Preparation And Application Of Polycationic Gene Delivery System Based On Modified Polyethylenimine

Non-viral gene vector can avoid the potential immunogenicity and toxicity of viral vector, and has many advantages such as the possibility of selected modifications, the ease of large scale manufacture, the capacity to carry large plasmid DNA (pDNA) inserts, and so on. However, the body’s systemic barriers, including serum proteins in blood stream, cell membrane, endosomal compartment and nuclear membrane, significantly reduce the effective therapeutic gene transfer and expression. Therefore, the design and preparation of low cytotoxicity, high transfection efficiency and targeting non-viral gene vectors has important research significance and applications. This thesis includes the following content:In the first chapter, the author outlined the status of gene therapy, introduced some commonly used polymer-based gene delivery system, and investigated the mechanism of cationic polymer gene delivery system, the main barriers of cationic polymer-mediated gene transfer and methods to overcome these barriers.In chapter 2, biocompatible and biodegradable cationic polymers, poly(L-succinimide)-graft-polyethylenimines (PSPs) were prepared as non-viral vectors. The branched polyethylenimine (PEI800) was grafted to poly(L-succinimide) (PSI) by one step reaction without any catalyst addition. Gel retardation assay results indicate that the mobility of PSP/pDNA complexes is entirely retarded at the ultra low N/P ratio of 0.42. Dynamic light scattering results suggest that PSP/pDNA complexes can form positively charged (10.7-19.5 mV) and nano-sized particles (150-300 nm). At the N/P ratio 0.84, PSPs can reach the highest transfection level with ten-fold enhancement in 293T cells and five-fold in HeLa cells as compared with PEI25k (Mw=25 kDa). As evidenced by fluorescent confocal microscopy, pGL-3 plasmids condensed by PSPs can be effectively transported into the nuclei of HeLa cells. Meanwhile, compared with PEI25k, the cytotoxicity of polymers significantly reduces, and the IC50 of PSPs is almost four times higher than that of PEI25k.In chapter 3, PEGylated poly(L-succinimide)-g-polyethylenimines (PSI-g-PEI-g-PEG) were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG, Mw=750 Da) to PSI-g-PEI. The physicochemical properties of PSI-g-PEI-g-PEGs including buffer capability, pDNA binding ability, cytotoxicity, complex zeta potential and particle size, in vitro gene transfection activity were investigated. The influence of PEGylation was discussed by the comparison between PSI-g-PEI-g-PEGs, PSI-g-PEI and PEI25k. SEM images demonstrate that PSI-g-PEI-g-PEG/pDNA particles have regular shape with the diameter less than 200 nm. PEGylation results in the reduction of polymer/pDNA complex size and inhibits the aggregation of complexes. PSI-g-PEI-g-PEGs exhibited lower cytotoxicity as compared with PSI-g-PEI and PEI25k. In 293T and HeLa cells, high transfection activity in vitro was observed for all the three vectors. PSI-g-PEI-g-PEGs can effectively transport pGL-3 plasmids into the nuclei of HeLa cells on the basis of fluorescent confocal microscopy. PSI-g-PEI-g-PEGs show great potential as the non-viral vectors for gene transfection.In chapter 4, a highly efficient gene transfer vector (PSI-g-PEI-g-LA) with hepatocyte-targeting function was designed and prepared. The transfection efficiency was purposely evaluated in two different cell lines including ASGP-R bearing HepG2 cell and ASGP-R-lacking HeLa cells. PSI-g-PEI-g-LA exhibits much higher cell-biocompatibility as compared with PSI-g-PEI and PEI25k. The hepatocyte-targeting function was elucidated based on the comparative studies towards three polymeric vectors including PSI-g-PEI-g-LA, PSI-g-PEI and PEI25k. Dynamic light scattering (DLS) reveals that PSI-g-PEI-g-LA can compactly condense pDNA resulting in complexes diameter in 95-203 nm. The in vitro transfection tests using different reporter genes indicate that relative to PSI-g-PEI and PEI25k, PSI-g-PEI-g-LA displays much better transfection activity in HepG2 cell via ligand-receptor recognition mechanism. The targeting effect is also partly supported by the finding that the best transfection efficiency of PSI-g-PEI-g-LA in HepG2 cell is relatively higher than that in HeLa cells. By means of western blotting analysis, flow cytometry and confocal laser scanning microscopy techniques, high p53 expression and strong p53-inducing apoptosis of HepG2 cells is distinctly detected in PSI-g-PEI-g-LA mediated transfection.