人源性低密度脂蛋白介导-pH/还原双敏感性二元聚合体胶束的构建及其基因递送逆转耐药机制研究

It is the hot issue that targeting therapy and reversing multidrug resistance(MDR) are used to improve therapeutic effect and reduce side effect. In view of co-transporting, reversing MDR and synergistic interaction of chemotherapeutics and gene therapeutic agents, a low density lipoprotein (LDL) modified co-transports system of siRNA and therapeutic drug was designed in our project. Carboxymethyl chitosan-lipoic acid polymer （CMCh-LA）micelle loaded doxorubicin(Dox) (Dox/micelle) was prepared based on pH/redox dual -stimuli responsive CMCh-LA, then coupled with siRNA/LDL complex to form siRNA/LDL modified Dox/micelle dualistic polymer (siRNA/LDL-Dox/micelle). The structure and characteristics of siRNA/LDL-Dox/micelle were evaluated such as particle size, morphology, entrapment efficiency and drug-loading amount. Tumor cell lines overexpressing LDL receptor were screened to study the uptake and sub-cellular localization of siRNA/LDL-Dox/micelle. A variety of technical means such as confocal microscope, atomic force microscope, flow cytometry and near-infrared small animal fluorescence imaging system are used to evaluate systematically the targeting effect of siRNA/LDL-Dox/micelle in vitro and in vivo. Some key factors affecting gene therapy such as anti-phagocytose, transmembrane and lysosomal escape are to be studied by hepatoma cell line and its drug-resistant cell line to optimize transfection conditions. The targeting efficiency, tissue distribution, antitumor effect and anti drug-resistance properties and mechanism of siRNA/LDL-Dox/micelle will be studied. Consequently, our project is aimed at constructing a novel co-transport drug delivery system which combines tumor-targeting and reversal of drug resistance.

肿瘤靶向和逆转耐药是提高抗肿瘤药物疗效和降低毒性的热点问题。课题基于化疗药物与基因联合递送、逆转耐药、协同增效的思路，利用羧甲基壳聚糖和硫辛酸基团在肿瘤内环境中的双敏感性以及人源性低密度脂蛋白（LDL）的天然靶向性，构建LDL配体介导的-pH/还原双敏感的羧甲基壳聚糖-硫辛酸二元聚合体胶束体系（LDL-micelle）。该递药系统疏水层包载抗肿瘤药物，表面偶联siRNA/LDL复合物。同时，以LDL受体过表达的肿瘤细胞模型，考察二元聚合体胶束的摄取过程和亚细胞定位，利用共聚焦显微镜、原子力显微镜和小动物荧光成像等技术考察二元聚合体胶束的体内外靶向性；以肝癌及其相关耐药细胞株模型，探讨影响基因疗效的关键因素，包括：巨噬细胞吞噬、跨膜机制及溶酶体逃逸等；优化转染条件，探讨其寻靶效率、肿瘤组织分布、抗肿瘤效果及抗耐药活性和机理等关键技术，构建与评价具有肿瘤靶向和抗耐药作用的新型纳米递药系统。

研究背景与目的： 采用LDL为配体，与壳聚糖衍生物偶联制备二聚体纳米聚合物胶束递送系统。从材料和技术，设计与构建高效、安全的抗肿瘤纳米药物新剂型；同时，对二聚体纳米聚合物胶束在分子、细胞、组织和整体动物水平上揭示摄取，转移和定位释放的机理；对siRNA诱导基因沉默机制进行了探索，介绍逆转耐药的相关机制；初步阐明LDL作为内源性载体具有良好靶向性和生物相容性，和逆转多药耐药和协同增效作用。.主要研究内容：提取纯化LDL，并包载基因并作为靶向配体，再与壳聚糖衍生物偶联制备二聚体纳米粒聚合物胶束，且构建载体具有pH/还原双敏感性，其表面偶联包载siRNA的LDL纳米颗粒及核心包载阿霉素（Dox）的共传输系统，能够发挥协同增效的抗肿瘤作用。LDL介导下，直接将siRNA和抗肿瘤药物输送到靶器官和靶细胞，并通过胞吞机制进入细胞，在肿瘤细胞内还原性物质谷胱甘肽（GSH）作用下，载体内S-S键断裂释药，从而发挥药物的化疗和siRNA诱导沉默的协同作用。并对耐DOX的细胞有限实现耐药反转，实现逆转耐药性。.重要结果：项目通过科学的分子设计，成功构建LDL为配体的靶向递送载体。.即由人血低密度脂蛋白包载siRNA，并共载抗肿瘤药物阿霉素（Doxorubicin, Dox）或紫杉醇（Paclitaxel, PTX）二聚体胶束纳米递送系统。利用肿瘤部位特异性LDL受体高表达，以及胶束EPR效应实现肿瘤主被动靶向。同时，利用载体pH/还原双敏感性特性，二聚体中基因/药物的摄取，转运和逃逸中起到重要作用，有效避免了基因与药物被体内溶酶体破坏，从而发挥药物的化疗和siRNA诱导沉默的协同作用。 .关键数据及科学意义：揭示LDL具有良好天然靶向性和生物相容性，构建的二聚体纳米递送系统是一种高靶向和低免疫原性的理想载体。 探讨二聚体药物的体内转运途径和定位释放药物的特性与规律，建立动力学模式，科学探索了基因/药物共转运逆转多药耐药和增强肿瘤靶向性的机制，为实现基因/药物共递送协同增效发挥抗肿瘤作用提供科学依据。.主要结果：项目通过科学的分子设计，成功构建LDL为配体的靶向递送载体。即由人血低密度脂蛋白包载siRNA，并共载抗肿瘤药物阿霉素（Doxorubicin, Dox）或紫杉醇（Paclitaxel, PTX）二聚体胶束纳米递送系统。利用肿瘤部位特异性LDL受体高表达，以及胶束EPR效应实