The Study Of A Novel Nuclear Targeting Non-viral Gene Delivery System

Both the advances in molecular biology and biotechnology, along with the completion of the Human Genome Project have led gene therapy to a new level, being an alternative treatment of genetic diseases, infections and cancer. The success of gene therapy is waiting for efficient delivery vectors. Non-viral carriers have advantages such as non-immunogenicity, low cytotoxicity and low cost, but poor transfection efficiency compared to viral carriers limits their application to clinical gene therapy. There were three main barriers in the intracellular delivery of genetic materials using non-viral gene carriers, namely, cellular membrane, endosomal membrane and nuclear membrane.In this work, glucocorticoid (GC) was conjugated with polycation to form novel steroid-polycation. In this polymer, GC was able to facilitate polymer to translocate into the nucleus as nuclear locatoin signal (NLS), and its transfection efficiency was enhanced accordingly. For its lipophilicity, the steroid-polycation was used to modify liposome to get a novel polycation liposome. Furthermore, ligand could also be used to modify the liposome, which makes the liposome target to both special cells and cell nucleus.Firstly, five kinds of glucocorticoid (GC) with 21-hydroxyl groups were selected, namely Betamethasone (BET), dexamethasone (DEX), methylprednisolone (MPL), prednisolone (PNL) and hydrocortisone (HC). To activate the five kinds of glucocorticoid, their 21-hydroxyl groups were substituted with mesylates using methanesulfonyl chloride. Then a one-pot reaction between PEI 1800, 2-iminothiolane (Traut's reagent) and glucocorticoid mesylate yielded GC-PEI polymer. The NMR and IR results indicated that GC was covalently conjugated to the backbone of PEI. The form of nano-size polyplexes with pDNA were investigated by agarose gel electrophoresis, Zeta-sizer and TEM. And their physicochemical properties resembled each other. MTT assay was performed to examine their cytotoxicity. Transfection efficiency was investigated in HEK 293 and HepG 2 cells with pEGFP-Nl and pGL-3 report gene. The GC-PEI polymer was labeled by FITC. The localization of the polymer in cell was observed using confocal microscopy. The result indicated that the physical-chemical properties of the polyplexes were similar with each other. Their cytotoxicities were significantly lower than PEI 25K. The transfection results indicated that the increase in transfection capability generally follows the order, HC-PEI<PNL-PEI<MPL-PEI<DEX-PEI<BET-PEI.And we assumed that the transgenic expression correlated linearly with GC potency. The higher was the potency of GC substituted on the polymer, the more effective was their transfection.Then, Triamcinolone Acetonide (TA) with more potent was conjugated with PAMAM (G4) dendrimer to synthesize PAMAM-TA to improve its transfection efficiency and reduce cytotoxicity. In order to examine the contribution of the substitution degree of TA, high and low substituted PAMAM-TA (PAMAM-TA-H and PAMAM-TA-L) was synthesized. The study showed that their physicochemical properties resembled each other. MTT assay showed that their cytotoxicity was lower than that of native PAMAM and PEI 25K significantly. The investigation of the polyplex localization showed that PAMAM polyplex was internalized into cytoplasm, but PAMAM-TA-H and PAMAM-TA-L polyplex both were found to be inside nuclei. This meant that TA conjugation to PAMAM dendrimer could accelerate intra-nuclear location, finally leading to satisfying transfection efficiency. The low substituted degree of TA to 0.22 did not interrupt its nuclear localization potency.Furthermore, TA was conjugated with various Molecular Weight PEI (Mw 600, 1800,25k). Their physicochemical properties, cytotoxicties, transfection efficiency and intracellular distribution were investigated. The result showed that the physicochemical properties of three polymers were no different significantly. The cytotoxicities of them were lower than that of native PEI 25K. The order of their transfection efficiency followed that PEI 1800-TA> PEI 600-TA> PEI 25K-TA. The investigation of transfection mechanism indicated that transgenic activity of PEI 1800-TA and PEI 600-TA was inhibited greatly in presence of TA while PEI 25K-TA did not affected significantly. The results suggested that PEI 600-TA and PEI 1800-TA might translocate into cellular nucleus, which could be competed by TA, but nor of PEI 25K-TA. The enhancing mechanism of low molecular weight PEI-TA for gene transfer activity may be ascribed to glucocorticoid receptor mediated nuclear translocation. High molecular weigh PEI may hardly to bind with glucocorticoid receptor and entered into nucleus due to its molecular structure. The result of confocal microscopy also confirmed this suggestion. When injected into the portal vein of mice, PEI 1800-TA and PEI 25k/pEGFP-N1 complexes showed a relatively high transgene expression in liver. Thus, the data strongly suggest that the transfection efficiency of PEI 1800-TA is much superior to that of PEI 25K. It was consistent with the result in vitro. The transgene expression of complexes was not found in other tissue.At last, PEI 1800-TA was incorporated with liposome and the liposome was modified by CHEDLA owned galactoside residue to prepare STL. Thus, STL has the capability of targeting nucleus and special cells and it suggested that STL could overcome the various barriers to present distinctive transgenic expression. The study of physicochemical property of STL showed that liposome could be decorated by PEI-TA and STL could condense efficiently pDNA to form nano-size and positive potential particles. Cytotoxicity of STL was lower significantly than that of PEI-TA, but its transfection efficiency was higher than the latter. STL, which was composed of lipid:PEI-TA at 6:4 (mol%) and 10% CHEDLA, had the best transgenic activity. the transfection efficiency of STL was greatly reduced in the presence of galactose added excessively, indicating that STL was absorbed via galactose receptor-mediated endocytosis. After portal vein administration in mice,10% CHEDLA STL showed better transgenic expression in liver than 0% CHEDLA STL, PEI 1800-TA and PEI 25K. There was no gene expression in other organs. It indicated that STL can delivery efficiently gene materials to target organ and carry out distinctive transfection efficiency. The goal of our study is completed. And it would be a beneficial attempt to construct multi-functional non-viral gene carrier.