“胞内触发”式还原敏感型药物联合基因靶向共传递体的构建

In this work, PEI-SS was synthesized using disulfide linkage(s) Disulfide bonds to form redox-sensitive polymers, which are stable in the mildly oxidizing extracellular milieu, may be prone to rapid cleavage through thiol-disulfide exchange reactions with intracellular reducing molecules, especially with glutathione (GSH). This could cause the intracellular drug release. Then PEI-SS was grafted to Hyaluronic acid (HA) to form PEI-SS-HA conjugate, which was used to make a complex with siRNA by electrostatic interaction. It was exploited for target-specific systemic treatment of cancers for HA receptors such as CD44 are abundantly presented in tumor cells. In the proposed researches, PEI-SS-HA was grafted to PLGA to form PLGA-PEI-SS-HA conjugates , which can be self-aggregated in water to form polymeric micellar nanoparticles. Modified with PLGA could increase the lipophilicify of the ternary micellar nanoparticles to enhence the transfection efficiency and its hydrophobic core could load the small molecular drug such as paclitaxel to form the combinatorial delivery of siRNA and lipophilic anti-cancer drugs. The structural, chemical characteristics, stability, redox-sensitive characteristic, in-vitro/in-vivo tumor-target ability of siRNA encapsulation of the ternary conjugates and the mechanism of cellular uptake will be evaluated. The project has a research fundament, it is expected to be a highly promising for the combinatorial delivery of siRNA and lipophilic anti-cancer drugs.

本项目以二硫键交联的低分子量聚乙烯亚胺（PEI-SS）为阳离子片段，并在其表面接枝透明质酸（HA）形成还原环境敏感型的二元聚合物PEI-SS-HA。该聚合物在细胞内高GSH条件下，二硫键断裂从而控制药物在胞内的释放。由于HA对肿瘤细胞表面过度表达的CD44能特异性结合，可实现靶向性传递siRNA的目的。同时在PEI末端氨基接枝PLGA构成三元聚合物（PLGA -PEI-SS-HA）非病毒载体，该载体在水中能自发形成胶束结构样的纳米粒。PLGA修饰可提高载体的亲脂性，提高转染效率，并且可载入疏水性小分子药物如紫杉醇，达到小分子和基因药物共传递的协同治疗作用。本项目通过评价载siRNA和小分子药物的还原敏感型载体在肿瘤细胞中的转染效率、肿瘤靶向性和抑瘤效果，从而构建出适合基因药物和小分子抗癌药物的还原敏感型的靶向共传递系统。

本研究拟构建共载针对COX-2的siRNA及具有抗癌作用的多西他赛药物于二硫键敏感的PEI-PLGA纳米粒，并进行透明质酸（HA）修饰，研究其理化性质的同时评价该载体对肿瘤的体内外靶向性，以期研制出一种高效、低毒、稳定的双载药载体。. 载DTX的siRNA/HA-PEIss-PLGA(HRPSP NPs)纳米粒的制备和物理化学性质评价：利用乳化溶剂挥发法制备载DTX的HA-PLGA-PEIss纳米粒,用透射电镜和原子力显微镜考察纳米粒的外观，均显示为类圆形，大小200nm左右，zeta电位为±20mv，最佳处方为40/1/2。. 载siRNA的HRPSP NPs的毒性，摄取和稳定性评价：HA包被的NPs由于表面负电荷呈现较小的毒性；肝素解离实验和抗核酸酶实验该载体能有效保护siRNA，使得RNA免受RNAase的降解，有利于siRNA在体内的稳定，而HRPSP NPs在GSH的存在下，稳定性降低；载体并在N/P/COOH为40/1/2时实现高转染率，胞内蛋白沉默效果达到40% 左右。. 摄取机制评价：受体抑制实验有效证明了HA可以提高纳米载体的靶向性主要通过CD44受体入胞，并且HRPSP NPs主要通过胞吞进入细胞，二硫键连接的PEI载体与市售PEI载体具有相似的溶酶体逃逸作用胀破溶酶体从而发挥细胞药效。. 体内评价：HRPSP NPs能显著增加在肿瘤部位的蓄积，减少在正常组织中的分布，能有效减少副作用，增强治疗作用。