Synthesis Of Novel Chitosan Derivatives And Their Properties For Gene Delivery

Developing safe and efficient gene delivery vectors is a key step for gene therapy. Chitosan gene vector has been known for its good biocompatibility, however the transfection efficiency of unmodified chitosan is low. While the modification of chitosan is considerably restricted by its solubility in non-aqueous solutions. 1-Butyl-3-methyl-imimidazolium acetate ([BMIM]Ac) ionic liquid can dissolve chitosan, accelerate the reaction, enhance the substitution degree, thus made novel chitosan derivatives achievable. Herein, three kinds of chitosan derivatives were synthesized in [BMIM]Ac and used in gene delivery.CS-g-PEI-x graft copolymers with different PEI graft ratios were synthesized in [BMIM]Ac, using carbonyldiimidazole (CDI) as selective activation reagent, through a two-step nucleophilic substitution reaction. The structures of as-synthesized CS-g-PEI-x copolymers were characterized by FTIR,1HNMR,13C-CP/MAS NMR and GPC. The DNA binding ability, and stability of CS-g-PEI-x/DNA polyplexes all increased with PEI graft ratios. The gene transfection and cytotoxicity in Hep-2 cells showed that CS-g-PEI-3 with PEI graft ratio (GR) of 4.5% gave the best gene transfection performance and low cytotoxicity.CS-g-mPEG-g-PEI-x graft copolymers were obtained by grafting mPEG-Nh2 and PEI onto CS by the method mentioned above. The structures were characterized by FTIR, 'HNMR, UV and GPC. When PEI GRs were similar, mPEG-NH2 GRs rarely affected the proton buffering capacity but reduced the DNA binding ability. CS-g-mPEG-g-PEI-x/DNA polylpexes with N/P ratio of 6 were stable in 0.10 ug/?L heparin and 2.0 U DNase I. CS-g-mPEG-g-PEI with mPEG-NH2 GR of 2.9% had the highest gene transfection efficiency in 10% FBS in Hep-2 cells.Stimulus-responsive chitosan (SRCS) cross-linked by acid-labile acylhydrazone bond and redox-labile disulfide bond was synthesized from CS oligosaccharide. Then SRCS-g-mPEG-g-PEI was obtained by grafting SRCS with mPEG-NH2 and PEI by the method mentioned above. The structure was characterized by 1HNMR,13C-CP/MAS NMR, FTIR, UV and GPC. SRCS-g-mPEG-g-PEI could effectively compact DNA, and SRCS-g-mPEG-g-PEI/DNA polyplex with N/P ratio of 10 was stable in 0.10 ?g/?L hepra,2.0 U DNase and 25% serum. DNA could be stimulus-responsively released in redox and acid environments from SRCS-g-mPEG-g-PEI/DNA polyplex. SRCS-g-mPEG-g-PEI gave an excellent gene transfection performance in 10% serum against Hep-2 cells, serving as a promising candidate for safe and efficient gene delivery.