青藤碱PLGA-TPGS纳米粒的制备及人肝癌HepG2细胞对其摄取、被其抑制的作用研究  

Study on the Preparation of Sinomenine-loaded PLGA-TPGS Nanoparticles,Uptake by Cells and Inhibitory Effect on Human Liver Cancer HepG2 Cells

作　　者：王洪刚[1] 高萌[2] 张成鸿[3] 徐静[3] 孙艺平[3] 徐红[3] 

机构地区：[1]大连医科大学附属第一医院药剂科,辽宁大连116011 [2]大连医科大学药学院,辽宁大连116044 [3]大连医科大学基础医学院,辽宁大连116044

出　　处：《中国药房》2016年第13期1811-1814,共4页China Pharmacy

摘　　要：目的:制备青藤碱乳酸羟基乙酸共聚物-水溶性维生素E(PLGA-TPGS)纳米粒(SPTN),研究其被人肝癌HepG2细胞摄取和其对细胞的抑制作用。方法:采用乳化-溶剂挥发法制备以香豆素-6(C6)为荧光标记物的SPTN(SCPTN)和青藤碱PLGA纳米粒(SPN)(SCPN),检测其粒径、Zeta电位和载药量;通过流式细胞仪研究加入不同抑制剂(叠氮化钠、蔗糖、细胞松弛素B、染料木黄酮、甲基-β-环糊精)和空白对照对SCPTN和SCPN被HepG2细胞摄取的影响;以5-氟尿嘧啶溶液(FS)为阳性对照药,采用水溶性四氮唑(WST-1)法测定10、20、40、80、160μg/ml的SPTN、SPN、青藤碱水溶液(SS)作用于Hep G2细胞24、48、72 h的生长抑制率(IR)。结果:SCPTN和SCPN的平均粒径为(192.1±2.4)、(389.3±2.2)nm,Zeta电位为(-21.5±2.6)、(-13.7±2.3)m V,青藤碱载药量为(9.3±1.7)%、(6.1±1.8)%(n=6)。与空白对照比较,叠氮化钠、蔗糖作用后Hep G2细胞对SCPTN和SCPN的相对摄取率均降低(P<0.01),染料木黄酮作用后HepG2细胞对SCPTN的相对摄取率降低(P<0.05),其余无明显变化。与SS比较,SPTN、SPN作用后HepG2细胞的IR呈浓度、时间依赖性升高,半数抑制浓度(IC50)降低(P<0.05或P<0.01);与FS比较,仅80μg/ml SPTN作用72 h和160μg/ml SPTN作用48、72 h后HepG2细胞的IR升高(P<0.05或P<0.01),作用72 h的IC50降低(P<0.05)。结论:成功制得SPTN,其主要通过网格蛋白介导的内吞途径进入细胞,对Hep G2细胞生长具有抑制作用。OBJECTIVE：To prepare Sinomenine（SIN）-loaded polylactic-co-glycolic acid-D-α-tocopheryl polyethylene glycol1000 succinate（PLGA-TPGS）nanoparticles（SPTN）,and to study its uptake by cells and inhibitory effect on human liver cancer HepG2 cells. METHODS：Emulsification-solvent evaporation method was used to prepare SPTN with coumarin-6（C6）as fluorescent marker（SCPTN）and sinomenine PLGA nanoparticles（SPN）with C6 as fluorescent marker（SCPN）. The particle size,Zeta-potential and drug-loading amount were detected,and flow cytometry was used to study the uptake of SCPTN and SCPN by HepG2 cells after adding different inhibitors（sodium azide,sucrose,cytochalasin B,genistein,methyl-β-cyclodextrin）and blank control. Using 5-fluorouracil solution（FS）as positive control,WST-1 was used to detect inhibitory rate（IR）of HepG2 cells 24,48 and 72 h after cultured with 10,20,40,80,160 μg/ml SPTN,SPN and sinomenine solutions（SS）. RESULTS：Mean particle size of SCPTN and SCPN were（192.1 ± 2.4）and（389.3 ± 2.2）nm,Zeta potential（-21.5 ± 2.6）and（-13.7 ± 2.3）m V and drug-loading amount of sinomenine（9.3 ± 1.7）% and（6.1 ± 1.8）%（n=6）. Compared with blank control,relative uptake rate of SCPTN and SCPN by Hep G2 cells decreased after treated with sodium azide and sucrose（P〈0.01）;relative uptake rate of SCPTN by HepG2 cells decreased after treated with genistein（P〈0.05）;no other obvious change was found. Compared with SS,IR of HepG2 cells increased in concentration-dependent manner after treated with SPTN and SPN,while IC50decreased（P〈0.05 or P〈0.01）;compared with FS,IR of HepG2 cells increased only after treated with 80 μg/ml SPTN for 72 h and 160 μg/ml SPTN for48 and 72 h（P〈0.05 or P〈0.01）,and IC50 of HepG2 cells decreased after treated for 72 h（P〈0.05）. CONCLUSIONS：SPTN has been prepared successfully,and cellular uptake mechanism of it by HepG2 cells might be clathrin-mediated endocytosis. SPTN can inhibit the growth of Hep

关 键 词：青藤碱 乳酸羟基乙酸共聚物-水溶性维生素E 纳米粒 HepG2细胞 体外 摄取 生长抑制率 

分 类 号：R284.6[医药卫生—中药学] R284.2[医药卫生—中医学]