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Well-Defined Polycation Gene Vectors Via Atrp For Gene Delivery

Posted on:2015-06-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y ZhuFull Text:PDF
GTID:1221330467971171Subject:Materials Science and Engineering
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Gene therapy shows much promise in therapies for various genetic diseases and cancers, viral infection, and cardiovascular disorders. For efficient gene therapy, a gene carrier or vector is needed to escort negatively charged nucleic acids through cell membranes. The most challenging task in gene therapy is the design of gene delivery vectors with low cytotoxicity and high transfection efficiency. Cationic polymers as the major type of non-viral gene delivery vectors show low host immunogenicity and high flexibility, and can be produced on a large scale.ATRP allows the preparation of well-controlled polymers of narrow molecular weight distribution, predetermined by the concentration ratio of the consumed monomer to the introduced initiator. The ATRP process does not require stringent experimental conditions and can tolerate a wide range of functional monomers except acidic monomers. Especially, this technique is less sensitive toward residual traces of oxygen. ATRP has been widely employed for the design of new polymeric gene vectors with different polymer architectures.Herein different ethanolamine(EA)-and ethylenediamine (ED)-functionalized PGMA (termed PGEAED) vectors, as well as ED-functionalized PGMA (termed PGED) vectors, are proposed and compared for efficient gene delivery. In addition to the cationic pendant secondary amine and hydroxyl groups of PGEA, PGEAED, and PGED also contain flanking primary amine groups. PGEA, PGEAED, and PGED were investigate with their ability to condense pDNA, cytotoxicity and transfection efficiency. Moreover, the flanking primary amine groups induced by ED could be readily functionalized by glycyrrhetinic acid or cholic acid to improve the biophysical properties of the gene vectors.HPCD-PGEAs with different ethanolamine-functionalized poly(glycidyl methacrylate)(PGEA) lengths were successfully prepared as gene carriers from the bromoisobutyryl-terminated2-hydroxypropyl-β-cyclodextrin (HPCD). Moreover, HPCD-PGEA/PDM vectors consisting of HPCD-PGEA and poly((2-dimethyl amino)ethyl methacrylate)(PDMAEMA) blocks were further prepared by ’click reaction’ from azide-terminated PDMAEMA and alkyne functionalized HPCD-PGEA to further enhance the gene transfection efficiency. The introduction of biocompatible HPCD into HPCD-PGEA vectors enhanced gene transfection efficiency. HPCD-PGEA/PDM vectors exhibited good ability to complex pDNA, enhanced cellular uptake ability, and improved gene transfection efficiencies in HepG2cell lines.Poly(DL-aspartamide)-based biomaterials with good degradability and excellent biocompatibility could be used as the potential backbones of gene vectors. Atom transfer radical polymerization (ATRP) was proposed to prepare the biocleavable and biodegradable combshaped poly(N-3-hydroxypropyl)aspartamide (PHPA)-based gene carriers. The bioreducible ATRP initiation sites were first introduced onto PHPA backbones. Then, the well-defined comb-shaped vectors (SS-PHPDs) consisting of degradable PHPD backbones and disulfide-linked cationic P(DMAEMA) side chains were produced for gene delivery. The P(DMAEMA) side chains were readily cleavable from the backbones under reducible conditions. The degradability of PHPA backbones would benefit the final removal of the gene carriers from the body.The ability to manipulate and control the surface properties of hydroxyapatite (HA) nanoparticles is of crucial importance for the design of HA-based carriers of therapeutic agents.Surface initiated atom transfer radical polymerization (ATRP) of (2-dimethyl amino)ethyl methacrylate (DMAEMA) is first employed to tailor the functionality of HA surfaces in a well-controlled manner and to produce a series of new cationic hybrids (termed as HA-PDM). The HA parts of HA-PDM were coated by different lengths of PDMAEMA chains. The HA-PDM exhibited a good ability to condense plasmid DNA (pDNA) with suitable particle size and zeta potential for gene transfection. Most importantly, in comparison with PDMAEMA homopolymers, the HA-PDM displayed considerably enhanced buffering capacity, and exhibited much higher gene transfection efficiencies in different cell lines, including osteoblast MC3T3and osteosarcoma MG63cells. In addition, good in vitro results of the ALP activity and calcium deposition osteogenic markers indicated that the HA-PDM/pDNA complexes could also greatly stimulate the differentiation of preosteoblast cells.
Keywords/Search Tags:gene therapy, cationic polymer gene delivery, ATRP, PGMA, PDMAEMA, 2-hydroxypropyl-β-cyclodextrin, poly(DL-aspartamide), hydroxyapatite
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