Studies On Chitosan And Alkylated Chitosan/DNA Complexes-Mediated Gene Transfection

In this thesis, the characteristics of chitosan(CS)/DNA and alkylated chitosan(ACS)/DNA polyelectrolyte complexes, and gene transfection mediated by these nonviral vectors were investigated. The main work includes three parts as follows:The properties of CS/DNA and ACS/DNA complexes were investigated by a variety of methods. The results of FTIR, 31P NMR, circular dichroism (CD) and fluorescence spectra indicated that the interaction of DNA with chitosan merely caused a slight perturbation of DNA bonds, and DNA still remained B-conformation in the complex; parts of chitosan was considered to be bound to the minor grooves of DNA. AFM images showed that the topological structures of CS/DNA complexes varied with charge ratios. Free DNA molecules existed at low charge ratios, whereas nanoparticles formed at higher charge ratios. Dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) was used as a model cell membrane, and atomic force microscopy (AFM) and differential scanning calorimetry(DSC) were employed to explore the transmembrane mechanism of the vectors by examining the thermodynamic behaviors of DPPC/CS and DPPC/ACS mixtures. The results revealed that chitosan and alkylated chitosan were capable of inducing the fluctuation of DPPC membrane, and the perturbation effects was enhanced with the increase in the hydrophobicity of chitosan. The analytical results of electrophoresis and DNase degradation demonstrated that a small quantity of ACS could condense DNA, and furthermore played the same shielding role as chitosan in protecting DNA from the DNase degradation. CS and ACS were used to transfer plasmid-encoding chloramphenicol acetyltransferase (CAT) into C2C12 cells. The transfection efficiency was evaluated by determining the contents of CAT expressed with ELISA, and the influence of hydrophobicity of alkyl side chains on the transfection efficiency was elucidated as well. The results indicated that CS and ACS could transfer pcDNA3.1/CAT into C2C12 cell lines. The transfection efficiency of chitosan vector was 4 times higher than that of naked DNA; the transfection level of ACS was further increased upon elongating the alkyl side chain. The transfection efficiency leveled off after the number of carbons in the side chain exceeded eight.DNA fragment coding both signal peptide and mature peptide of insulin-like growth factor-â… (IGF-â… ) was subcloned into plasmid pTracer-CMV/Bsd, and the effect of IGF-â… on cell proliferation was discussed. It was observed by fluorescence microscope that plasmid DNA could be delivered into C2C12 cell lines, and the genes were effectively expressed in receptor cells. The selected stable trans-IGF-â… cell lines had a higher proliferating rate than the untransfected ones.