Synthesis And Characterization Of Acid-labile Poly(Ortho Ester Amino Alcohols) For Gene Delivery

Gene therapy, as a new way of treating disease to cure completely the inherited or acquired diseases through delivering therapeutic genes to patient tissues and cells and replacing or repairing the disorder genes, has attracted more and more attention. Nowadays,those diseases that seriously threaten human life could be cured by gene therapy, such as cancer, hemophilia, cardiovascular disease, and viral infection, etc. However, the main obstacles to gene therapy are the lack of safe and highly efficient gene delivery vectors. Compared to viral vectors, cationic polymers as a major category of non-viral gene vectors have displayed many advantages including high security, flexibility of design, and ease of large-scale production. So far, a large variety of polycationic carriers such as poly(2-dimethylaminoethylmethacrylate) (pDMAEMA), polyethylenimine (PEI) and poly-L-lysine (pLL) have been developed to efficiently condense DNA into nanoparticles, and exhibit relatively high transfection efficiency in in vitro experiments. However, high molecular weight (Mw) and highly positive charge densities of these non-degradable cationic polymers also lead to increased cytotoxicity and restricted DNA release. In addition, these polymeric vectors/DNA complexes failed to exhibit similarly high transfection efficiency in in vivo due to their non-specific interaction with blood components.The aim of this study is to prepare a new type of linear cationic copolymers--poly(ortho ester amino alcohols) (POEAAs). POEAAs has been synthesized by ring-opening polymerization between amine monomer with ortho ether bond (AOE) and ethylene glycol diglycidyl ether (EGDE) and poly(ethylene glycol) diglycidyl ether (PEGDE), respectively. POEAA1 and 2 containing different diglycidyl ether moieties were prepared to regulate their hydrophilicity and DNA condensation ability. The uniform distribution of cationic tertiary amines in main chains and acid-cleavable cationic tertiary amines in side chains were expected to precisely protect and control DNA release from the POEAAs/DNA polyplexes with improved gene transfection efficiency. In detail, the cleavage of acid-triggered cationic tertiary amines in side chains would facilitate DNA to escape from endosome and to release in cytosol, while the cationic tertiary amines intact in main chains would still somewhat condense and prevent plasmid DNA from too fast release and degradation by enzymes. Abundant hydroxyl groups in main chains of POEAAs were expected to be able to improve biocompatibility and serum-tolerant transfection efficiency. The following works were finished mainly in this thesis:In Chapter 1, the overview of gene therapy and gene delivery system was introduced. The commonly used non-viral gene delivery systems and stimuli-responsive polycationic vectors were reviewed. The obstacles of gene delivery mediated by polycationic gene vectors were described, and the solution was put forward.In Chapter 2, POEAA1 and POEAA2, were synthesized by ring-opening polymerization between AOE and EGDE and PEGDE, respectively. The structure, molecule weight and polydispersity of POEAAs were characterized, and pH-dependent hydrolysis of POEAAs was studied. Ortho ester hydrolysis of POEAAs followed the exocyclic path and the degree of hydrolysis increased with extension of time.In Chapter 3, the binding capacity of POEAAs/DNA complexes were evaluated. The stability of POEAAs/DNA polyplexes in the presence of serum components was tested. The particle sizes and zeta-potentials of POEAAs were measured by dynamic light scattering assay (DLS).Whether the hydrolysis of ortho ester side-chains of POEAAs regulates the release of DNA has been investigated. Results indicated that POEAAs exhibited good DNA binding ability. POEAA2 with longer ethylene glycol bridge in the main chain displayed stronger condensation strength towards DNA than POEAA1. The sizes of POEAA1/DNA and POEAA2/DNA complexes decreased with increase of N/P ratios, and the average particle sizes of POEAAs/DNA stabilized around 150-220nm. The zeta-potentials of POEAAs/DNA complexes increased with increase of N/P ratios and stabilized at around 9 and 23mV, respectively. Compared to 25 KDa bPEI/DNA complexes, POEAAs/DNA complexes showed stronger anti-enzyme ability, anti-polyanionic ability, and adsorption resistance ability, which thus were demonstrated to have excellent stability in the presence of serum components. POEAAs/DNA complexes were stable under physiological condition and released DNA under mild acid condition. The DNA release rate of POEAAs increased with extension of time and POEAA1/DNA polyplexes at acid pHs released faster and more DNA than POEAA2/DNA.In Chapter 4, cytotoxicity of POEAAs was evaluated on 293T cells. The transfection efficiency of POEAAs and the effect of serum condition were detected. Compared to 25 KDa bPEI as positive control, POEAAs showed much lower cytotoxicity even when the concentration was high as 1 mg/ml. In addition, POEAA1 with lower positive charge showed slightly higher cell viability than POEAA2. Compared to 25 KDa bPEI with 26.3% percentage of positive cells, POEAAs showed lower transfection efficiency (4.12% percentage of positive cells) in the absence of serum. Interestingly, the percentage of positive cells induced by POEAAs at the optimal N/P ratio were almost equal to that of 25 KDa bPEI, and even obviously increased to be as high as 4.93% in the presence of 10% serum.In conclusion, POEAAs possessed the precisely regulation of DNA release, excellent biocompatibility, and improved serum tolerance.So POEAAs have great potential to be good gene delivery vectors for in vivo research and application.