基于热激活细胞穿透肽与胞内触发释药的新型siRNA肿瘤靶向长循环脂质体的研究

How to efficiently deliver siRNA to the targeted tumor cells play a key role in gene silencing efficiency of siRNA against cancer. In this work, a novel drug delivery system based on thermal sensitive liposome (TSL), the active targeting ability of Asparagines-Glycine-Arginine (NGR) peptide and “thermal switch” controlled cell penetrating activity of cell penetrating peptides (CPPs) will be constructed to transport the small interfering RNA (siRNA) for tumor-specific therapy. In which, the delivery of siRNA in the system will be triggered by the intracellular environment. The delivery system can actively targeting to human fibrosarcoma cells (HT-1080 cells) with CD13 overexpressed on the surface and deliver siRNA into the cytoplasm of HT-1080 cells to downregulation c-myc oncogene. The system will be constructed as follows: CPPs-siRNA conjugate will be synthesized via the disulfide-linked, which can be restored by Glutathione (GSH) existing in the cytoplasm of the tumor cells. Then we firstly propose a novel method to shielding the cell penetrating function of CPPs and protect them from degradation before their arrival at the target cells. In which, CPPs-siRNA will be encapsulated into TSL in accordance with the “thermal switch” designing ideas. Meanwhile, NGR will be modified on the surface of TSL to achieve the specific tumor targeting of the vesicles. At last, a smart vehicle of CPPs-siRNA/NGR-TSL for the delivery of siRNA will be constructed. When the vesicle is located on the targeting cite, CPPs-siRNA can be delivered from TSL under the heat stimulus based on the thermal sensitive drug release characteristic of TSL. The activated CPPs-siRNA will penetrate the tumor cell membrane with the help of CPPs, then the disulfide-linked of CPPs-siRNA can be restored by GSH in the cytoplasm of HT-1080 cells and deliver enough siRNA to against c-myc oncogene. The project will mainly focus on improving the encapsulation efficiency and loading capacity of CPPs-siRNA into the TSL. Meanwhile, heat stimulus conditions such as temperature and time on the properties of CPPs-siRNA/NGR-TSL and the gene silencing efficiency of the delivered siRNA both in vitro and in vivo will be also systematically studied. We aim to provide a better protection for siRNA, a better target for tumor and a better gene silencing ability by the smart vesicle constructed in the study.

将siRNA高效、靶向递送至肿瘤细胞发挥基因沉默功能是肿瘤基因治疗成功的关键。本项目拟构建一种转运siRNA的新型热敏脂质体(TSL),该载体综合冬酰胺-甘氨酸-精氨酸(NGR)主动寻靶、“热开关”控制细胞穿透肽(CPPs)活性和细胞内还原环境触发释药等多种作用机制，可将siRNA靶向递送入表面有CD13高表达的人纤维肉瘤的细胞质内沉默致癌基因c-myc。根据TSL热敏释药特性，TSL包载二硫键连接的CPPs-siRNA以掩蔽CPPs达靶前的穿膜活性并保护其不被酶降解；NGR引导载体在靶部位聚集后，对靶部位加热以定时、定位和定量激活CPPs-siRNA穿透细胞膜进入细胞质中；CPPs-siRNA的二硫键被胞质中的谷胱甘肽还原，游离出活性siRNA沉默c-myc基因。重点研究载体的载药效率和体外热激活条件对siRNA体内/外递送效率及基因沉默规律的影响，为高效递送siRNA提供理论基础。

将siRNA高效、靶向递送至肿瘤细胞发挥基因沉默功能是肿瘤基因治疗成功的关键。本项目构建了一种转运siRNA的新型热敏脂质体(TSL),该载体综合冬酰胺-甘氨酸-精氨酸(NGR)主动寻靶、“热开关”控制细胞穿透肽(CPPs)活性和细胞内还原环境触发释药等多种作用机制，可将siRNA靶向递送入表面有CD13高表达的人纤维肉瘤的细胞质内沉默致癌基因c-myc。根据TSL热敏释药特性，TSL包载二硫键连接的CPPs-siRNA以掩蔽CPPs达靶前的穿膜活性并保护其不被酶降解；NGR引导载体在靶部位聚集后，对靶部位加热以定时、定位和定量激活CPPs-siRNA穿透细胞膜进入细胞质中；CPPs-siRNA的二硫键被胞质中的谷胱甘肽还原，游离出活性siRNA沉默c-myc基因。结果显示，所制备的基因递送载体粒径为100nm左右，外形为圆形纳米粒子，其与体外热疗相结合，可以实现有效的体外基因沉默和诱导细胞凋亡的能力；在体内可以有效的靶向肿瘤组织，具有良好的体内肿瘤抑制效果和体内基因沉默效率，有望成为siRNA有效递送的潜力载体。