叶酸-脂质体制备及对HeLa细胞靶向作用  

Preparation of Folate Liposome and Its Delivery into Cultured HeLa Cells

作　　者：陆伟跃[1] 刘敏[1] 潘俊[1] 力弘 马俊 

机构地区：[1]上海医科大学药学研究所药物靶向制剂研究室,上海200032 [2]上海医科大学药学研究所药学院药理学教研室,上海200032

出　　处：《上海医科大学学报》2000年第1期4-8,共5页Journal of Fudan University(Medical Science)

基　　金：国家自然科学基金!39670859;高等学校博士学科点专项科研基金!9506183

摘　　要：目的　研制一种能通过叶酸受体途径靶向肿瘤细胞的叶酸脂质体。方法　将叶酸（Ｆ）、聚乙二醇二胺（ＮＨ２ＰＥＧＮＨ２）、琥珀酸酐（ＳＵＣ）和卵黄磷脂酰乙醇胺（ＥＰＥ）按序共价连接，并与胆固醇（ｃｈｏｌ）、卵黄磷脂酰胆碱（ＥＰＣ）以１０∶４０∶５６配比，用成膜水化结合冷冻熔融超声法制备内含钙黄绿素叶酸脂质体，以负染色电镜法确定其粒径；用荧光显微镜观察ＨｅＬａ细胞摄取叶酸脂质体与脂质体的差异；以荧光定量法确定浓度、时间、游离Ｆ、磷脂酶Ｄ（ＰＬＤ）和磷脂酰肌醇特异磷脂酶Ｃ（ＰＩＰＬＣ）对ＨｅＬａ细胞摄取叶酸脂质体影响程度。结果　（１）ＦＰＥＧＳＵＣＥＰＥ连接物为色谱纯；叶酸脂质体平均粒径为２００ｎｍ。（２）叶酸脂质体进入ＨｅＬａ细胞质内量明显高于脂质体。（３）浓度增大、时间延长，叶酸脂质体摄取量递增幅度明显减缓；ＨｅＬａ细胞摄取叶酸脂质体量均在脂质体的４倍以上。（４）５０μｍｏｌ／Ｌ游离Ｆ可使叶酸脂质体摄取量降低５０％；ＰＩＰＬＣ（０．２Ｕ／ｍｌ）和ＰＬＤ（０．５ｍｇ／ｍｌ）处理后，叶酸脂质体摄取量分别降低７１％和７０％。Purpose To prepare folate liposomes targeting tumor cells via folate receptor mediated endocytosis. Methods Folate polyethyleneglycol succinyl egg phosphatidylethanolamine(F?PEG?SUC?EPE) was synthesized by reacting F?PEG?NH 2 (conjugate prepared by folic acid and PEG bis amine) with SUC?EPE (conjugate prepared by succinic anhydride and egg phosphatidylethanolamine).Folate liposomes composed of F?PEG?SUC?EPE/cholesterol/egg phosphatidylcholine(10∶40∶56) were prepared by rehydortion of lipid film in PBS containing calcein,and subjected to five cycles of freezing,thawing and ultrasonic treating.Negative stainelectron microscopy was utilized to examine size distribution of folate liposomes.The difference that cultured HeLa cells took up folate liposomes and liposomes was viewed by fluoresence microscopy.The extent that concentration,incubated time,free folate,phospholipase D (PLD) and phosphatidylinositol speci fic phospholipase C (PIPLC) influenced uptake of folate liposomes by HeLa cell was determined respectively using fluorescence spectrophotometer. Results (1) F?PEG?SUC?EPE was identified to a pure conjugate by thin layer chromatography.The average size of folate liposomes was 200 nm.(2) Folate liposomes entering the cytoplasm of HeLa cells was significantly more than liposomes.(3) Uptake of folate liposomes by Hela cells in 4 hours or at different concentrations were both over 4 times more than that of liposomes.Yet the increasing extent of HeLa cell associated folate liposomes was gradually declined while extending incuba ted time or increasing concentration of folate liposomes.(4) 50 μmol/L free folate may cause the reduction of HeLa cell associated folate liposomes to half its normal value.Uptake of folate liposomes was also significantly reduced by 71% and 70% after treating HeLa cells with PIPLC(0.2U/ml) and PLD(0.5 mg/ml), res pectively . Results (1) F?PEG?SUC?EPE was identified to a pure conjugate by thin layer chromatography.The average size

关 键 词：叶酸-脂质体 HELA细胞 叶酸受体 靶向作用 

分 类 号：R962[医药卫生—药理学] R944.9[医药卫生—药学]