FRET技术研究PEG-PCL胶束跨MDCK细胞单层转运的完整性  

The integrity study on PEG-PCL micelles transcellular transported across MDCK epithelial cell monolayer using FRET technology

作　　者：李瑞[1] 宋晓宁[1] 张华[1] 代文兵[1] 何冰[1] 郑颖[2] 张强[1] 王学清[1] 

机构地区：[1]北京大学药学院药剂学系,北京100191 [2]澳门大学中华医药研究院中药质量研究国家重点实验室

出　　处：《药学学报》2016年第8期1316-1324,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金面上项目(81473159);国家自然科学基金-海外及港澳学者合作研究基金(81528021)

摘　　要：本文旨在研究聚乙二醇-聚ε-己内酯[poly(ethylene glycol)-co-poly(ε-caprolactone),PEG-PCL]胶束跨犬肾极性上皮细胞(madin-darby canine kidney,MDCK)单层转运后的完整性。通过化学键合的方法将异硫氰酸荧光素(fluorescein isothiocyanate isomer I,FITC)连接于PEG-PCL上,制备荧光标记的载体材料PEG-PCLFITC,并采用荧光光谱法进行鉴定。采用薄膜水化法制备两种荧光标记的胶束:一种是同时物理包载荧光共振能量转移(F?rster resonance energy transfer,FRET)荧光对3,3'-dioctadecyloxacarbocyanine perchlorate(DiO)与1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate(Di I)的胶束(DiO-DiI-M);另一种是含有部分PEG-PCL-FITC载体材料并物理包载Di I的胶束(FITC-DiI-M)。采用动态光散射法测定胶束的粒径,荧光光谱法测定胶束在孵育介质中的稳定性。采用激光共聚焦扫描显微镜和FRET技术研究这两种荧光标记的胶束跨MDCK极性上皮细胞单层的完整性。结果表明,FITC与PEG-PCL被成功连接。DiO-DiI-M与FITC-DiI-M的粒径大小一致,均约为30 nm,多分散度良好,且两种胶束在4 h内可在孵育介质中以稳定形式存在,基本无释放。通过比较Transwell层和接收层的FRET效率和皮尔森共定位系数(Person’s coefficient)可知,1 h时,两种胶束的FRET效率与Person’s系数在Transwell层和接收层间基本不变,但4 h时,接收层FRET效率与Person’s系数明显下降,同时FITC-DiI-M下降的幅度大于DiO-DiI-M,说明1 h时胶束基本保持完整且能以完整形式从极性上皮细胞基底侧排出并被接收室中的细胞摄取,4 h时部分胶束已在与细胞单层相互作用的过程中被破坏,以完整形式跨膜的比例有所下降。The integrity of poly（ethylene glycol）-co-poly（ε-caprolactone）（PEG-PCL） micelles transcellular transported across madin-darby canine kidney（MDCK） epithelial cells was investigated. Fluorescein isothiocyanate isomer I（FITC） was conjugated to PEG-PCL and the product PEG-PCL-FITC was identified by fluorescence spectra. Two micelles were prepared using the thin-film hydration method： 3,3＇-dioctadecyloxacarbocyanine perchlorate（DiO） and 1,1＇-dioctadecyl-3,3,3＇,3＇-tetramethylindocarbocyanine perchlorate（DiI） co-loaded PEG-PCL micelles（DiO-DiI-M）, DiI loaded and PEG-PCL-FITC contained micelles（FITC-DiI-M）. The size of the micelles was characterized by dynamic light scattering analysis using a Malvern Zetasizer Nano ZS and it turned out that the particle sizes of both micelles were about 30 nm with identical polydispersity index（PDI）. The stability of the micelles in phosphate buffer saline（PBS） was monitored using fluorescence spectra and both micelles were stable within 4 h in PBS. The integrity of PEG-PCL micelles in the transcellular process across MDCK epithelial cell monolayer at 1 and 4 h was investigated using laser confocal scanning microscope and F？rster resonance energy transfer（FRET） technology. The Person＇s coefficient and FRET efficiency of both Transwell layer and Receive layer were recorded. The results show that the FRET efficiency and Person＇s coefficient of the Receive layer was consistent with that of Transwell layer for both the micelles at 1 h, but decreased at 4 h and FITC-DiI-M decreased more significantly than Di O-DiI-M. The results indicated that the micelles could transport across the MDCK monolayer intactly at 1 h but some of them were disassembled during the 4 h transportation process.

关 键 词：胶束 完整性 荧光共振能量转移 穿细胞转运 

分 类 号：R943[医药卫生—药剂学]