Gal/GalNAc配体中连接臂的亲水/疏水性对其修饰脂质体肝靶向性的影响  

Effect of Hydrophilicity/Hydrophobicity of Linker in Gal/GalNAc-containing Ligands on Liver Targeting of Its Modified Liposomes

作　　者：聂华[1] 刘宝雯 赵莹[1] 叶小玲[1] 何泽和 袁思颖 刘小敏 NIE Hua;LIU Bao-wen;ZHAO Ying;YE Xiao-ling;HE Ze-he;YUAN Si-ying;LIU Xiao-min(Medical College of Jiaying University,Meizhou 514031,China;Institute of Hakka Medicinal Biological Resources,Medical College of Jiaying University,Meizhou 514031,China)

机构地区：[1]嘉应学院医学院,广东梅州514031 [2]嘉应学院医学院,客家药用生物资源研究所,广东梅州514031

出　　处：《中国药学杂志》2023年第1期65-76,共12页Chinese Pharmaceutical Journal

基　　金：广东省自然科学基金-面上项目资助(2022A1515011529);梅州市应用型科技专项资金项目资助(2020B0205003);广东省普通高校特色创新项目资助(2020KTSCX137);广东省医学科研基金项目资助(A2020372);2020年大学生创新创业训练计划国家级项目资助(202010582019);2020年“攀登计划”广东大学生科技创新培育专项资金资助(pdjh2020b0554);2021年“攀登计划”广东大学生科技创新培育专项资金资助(pdjh2021b0469)。

摘　　要：目的 本实验研究Gal/GalNAc-胆固醇(cholesterol, CHS)配体中连接臂的亲水/疏水性及其修饰的脂质体表面荷电性对去唾液酸糖蛋白受体(asialoglycoprotein receptor, ASGPR)亲和力的影响,为构建高效的Gal/GalNAc配体修饰的靶向肝癌细胞递药载体提供理论指导和实验依据。方法 采用酶促法合成具有疏水性连接臂的Gal/GalNAc-CHS配体:CHS-C8-GalNAc、CHS-C8-Gal、CHS-C8-Lac和具有亲水性连接臂的Gal/GalNAc-CHS配体:CHS-DIO-GalNAc,其中CHS-DIO-GalNAc为首次报道。将hydrogenated soya phosphatide(HSPC)、CHS和各种配体按(6∶3∶1)摩尔比例混合(如制备阴离子脂质体则加入脂质总质量1%的DSPG-Na),然后采用薄膜分散-高压挤出-硫酸铵梯度法制备配体修饰载多柔比星脂质体。以小鼠为实验对象,采用尾iv给药,对比4种Gal/GalNAc配体修饰的多柔比星脂质体在小鼠体内血液和肝组织分布特征。结果 CHS-DIO-GalNAc经MS、NMR和HMBC鉴定为目标产物,产率>90%;制得脂质体外观圆整,边缘清晰,粒径介于60~75 nm, Zeta电位介于-6~+5 mV(阴离子脂质体Zeta电位介于-4~-17 mV),polydispersity index<0.1,包封率> 98%,泄露率<3%。小鼠体内实验结果显示CHS-DIO-GalNAc修饰的脂质体在血液中的消除速率、肝脏组织的蓄积率均显著高于由CHS-C8-GalNAc、CHS-C8-Gal和CHS-C8-Lac修饰的脂质体。通过体内ASGPR竞争性抑制实验,证明小鼠体内肝脏对CHS-DIO-GalNAc和CHS-C8-GalNAc配体修饰脂质体的高摄取率是由肝实质细胞膜表面ASGPR介导的主动内吞作用。结论 Gal/GalNAc配体结构中连接臂的亲水性越强,ASGPR对脂质体表面修饰的Gal/GalNAc的识别效率越高。脂质体中加入负电荷磷脂可协同增强ASGPR对脂质体表面修饰的Gal/GalNAc配体亲和力。本研究成果将为设计ASGPR高亲和力的Gal/GalNAc配体提供有益的指导。OBJECTIVE To investigate the effect of the hydrophilic/hydrophobicity of linker in the Gal/GalNAc-cholesterol(CHS)ligand and its modified liposome surface charge on the affinity of asialoglycoprotein receptor(ASGPR), so as to provide theoretical guidance and experimental basis for construction of high-efficiency Gal/GalNAc ligand-modified drug delivery vector for targeting liver cancer cells. METHODS The enzymatic method was adopted to synthesize Gal/GalNAc-CHS ligands with hydrophobic linker: CHS-C8-GalNAc, CHS-C8-Gal, CHS-C8-Lac, and Gal/GalNAc-CHS ligand with hydrophilic linker: CHS-DIO-GalNAc.Among them, CHS-DIO-GalNAc was reported for the first time. Hydrogenated soya phosphatide, cholesterol and various ligands were mixed in a molar ratio(6∶3∶1), and 1% distearoyl phosphatidylglycerole sodium salt(DSPG-Na) was added to the total lipid mass if anionic liposomes were prepared. Then, ligand-modified liposomal doxorubicin(DOX) was prepared by thin film dispersion-high pressure extrusion-ammonium sulfate gradient method. With mice as the experimental subjects, the liposomal DOX was administered by tail vein, and the distribution characteristics of four kinds of Gal/GalNAc ligand-modified liposomal DOX in blood and liver tissues of mice were compared. RESULTS CHS-DIO-GalNAc was identified as the target product by MS, NMR and HMBC.In addition, the yield was > 90%. The obtained liposomes had rounded appearance, and clear edges, with the particle sizes ranging from 60 nm to 75 nm, and Zeta potential ranging from-6 mv to +5 mV(-4 mV to-17 mV for anionic liposomes added with DSPG-Na), polydispersity index<0.1, encapsulation rate > 98% and leak rate<3%.According to the results of biological distributions experiments in mice, the elimination rate in blood and the accumulation rate in liver tissue of liposomal DOX modified by CHS-DIO-GalNAc were significantly higher than those modified by CHS-C8-GalNAc, CHS-C8-Gal and CHS-C8-Lac.Moreover, the competitive inhibition experiment of ASGPR in vivo, proved that the liver h

关 键 词：半乳糖-胆固醇缀合物 酶催化 去唾液酸糖蛋白受体 多柔比星 肝靶向 抗肿瘤药物 药动学 

分 类 号：R944[医药卫生—药剂学] R965[医药卫生—药学]