基于ASGPR受体与GA结合位点的肝靶向斑蝥素双靶点脂质体的制备及抗肝肿瘤作用的研究

Liver cancer is a disease of serious damage to human health, and due to the limitations of current surgical treatment and the serious toxic and side effects of antitumor drugs, the research and development of new type of liver cancer targeting preparation become inevitable. This project will select cantharidin as a model drug,which is one of active ingredients of Chinese medicine cantharis and has exact curative effect on primary liver cancer but the same time large toxic and side effects on human body, designing to jointly modify liposome surface with galactose ligand and 11-deoxyglycyrrhetinic acid ligand based on the ASGPR receptors and glycyrrhetinic acid (GA) binding sites on the liver cell membrane, and preparing the double ligands modified "double targets liposome" of cantharidin available for acting on two targets and with stronger liver targeting, and then exploring and illuminating the feasibility of above double targets liposome as a new type of liver targeting drug delivery carrier for developing new liver targeting preparations with high efficient and low toxicity through the research of the physical and chemical properties, the rat in vivo liver targeting and the effectiveness and possible mechanism of in vivo and in vitro anti liver tumors on it, and in comparison with the general liposome and different single ligand modified liposomes of cantharidin. It has an important practical significance on solving the clinical problems of the enhancing effect and reducing toxicity of anti liver cancer drugs, and promoting the modernization of traditional Chinese medicine preparation, meanwhile it will also provide experimental basis for the secondary development of cantharidin preparations.

肝癌严重危害着人类健康，由于目前手术治疗的局限及抗肿瘤药物的严重毒副作用，研发新型抗肝癌靶向制剂成为必然。本项目选择对原发性肝癌具有确切疗效但毒副作用较大的中药斑蝥有效成分——斑蝥素作为模型药物，基于肝细胞膜上的ASGPR受体与甘草次酸（GA）结合位点，设计用半乳糖配基与11-脱氧甘草次酸配基共同对脂质体表面进行修饰，制备可作用于二个靶点、肝靶向性更强的双配基修饰的斑蝥素“双靶点脂质体”，通过对其理化性质、大鼠体内的肝靶向性及体内外抗肝肿瘤的有效性与可能机制的研究，并与斑蝥素普通质脂体及不同单配基修饰的斑蝥素脂质体的对比研究，探索并阐明其作为新型肝靶向给药载体用于开发高效低毒的抗肝癌靶向新制剂的可行性。这对于解决目前临床上抗肝癌药物的“增效减毒”问题及促进中药制剂的现代化具有重要的现实意义，同时也将为斑蝥素制剂的二次开发提供实验依据。

本研究根据脂质体的结构及肝细胞膜表面的ASGPR受体和GA受体识别特点，设计了新型的导向分子18-GA-Suc、11-DGA-Suc、SA-Gal及11-DGA-3-O-Gal，制备了导向分子修饰的CTD脂质体18-GA-Suc-CTD-lip、11-DGA-Suc-CTD-lip, SA-Gal-CTD-lip, 11-DGA-3-O-Gal-CTD-lip 和 (11-DGA-Suc+SA-Gal)-CTD-lip，采用细胞增殖抑制、细胞迁移、周期分析和凋亡试验，对比评价各CTD脂质体的抗肿瘤作用。结果表明，经导向分子修饰后，各CTD脂质体均对HepG2细胞增殖和迁移有较强的抑制作用并能促进HepG2细胞的凋亡。细胞增殖抑制实验中，各脂质体的抑制效果排序为：11-DGA-Suc-CTD-lip＞SA-Gal-CTD-lip＞18-GA-Suc-CTD-lip＞(11-DGA-Suc+SA-Gal)-CTD-lip＞11-DGA-3-O-Gal-CTD-lip。各CTD脂质体细胞周期分析显示：在IC50作用浓度下，CTD-lip及11-DGA-3-O-Gal-CTD-lip阻滞细胞周期于G2期，而其余四种修饰脂质体阻滞细胞周期于G1期。体内抗肿瘤试验表明，在60μg/mL剂量下，18-GA-Suc-CTD-lip的抑制肿瘤生长效果强于CTD-lip和CTD。在大鼠药动学与组织分布试验中，11-DGA-Suc-CTD-lip在肝脏的Re值（1.89）、Ce值（3.39）最大，提示11-DGA-Suc-CTD-lip肝靶向性最好。基于上述试验结果，11-DGA-Suc-CTD-lip作为有毒中药的肝靶向性载体，有潜能达到增效减毒的目的。本研究为治疗肝脏疾病及肝肿瘤新型靶向纳米制剂的研究奠定了基础。