Synthesis Of Fluorinated Polypeptides Copolymers And Their Gene Delivery Applications

Gene therapy has attracted extensive attention during the past three decades.Gene therapy is the therapeutic delivery of exogenous genes into the targeting cells to treat a broad variety of human diseases caused by genetic defects or abnormalities,such as genetic disorders,severe combined immunodeficiency and cancer.Free genes can't enter cells directly due to the rapid degradation by endonucleases in the physiological fluids and extracellular environment.Hence research efforts in the field are focused on designing safe and effective gene delivery vectors.Recently cationic polymers get more and more attention as promising gene carriers due to their biocompatibility,flexibility of chemical design and potential for large-scale production.Cationic polymers can condense negatively charged DNA into complexes(polyplexes)through electrostatic interaction and then deliver them into the targeting cells.However,polyplexes tend to break apart or aggregate in biological fluids which contain serum components and salts resulting in reduced gene transfection efficacy and increased toxicity.Moreover,the conventional cationic polymers can induce the increase of intracellular reactive oxygen species(ROS)concentration and oxidative stress,which reduces the gene transfection efficacy.Hence,it is quite important to design gene delivery carriers with good physiologic stability and biocompatibility.In this dissertation,a PEGylation bioreductive polylysine(PLL)polymers modified with fluorocarbon chains was synthesized and we studied the physiologic stability and the serum-resistance of the polyplexes.We also created a new class of responsive thioether dendron-branched polyethyleneimine(PEI)conjugate nanomicelles and discover their ROS self-scavenging ability.Specifically,the dissertation can be classified into the following two parts:We synthesized a novel PEGylation polypeptide,poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-cysteine)(PEG-PLL-PCys)triblock copolymer via the sequential ring-opening polymerization of amino acid N-carboxyanhydrides(NCAs)initiated by methoxypolyethylene glycol amine(mPEG-NH2).Subsequently,the obtained polypeptide is partially conjugated with fluorocarbon chains via disulfide exchange reaction.PLL segment can condense plasmid DNA(completely condense DNA at a low N/P ratio of 2)through an electrostatic force to form a complex core,PEG segment surrounding the complex like a corona can prevent the complex from precipitation and reduce the adsorption of serum,while PCys segment with fluorocarbon can significantly enhance the cellular uptake and the stability of the formed polyplex micelles in physiological conditions.The disulfide bond could be cleaved at an intracellular glutathione level leading to DNA release.Experiment results exhibit that the fluorinated polypeptides have good gene transfection efficacy(293T cells,35%)even in medium containing 50%FBS.The fluorinated polypeptides also show low cytotoxicity and good biocompatibility.We also created a novel polymer conjugate of thioether dendron-branched PEI which can self-assemble into bioreducible nanomicelles,and the nanomicelles exhibit excellent ability of scavenging the intracellular generated ROS and condense DNA at a low N/P ratio of 1,thereby highly improving gene transfection efficiency(293T cells,about 90%).