Studies On Gene Delivery And Transfection System Based On Non-viral Vector N-methylene Phosphonic Chitosan

In this study, N-methylene phosphonic chitosan (NMPCS), an amphiphilic macromolecule with powerful chelating ability of Ca2+ ions, was synthesized and characterized, the interactions between NMPCS and plasmid DNA, the gene transfection and delivery mediated by NMPCS/DNA complexes, and cell cytotoxcity of the derivative and their complexes were also investigated. The main work is summarized as follows:(1) N-methylene phosphonic chitosan (NMPCS) was synthesized using a one-step reaction that allowed homogeneous modifications without any sharp decrease in its properties. FT-IR, 13CNMR, and element analysis permitted the identification of the structure and the degree of substitution of NMPCS.(2) NMPCS/DNA complexes were obtained using a complex coacervation process, characterized by agarose gel electrophoresis retardation assay for their stabilities, atomic force microscopy (AFM), transmission electron microscopy (TEM) and laser size analysizer observation for morphology and size, results showed that at charge ratio 2:1 or above, DNA could be completely entrapped and spherical complexes with mean size of 80~210nm were formed, CD spectra verified that the interaction between DNA and NMPCS caused a slight perturbation on DNA bonds and DNA still remained B-conformation within the complexes.(3) Complexes were further employed to transfect HeLa and HepG2 cells, and transfection efficiency was investigated using the pGL3 luciferase reporter gene in cell culture medium with different pH values. The results indicated that the transfection efficiencies were strongly dependent on the charge ratio and pH value of culture medium, for HeLa cells, the highest efficiency obtained at ratio of 4:1 and pH 6.2 was greatly higher than that from CS/DNA complexes or naked DNA and approximate to that from PEI/DNA complexes, although HepG2 cells were less efficiently transfected. Polymer-inorganic salt type NMPCS-Ca vector showed some similarities with traditional Ca-P, giving high efficiencies probably owe to positive role of Ca2+ ion channel. Synergistic effect of 16 N-dodecylated chitosan(16-CS)on the transfection efficiency of NMPCS /DNA complexes indicated that hydrophobic and hydrophilic interactions influenced the formation of the complexes and thus the transfection process.(4) The cytotoxity of derivative alone and its complexes with plasmid DNA were determined by 3-(4, 5-dimethylthiazd-2-yl)-2, 5-diphenylt-entrazolium bromide (MTT) assay both on HepG2 and HeLa cells, the results suggested that the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively, and Nevertheless, both NMPCS alone and its complexes also showed a dose-dependent cytotoxicity.