一种体内安全、靶向递送外源性microRNAs新方法的建立与评价

MiRNAs are a class of non-coding small RNAs with a global regulatory role, can be used as a novel therapy target of the diseases, how to deliver miRNAs to targeted region safely is still obscure. In our previous research, we synthesized a nano lipidoid vector with a high transfection efficiency, good gene-load ratio, biodegradable, biological safety and poor-targeted, which composed to a negative polymer as headgroup and a neutral modified lipid as an auxiliary base, showed a good clinical application prospect. This project will modify the novel lipidoid material to be nanocrystallization, microvesicle and initiative target. Based on principles of physics, chemistry and immunology, realize the effective connection between targeted antibody and lipidoid depend on the electrostatic effect avoid to destroy the antibody protein spatial structure, and reduce miRNAs degradation and protein adsorption in the blood vessel. Then we will explore the endocytosis mechanism of antibody-mediated nano-lipidoid vector and the various parameters among the synthesis and optimization of the new carrier. And then asses the biological safety of this carrier through cellular and animal toxicity tests, discuss the targeted delivery ability of miRNAs to a target region with in vivo and in vitro experiments. Our object would be to develope a nano-lipidoid vector to deliver miRNAs with high load ratio and transfection efficiency, biological safety, target-selectivity, and to offer a novel delivery system and new thinking for miRNAs targeted therapy.

miRNAs是一类具全局调控作用的非编码小分子，可作为疾病治疗新靶点，建立一种体内低毒有效递送miRNAs新方法是其治疗的关键。前期预研中我们以阴性高分子聚合物为头基、中性的修饰脂质为辅基，合成了一基因负载率与转染效率高、可生物降解、低毒的新型类脂纳米载体，表现出良好的临床应用前景，但靶向性较差。本项目拟将该类脂载体纳米化、微粒化、靶向化，依据物理化学、免疫学原理，以静电相互作用实现胺基的锚定及靶向抗体的有效连接而不破坏蛋白的四级结构，模拟微粒的膜性结构减少miRNAs降解与血管内蛋白吸附，研究抗体靶向性类脂纳米载体合成及优化的各种参数，探讨其内吞机制，细胞和动物毒性实验检测该载体的生物毒性与缓释效果，体内体外实验评价其靶向递送miRNAs能力，最终研发出一负载率高、转染效率高、 生物安全性高、靶向选择性强和制备简便的纳米miRNAs递送系统，为miRNAs治疗提供新型靶向给药系统与新思路

近年来，肠癌发病率增高趋势明显，然而其治疗，特别是中晚期肠癌的治疗依然不理想，亟需开发新的治疗策略。MicroRNA是一类具全局调控作用的非编码小分子，可作为疾病治疗的靶点，然瓶颈是缺乏稳定安全有效的体内递送方式，本研究开发了一种可靶向肠癌细胞的新型类脂载体，用于包封miR-145，评价该载体靶向性等各种性能指标。98N12-5(1)、PEG2000修饰的12/14碳脂质与胆固醇组成的基础型载体，粒径约90 nm，Zeta电位为-8±6 mv，载体毒性很小，对其包封的核酸具有很强的保护作用，稳定性较高，能较好地递送核酸至靶器官（主要集中于肠癌等癌组织），且探明该载体为能量依赖的脂筏内吞途径。进一步实验证实，单层类脂纳米载体包被增殖诱导配体（APRIL）siRNA可有效治疗DMH（二甲基肼）诱导的原位肠癌小鼠模型，显著延长肠癌小鼠模型的总体生存率。然后，我们将单层类脂载体制成生物靶向性类脂纳米载体，变被动靶向为主动靶向。先锚定鱼精蛋白（静电互作），再用鱼精蛋白的胺基与villin单抗的羧基发生缩合反应，从而实现靶向性。流式细胞术检测发现，靶向载体成功构建。其粒径约为351.0 nm，表面电荷平均约为6.8 mv。该研究为开发靶向性系统型递送载体提供了新视角，且为肠癌治疗提供了一个可能的新药靶。