多功能高分子-姜黄素轭合物混合胶束的微结构调控及抗癌药物组合递送研究

Background: The tumor microenvironment is characteristic of the over-expression of certain receptors, vascular abnormalities, low pH in the intracelluar endosome/lysosome and excellular matrix, and the high redox potential in the cytosol and cell nucleus. Chemotherapy has been suffering from the toxic side-effects to the healthy tissues and the multidrug resistance. Curcumin has been reported as a pleiotropic anticancer agent and chemosensitizer because of its ability to interfere with multiple cell signaling pathways including angiogenesis, inflammation, proliferation, metastasis, and apoptosis etc. Hypothesis: It was hypothesized that the co-delivery of curucmin with other anticancer agents (e.g. doxorubicin) in a multifuntional micellar nanocarrier can address the side-effects and multidrug resistance issues of chemotherapy. Purpose: The aim of this study was to generate, evaluate and optimize the multifunctional micelles with the capability of active targeting, dual pH/redox- reponsive drug release, multidrug resistance reversal, and combinational synergism. Materials & Methods: Hydrophilic poly(ethylene glycol) and hydrophobic poly(lactic acid) was linked via the disulfide bond to generate the amphiphilic copolymer. Curcumin was modified with levulinic acid producing two types of curcumin derivatives, which together with doxorubicin was connected to the copolymer via a pH-sensitive hydrazone bond. The targeting molecule, folate was also linked to the copolymer. These polymer conjugates were selected to generate mixed micelles via co-assembly. The micelles were characterized in terms of size, zeta potential, drug loading, physical stability, and drug release via standard protocals and validated methods. The intracellular uptake, cytotoxicity and apoptosis study were carried out to evaluate the delivery efficiency of micelles and chemosensitizing effect of curcumin. The western blot analysis was employed to assess the multidrug resistance. The "combination index" was used to judge the synergistic effects of curcumin and doxorubicin coadministration. The micelle composition and architecture was correlated with its physicochemical characteristics and drug delivery performance to optimize the conjugate/micelle structure and the dose ratio of cucumin and doxorubicin.This study will add value to the spectrum of anticancer drug delivery systems available for reduced side-effects and improved efficacy.

本课题的创新点在于：针对肿瘤化疗的毒副作用和多药耐药问题，利用姜黄素增敏剂的多靶点和多效能特性，设计具有长循环、可降解、主动靶向、可控释放和逆转耐药的纳米胶束组合给药系统。以阿霉素为模型药物，叶酸为靶向分子，分别合成姜黄素、阿霉素和叶酸与高分子(PEG-PLA)的轭合物。利用不同类型轭合物的共同自组装构建系列姜黄素/阿霉素共包载纳米混合胶束，通过轭合物分子内二硫键和腙键实现肿瘤微环境条件下药物的pH/redox应答释放。探讨混合胶束组成、微结构、载体物化和递送性能之间的内在关系；通过细胞摄取、细胞毒性和凋亡实验在细胞水平揭示胶束结构和性能对其生物转运和药效的作用规律；利用蛋白免疫印迹法研究混合胶束特性以及姜黄素/阿霉素剂量比例对阿霉素逆转耐药的影响规律；利用"组合指数"评价姜黄素和阿霉素共递送的协同效应及其剂量比依赖关系。本课题将为环境应答型多功能抗肿瘤纳米给药系统的设计和开发提供新思路。

本项目主要针对肿瘤化疗的毒副作用和多药耐药问题，利用多效分子姜黄素的多靶点和多功能特性，将其与可生物降解的两亲性共聚物“聚乙二醇-聚乳酸”通过腙键或二硫键共价连接，然后利用轭合物在水相中的自组装物理包载模型抗癌药物阿霉素，这一设计具有血液循环时间长、载药量高、载体可降解、药物应答控释等优点。主要研究内容涉及姜黄素的化学修饰、共聚物与轭合物的合成、纯化与表征、纳米载体的构建与理化性能与药剂性能评价、在细胞水平考察药物共递送的协同性。实验结果证明：姜黄素与阿霉素的共递送可以实现协同增效（组合指数 < 1）；腙键及二硫键连接的纳米载体均可在细胞内部微环境下实现药物的pH/Redox应答释放；轭合物的微结构由于可显著影响其自组装特性，因此可用于调控纳米载体的理化和递送性能。本项目还首次发现腙键连接的纳米载体释药后由于含有阳离子基团可产生细胞毒性；由于谷胱甘肽的扩散屏障，二硫键连接的纳米载体的释药速率显著低于腙键连接的载体；此外，由于物理包载药物通常的载药量都低于5%，因此较难调控分别通过化学和物理方式包载的不同药物的剂量比例。本课题为环境应答型多功能抗肿瘤纳米给药系统的设计和开发提供新思路。