基于iRGD类脂靶向载体的可控构建及其siRNA转运研究

Delivery of siRNA into cells is a key step for RNAi, among these vectors peptides based lipids could provide a promising solution,but their site recognition and penetration properties need to be improved. Therefore, iRGD lipids were designed and planned to be synthesized aiming to lower the toxicity of traditional lipids, increase the transfection efficiency of peptide lipids and suppress the intrinsic shortcomings of liposomes. It has been supposed that these iRGD lipids are both of site-recognized and cell-penetrated. Calcium phosphate@siRNA nano-particles will be used as the templates for adsorbing iRGD lipids, tri-peptide lipids and DSPE-PEG2000 to construct hybridized siRNA vectors. The physicochemical and biological properties will be researched through the measurement of particle size and zeta potential, of siRNA encapsulation efficiency, and of transfection efficiency and cytotoxicity. Site-recognition and cell-penetration will also be studied through both of in vitro and in vivo methods. Based on the study of this project, hybridized vectors of RGD-target and cell-penetration and a novel method for siRNA delivery could be obtained and the theoretical reference for siRNA delivery could also be provided.

肽类脂是一类重要的siRNA转运载体，其良好的生物相容性为克服季铵盐型阳离子类脂较高的毒性提供了解决途径，但其位点识别和穿透能力差，严重制约了其基因转运效果的提高和临床应用。本项目拟将iRGD肽作为阳离子类脂的头部结构，合成具有位点识别和穿透功能、生物相容性好及siRNA转运效率高的iRGD阳离子类脂。同时，为克服传统脂质体/siRNA复合物粒子多尺度分布的缺点，以磷酸钙@siRNA纳米粒子为模板，自组装iRGD类脂、三肽类脂和DSPE-PEG2000等组分，构建粒径和形态可控的iRGD复合载体。考察复合载体的粒子形态及其siRNA包封率，评价复合载体运载siRNA的体内和体外效率及其毒性，研究其位点识别能力和穿透功能，探讨iRGD在复合载体进入细胞及胞内传递过程中的作用。本项目可实现siRNA的可控组装和定向转运，为构建高效低毒复合载体开辟新思路，为运载siRNA机制研究提供理论参考。

肽类脂作为一种重要的siRNA转运载体，具有良好的生物相容性，可克服季铵盐阳离子脂类的高毒性，但其位点识别和穿透能力差，严重制约了其基因转运效果的提高和临床应用。本项目合成了以RGD肽为头部的肽阳离子类脂，利用RGD的主动靶向肿瘤和穿透功能的特性，构建了一类新型的肽靶向脂质体，用于siRNA的高效转运研究。.首先使用链状RGD肽和环状cRGD肽通过酰胺缩合反应与类脂中间体连接，分别得到链状RGD类脂和环状cRGD类脂。通过质谱、红外光谱和核磁共振波谱证实两种RGD类脂结构正确，以高效液相色谱测得其纯度分别达到95%和97%。然后使用三肽类脂CDO14、助类脂DOPE和两种RGD靶向类脂，制备了不同靶向结构和修饰密度的RGD肽靶向脂质体。该肽靶向脂质体颗粒分散均匀，呈球形或椭球型结构，粒径为50-100 nm，电位为40-60 mV。实验证明其非特异性蛋白质吸附较少，且靶向修饰密度对其蛋白吸附无明显影响。通过凝胶电泳、体外转染及基因沉默研究验证了RGD肽靶向脂质体具有较好的基因结合和运载能力，基因沉默效率高达50%。载IGF-1R siRNA的RGD肽靶向脂质体通过主动靶向进入肿瘤细胞，诱导细胞凋亡，能够有效抑制肿瘤生长。其中cRGD(5%)/CD@IGF-1R siRNA的体内抑瘤效果最佳，其肿瘤体积比生理盐水对照组小6.94倍。载IGF-1R siRNA 的RGD肽靶向脂质体对小鼠的正常组织和器官无明显的毒副作用。.本项目设计合成了一类新型RGD肽阳离子类脂，不仅较传统阳离子类脂具有良好的生物相容性，而且具有肿瘤靶向和组织穿透功能，有效提高了siRNA的基因沉默效率。此外通过研究RGD肽靶向脂质体的靶向性和穿透性，阐明了RGD对肽靶向脂质体进入细胞及释放siRNA的作用机制。本项目为构建高效低毒转运载体开辟了新思路，为运载siRNA机制研究提供了理论参考。