荧光探针示踪pDNA/壳聚糖纳米粒在细胞内转运

 目的采用荧光探针追踪质粒DNA(pDNA)/壳聚糖(CS)纳米粒在细胞内转运轨迹,分析CS介导的细胞转染屏障。方法绿色荧光蛋白质粒基因作为报告基因进行体外转染实验,用异硫氰酸荧光素分别标记pDNA和CS,荧光显微镜下观察细胞对pDNA/CS纳米粒的吸附,流式细胞技术测定细胞对pDNA/CS纳米粒的摄取率,激光扫描共聚焦显微镜下观察pDNA/CS纳米粒在细胞内转运和入核过程。结果pDNA/CS纳米粒以小聚集体形式吸附在细胞表面,细胞吸附受介质pH值的影响。流式细胞仪定量分析表明,细胞对pDNA/CS纳米粒有高摄取率,共温孵4h,细胞摄取率为88.8%。激光扫描共聚焦显微图像显示,pDNA/CS纳米粒依赖细胞内吞功能经细胞(内外)转运通道进入细胞。pDNA/CS纳米粒与细胞共温孵3h,CS主要分布在胞浆中,pDNA不仅分布在胞浆中,核内也清楚可见荧光标记的pDNA。结论荷正电的pDNA/CS纳米粒以小聚集体形式被细胞吸附,通过细胞胞吞作用进入细胞。pDNA/CS纳米粒主要在胞浆中解离,pDNA入核。实验提示,增加pDNA/CS纳米粒在胞浆中解离将有助于提高CS介导的转染效率。

Study on Intracellular Pathway of pDNA/Chitosan Nanoparticles for Gene Delivery by Fluorescence Probe

OBJECTIVE To track the intracellular pathway of pDNA/chitosan nanoparticles by fluorescence probe to analysis of the obstacles for the gene delivery mediated by chitosan.METHODS The plasmid DNA(pDNA) and chitosan were labeled with fluorescein isothiocyanate respectively.The cell adhesion of pDNA/chitosan nanoparticles was observed using a fluorescence microscope,the cell uptake of nanoparticles were analyzed with a flow cytometer,and the intracellular pathway and nucleus localization of the nanoparticles were visualized with a confocal laser scanning microscope.RESULTS The pDNA/chitosan nanoparticles were found to adhere to the cell surface as small aggregates was affected by the pH of medium.The cell uptake of the nanoparticles were obtained as high as 88.8% after 4 h of incubation with HeLa cells using flow cytometric analysis.The confocal laser scanning microscope images showed that the pDNA/chitosan nanoparticles were transcelluarly transported into cells via endocytosis.When the cells were incubated with nanoparticles for 3 h,the chitosan were detected predominantly throughout the cytoplasm,while the pDNA not only appeared in the cytoplasm but accumulated within the nucleus.CONCLUSION The positively-charged pDNA/chitosan nanoparticles adhered to the cell surface as small aggregates,and subsequently entered the cells by endocytosis.The dissociation of pDNA from pDNA/chitosan nanoparticles occured in the cytoplasm prior to nuclear entry of pDNA.The results suggested that the increase of intracellular dissociation of pDNA/chitosan nanoparticles seem to be favorable for enhancement of the chitosan-mediated transfection efficiency.