Synthesis And Characterization Of PEGylated Or Polyphosphoester-based Brush-type Copolymer For Fabricating Multifunctional Gene Vector

Gene therapy has been generally acknowledged as a promising treatment fornumerous hard curable diseases, including genetic diseases, infectious diseases, andcancer. Safety, efficiency and stability play the most challenging role in gene therapy.Preparation and controlled release of gene vector concern the materials science,molecular biology, medicine and polymer chemistry and other fields with importanttheoretical significance and potential applications. Non-viral cationic gene vectors arepromising due to their easily-adjustable and controlled structures, high condensationability. This thesis is mainly focused on the design and synthesis of PEGylated orpolyphosphoester (PPE) based brush-type copolymers for fabricating multi-responsivegene vectors, combining the advantages of brush-type copolymers, disulfide bond,magnetic nanoparticles, biodegradable PPE and fluorescent coumarin. It is reportedthat brush-type hydrophilic units on the surface of vectors can enhance their resistanceagainst self-aggregation, and nonspecific adsorption with proteins or serum-drivencomponents, also it can enhance the solubility of polyplexes and prolong the lifetime inblood circulation because the abundant periphery highly flexible chains stericallyprevent undesirable interactions of the vectors with negatively-charged components inbloodstream. The present work can be summarized as follows:(1) A novel magnetic-responsive complex composed of polycation, DNA andpolyanion has been constructed via electrostatic interaction. The magneticnanoparticles (MNPs) were first coated with a polycation, poly[2-(dimethylamino)ethyl methacrylate] end-capped with cholesterol moiety(Chol-PDMAEMA), and then binded with DNA through electrostatic interaction, thecomplexes were further interacted with the brush-type polyanion, namelypoly[poly(ethylene glycol)methyl ether methacrylate]-block-poly[methacrylic acidcarrying partial mercapto groups](PPEGMA-b-PMAASH). The resulting magneticparticle/DNA/polyion complexes could be stabilized by oxidizing the mercapto groupsto form cross-linking shell with bridging disulfide (S-S) betweenPPEGMA-b-PMAASHmolecular chains. The interactions among DNA,Chol-PDMAEMA coated MNPs and PPEGMA-b-PMAASHwere studied by agarosegel retardation assay. The complexes were fully characterized by means of zetapotential, transmission electron microscopy (TEM), dynamic light scattering (DLS)measurements, cytotoxicity assay, anti-nonspecific protein adsorption and in vitrotransfection tests.(2) A novel kind of pH-sensitive brush-tpye copolymer,[poly(2-hydroxyethylmethacrylate)-graft-poly(ethylethylene phosphate)]-block-poly[2-(dimethylamino)ethyl methacrylate][(PHEMA-g-PEEP)-b-PDMAEMA] with biodegradablepolyphosphoester as the side chains were synthesized via a combination ofsingle-electron transfer living radical polymerization (SET-LRP) and ring-openingpolymerization (ROP). The chemical structures were characterized by1H NMR,31PNMR, FT-IR, and GPC measurements.(PHEMA-g-PEEP)-b-PDMAEMA canself-assembled into aggregates served as a gene carrier. The brush-like PPE chainsprovide the carrier with good biocompatibility and biodegradability, as well as longcirculation lifetime. The interaction of (PHEMA-g-PEEP)-b-PDMAEMA and DNAwas studied by agarose gel retardation assay, and the formed complexes were furtherinvestigated by means of zeta potential, DLS and TEM measurements. In addition, thein vitro cytotoxicity and transfection tests were also investigated. (3) In this part, we report on a novel acid-cleavable and fluorescent cationic blockcopolymer CE-PCL-a-(PDMAEMA-co-PPEGMA) by a combination of atom transferradical polymerization (ATRP), ring-opening polymerization (ROP) and “Click”reaction. The cationic block copolymer CE-PCL-a-(PDMAEMA-co-PPEGMA) canself-assemble into micelles to load drug and DNA simultaneously to form complexeswith the brush-type PPEGMA on the surface. The drugs and DNA can be releasedafter the acetal linkage was cleaved under intracellular acid conditions. We studiedthese micelles as the drug and gene co-delivery vector by employing gel retardationassay, zeta potential, DLS, TEM, and subsequently its in vitro drug release, in vitrocytotoxicity and transfection efficiency were tested. Fluorescence spectrometer wasused to evaluate the fluorescence of complex for detecting and locating the carrier.(4) A series of brush copolymer P(PEGMEMA-co-PEGMAGal)-b-PDMAEMAmodified by liver targeted galactose were prepared by two-step ATRP technique andesterification reaction activated by N, N’-carbonyldiimidazole (CDI) and furthermodified by galactosamine. The brush-type hydrophilic polymer can enhance theresistance against self-aggregation, prolong the lifetime in blood circulation andprevent non-specific adsorption of proteins. Meanwhile, the pH responsivePDMAEMA segment can be protonized to carry positive charges to condense DNA. Inaddition, the galactose ligands can enhance the endocytosis of complex by hepatocyteto increase the transfection efficiency. The chemical structures ofP(PEGMEMA-co-PEGMAGal)-b-PDMAEMA were characterized by1H NMR, FT-IR,and GPC measurements. The binding capacity of this cationic copolymer with DNAwas investigated by agarose gel electrophoresis, zeta potential, in vitro cytotoxicity andtransfection assay was tested.