delivery.

本项目是已结题基金项目（No：81072595）的连续申请。已结题项目以多功能甘露糖化壳聚糖季铵盐-半胱氨酸（MTC）为载体，经三聚磷酸钠（TPP）交联制得包载siRNA的MTC/TPP纳米粒，可高效递送siRNA至巨噬细胞，肝损伤小鼠口服50μg/kg siRNA可获得较好的抗炎效果，这与其以独特的类病毒式的巨胞饮和小窝蛋白介导内吞进行胞内转运密切相关。为阐明MTC/TPP纳米粒巨噬细胞胞内转运的分子机制并获得siRNA高效递送的分子水平策略，本项目使用多种实验方法从不同角度系统研究MTC/TPP纳米粒递送siRNA至巨噬细胞的内吞途径、胞内定位和胞吐路径，揭示影响其入胞的关键信号通路因子，经体内外试验获得可调节巨噬细胞胞内转运的主要蛋白分子，并明确巨胞饮和小窝蛋白介导内吞间的相互作用及各自对siRNA递送的贡献。为从分子水平合理设计具临床应用潜力的siRNA递送载体提供重要的理论依据。

小分子干扰RNA（small interfering RNA, siRNA）可选择性沉默致病基因，已成为抗炎、抗病毒、抗遗传性疾病及抗肿瘤新药研发的热点之一，相关siRNA药物不断获批上市或进入临床研究。虽然siRNA药物的临床应用已取得了突破性进展，但因siRNA稳定性差且难以进入靶细胞，将其递送至靶细胞发挥RNA干扰（RNA interference, RNAi）功效仍面临着巨大挑战。为解决这一难题，需借助载体克服递送过程中的多个生理屏障。现有的大量研究均通过从宏观层面调控聚合物纳米粒的理化性质和组成来克服siRNA递送过程中多重屏障，介导高效的基因沉默效率。而从分子水平发现提高siRNA递送的策略的相关研究较少，严重制约siRNA纳米递送载体的合理设计。本项目以可高效递送siRNA至巨噬细胞的MTC/TPP纳米粒为研究平台，系统解析其自细胞内吞、胞内定位、胞内运输至胞吐的整个过程的分子机制，确定参与其细胞加工的关键蛋白分子。研究结果表明MTC/TPP纳米粒细胞内吞受PAK1、Scr激酶、PKC、TK和PI(3)K的调节，需要发动蛋白、Na+/H+转运体和胆固醇等脂质分子参与，要求高尔基体和内质网结构完整并涉及微丝和微管介导的细胞膜动态变化、胞质流动和囊泡运输；入胞后2 h内MTC/TPP纳米粒主要分布于小窝蛋白包被囊泡，随后沿着微管或自由移动至高尔基体和内质网，4 h后主要分布在高尔基体和内质网，高尔基体和内质网为RNAi发挥作用的关键细胞器；部分MTC/TPP纳米粒经Rab4/11和Rab27调节分别经循环内涵体和外泌体分泌至胞外；与小窝蛋白介导内吞途径密切相关的caveolin-1、syntaxin6和NPC1下调可显著抑制MTC/TPP纳米粒体内外巨噬细胞摄取及基因沉默效率，首次系统证实小窝蛋白介导内吞为聚合物纳米粒递送siRNA进入巨噬细胞的有效途径。这些结果可为siRNA高效递送提供分子层面的理论指导，如需考虑某些疾病状态下caveolin-1、syntaxin6和NPC1表达异常对siRNA高效递送的影响。