EGFR受体介导的肺肿瘤靶向siRNA类脂质载体的构建及转运调控机制研究

Pulmonary delivery of siRNA has strong potential for the treatment of respiratory disease, but its application was limited by short of suitable carriers and delivery systems. The aim of this study is to clarify the control mechanism of cellular uptake and intracullular fate of a siRNA complex with a novle lipid like compound,epoxy alkyl-amine derivatives(EAADs). After that, two siRNA with different mechamism for cancer therapy(survivin-siRNA/MRP1-siRNA) will be loaded in the carrier modified with EGFR mAb for lung cancer targeting.Dried powder inhalation will be prepared using spray freeze drying method. The cellular uptake pathway and intracelluar fate of EAADs-siRNA complex including escape from endosomes and lysosomes,deaggregation and combinding with RISC, the relationship with target gene silencing efficiency will be investigated throughly.Finally, in vivo distribution of siRNA and carriers, accompany with the expression of target mRNA gene and protein will be detected after pulmonary delivery of siRNA dry powder inhation.

siRNA肺部给药在呼吸道疾病治疗方面具有独特的优势，但输送载体和给药系统设计限制了其临床应用。本研究旨在阐明新型siRNA负载类脂质化合物（1,2-环氧烷胺衍生物，EAADs）的细胞摄取与细胞内动态调控机制的基础上，将二个不同抗肿瘤机制的siRNA（survivin-siRNA/肿瘤细胞凋亡抑制,MRP1-siRNA/肺肿瘤细胞耐药性）同时包载于EGFR受体介导的肺肿瘤细胞靶向类脂质载体的干粉吸入剂。通过考察siRNA-EAADs复合物细胞摄取通路、细胞内动态调控（内涵体与溶酶体逃逸、siRNA-EAADs复合物的解聚、RISC结合）机制及其与相关基因沉默效率的相关性。制备抗EGFR mAb修饰长循环脂质体后，采用喷雾冷冻干燥法制备干粉吸入剂。经荷瘤小鼠肺部给药，动态观察荧光标记siRNA和载体的体内组织分布，考察靶基因mRNA与蛋白质表达以及抗肿瘤效果，阐明影响siRNA肺部给的因素。

siRNA肺部给药在呼吸道疾病治疗方面具有独特的优势，但输送载体和给药系统设计限制了其临床应用。该项目在阐明新型siRNA负载阳离子类脂质化合物（1,2-环氧烷胺衍生物，EAAD）的细胞摄取与细胞内动态调控机制的基础上，成功将二个不同抗肿瘤机制的siRNA（survivin-siRNA/肿瘤细胞凋亡抑制,MRP1-siRNA/肺肿瘤细胞耐药性）共包载于EGFR受体介导的肺肿瘤细胞靶向类脂质载体的干粉吸入剂。考察和系统研究了siRNA-EAADs复合物细胞摄取通路、细胞内动态调控（溶酶体逃逸、siRNA-EAADs复合物的解聚、RISC结合）机制及其与相关基因沉默效率的相关性等。通过制备抗EGFR mAb修饰长循环脂质体，并采用喷雾冷冻干燥法制备可用于肺部给药的多孔性干粉吸入剂，实现逃避巨噬细胞的摄取，并特异性靶向肿瘤的目的。原位荷瘤小鼠肺部给药后的组织分布与药效学研究结果，初步证实了本研究所制备siRNA纳米载体具有良好的应用前景。