| Chitosan and its derivatives have good biocompatibility, biodegradability, and good combination capability of DNA, which are widely concerned in the field of gene vector. But the poor solubility of chitosan and its low gene transfection efficiency restricts its further development and application greatly. How to improve the efficiency of chitosan as gene vector has always been a challenge that urgently to be solved by the researchers. This study synthesized arginine modified chitosan (CS-Arg) and ploy-arginine modified chitosan (CS-5Arg), and then combined them with plasmids separately to form complex nanoparticles. The physicochemical and biological property of CS-Arg and CS-5Arg was analyzed, and their gene transfection efficiency was also studied.Firstly, chitosan was used as raw material and reacted with arginine and pentapeptides arginine to form CS-Arg and CS-5Arg products. The chemical structure of the products was characterized by 1H NMR. Then, the CS-Arg and CS-5Arg combined with plasmids by electrostatic interactions to form stable particles. The morphology features, Zeta potential, buffering capacity of the complex particles was analyzed. By the agarose gel retardation assay, the combination ability with DNA and the protection ability to DNase I was studied. Finally, by using of human embryonic kidney cell HEK-293, the cytotoxicity of the complex particles and their gene transfection efficiency in vitro was studied.The results showed that the CS-Arg and CS-5Arg had been synthesized successfully and their water solubility was improved greatly. The graft percents of the amino acids on chitosans were approximately calculated as 6.45% in CS-Arg and 4.9% in CS-5Arg from the 1H NMR spectra. According to the graft rates and the molecular weight of unmodified chitosan, the estimated molecular weights of CS-Arg and CS-5Arg were 21 k and 23.8 k, respectively. The buffering capacity of CS-Arg and CS-5Arg was obviously enhanced compared with chitosan, the buffer capacity of CS-Arg was 4-fold higher compared with that of unmodified chitosan, and the buffer capacity of CS-5Arg was 2-fold higher than that of unmodified chitosan. The morphologies of CS-Arg/pDNA and CS-5Arg/pDNA complexes were observed by TEM and AFM. Both of the two complexes were nanoparticles with the size around 100 nanometers and with the height of 6-8 nanometers, which met the requirements of the nanoparticles characteristics. Both of the two products were positively charged, and their zeta potentials ranged from +21 to +31 mV. In agarose gel retardation assay, both CS-Arg and CS-5Arg could combine with plasmids completely at the N/P ratio of 8 and 6, and showed good protection ability of DNA, avoiding the degradation caused by DNase I.The CCK-8 assay was used to investigate the cytotoxicity of CS-Arg and CS-5Arg on HEK293 cell line. The results showed that the cell viability of each group was above 90% when the concentrations ranged from 50 to 200 μg/mL, which demonstrated that both CS-Arg and CS-5Arg were nontoxic. The cellular uptake results showed that the modified chitosan had better cell uptake ability than chitosan, which had time dependence property. The in vitro gene transfection of CS-Arg/pDNA and CS-5Arg/pDNA complexes was performed on HEK293 cells with pEGFP-C1 and pGL3-Luc as reporter genes. The results showed that the gene transfection efficiency could be enhanced greatly by the modification on chitosan, which is similar to PEI. But there was no difference between CS-Arg and CS-5Arg. Intracellular tracking results showed that the CS-Arg/pDNA and CS-5Arg/pDNA complexes were dissociated to release the condensed DNA in cytoplasm and both CS-Arg and CS-5Arg could penetrate into nuclei.The research results show that using anginine and poly-arginine to modify chitosan is an effective method for improving gene transfection efficiency, which provides a new method for the development of chitosan based nanoparticles as gene vector. |
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