Synthesis, Characterization And Gene Transfection Efficiency Of Phosphonium Chitosan As Novel Non-Viral Vector

As a new way of treating diseases, gene therapy provides newpathway to a lot of difficulties in clinical treatment, such as cancer, autoimmune disease, and infectious diseases. However, the deficiency of safeand efficient gene delivery vector is the main obstacle of the application ofgene therapy.In this study, chitosan derivatives, N-phosphonium chitosans (NPCSs)were prepared as novel gene vectors using chitosan which has goodbiocompatibility but low gene transfection efficiency. Interaction betweenNPCS and plasmid DNA and the gene transfection efficiency of NPCSwere then investigated in detail.Low molecular phosphoniums were grafted to the main chain ofchitosan using chemical synthesis and then the water solubility of NPCSwas investigated. The results of Fourier-transformed infrared spectra and~1H-NMR spectra showed that N-phosphonium chitosan with two degrees of substitution (DS) were successfully synthesized. Compared withchitosan, NPCSs showed an enhanced water solubility and aqueoussolution stability upon pH.The polymer/DNA complexes at various charge ratios wereformulated and characterized. The particle sizes of NPCS/DNA complexesat different pH were between120and200nm as determined by dynamiclight scattering (DLS). The Zeta potentials indicated that surfaces of thesecomplexes were all positively charged. The cytotoxicity assay showed thatthe synthesized polymers were less toxic than that of branched25k DaPEI.Furthermore, gene transfection efficiencies of NPCS/DNA complexeswere also investigated, using the plasmid DNA encoding green fluorescentprotein (pEGFP-N1) on HEK293and HeLa cells. The results showed thatthe gene transfection ability of the copolymer was better than that ofchitosan and NPCS with a DS of21.5%had comparative gene transfectionefficiency to PEI on HeLa cells as the medium pH was adjusted to6.5.To improve the stability of complexes, monomethoxy polyethyleneglycol (MPEG) and NPCS were used to synthesize PEG-NPCS. FT-IR and~1H-NMR spectra showed that MPEG were successfully grafted to NPCS.Results of MTT essay indicated that PEG-NPCS were less toxic thanNPCS. Stability of PEG-NPCS/DNA complexes was investigated. DLSand Zeta potentials showed that PEG-NPCS/DNA complexes werepositively charged and the particle sizes were about145nm. Thecomplexes were stable within72hours.