cRGD修饰性共转运抗肿瘤药物与siRNA自组装聚合物胶束递药系统的研究

It is the hot issue that targeting therapy and reversing multidrug resistance effect are to improve the anti-tumor efficacy and reduce recurrence. Based on the mechanisms of tumor resistance, a cRGD mediated polymeric micelles drug delivery system, which cotransports siRNA and anti-cancer drugs was designed in our project. N-trimethyl chitosan was synthesized firstly，then coupled with cRGD and doxorubicin grafted palmitic acid to form cRGD-mediated doxorubicin-trimethyl chitosan-palmitic acid amphiphilic polymer. siRNA is encapsulated in the amphiphilic polymer to establish siRNA and anti-cancer drugs cotransported, self-assembled polymer micelles drug delivery system.The structure and quality of the delivery system will be characterized and evaluated in terms of particle size, entrapment efficiency(EE), drug-loading (DL) amount and so on. Sensitive tumor cell lines are to be filtered as the objects of study to inspect the uptake rate and to observe subcellular localization of the polymer micelles to explore the uptake mechanism. The tumor targeting properties of the polymer micelles will be systematically evaluated using a variety of technical means, such as fluorescent marking, radionuclide tracing, near-infrared small animal in vivo imaging, confocal microscopy and flow cytometry. Breast cancer cell lines MCF and its drug-resistant cell line MCF/DOX are to be chosen to assess the tissue distribution, anti-tumor activity and anti-drug resistance properties of polymer micelles. On this account, the aim of current study is to develop a novel polymeric micelles drug delivery system which combines tumor targeting and drug-resistance reversing, to investigate its tumor-inhibitory activity and then confirm its targeting and drug-resistance reversing characteristics.

肿瘤的靶向治疗与抗耐药性是提高抗肿瘤疗效，降低肿瘤复发的热点课题。本课题基于肿瘤耐药性机制，协同递送小分子化疗药物和基因药物，组合具有cRGD 介导的siRNA 与抗癌药物共转运递药系统。以合成三甲基壳聚糖为基础，偶联cRGD 介导的阿霉素接枝软脂酸形成cRGD 介导的阿霉素-三甲基壳聚糖-软脂酸两亲性聚合物，并构建SiRNA 与抗癌药物共转运自组装聚合物胶束递药系统及其质量评价体系。以敏感肿瘤细胞株模型，考察对聚合物胶束的摄取率，探索摄取的机制，观察在胞内的分布及定位；利用荧光标记，核素示踪等手段，通过近红外小动物活体成像、共聚焦显微镜及流式细胞技术，系统研究药物对肿瘤组织的靶向性。以乳腺癌细胞株及其耐药细胞株模型，考察其体内的组织分布及药效学，评价其肿瘤抑制效率及抗耐药性活性和机理，探索建立肿瘤细胞靶向及抗耐药的新型功能性高分子胶束递送系统。

研究目的：采用自组装胶束释药系统递送材料和技术，设计、构建高效、安全的抗肿瘤纳米药物新剂型；在分子、细胞、组织和整体动物水平上揭示纳米药物新剂型降低肿瘤耐药性的药效及其机理；初步阐明纳米材料、传统小分子化疗药物和基因药物组成的载药系统、协同增效后影响其体内活性的关键因素。..研究内容：合成生物相容，两亲性的高分子载体材料，并对其进行结构修饰，化学键键合靶向配体cRGD，实现材料功能化的设计与构建；对带负电荷的小干扰 RNA 进行复合，利用自组装纳米技术载入合成的功能性载体材料中，实现化疗药物与基因药物协同释药系统新剂型的设计与构建；建立生物发光的原位非小细胞肺癌模型和肿瘤耐药细胞模型，对肿瘤靶向性和耐药性进行评价与机理研究。 ..研究结果：项目通过合理的分子设计成功构建了针对原位非小细胞肺癌的靶向递送载体，该载体以亲水性材料琥珀酸壳聚糖为基础，接枝疏水基团软脂酸包载紫杉醇后形成纳米胶束，利用自由羧基在胶束表面连接cRGD实现主动靶向，以达到增加在血液中循环循环时间和肺癌肿瘤部位富集的目的。体外试验证明该载药胶束具有一定的pH响应性和缓释性，能够在肿瘤酸性环境中快速释药。同时，cRGD介导的受配体增强肿瘤细胞对于胶束摄取，提高药物在肿瘤组织及细胞内的浓度，增加细胞毒性；体内证实该递送系统对于皮下肿瘤和原位肺肿瘤都具有较好的主动靶向性，减少了肝脏的摄取，显示出明显的增效减毒作用。构建了共转运siRNA 和抗癌药物Dox的自组装聚合物胶束递药系统，并考察其体外细胞摄取和亚细胞分布特性及其药物在细胞内的摄取消除动力学。探讨了耐药蛋白在细胞间的转移对化疗药物药效学的影响，从逆转耐药和增强肿瘤靶向方面提高共转运胶束协同增效的抗肿瘤作用。..科学意义：揭示cRGD介导的siRNA 与抗癌药物共转运新型胶束递药系统降低肿瘤耐药性的机理及其协同增效的新思路，构建安全、可控并具有同时降低肿瘤耐药机制作用的纳米胶束药物新剂型，在改善肿瘤治疗效果的技术上取得突破。