Development Of A Novel Targeted Polyethylenimine Gene Delivery System

The prospect of curing inherited and acquired diseases through gene therapy has engendered considerable effort toward the development of gene transfer vectors. Most ongoing human gene therapy protocols rely on recombinant retroviral and adenoviral vehicles, which risk encountering acute safety and immunological problems with large scale or repeated use, besides their limited carrier capacity. Synthetic vectors, although currently orders of magnitude less efficient than biological vectors, are increasingly being considered to be possible solutions as well.Polyethylenimine (PEI) is the most common used polycation gene delivery vector, which can condense plasmids DNA into the particles of hundreds of nanometer or so. PEI/DNA particles abundant with positive charge adhere to negative charged mucoproteins on the surface of the cells, then passively pinocytized into the cells. PEI can not be decomposed by enzymes in the endosome, has a "proton sponge effect" which make endosome osmotic swelling to rupture, and help DNA escape into the cytosol from the endosome. Receptor targeted PEI can enhance PEI's transfection efficiency. In this study, the oligopeptides of basic fibroblast growth factor (bFGF) and RGD oligopeptide were coupled with PEI, aiming at integrins and fibroblast growth factor receptor (FGFR) which are highly expressee on neoplasm neovasculature.The study was divided into two parts: 1) To determine the related parameters affecting PEI/DNA transfection and to optimize the PEI/DNA transfection efficiency; 2) To design and synthesize bFGF oligopeptide and RGD oligopeptide binding integrins, to conjugate oligopeptide with PEI, and to assay their transfection efficiency. At the same time, we investigated the effect of nuclear locational peptide (NLS) on PEI/DNA transfection efficiency. Additionally, we developed a novel bFGF oligopeptide/RGD peptide dual-targeted PEI gene delivery vectors and investigated its transfection efficiency in vitro and in vivo.Part I : The optimization of PEI/DNA transfection efficiencyObjective: We chose branched 25 kDa PEI as plasmids DNA delivery vector. In this part, parameters related to PEI/DNA transfection were investigated and PEI/DNA transfection efficiency was optimized, and the data were used for the synthetic targeted PEI vectors.Methods: PEI's cytotoxicity was determined by MTT method. The binding capacity of PEI and DNA was determined through electrophoresis gel shift assay. With PEI transfection of pEGFP plasmid coding enhanced green fluoroscene protein (EGFP), pSVp plasmid and pcDNA3.1β coding β-galactosidase(p-gal), the effect of influencing factors (including albumin, serum, dilution medium, endosome inhibitor-chioroquine, etc.) were determined. The manipulation methods (including transfection times, level shaking, incubation times, etc.) and plasmids factors (including the compatibility between the plasmid and the target cells, purityand quality of plasmids, plasmids concentration, etc.) were investigated.Results: PEI concentration above 7~8 g/ml was significantly cytotoxic. N/P molar ratio 2.5-3.0 could completely retard DNA migration in the agarose gel electrophoresis. N/P molar ratio in the range of 7.5-10 is optimal for a variety of cells, not concomitant with significant toxicity. Dilution medium of PEI/DNA influenced the transfection efficiency, and physiological solution as dilution medium excelled over 5% glucose solution. Endosome inhibitor chloroquine decreased PEI's transfection efficiency and enhanced PEI's cytotoxity. Albumin and serum in the culture medium decreased the polycation nonviral vector's transfection efficiency.In 12 h after PEI/DNA transfection, expressed products of reporter gene could not be detected. 24 h after the transfection, the reporter gene commenced to express, culminated in 36 h, maintained several days and later the expression amounts began to lower. The transfection efficiency was improved with additional transfection, double transfection was optimal. Level shaking of the culture plates for 30 min afte...