用于乳腺癌联合化疗的双层纳米药物输送体系的研究

Tumor angiogenesis is critical for tumor growth, invasion and metastasis such as breast cancer. According to previous studies by our group，double-layer core-shell structure nanoparticles could serves as carriers for targeting co-delivery of chemotherapeutic drug and anti-angiogenesis drugs. In this continuing project search for effective treatments for cancer, the model is the combination of traditional combination chemotherapy with anti-angiogenesis gene therapy that inhibit blood vessel growth. To address this challenge we designed a novel double-layer core-shell structure nanoparticle delivery system, comprising a nanoscale self-assembly mPEG-g-CS envelope coating on a nuclear PLGA nanoparticle. A combination chemotherapeutic agents are loaded in the core PLGA nanoparticle and an antiangiogenesis agent VEGF-siRNA is conjugated to the mPEG-g-CS as outer envelope. We proposed that the disruption of this envelope inside a tumour would result in a rapid deployment of the anti-angiogenesis agent, leading to vascular collapse and the intra-tumoural trapping of the nanoparticles. The subsequent slow release of the cytotoxic agents from the nanoparticles should then kill the tumour cells. To explore the combination chemotherapy anti-tumor activity by inhibiting angiogenesis and subsequently tumor cell of this novel nanoparticle system and explain the ‘integrative’ mechanism approach in cancer therapy, we characterize the nanoparticle for physical/chemical properties, controlled drug release kinetics, finally evaluated in vitro and in vivo anti-tumor activities. This novel nanoparticle system may represent a new way of combining traditional combination chemotherapy and anti-angiogenesis gene therapy for controlled drug delivery applications. The multifunctional double-layer core-shell structure nanoparticle system may represent a new method for the future treatment of cancer.

恶性实体瘤如乳腺癌的生长和转移依赖于血管生成，纳米药物输送体系可以作为抗肿瘤血管新生及化疗药物的载体发挥双重抗肿瘤作用。据此本项目提出研制一种多功能双层纳米输送体系，发挥抗血管新生的靶向基因治疗和联合化疗的协同抗乳腺癌的作用。双层纳米粒外层为聚乙二醇单甲醚接枝壳聚糖（mPEG-g-CS）自聚集纳米粒，复合抗VEGF-siRNA包载于外层作为抗血管内皮细胞进行靶向基因治疗，首先从纳米粒中释放达到抑制肿瘤血管生成的效应，切断肿瘤细胞的营养运输，并防止化疗药物外渗；联合化疗药物包载于聚乳腺羟基乙酸（PLGA）纳米粒作为双层纳米粒的内层，随后释放，发挥抑制肿瘤实体细胞的作用。通过研究双层纳米粒的理化性质及载药性质，体外抑瘤效应及动物体内抑瘤效应，探索其达到顺序抑制肿瘤血管及实体瘤细胞的联合化疗作用，对阐明其综合治疗肿瘤的机制有重要意义，为新型纳米输送体系作为临床抗肿瘤治疗提供新的思路。

本项目探索了多功能双层纳米粒制剂作为基因与化疗用药物载体的可行性；并阐明了双层纳米粒靶向抑制肿瘤血管及乳腺癌的作用，发挥系统抑制肿瘤的体内外作用机制。本项目研制了一种新型双层纳米粒，其外层为复合抗血管内皮生长因子（VEGF）-siRNA 的聚乙二醇单甲醚接枝壳聚糖（siRNA-mPEG-g-CS），发挥抑制肿瘤血管生成的效应；包载表阿霉素（EPI）的聚乳腺羟基乙酸（PLGA）纳米粒作为双层纳米粒的内层，随后释放，达到发挥抑制肿瘤实体细胞的作用。通过对双层纳米粒理化性质，体外释药物行为，体内外抑瘤效应等实验评价其作为基因与化疗用药物载体的可行性。体外细胞毒实验结果显示，三种空白纳米粒（双层纳米粒，PLGA纳米粒和mPEG-g-CS纳米粒）在浓度0.5~1000 µg/mL范围内，分别与MCF-7细胞和HUVEC细胞共孵育24h或48h时，对细胞的增殖无显著影响。激光共聚焦以及细胞流式结果显示双层纳米粒分别与MCF-7细胞和HUVEC细胞共孵育2h时，细胞摄取量较高。琼脂糖凝胶电泳阻滞实验结果表明，mPEG-g-CS能有效的结合pDNA形成稳定的复合物，细胞转染实验证实其可成功转染并表达绿色荧光蛋白。动物体内药物分布表明双层纳米粒可以延长体内循环时间，有一定的肿瘤靶向性，体内抑瘤实验表明双层载药纳米粒与两种单层载药纳米粒相比，抑瘤效果最佳，且可显著降低肿瘤组织中的微血管密度。综上所述，此双层纳米粒作为基因与化疗用药物载体具有长循环性和肿瘤靶向性，有显著的体内抑瘤效果；用于乳腺癌联合化疗的双层纳米药物输送体系，具有良好的应用前景。