微环境响应的聚合物载体靶向高效递送siRNA治疗肝纤维化的研究

There are no effective drugs for the treatment of liver fibrosis in clinic. Activated hepatic stellate cells (HSC) contribute to excessive deposition of extracellular matrix collagen fibers and have been become an ideal target for anti-liver fibrosis. RNA interference shows the advantages of high efficiency and specificity in silencing targeted mRNA, thus being widely used in the research for the treatment of liver fibrosis. The key that achieves effective treatment lies in the biocompatible carrier that can overcome multiple obstacles in vivo and efficiently deliver siRNA into the cytosol of HSC. Though many carriers have been constructed in recent years, developments of the HSC-targeted siRNA delivery carrier remain a challenge and are of great significance. This proposal aims to establish a polymeric nanodrug for efficiently siRNA delivery through combining the strategies of specifically capturing endogenous targeting ligand, crosslinking bound to improve its stability and microenvironment-responsive drug release. This tailor-made nanodrug based on the extracellular and intracellular microenvironment of HSC is endowed with the properties of good biocompatibility, negative charge for long blood circulation, small particle size for passing through sinusoidal endothelial fenestrations, targeting HSC, lysosomal escape and cytosol microenvironment-responsive siRNA release. The activation, proliferation, differentiation and collagen secretion of HSC will be inhibited through RNA interference, and collagen fiber will be degraded, thus efficiently attenuating liver fibrosis. Its success will facilitate highly efficient RNA interference effect and provide research basis for the clinical application of gene therapy in the treatment of liver fibrosis.

目前临床上缺乏治疗肝纤维化的有效药物。活化的肝星状细胞（HSC）导致细胞外基质胶原纤维过度沉积，成为治疗的理想靶细胞。RNA干扰具有高效和特异性沉默靶基因的优点，被广泛用于治疗肝纤维化的研究，实现有效治疗的关键在于制备一种能够克服体内递送的多重障碍将siRNA高效递送至HSC胞质的理想载体。本项目针对现有载体的不足及高效递送面临的难点，结合申请人的研究基础，基于HSC内外微环境的特点，拟联合特异性捕获内源靶向配体、交联捆绑提高稳定性和微环境响应定点药物释放策略，“量体裁衣”构建高效递送siRNA的负电聚合物纳米药物。其不仅具有血液长循环、较小粒径穿过肝窦内皮细胞层窗孔和溶酶体逃逸性能，还具有靶向HSC和胞质完全释放siRNA性能。通过RNA干扰抑制HSC活化和促进胶原纤维降解，有效治疗肝纤维化。通过本项目的研究，有望实现高效的RNA干扰效果，为基因疗法治疗肝纤维化走向临床应用提供新的思路。

临床统计表明部分肝纤维化会发展为癌症，然而尚无强效的治疗药物用于这两个阶段疾病的治疗。纳米递药系统可响应病灶局部微环境释放药物，可提高药物的治疗指数。构建用于肝纤维化及其后期癌症治疗的siRNA靶向递送系统或者药物联合递送系统具有重要的研究价值。本项目成功合成了一种携-N3侧基和邻二醇响应的阳离子三嵌段聚合物维生素A(VA)-PEG-PAsp (N3)-PAsp (BBA-DEAP)。该聚合物在微酸性溶液中复合siRNA，其后加入ROS敏感的交联剂Tk-DBCO2，成功制备了pH 7.4溶液中带有负电荷的交联捆绑的纳米复合物（简称为T-C-siRNA）。细胞水平的实验结果表明，该纳米复合物展现出良好的血清稳定性和靶向肝星状细胞（HSC）高效递送siRNA的性能。T-C-siPDGFR-β纳米粒子在细胞和小动物水平均有效抑制了HSC的PDGFR-β蛋白的表达、活化和促肝纤维化相关蛋白（COL1A1和TIMP1等）的表达。T-C-siPDGFR-β减少了CCl4模型小鼠肝脏胶原纤维形成，有效缓解了肝纤维化。最后，本项目还开发了用于siRNA和化疗药物共载共递、大分子抗体和小分子药物共载共递的纳米药物。通过采用隐藏式递送的方式，提高了药物在病灶组织的浓度。在siRNA和化疗药物的协同作用或免疫协同作用下，小鼠肿瘤的生长得到明显抑制。本项目为抗肝纤维化等的基因治疗及其联合治疗提供了载体的制备方法和技术手段。