Synthesis And Application Of Novel Cationic Copolymers As Nonviral Gene Vectors

Gene therapy can be defined as the treatment of human disease by the transfer of genetic material into specific cells of the patient. Over the past decades, advances in molecular biology and biotechnology, and the completion of the Human Genome Project, have led to the identification of numerous disease-causing genes, which makes it possilble to the treatment of genetic diseases such as haemophilia, muscular dystrophy, cystic fibrosis or cancer by the replacement of errant genes within the affected cells. Gene therapy has been received more and more attention, but the absence of an efficient delivery system has been identified one of major obstacles in progress of gene therapy. Generally, gene-delivery vehicles can be divided into two categories:recombinant viruses and non-viral vectors. Viral vectors have been employed in clinical trials for their high transfection efficiency. However, the drawbacks in safety, target-cell specificity and the costs of manufacturing viral-based gene therapies limit their applications in clinical trial. Non-viral vectors provide opportunities for improved safety, greater flexibility and more facile manufacturing. Nowadays, polycations as non-viral gene delivery vectors have been the hotspot in this field.Polycations have been identified as the promising non-viral gene delivery vectors, but their poor gene-transfer efficiency and high cytotoxicity have limited their further application. Inefficient endosomal release, cytoplasmic transport and nuclear entry of plasmids are amongst some of the key limiting factors in the use of polycations as effective non-viral gene delivery vectors. Therefore, it is important to design novel polycationic vectors with little cytotoxicity and high delivery efficiency for gene therapy.In chapter 1, we summarize the progress in gene therapy and list a detailed review of the main polycationic vectors. We also explain the barriers along the plasmid DNA delivery pathway and summarize the corresponding solutions. In the next five chapters, we design and perpare a series of novel polycations as gene vectors. Their physicochemical and biological properties are also evaluated.In chapter 2, well-defined diblock copolymers, poly(ethylene glycol)-block-poly (glycidyl methacrylate)s (PEG-PGMAs), with different poly(glycidyl methacrylate) (PGMA) chains, were prepared via atom transfer radical polymerization (ATRP) from the same macromolecular initiator 2-bromoisobutyryl-terminated poly(ethylene glycol). Ethyldiamine (EDA), diethylenetriamine (DETA), triethylenetetramine (TETA), and polyethyleneimine (PEI) with an Mw of 400 (PEI400) were used to decorate PEG-PGMAs to get the cationic polymers PEG-PGMA- oligoamines. The results of 1H NMR and elemental analysis demonstrated inter or intra PEG-PGMA cross-linking occurred when oligomines were grafted, and cross-linking degree might increase with the increase of the length of the oligoamines. These cationic polymers possessed high buffer capability and could condense DNA into nanoscaled complexes of 125-530 nm. These complexes showed the positive zeta potential of 20-35mV at N/P ratios of 10-50. Most of them exhibited low cytotoxicity and good transfection efficiency in 293T cells. The presence of the serum medium did not decrease the transfection efficiency due to the steric stabilization of the PEG chains. The transfection activity of PEG-b-PGMA-oligoamines was accordance with their buffer capability.In chapter 3, in order to improve the transfection activity of PGMA derivatives, well-defined BAB triblock copolymer, poly(glycidyl methacrylate)-b-poly(propylene oxide)-b-poly(glycidyl methacrylate) (PGMA-PPO-PGMA) was prepared via atom transfer radical polymerization (ATRP), and modified by different ratios of ethylenediamine (EDA) and 1-(3-aminopropyl) imidazole (API) to obtain cationic amphiphilic polymers. We hoped that introducing of biocompatible hydrophobic PPO chain could enhance the complex-plasma membrane interactions, and adjusting the ratio of EDA and API on the side chains of PGMA could achieve gene carriers with high transfection efficiency and low toxicity. The ratios of EDA and API could be alculated based on the results of 1HNMR spetra, demonstrated that the reaction between PGMA and oligoamines processed in a quantifiable manner. The difference in the ratio of EDA and API could have influence on DNA binding capability, size, and zeta potential of PGMA-PPO-PGMA derivatives. All the cationic amphiphilic polymers exhibited rather low cytotoxicity and good transfection activity in comparison with 25kDa PEI due to their special architecture. The optimal polymer, with 89% API and 11% EDA, showed the highest transfection efficiency among these polymers. Its luciferase expression at N/P ratio of 30 was comparable to that of 25kDa PEI in serum-free medium and higher than that of 25kDa PEI by roughly an