酶促合成3种半乳糖-胆固醇配体及其与肝靶向性的构效关系研究

目的酶促法构建3种不同类型的半乳糖-胆固醇配体修饰的阿霉素(doxorubicin,DOX)脂质体,研究其在小鼠体内的药动学及组织分布特征。方法利用脂肪酶在非水相中合成了3种不同结构的半乳糖-胆固醇配体:CHS-C8-Gal NAc、CHS-C8-GAL、CHS-C8-LA;利用MS、NMR鉴定产物结构;采用薄膜分散-梯度载药法制备DOX脂质体并对其理化性质进行表征;以小鼠为实验对象,采用尾iv给药,通过比较3种配体修饰的DOX脂质体在小鼠体内的药动学与组织分布特征来阐明配体中半乳糖基的结构与去唾液酸糖蛋白受体之间的构效关系。结果产物经MS、NMR鉴定均为目标产物,产率均90%;采用薄膜分散-梯度载药法制备的DOX脂质体粒径90 nm,PDI0.1,包封率99%,24 h渗漏率5%,Zeta电位接近于0;3种配体分子对肝脏亲和力大小依次为CHS-C8-Gal NAcCHS-C8-LACHS-C8-Gal;肝脏对CHS-C8-Gal NAc修饰的脂质体的摄取几乎完全被预注射的asialofetuin所抑制(P0.01),而对CHS-C8-Gal及CHS-C8-LA修饰的脂质体无显著的抑制效果(P0.05)。结论含有N-乙酰半乳糖胺基(N-acetylgalactosamine,Gal NAc)为末端的配体分子能被去唾液酸糖蛋白受体高效识别,而含有D-半乳糖基(D-galactose,Gal)或乳糖醇基(lactitol,LA)为末端的配体分子易被肝枯否细胞上的半乳糖粒子受体所竞争性结合。

Enzymatic synthesis of three kinds of galactose-cholesterol ligands and their structure-activity relationship with liver targeting

Objective To construct three kinds of doxorubicin liposomes modified with cholesterol-galactose ligand by lipase-catalyzed method and compare their characteristic of pharmacokinetics and tissue distribution in vivo. Methods Three types of cholesterol-galactose ligands, CHS-C8-GalNAc, CHS-C8-GAL, and CHS-C8-LA were synthetized by lipase-catalyzed method in nonaqueous phase. The structure characterizations of products were obtained by MS and NMR. Conventional liposomes (CL DOX) and ligand-coupled liposomes (NGal-LP DOX, Gal-LP DOX, and LA-LP DOX) were prepared by thin film dispersion-ammonium sulphate gradient method. Structure-activity relationship between asialoglycoprotein receptor (ASGPr) and the chemical structure of the glycolipids was explored through the pharmacokinetics and tissue distribution parameters of ligand-coupled liposomes in vivo. Results The desired compounds with a high yield of above 90% were confirmed by MS and NMR. The liposomes average size was lower than 90 nm, polymer dispersity index was lower than 0.1, encapsulation efficiency was greater than 99%, leakage rat was lower than 5% with 24 h, and zeta potential closed to zero. The affinity of the three ligand molecules to liver was the following order:CHS-C8-GalNAc > CHS-C8-LA > CHS-C8-Gal. However, only the liposomes modified with CHS-C8-GalNAc could significantly be inhibited by the preinjection of asialofetuin for hepatic uptake rate (P < 0.01), but the liposomes modified with CHS-C8-LA and CHS-C8-Gal could not be inhibited by the preinjection of asialofetuin for hepatic uptake rate (P > 0.05). Conclusion The ligand with N-acetylgalactosamine residue showed high targeting efficiency for hepatocytes, while the ligand with D-galactose (Gal) or lactitol residue could competitive bind with Gal particle receptor on kupffer cells.