CAJ | 학술논문

目的：叶酸受体在某些肿瘤细胞表面的高表达给靶向治疗提供了理论依据。文中制备肿瘤靶向性自组装纳米药物载体，探讨其体外细胞抑瘤效应及其细胞摄取途径，评价作为靶向纳米药物载体的可行性。方法透析法制备包载表柔比星的乙酰普鲁兰叶酸偶合体（fola te con juga ted PA， FPA）纳米粒（FPA／EPI NP），MTS法考察该抗肿瘤药物载体对于肝癌细胞（HepG2，叶酸受体阴性细胞株）和宫颈癌细胞（Hela，叶酸受体高表达细胞株）的抑瘤效应。细胞实验分为纳米粒对照组、氯丙嗪组、氯喹组、阿米洛利组和叶酸组。对照组不经各种抑制剂预处理，直接加入FPA／EPI NP悬液，37℃孵育2 h，用流式细胞分析仪检测荧光强度。结果纳米粒呈球形，FPA NP粒径为（204．2±10．9） nm，FPA／EPI NP粒径（273．4±11．0） nm，FPA／EPI NP载药量和包封率分别为（6．45±1．04）％和（72．45±11．50）％。当HepG2和Hela细胞分别与5、40、200、400和1000μg／mL的FPA NP孵育24 h时，细胞存活率均＞95％；孵育72 h时细胞存活率高达90．0％。 FPA／EPI NP作用HepG2细胞24 h时，存活率分别是（92．3±5．2）％、（70．4±4．6）％、（54．0±4．0）％、（41．1±4．1）％和（27．0±3．6）％。与纳米粒对照组相比，用氯丙嗪、阿米洛利、叶酸分别预处理Hela细胞，纳米粒的摄取量均减少（ P＜0．05）；用氯丙嗪、阿米洛利分别预处理HepG2细胞，纳米粒的摄取量均下降（ P＜0．05）。 FPA／EPI NP作用HepG2和Hela细胞72 h时半数抑瘤浓度分别为168μg／mL和105μg／mL。结论对于HepG2细胞，FPA／EPI NP主要通过网格蛋白介导的内吞以及巨胞饮途径进入细胞，对于Hela细胞，主要通过网格蛋白介导的内吞以及叶酸受体介导的途径进入细胞。 FPA NP有望成为一种新型的肿瘤靶向药物载体。
목적：협산수체재모사종류세포표면적고표체급파향치료제공료이론의거。문중제비종류파향성자조장납미약물재체，탐토기체외세포억류효응급기세포섭취도경，평개작위파향납미약물재체적가행성。방법투석법제비포재표유비성적을선보로란협산우합체（fola te con juga ted PA， FPA）납미립（FPA／EPI NP），MTS법고찰해항종류약물재체대우간암세포（HepG2，협산수체음성세포주）화궁경암세포（Hela，협산수체고표체세포주）적억류효응。세포실험분위납미립대조조、록병진조、록규조、아미락리조화협산조。대조조불경각충억제제예처리，직접가입FPA／EPI NP현액，37℃부육2 h，용류식세포분석의검측형광강도。결과납미립정구형，FPA NP립경위（204．2±10．9） nm，FPA／EPI NP립경（273．4±11．0） nm，FPA／EPI NP재약량화포봉솔분별위（6．45±1．04）％화（72．45±11．50）％。당HepG2화Hela세포분별여5、40、200、400화1000μg／mL적FPA NP부육24 h시，세포존활솔균＞95％；부육72 h시세포존활솔고체90．0％。 FPA／EPI NP작용HepG2세포24 h시，존활솔분별시（92．3±5．2）％、（70．4±4．6）％、（54．0±4．0）％、（41．1±4．1）％화（27．0±3．6）％。여납미립대조조상비，용록병진、아미락리、협산분별예처리Hela세포，납미립적섭취량균감소（ P＜0．05）；용록병진、아미락리분별예처리HepG2세포，납미립적섭취량균하강（ P＜0．05）。 FPA／EPI NP작용HepG2화Hela세포72 h시반수억류농도분별위168μg／mL화105μg／mL。결론대우HepG2세포，FPA／EPI NP주요통과망격단백개도적내탄이급거포음도경진입세포，대우Hela세포，주요통과망격단백개도적내탄이급협산수체개도적도경진입세포。 FPA NP유망성위일충신형적종류파향약물재체。
Objective Folate receptors ( FRs) , overexpressed on the surface of a variety of tumor cells , are potential targets for tumor targeting therapy .This study aimed to prepare an FR-mediated drug nanocarrier with folate conjugated pullulan acetate ( FPA) to target chemotherapeutic agents to FR-overexpressed tumor cells and investigate its tumor-suppressing effect in vitro. Methods The cytotoxicity of epirubicin-loaded FPA nanoparticles ( FPA/EPI NP) against HepG2 and Hela cells was evaluated by MTS assay.The HepG2 and Hela cells were divided into five groups to be treated with NPs (NP control), chlorpromazine, chloro-quine, amiloride, and folate, respectively, followed by detection of the fluorescence intensity by flow cytometry . Results FPA/EPI NP was successfully formulated into NPs , with the mean particle size of (268.5 ±12.0) nm, by dialysis with an almost spherical shape . The drug-loading rate and entrapment rate of FPA/EPI NP were (6.45 ±1.04) and (72.45 ±11.50) %, respectively.The survival rates of the HepG2 and Hela cells were both >95%after 24 hours of incubation with FPA NP at 5, 40, 200, 400 and 1000μg/mL and 90.0%after 72 hours.The survival rates of the HepG2 cells treated with 5, 40, 200, 400 and 1000μg/mL FPA/EPI NP for 24 hours were (92.3 ±5.2), (70.4 ±4.6), (50.0 ±4.0), (41.1 ±4.1) and (27.0 ±3.6) %, respectively.Compared with the NP control group, the Hela cells of the chlorpromazine , amiloride and folate groups showed a significantly lower rate of NP uptake (P<0.05), and so did the HepG2 cells pretreated with chlorpromazine or amilo-ride (P<0.05).At 72 hours, the half maximal inhibitory concentrations (IC50) of FPA/EPI NP against HepG2 and Hela cells were 168 and 105μg/mL, respectively . Conclusion Clathrin-mediated endocytosis and macropinocytosis are involved in the internaliza-tion of FPA/EPI NP in HepG2 cells, while clathrin-and FR-mediated endocytosis in that of Hela cells .FPA NP may serve as a new drug carrier for tumor-targeted therapy .