order of magnitude in medium with serum.According to the results in chapter 2 and chapter3, PGMA derivatives showed some special properties. We thought this might be attributed to the influence of hydroxyl groups at the side chains of PGMA derivatives. In chapter 4, poly(aminoethyl methacrylate) (PAEMA), poly(3-amino-2-hydroxypropyl methacrylate) (PAHPMA), poly 2-(2-aminoethylamino)ethyl methacrylate) (PAEAEMA) and poly(3-(2-aminoethylamino) 2-hydroxy propyl methacrylate) (PAEAHPMA) were synthesized using atom transfer radical polymerization to evaluate the effect of hydroxyl groups on the relative properties of cationic polymeric gene vectors. The results of heparin displacement assays showed that PAHPMA possessed a stronger binding capacity than PAEMA. PAHPMA/DNA complexes and PAEAHPMA/DNA complexes had lower zeta potentials than those of PAEMA and PAEAEMA. MTT assay results indicated that PAHPMA and PAEAHPMA exhibited obviously lower cytotoxicities than PAEMA and PAEAEMA. Subsequently, in vitro gene transfection studies in 293T cells without serum showed that PAHPMA exhibited a lower transfection efficiency than PAEMA and PAEAHPMA/DNA complexes possessed a similar transfection efficiency to PAEAEMA/DNA complexes. Moreover, PAHPMA and PAEAHPMA retained similar transfection efficiencies in DMEM with 10% serum, but PAEMA and PAEAEMA showed slightly lower transfection efficiencies than in the absence of serum. The reason for these phenomena might be attributed to the introduction of hydroxyl groups into PAHPMA and PAEAHPMA, i.e. the existence of hydroxyl groups might increase the binding capacity to DNA and at the same time decrease the surface charge of the polymer/DNA complexes due to the formation of hydrogen bonds between the polymers and DNA. Therefore, a lower zeta potential and stronger binding ability may result in lower gene transfection efficiency. This effect of hydroxyl groups decreased with increasing amino group density on the polymer.In chapter 5, amphiphilic triblock copolymers monomethoxyl poly(ethylene glycol) (mPEG)-b-poly(s-caprolactone) (PCL)-b-poly(aminoethyl methacrylate)s (PAMAs) (PEG-PCL-PAEMA) were synthesized as gene delivery vectors. PEG-PCL-PAEMA could form micelles with the hydrophobic PCL core and the hydrophilic shell consisting of nonionic PEG chains and cationic PAMA chain. The micelles showed good DNA binding capability, and could condense DNA into nanoparticles with the size of 320±40 nm and zeta potential of 35±10 mV. The polycations exhibited lower cytotoxicity and higher transfection efficiency in COS-7 cells in presence of serum compared to 25 kDa bPEI. The influence of PEG and PCL segments in PEG-PCL-PAEMA was evaluated by comparing with corresponding diblock copolymers. The studies showed the incorporation of hydrophobic PCL segment in triblock copolymers might make a little effect on the binding capability to DNA and surface charges of complexes due to the formation of micelles increasing the local charges. The presence of PEG segment in gene vector decreased the surface charges of the complexes and increased the stability of the complexes in serum for its steric hindrance. It was also found that the combination of PEG and PCL segments into one macromolecule might lead to synergistic effect for better transfection efficiency.In chapter 6, a novel multiple functional ternary complex biotinylated transferrin-avidin-biotin-poly (ethylene glycol)-poly (gama-benzyl-L-glutamate acid)/ poly(2-(2-aminoethylamino)ethyl methacrylate)/DOX-poly(L-aspartic acid)/DNA was constructed by electrostatic interaction and avidin-biotin bridge for the first time.A series of self-assembled polyionic complexes (PICs) were firstly prepared via electrostatic attraction between PASP (or DOX-PASP) and PAEAEMA. The size of the PICs measured by Nano-ZS ZEN3600 was around 180 nm-240 nm at different weight ratios. At weight ratios of 4/1 and 5/1, PICs showed good DNA binding capability for their high positive surface charge. The cytotoxicity study indicated that PICs had no obvious cytotoxocity compared to PEI, and DOX-PICs could suppress the growth of HeLa cells and HepG2 cells. Luciferase expression study indicated PICs had good transfection ability at lower weight ratios. Based on the high gene expression level and little cytotoxicity, PICs-4/DNA at weight ratio of 15/1 was chosen to form TAB/PICs/DNA ternary complex. The ternary complex was characterized via size and zeta potential measurement and gel retardation assay. Cytotoxicity assay showed TAB/PICs-D/DNA could delivery DOX-PICs through receptor endocytosis way, and luciferase expression assay demonstrated that TAB/PICs-D/DNA could achieve targeted transfection. Furthermore, confocal laser scanning microscopy showed that DOX and gene could be expressed simultaneitily by TAB/PICs-D/DNA. The formation of the ternary complex provides a new approach to construct multiple functional vectors co-delivery drug and gene.