Characterization Of Chitosan-DNA Or Modified Chitosan-DNA Nanoparticles And Their Effects To L02 Cells

Chitosan, the deacetylated derivative of chitin, is a cationic polysaccharide, which has been well defined with characteristics that include good biocompatibility and biodegradation. The cationically charged chitosan can efficiently condense negatively charged DNA to form nano scale particles via electrostatic interaction, and protect DNA from nuclease degradation. As a potential gene delivery vector, it has been drawing widely attention from scientists. The major drawback of being a gene delivery vector is the low efficiency of transfection, so the scientists have been trying to increase the efficiency of chitosan with the methods of modifications. The previous research work of our lab has demonstrated that the arginine modified chitosan and hexadecylene modified chitosan could improve the gene transfection efficiency obviously. However, the effects of these modified chitosan-DNA nanoparticles on cells have not been identified.In our research work, chitosan-DNA nanoparticles (CNP), arginine modified chitosan-DNA nanoparticles (ANP) and hexadecylene modified chitosan-DNA nanoparticles (HNP) were selected for the evaluation of their effects on L02 cells, which is a human normal liver cell line. The research work includes the following two parts.1. Endotoxin-free plasmid DNA was prepared and used to form nanoparticles with chitosan, arginine mdified chitosan, and hexadecylated chitosan, respectively. The nanoparticles were prepared using complex coacervation method and the N/P ratio was 4:1. The size and zeta potential of the gene nanoparticles were measured using Zetasizer nanoZS. Results: The zeta potential of the gene nanoparticles ranged from 12.1 to 14.63 mV, and their diameters ranged from 148.07 to 179.47 nm.2. L02 cells were incubated with three kinds of nanoparticles at the concentration of 5, 10, 30, 50μg/ml (based on the content of DNA) for 24h. The cell viability was evaluated by MTT assay, the effect of the gene nanoparticles on the cell apoptosis was analyzed by both flow cytometry and inverted fluorescent microscopy. The quantity of ROS was detected by fluoroanalyzer. The secretion of Cyt C was detected using Western-blot method. The results indicated that below the concentration of 30μg/ml, the gene nanoparticles neither showed obvious cytotoxicity to L02 cells nor induced cell apoptosis. However, at a relatively higher concentration (50μg/ml), they showed some cytotoxicity. Combined together with our previous data, our study demonstrated both arginine modified chitosan and hexadecylated chitosan may be relatively safe gene carriers, since they did not show obvious cytotoxicity to L02 cells.