Study on Intracellular Pathway of pDNA/Chitosan Nanoparticles for Gene Delivery by Fluorescence Probe
YANG Xiao-rong，ZONG Li*，ZHU Min-yan，YUAN Xi-ying，WU Zheng-hong
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Institute of Pharmaceutics，China Pharmaceutical University，Nanjing 210009，China
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Received
Published
2007-04-14
2008-05-10
Issue Date
2008-05-10
Abstract
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.
YNG Xio-rong;ZONG Li;ZHU Min-yn;YUN Xi-ying;WU Zheng-hong.
Study on Intracellular Pathway of pDNA/Chitosan Nanoparticles for Gene Delivery by Fluorescence Probe [J]. Chinese Pharmaceutical Journal, 2008, 43(10): 727-731
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