Preparation And Study Of Modified Polyethylenimine Gene Carrier

Gene therapy is the treatment of diseases by delivery of normal genes or therapeutic genes into specific cells of patients to correct or supplement defective genes responsible for disease development. The tools that delivery the genes are called vectors. For critical elements are involved in the manipulation of gene therapy: the functional genes, gene vectors, targeting cells and suitable delivery routes. Now, the vectors are divided into two kinds, including viral and non-viral vectors, with viral vectors more commonly employed. Though viruses are quite effective gene delivery vectors, it should also be noted that there are still considerable problems such as immunogenicity of viruses, the limited carrying capacity and carcinogenicity, during the past two decades, more scientists have shifted their interest to develop gene delivery systems based on non-viral vectors. The non-viral vectors include liposomes,nanoparticles and colloidal particles with the advantages of low cost, simple synthesis, easily manipulation, large carrying capacity and high safety. However, there are some disadvantages with non-viral vectors such as relatively low gene delivery efficiency and no specific cells or tissues targeting abilities.Polyehtylenimine (PEI) is a kind of polymer commonly used in the paper manufacture, formed with the polymerization of aziridine monomers with the catalysis of acids. Due to the difference of the synthesis, there 2 forms of PEI: linear and branch ones. Under the physiologic pH conditions, only 1 nitrogen of 5~6 amines of PEI is protonated and with the combination of nucleic acid, 1 nitrogen of 2~3 amines of PEI can be protonated. When PEI enters into endosomes, the nitrogen of the amines can be further protonated with the decreased pH. The more protons are captured by PEI and cause the Cl- inflow over the membranes of endosomes, resulting that the endosomes are disrupted by the osmotic pressure and the plasmids DNA are released into cytoplasm. PEI is one of the non-viral vectors. However, the disadvantages such as lower transgene efficiency compared with viral vectors, high cytotoxicity with high moleculzr weight PEI, non-degradable characters and having no specific cells or tissues targeting ability limit the usage of PEI especially in vivo. In order to improve the characters of PEI for better behaviors in gene delivery and get ideal nonviral vectors, it is necessary to modify PEI.The aim of our studying is to modify PEI to obtain higher gene transfection efficiency and lower cell toxicity. To solve the contradiction of PEI that existing in itself, we using some crosslinkers crosslink low molecular weight PEI to higher molecular weight. This will keep low molecular weight PEI with low cell toxicity, and at the same time have higher gene transfection efficiency. With hyperbranched PEI as a macroinitiator, a novel amphiphilic hyperbranched multi-arm polyethylenimine-poly(γ-benzylL-glutamate) (PEI-PBLG) copolymer was synthesized by ring-opening polymerization (ROP) ofγ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA), with the inducing of PBLG, the cell toxicity is further lowed. The whole study is including two aspects. On the basis of successful synthesis of vectors, the phy sicochemical characters and cytotoxicities of the vector, the interation with plasmids DNA, the in vitro gene delivery efficiencies were investigated.In my first part, the bond of sulfur and gold is effectively to join PEI or PEIPBLG to nano Au, we abtain the carrier with starlike structure. This kind of structure make PEI or PEIPBLG connecting with Au not so dense, so the cytotoxicities is lowed. We also found with the decrease of Au content, the transfection efficiency is enhanced. This maybe interrelated with amount of PEI or PEIPBLG joined with Au. The size of particle is no more than 100nm studying by DLS and TEM. When forming the complex with DNA, the size is still below 200nm. This is suited for gene carrier.In the first part, we have got carrier with low cytotoxicities and high gene transfection. But it still has the limitation is, it can not be broken down in cells. So, in the second part, we study and synthesize another carrier, that can be disassembled in cells. A series of crosslinked PEI (PEIPBLG) (CLP(P)) was specially designed via crosslinking reaction between low molecular weight PEI (PEIPBLG) and N,N′-cystaminebisacrylamide(CBA)which contain disulfide bonds. And the disulfide bonds can be broken at the intracellular concentration of the reductive glutathione (GSH). The result indicated that CLP(P) showed best DNA condensation ablity and suggested to increase endosomal rease of DNA complexes into the cytoplasm. The polyplexes were unpacked due to the cleavage of the disulfide bonds in CLP(P) at intracellular GSH. As a result the gene vector significantly reduced the cytotoxicity and showed effective transfection.