触发式细胞靶向多功能纳米递药系统的构建及抗肿瘤机制的研究

Paclitaxel (PTX) is limited in the practical applications due to its toxicity, short half-life, poor solubility and low targeting. Polymer-drug conjugates, a novel drug delivery system is promising to improve the situation of the anticancer drugs. It has been an important strategy for targeting drug delivery system. In the previous work, targeting conjugates of HA-PTX were synthesised by chemical conjugation of an amino acid-modified PTX to the hyaluronic acid (HA) backbone contained multi-functional groups. HA-PTX conjugates showed better properties in drug loading, tumor targeting and efficacy, compared to PTX. In the present study, by designing and synthesizing a new polymer-drug conjugate, a multifunctional nano targeted drug delivery system is developed for triggered intracellular drug release. Intelligent conjugates (HA-ss-PTX) is obtained by binding PTX to targeting ligand of HA via disulfide which is sensitive to GSH and rapidly break down to release drug in tumor cells. The conjugates self-assemble into compound nanoparticles (HAPEL-NPs) with amphiphilic polymers of mPEG-PLA in aqueous media. The nanoparticles not only possess all merits of classic physically entrapped-drug nanoparticles, but also exhibit additional advantages, namely, enhanced stability. The mechanism of cellular uptake and multidrug resistance reversal and cell metabolism kinetics are systematicly researched. The informations of targeted nanoparticles into tumor cells, drug release from the compound nanoparticles and the distribution of drug within the cells are real-time monitored by using fluorescent labeling technology, high content live cell workstations and confocal laser scanning microscope. This will provide some grounds for experimental basis and clinical therapy in the context of breast cancer, ovarian cancer and other carcinomas. It is great significant for exploring the targeting mechanism of polymer prodrug.

针对抗癌药紫杉醇（PTX）溶解度低，体内循环时间短、靶向性低和毒副作用大等问题，构建的高分子偶联物已成为靶向递药系统的重要新兴战略领域。在前期研究中发现，利用透明质酸（HA）多功能基团化学修饰的PTX（HA-PTX），具有载药量高、肿瘤靶向性和药效好等优势。本项目拟设计合成新型聚合物-药物偶联物，构建触发释药的多功能抗肿瘤纳米靶向递药系统。将靶向配体HA与PTX通过GSH敏感的二硫键结合，制成在肿瘤细胞内迅速释放药物的智能偶联物（HA-ss-PTX）。利用自组装技术，偶联物与两亲性载体mPEG-PLA自组装复合纳米粒（HAPEL-NPs）。系统研究复合纳米粒的细胞摄取、肿瘤耐药逆转机理、细胞代谢动力学等。采用荧光标记技术，高内涵活细胞工作站和激光共聚焦显微镜实时监测靶向纳米药物入细胞情况，药物从复合纳米粒中释放及在细胞内的分布情况。课题的研究对探索聚合物偶联物靶向机理具有重要意义。

根据药物递送中的生物屏障和肿瘤组织的病理特征，针对抗癌药紫杉醇（PTX）溶解度低，靶向性差和毒副作用大等问题，本项目设计合成胞内触发式聚合物-药物偶联物（HA-ss-PTX ），研究不同连接臂、HA分子量和取代度对偶联物稳定性、释药速率与抗肿瘤活性的影响，建立结构-活性关系，其中HA9.5-ss-PTX的载药量为8.8-18.6%，平均粒径为184 nm；项目合成表征还原敏感糖脂接枝共聚物（HA-ss-SA，HSSA），通过协同组装制备壳交联还原敏感纳米粒PTX@PHSSA，载药量高达38.7%。细胞免疫荧光，摄取和胞内释药机制研究表明，HA9.5-ss-PTX和PHSSA纳米粒的摄取与CD44表达有关，在胞内GSH作用下触发释放药物。HA9.5-ss-PTX纳米粒主要通过小窝蛋白介导的内吞作用进入细胞，而壳交联纳米粒的内吞则与小窝蛋白和微管蛋白相关。体内药效学研究表明HA9.5-ss-PTX和PTX@PHSSA纳米粒的对乳腺癌的抑瘤率分别为89.22%和87.78%。荷瘤鼠体内生物分布研究表明两种纳米粒均能显著延长体内滞留时间，提高肿瘤靶向性。采用LC-MS/MS技术检测壳交联纳米粒在细胞浆、细胞核、线粒体的摄取和分布，阐明其细胞药代动力学处置行为，为还原刺激响应纳米递药系统相关基础研究提供了新的研究思路。. 本项目设计合成透明质酸-二硫键-聚乳酸新载体（HA-ss-PLA），与P-gp抑制剂TPGS组装成复合纳米粒（HSPT），应用细胞药代动力学技术阐明该复合纳米粒在细胞及亚细胞器内药代行为特征及逆转肿瘤多药耐药机制，为科学评价新型纳米靶向制剂提供了示范性研究体系。本项目对推动国内外新型药用生物材料的发展和靶向制剂的应用，具有十分重大的挑战意义；将为抗肿瘤药物靶向制剂的构建、筛选和安全性评价提供新的理论指导。