Intracellular Transport Process of DNA Delivered by Lipid Nanoparticles
Biotin-streptavidin-fluorescein isothiocyanate labeled nucleic acid was achieved by PCR and immunofluorescence,while LNP was labeled with 0.1%(mole percentage)ATTO-647 DOPE(SM-102∶DSPC∶Cholesterol∶PEG2000-DMG=50∶10∶38.5∶1.5,mole percentage).Firstly,investigated the physical and chemical properties and encapsulation of the LNP-DNA preparations.Secondly,in vitro Hela cell experiments,specific antibodies were used to label relevant vesicles in endocytic pathways such as endosomes/lysosomes;LNP endocytosis and its intracellular transport were investigated under inverted fluorescence microscope or high-content imaging system.In addition,the GFP expression plasmid was used as the delivery cargo to examine the efficiency of green fluorescent protein expression after transfection.The results showed that the naked DNA,after endocytosis,forms endosomal granules in the periphery of the cell and does not be further transported into the cell.Transfection time is one of the main factors affecting the transfection efficiency of LNP-DNA.LNP-DNA enters cells through endocytosis,and most of LNP-DNA is taken up by early endosomes to form LNP-DNA endosomes,which are then transported around the nucleus and partially to lysosomes,during which DNA escape is realized.
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维普
