Construction, Preparation And Characterization Of High Efficient Gene Vectors Based On Methacrylate

With the development of human society, the incidence of genetic diseases, cancer and cardiovascular disease has been rising year by year. Traditional treatment is very difficult to completely cure these diseases. By transferring the exogenous genes into target cells, gene therapy opens up a new treatment for these diseases. At present, the key of gene therapy is safe and efficient gene carriers. Researchers now focus on the development of non-virus gene vector due to the poor biological safety of viral gene vector. Cationic polymers are most widely researched non-virus gene vectors. Atom transfer radical polymerization (ATRP) which has the advantages of moderate reaction temperature, applicable to a variety of monomers is very suitable for preparation of multifunctional cationic polymers. In this dissertation, according to the current development trend of the non-virus gene carrier materials and the scientific problems of low transfection efficiency and high toxicity, a series of high-performance cationic gene carrier based on methacrylate were prepared. The gene transfection efficiency of these carriers was evaluated by cell and animal experiments. This dissertation has important effects on the promotion of the safe and efficient non virus gene vectors.Recently, we reported that ethanolamine (EA)-functionalized poly(glycidylmethacrylate) (or PGEA) can produce good transfection efficiency, while exhibiting very low toxicity due to its plentiful secondary amine and nonionic hydroxyl units. Herein, the low-toxic PGEA was proposed to be conjugated with PBIs via facile atom transfer radical polymerization for the well-defined highly fluorescent cationic bifunctional conjugate (PBI-PGEA). The obtained PBI-PGEA exhibited good water-solubility properties, characteristic spectroscopic patterns of PBIs, and excellent photostability. The PBI-PGEA conjugate can be used as an efficient cell bio-dye for rapid (2-5 min) cell labeling at low concentrations (0.06-0.12 mg·mL-1). Such a fast labeling process did not induce obvious cytotoxicity, avoiding possible side-effects to the cells. In addition, the PBI-PGEA still possessed good gene transfection efficiency in different cell lines. With the strong fluorescence in water and good transfection properties, the developed bifunctional PBI-PGEA should possess more potential in bioimaging and gene delivery.The introduction of reduction-sensitive disulfide bond will improve the transfection efficiency of gene vectors, according to some reports. In this dissertation, a tailor-made biocleavable pullulan-based gene vector (PuPGEA) with good hemocompatibility was successfully proposed via atom transfer radical polymerization (ATRP) for efficient liver cell-targeting gene delivery, using the specificity for liver of Pullulan and the reduction-sensitivity of disulfide bond. A two-step method involving the reaction of hydroxyl groups of pullulan with cystamine was developed to introduce reduction-sensitive disulfide-linked initiation sites of ATRP onto pullulan. The poly(glycidyl methacrylate) (PGMA) side chains prepared subsequently via ATRP were functionalized with ethanolamine (EA) to produce the resultant biocleavable comb-shaped PuPGEA vectors consisting of nonionic pullulan backbones and disulfide-linked cationic EA-functionalized PGMA (PGEA) side chains with plentiful secondary amine and nonionic hydroxyl units. The cationic PGEA side chains can be readily cleavable from the pullulan backbones of PuPGEA under reducible conditions. Due to the liver targeting performance of pullulan backbones, such PuPGEA vectors exhibited much higher gene transfection efficiency and cellular uptake rates in HepG2 cell lines than in Hella cell lines. In addition, in vitro transfection efficiency and uptake mechanism of polyplex in HepG2 cells were evaluated in the presence of different endocytosis inhibitors, indicating that the asialoglycoprotein receptor was involved in transfection process of hepatocytes. More importantly, in comparison with gold standard polyethylenimine (PEI,25 kDa), PuPGEA vectors possessed excellent hemocompatibility without causing undesirable hemolysis.In the basement of preparation of high efficient gene vector with very low cytotoxicity, we produced a series of bifunctional vector which can deliver genes and carry anti-cancer drugs. In this work, a flexible two-step method was first developed to introduce the atom transfer radical polymerization (ATRP) initiation sites containing disulfide bonds onto GO surfaces. Surface-initiated ATRP of (2-dimethyl amino) ethyl methacrylate (DMAEMA) was then employed to tailor the GO surfaces in a well-controlled manner, producing a series of organic-inorganic hybrids (termed as SS-GPDs) for highly efficient gene delivery. The GO sheets and SS-GPD nanoparticles were characterized by X-ray photoelectron spectroscopy (XPS) and atom force microscopy (AFM) respectively. Compared with the pristine GO sheets and PDMAEMA homopolymers, well-defined cationic SS-GPDs consisting of GO backbones and disulfide-linked cationic PDMAEMA chains were expected to provide much more flexibility to condense pDNA, lower cytotoxicity and higher transfection efficiency. Under reducible conditions, the PDMAEMA side chains can be readily cleavable from the GO backbones, benefiting the resultant gene delivery process. Moreover, due to the conjugated structure of the graphene basal plane, SS-GPD can attach and absorb aromatic, water insoluble drugs, such as 10-hydroxycamptothecin (CPT), producing SS-GPD-CPT. The MTT assay and the simultaneous double staining procedure revealed that SS-GPD-CPT possessed a high potency of killing cancer cells in vitro. With a high aqueous solubility and coulombic interaction with cell membrane, SS-GPDs may have great potential in gene and drug delivery fields.Cardiovascular disease remains the primary cause of morbidity and mortality worldwide. Despite the therapeutic benefits of numerous treatment options, including statins, angiotensin-converting enzyme inhibitors, beta blockers and other drugs, the prevalence of cardiovascular disease continues to increase, underscoring the need for new therapeutic strategies. Recently, research results show that downregulation of the miR-29 family has been shown to be associated with the pathogenesis of tissue scarring including ischemic heart disease. Overexpression of miR-29b is capable of attenuating fibrosis in chronic heart disease and lung fibrosis. Our previous research has shown that EA functionalization of PGMA (PGEA) has a large amount of secondary amine groups and nonionic hydroxyl units which bring it good gene transfection efficiency and low cytotoxicity. Special shape (like a star or comb) cationic polymer compared with the same molecular weight linear polymers, have lower cytotoxicity and higher transfection efficiency. Using the pentaerythritol (PER) functionalized by BIBB as the initiator, through ATRP and the subsequent EA functionalization as we mentioned before, we compound the star-like PGEA (s-PGEA) gene carrier. The s-PGEA carrier can form uniform nanoparticles with miR-29b. Through intravenous injection, miR-29b can be successfully brought into the heart of mice and effectively alleviate angiotensin Ⅱ-mediated cardiac fibrosis in vivo. In addition, in comparison with PEI, s-PGEA vectors possessed longer circulation time in vivo and better therapeutic effect due to its good biocompatibility and protein resistance ability.To sum up, grafting methacrylate polymers on different functional matrix material through atom transfer radical polymerization method is of great significance for the construction of novel multifunctional cationic gene carrier.