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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 purpose of this study was to investigate the effects of poly (ethylene glycol) -co-poly (ε-caprolactone) and PEG-PCL micelles on canine canine kidney epithelial cells , MDCK) monolayer transport integrity. Fluorescein isothiocyanate isomer I (FITC) was attached to PEG-PCL by chemical bonding, and fluorescence-labeled carrier material PEG-PCLFITC was prepared and identified by fluorescence spectroscopy. Two kinds of fluorescence-labeled micelles were prepared by using the membrane hydration method. One was the simultaneous fluorescence analysis of 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO) with 1, Tetramethylindocarbocyanine perchlorate (DiI) micelles (DiO-DiI-M); the other is containing part of the PEG-PCL-FITC carrier material and physically encapsulated Di I micelles (FITC-DiI-M). The particle size of micelles was determined by dynamic light scattering and the stability of micelles in the incubation medium was measured by fluorescence spectroscopy. The integrity of the two fluorescently labeled micelles across the monolayer of MDCK polar epithelial cells was investigated using laser confocal scanning microscopy and FRET techniques. The results showed that FITC and PEG-PCL were successfully linked. The sizes of DiO-DiI-M and FITC-DiI-M were about 30 nm with good polydispersity, and the two micelles could exist in a stable form in the incubation medium within 4 h with almost no release . By comparing the FRET efficiency and Pearson’s co-location coefficient of Transwell layer and receiving layer, we can see that the FRET efficiency and Person’s coefficient of the two micelles are basically unchanged between Transwell layer and receiving layer at 1 h, but the 4 h , The FRET efficiency and Person’s coefficient of the receiving layer decreased significantly, while FITC-DiI-M decreased more than DiO-DiI-M, indicating that the micelles remained intact and could be completely excreted from the basal side of the polar epithelial cells And was taken up by the cells in the receiving compartment. At 4 h, some of the micelles were destroyed during the interaction with the cell monolayer, and the proportion of intact form of transmembrane decreased.