Novel Poly(histamine Acrylamide) (PHA) Based Cationic Polymers For Non-viral Gene Delivery

Gene therapy is promising for the treatment of various human diseases including genetic diseases and different types of cancers. In recent years, polymer-based non-viral vectors have received increasing attention, as they provide several advantages over their viral counterparts such as low immunogenicity, improved safety, great DNA condensation ability, and good reproducibility. Among those cationic polymers, polyethylenimine (PEI), poly(amido amine) (PAMAM) dendrimer, and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) are the most extensively studied for non-viral gene delivery. Their superior transfection activity is most likely associated with their high endosomal pH buffer capacity which is hypothesized to facilitate the endosomal escape of DNA complexes via the"proton sponge effect". In consistence with this, modification of high pKa cationic polymers (such as polylysine and chitosan) with histidine or imidazole group that is known to possess a superior endosomal pH buffer capacity has been reported to bring about dramatic enhancement of transfection efficiency. Cationic vinyl polymers like PDMAEMA are a particularly versatile family of non-viral gene carriers. Unlike other cationic polymers including PEI and PAMAM dendrimer, vinyl-type cationic copolymers can be conveniently prepared with controlled macromolecular structures, molecular weights and compositions by controlled radical polymerization techniques. The unique control over synthesis renders cationic vinyl polymers remarkably versatile in vector design. The first chapter of this thesis gives a general introduction to polymer-based non-viral gene vectors.The second chapter describes cationic poly(histamine acrylamide) (PHA) polymers with controlled structures and molecular weights obtained through radical polymerization of histamine acrylamide (HA) for gene delivery. The buffer capacity of all PHA polymers had a remarkably high buffer capacity in a pH range from 5.1 to 7.2, which were five-fold higher than 25 kDa PEI. 12.7, 17.5 and 28.7 kDa PHAs were able to condense DNA into nano-sized particles (<220 nm) with positive Zeta potential (> +13 mV) at and above an N/P ratio of 10/1. The PHA polymers were able to transfect COS-7 cells using the pEGFP-C1 and pCMV-Luc plasmid DNA as reporter genes. It is remarkable that low molecular weight PHA (e.g. 12.7 kDa PHA) presents a transfection activity comparable to 25 kDa PEI control, and CCK assays indicated that polyplexes of PHA were non-toxic to COS-7 cells.The third chapter describes PHA copolymers containing different amounts of imidazole ring and primary amino groups in side chain for gene delivery. Two different types of acrylate copolymers, poly((histamine acrylamide)/aminoethyl acrylate) [P(HA/AEA)] and poly((histamine acrylamide)/aminohexyl acrylate) [P(HA/AHA)], were obtained by radical copolymerization of HA with N-(tert-butoxycarbonyl)aminoethyl acrylate (Boc-AEA) or N-(tert-butoxycarbonyl)aminohexyl acrylate (Boc-AHA) followed by acid deprotection. The copolymer compositions were well controlled by monomer feed ratios, and mole fraction of PHA in the copolymers was varied from 70 %, 80 % to 90 %. These copolymers were designed with HA as the major component to retain a good buffer capacity at endosomal pH. The incorporation of primary amino functions was intended to increase their charge density at physiological pH, which may improve DNA condensation ability, enhance water solubility and colloidal stability of DNA polyplexes. The polyplexes of P(HA/AEA) and P(HA/AHA) copolymers had smaller sizes and higher positive surface charges than those of PHA. The in vitro cytotoxicity and transfection studies indicated that polyplexes of all PHA copolymers were non-toxicity to cells, and notably P(HA/AHA) had significantly higher transfection activity than PHA and 25 kDa PEI control.