两种叶酸偶联物的合成及体外靶向性研究

Synthesis of two folate conjugates and their targeting effect in vitro

合成叶酸偶联物并将其应用于叶酸靶向脂质体制备,考察其在体外肝癌HepG2细胞中的靶向性。通过酰胺反应将叶酸、胆固醇琥珀酸单酯与两种不同相对分子质量的聚氧乙烯二胺材料连接,合成两种两亲性的叶酸-聚乙二醇-胆固醇琥珀酸单酯(Folate-PEG_(2000)-CHEMS和Folate-PEG_(4000)-CHEMS),并利用核磁共振氢谱(^(1)H NMR)和超高分辨率复杂体分离鉴定质谱系统对其进行了结构表征。选取钙黄绿素为模型药物,采用薄膜分散法分别利用Folate-PEG_(2000)-CHEMS和Folate-PEG_(4000)-CHEMS制备了钙黄绿素脂质体FA-PEG_(2000)-L与FA-PEG_(4000)-L。利用激光粒度仪检测FA-PEG_(2000)-L与FA-PEG_(4000)-L的粒径、Zeta电位;利用流式细胞仪和激光共聚焦扫描显微镜,考察了脂质体FA-PEG_(2000)-L与FA-PEG_(4000)-L在HepG2细胞体外摄取实验中的药物递送效果。结果显示,钙黄绿素脂质体的平均粒径为(205.8±10.2)nm,经电位测试脂质体的Zeta电位为-(1.19±0.31)mV。FA-PEG_(4000)-L靶向脂质体的荧光强度分别约为普通脂质体、FA-PEG_(2000)-L的3.6和3.1倍(P<0.01),FA-PEG_(4000)-L在HepG2细胞中的递送效率明显高于FA-PEG_(2000)-L与非靶向组,说明Folate-PEG_(4000)-CHEMS可应用于脂质体制备,并可促进体外HepG2细胞对脂质体的摄取。

The aim of this study was to study the synthesis of two folate conjugates and their application in the preparation of folate targeted liposome,and to investigate their targeting effect in hepatocellular carcinoma HepG2 cell line in vitro.In this study,Folate-PEG-Cholesteryl hemisuccinate(Folate-PEG_(2000)-CHEMS and Folate-PEG_(4000)-CHEMS)were synthesized by linking folate and cholesterol succinate with two kinds of PEG materials.Structures of Folate-PEG_(2000)-CHEMS and Folate-PEG_(4000)-CHEMS were characterized by ^(1)H NMR and ultra-high resolution mass spectrometry.Calcein was selected as the model drug,and calcein liposomes FA-PEG_(2000)-L and FA-PEG_(4000)-L were prepared by film dispersion method using Folate-PEG_(2000)-CHEMS and Folate-PEG_(4000)-CHEMS,respectively.The particle size and Zeta potential of FA-PEG_(2000)-L and FA-PEG_(4000)-L were measured by laser particle size analyzer.The drug delivery effect of FA-PEG_(2000)-L and FA-PEG_(4000)-L was evaluated by cellular uptake experiment in HepG2 cell line in vitro.Flow cytometry and laser confocal scanning microscope were used to determine fluorescence in HepG2 cells in vitro.The results showed that the average particle size of calcein liposome was(205.8±10.2)nm,and the Zeta potential of calcein liposome was-(1.19±0.31)mV.There was no significant difference in particle size and Zeta potential between FA-PEG_(2000)-L and FA-PEG_(4000)-L.The fluorescence intensity of FA-PEG_(4000)-L liposome group was about 3.6 and 3.1 times higher than that of non-targeted liposome group and FA-PEG_(2000)-L liposome group,with statistically significant difference(P<0.01).The drug delivery efficiency of FA-PEG_(4000)-L group in HepG2 cells was higher than that in FA-PEG_(2000)-L and non-targeted groups,and the results indicated that Folate-PEG_(4000)-CHEMS can promote the uptake of liposome by HepG2 cells in vitro.All in all,Folate-PEG_(4000)-CHEMS could be applied in the preparation of folate targeted liposome,which could promote the uptake of liposome by HepG2 cells.