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。
Abstract
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 high uptake rate of liposomal DOX modified by CHS-DIO-GalNAc or CHS-C8-GalNAc ligand was the active endocytosis mediated by ASGPR on the surface of liver parenchymal cell membrane. CONCLUSION The stronger the hydrophilicity of the linker in the Gal/GalNAc ligand structure is, the higher the recognition efficiency of ASGPR for Gal/GalNActarget modified on the liposomes surface becomes.At the same time, the addition of negatively charged phospholipids to liposomes can synergistically enhance the affinity of ASGPR for the liposomes modified with Gal/GalNAc ligands.The results of the study will provide beneficial and helpful guide to the design of Gal/GalNAc ligand with high affinity for ASGPR.
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
{{custom_keyword}} /
Key words
galactosylated cholesterol /
enzyme catalysis /
asialoglycoprotein receptor /
doxorubicin /
liver targeting /
anti-tumor drug /
pharmacokinetics
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] HUBBARD A L, WILSON G, ASHWELL G, et al. An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. I. Distribution of 125I-ligands among the liver cell types.[J]. J Cell Biol, 1979, 83(1):47-64.
[2] E SILVA A T M, MAIA A L C, DE OLIVEIRA SILVA J, et al. Synthesis of cholesterol-based neoglycoconjugates and their use in the preparation of liposomes for active liver targeting[J]. Carbohyd Res, 2018, 465:52-57.
[3] LUO L H, ZHENG P J, NIE H, et al. Pharmacokinetics and tissue distribution of docetaxel liposome mediated by a novel galactosylated cholesterol derivatives synthesized by lipase-catalyzed esterification in non-aqueous phase[J]. Drug Deliv, 2016, 23(4):1282-1290.
[4] UEKI A, UN K, MINO Y, et al. Synthesis and evaluation of glyco-coated liposomes as drug carriers for active targeting in drug delivery systems[J]. Carbohyd Res, 2015, 405:78-86.
[5] HU A, NIE Y I, et al. Lipase-catalyzed synthesis of novel galactosylated cholesterol[J]. Chin Chem Lett, 2015, 26(5): 543-546.
[6] PATHAK P O, NAGARSENKER M S, BARHATE C R, et al. Cholesterol anchored arabinogalactan for asialoglycoprotein receptor targeting:synthesis, characterization, and proof of concept of hepatospecific delivery[J]. Carbohydr Res, 2015, 408:33-43.
[7] ZHOU X, ZHANG M, YUNG B, et al. Lactosylated liposomes for targeted delivery of doxorubicin to hepatocellular carcinoma[J]. Int J Nanomed, 2012, 7:5465-5474.
[8] MUKTHAVARAM R, MAREPALLY S, VENKATA M Y, et al. Cationic glycolipids with cyclic and open galactose head groups for the selective targeting of genes to mouse liver[J]. Biomaterials, 2009, 30(12):2369-2384.
[9] NIE H, QIU B, YANG Q, et al. Enzymatic synthesis of three kinds of galactose-cholesterol ligands and their structure-activity relationship with liver targeting[J]. Chin Tradit Herb Drugs(中草药), 2018, 49(16):3804-3814.
[10] D'sOUZA A A, DEVARAJAN P V. Asialoglycoprotein receptor mediated hepatocyte targeting — Strategies and applications[J]. J Controlled Release, 2015, 203:126-139.
[11] SOENEN S J, BRISSON A R, JONCKHEERE E, et al. The labeling of cationic iron oxide nanoparticle-resistant hepatocellular carcinoma cells using targeted magnetoliposomes[J]. Biomaterials, 2011, 32(6):1748-1758.
[12] WANG L J, LIU Z, GUO D, et al. Study on the synthesis of vinyl pelargonate by transvinylation[J]. Thermosetting Resin(热固性树脂), 2012, 27(4):29-32.
[13] WANG S N, DENG Y H, XU H, et al. Synthesis of a novel galactosylated lipid and its application to the hepatocyte-selective targeting of liposomal doxorubicin[J]. Eur J Pharm Biopharm, 2006, 62(1):32-38.
[14] NIE H, QIU B, YANG Q, et al. Effect of gal/GalNAc regioisomerism in galactosylated liposomes on asialoglycoprotein receptor-mediated hepatocyte-selective targeting in vivo[J]. J Liposome Res, 2021, 31(1):79-89.
[15] LIU X, HAN M, XU J, et al. Asialoglycoprotein receptor-targeted liposomes loaded with a norcantharimide derivative for hepatocyte-selective targeting[J]. Int J Pharm, 2017, 520(1-2):98-110.
[16] MANAGIT C, KAWAKAMI S, YAMASHITA F, et al. Effect of galactose density on asialoglycoprotein receptor-mediated uptake of galactosylated liposomes[J]. J Pharm Sci, 2005, 94(10):2266-2275.
[17] SHIMADA K, KAMPS J A, REGTS J, et al. Biodistribution of liposomes containing synthetic galactose-terminated diacylglyceryl-poly(ethyleneglycol)s[J]. Biochim Biophys Acta, 1997, 1326(2):329-341.
[18] KEPCZYNSKI M, NAWALANY K, KUMOREK M, et al. Which physical and structural factors of liposome carriers control their drug-loading efficiency?[J]. Chem Phys Lipids, 2008, 155(1):7-15.
[19] NIE H, ZHENG P J, LUO L H, et al. Optimization of lipase-catalyzed synthesis of novel galactosyl ligands for selective targeting of liposomes to the asialoglycoprotein receptor[J]. Biocatal Biotransform, 2015, 33(2):130-139.
[20] CHOPINEAU J, MCCAFFERTY F D, THERISOD M, et al. Production of biosurfactants from sugar alcohols and vegetable oils catalyzed by lipases in a nonaqueous medium[J]. Biotechnol Bioeng, 1988, 31(3):208-214.
[21] MIURA Y, IKEDA T, KOBAYASHI K. Chemoenzymatically synthesized glycoconjugate polymers[J]. Biomacromolecules, 2003, 4(2):410-415.
[22] WU J, ZERN M A.Modification of liposomes for liver targeting.[J]. J Hepatol, 1996, 24(6):757.
[23] MAYER L D, TAI L C, KO D S, et al. Influence of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice[J]. Cancer Res, 1989, 49(21):5922-5930.
[24] RENSEN P C, SLIEDREGT L A, FERNS M, et al. Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo[J]. J Biol Chem, 2001, 276(40):37577-37584.
[25] MEIER M, BIDER M D, MALASHKEVICH V N, et al. Crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein receptor[J]. J Mol Biol, 2000, 300(4):857-865.
[26] KINBERGER G A, PRAKASH T P, YU J, et al. Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes[J]. Bioorg Med Chem Lett, 2016, 26(15):3690-3693.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
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)
{{custom_fund}}
PDF(2620 KB)
