| Gene therapy has emerged as a potential new approach treating genetic disorders by delivering therapeutic genes to targeted diseased tissues. However, its wide use in clinical trial has been hampered by several challenges, most prominently in gene delivery.Viral vectors and nonviral vectors are two common carriers used in gene delivery. Compared to viral vectors, nonviral vectors demonstrate advantages of safety and capability of large-scale application, but disadvantages of low transfection efficiency. The objective of this thesis is to design polymer gene delivery systems for in vivo gene therapy.Polyethylenimine (PEI) is one of the best studied nonviral vectors with high gene transfection efficiency in vitro, but suffers high toxicity and non-specific interaction with serum proteins due to its positive charges. Coating negatively charged polymers on its polyplexes PEI/DNA can solve the problems, but the coating destabilizes the polyplexes because the negatively charged polymers compete with DNA to bind PEI, making it instable in vivo gene delivery.We proposed that crosslinking the PEI/DNA polyplexes via intracellular cleavable bonds would stabilize the polyplexes even after being coated with negatively charged polymers and make the coated particles stable for in vivo gene delivery. On the other hand, after the particles are taken up into the cell and the crosslinkers are cleaved, the coated negatively charged polymers bind PEI, facilitating the release of DNA and thus gene transfection. To this end, in this thesis, three gene delivery systems with high stability and negatively charged surface based on PEI/DNA complexes were developed in order to achieve in vivo gene delivery.The first gene delivery system is reversely cross-linked PEI/DNA complexes coated with heparin (HP). This vector was prepared by crosslinking of PEI/DNA complexes via disulfide bonds and then being coated by heparin. This HP/PEI/DNA complex had a hydrodynamic diameter of 100 nm and low cytotoxicity with high stability both in serum and in high concentration salt solutions. The in vivo transfection efficiency of the Sandwich-like particles was also tested. The plasmid encoding EGFP demonstrated high expression in the tumor site of nude mouse bearing xenografted breast tumor (BCap37), implicating great potential in in vivo use.The second gene delivery system is hyaluronic acid (HA) coated and dexamethasone (Dexa)-functionalized Sandwich-like polyplexes. Dexamethasone (Dexa) was conjugated to PEI (MW25 kDa) for better nuclear localization due to the Dexa's nuclear lozalization ability. The impacts of dexamethasone on cytotoxicity and gene transfection efficiency were investigated. After modification with Dexa, the cytotoxicity of PEI (MW25 kDa) reduced to a certain degree, but the transfection efficiency of the polyplexes was not improved. HA was coated to the polyplexes because HA receptors are overexpressed in many tumor cells and HA can be used as a targeting group while shielding the positive charges. The HA/PEI-Dexa/DNA complex was obtained by self-assembly of PEI-dexa/DNA complexes with HA via electrostatic interaction. The complexes had higher gene transfection efficiency to HA receptor positive cell lines compared with HA-receptor negative cell lines.The lipid membrane coated on particles may fuse with cell membrane and thus facilitate cellular entry of the nanoparticles. PEI-Dexa/DNA polypexes coated with a lipid layer was thus preliminary explored for gene delivery. The formed lipid/PEI-Dexa/DNA polyplexes displayed a suitable size and negatively surface charge, implicating potentials in in vivo applications. But the transfection efficiency was very low, even after introducing folic acid as targeting group. Therefore, in order to enhance gene transfection efficiency, the kind of lipid and some other elements of this gene delivery system need further optimization. |
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