用于动脉粥样硬化治疗的microRNA肝靶向纳米递送系统研究

IMB-185 is a safer and more efficient drugs against atherosclerosis, with independent intellectual property rights and novel mechanism in blood lipid regulating, which could meet the urgent need of atherosclerosis treatment. Lack of safe and effective targeting delivery system in vivo has severely hindered wide application of nucleic acid drugs, such as IMB-185. This project intends to formulate the hepatocyte targeting non-positive liposome encapsulating IMB-185, introducing N-acetyl-D-amino galactose (GalNac) and pH low insertion peptide (pHLIP)..The passive targeting of liposomes and GalNac, decorating the surface of liposomes, mediating active endocytosis improve the uptake of IMB-185, collectively. Depending on the property of forming α-helix in acidic conditions that could insert into lipid film, the conjugation of pHLIP and IMB-185 is embedded in liposomes with the acid internal core. Adding hyaluronic acid and DOPE conjugation (DOPE-HA), which protects pHLIP from dissociating from liposomes under physiological condition by forming the protection layer. In lysosomes, pHLIP continues to pave the way for IMB-185 entering into cytoplasm by inserting film in lysosomes. Finally, the plenty of reduced glutathione (GSH) in hepatocyte reducing disulfide bond, and carriers remain in lysosomes for degradation and removal. Different from pore forming agent and penetration enhancer, PHLIP is depending on the inserting membrane by α-helical structure, which has little effect on the stability of the lipid membrane. In conclusion, liposomes encapsulating IMB-185 is safe and effective, and harbors promising potential of clinical transformation.

反义核酸药物IMB-185是新型调血脂药，抗动脉粥样硬化效果显著，具有自主知识产权。IMB-185作用位点在肝实质细胞，但核酸药物的肝靶向递送是制约其应用的难题。拟构建肝靶向非阳离子脂质体，用于IMB-185的肝靶向递送。脂质体对肝的被动靶向和N-乙酰基-D-氨基半乳糖GalNac介导的高效内吞，协同提高IMB-185的细胞摄取。引入pH敏感肽pHLIP实现核酸药物的脂双层包载和溶酶体逃逸。pHLIP在酸性环境中变成α-螺旋结构，插入脂膜，将pHLIP与核酸偶联物镶嵌在脂双层中。掺入透明质酸与DOPE偶联物DOPE-HA，形成表面保护层，避免pHLIP在生理条件下解离。溶酶体中，HA水解，pHLIP继续发挥螺旋插膜作用，将IMB-185递送到细胞质。构建IMB-185脂质体递送肝靶向核酸药物治疗动脉粥样硬化，临床转化潜力较大。

核酸药物的体内递送至今仍面临挑战，分子水平上核酸易被核酸酶降解失活；细胞水平上其分子量大且带有大量负电荷，细胞渗透性差，入胞后很难从溶酶体中逃逸到细胞浆中，因此需要借助纳米递送系统来提高胞内递送效率。本项目拟构建非阳离子脂质体，实现核酸药物的安全、有效递送。磷酸钙具有良好生物相容性和生物可降解性，是基因转染的天然载体，并且磷酸钙在溶酶体等酸性环境中可以迅速溶解，引起溶酶体内渗透压增加，溶酶体涨破，将核酸释放到细胞质中，实现核酸的细胞质递送。载脂蛋白A-1（apolipoprotein A-1，ApoA1）在高密度脂蛋白与肝细胞表面清道夫受体B1受体的结合的过程中起到关键作用，因此希望借助ApoA1实现脂质体的肝靶向。. 因此本项目利用核酸、磷酸基团与钙离子之间的相互作用，借助磷酸钙内核固缩核酸药物IMB185，并通过核酸固缩能力的评价，对处方中磷酸根离子和钙离子浓度进行优化。采用薄膜分散法包裹磷酸钙/ IMB185内核，在脂质体表面修饰载脂蛋白ApoA1构建肝靶向IMB185脂质体ApoA1-LP/IMB185，实现了IMB185的体内外有效递送。经过处方筛选与工艺优化，获得了粒径均匀（150 nm），表面电荷为负（-30 mV）的肝靶向脂质体。体外摄取和转染结果表明，与无靶向修饰的被动脂质体（LP/IMB185）相比，主动靶向脂质体（ApoA1-LP/IMB185）在HepG2细胞上的摄取显著增多，在HepG2-miR185-Luc细胞模型上的转染效率显著提高，并且脂质体处方对细胞摄取和转染效率有显著影响。活体成像结果显示，ApoA1-LP/IMB185在小鼠体内有肝靶向效果。本项目的成功实施，不仅能实现IMB185的高效递送，而且将大大降低由阳离子材料引起的毒性，提高核酸递送系统的安全性，对推进微小核酸药物的研发起到积极作用。

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