| Summary: | 碩士 === 國立臺灣科技大學 === 化學工程系 === 96 === Gene therapy has been deemed its potential to treat a lot of types of diseases. A major aim of gene therapy is efficient delivery and release of therapeutic gene into specific target cells. At the present, viral vectors have more significantly efficient gene expression than non-viral vectors. However, viral vectors has cruelly limited such as immune response and toxic reaction in vivo gene delivery system. Non-viral vectors are not expected to induce specific immune response and therefore are looked to as the future of gene delivery system. Non-viral vectors include lipid-based encapsulation of DNA as well as the delivery of naked DNA into cells. Furthmore, DNA is an anionic polymer and cationic liposomes are capable of conjugating DNA by condensation. Therefore, cationic liposomes are potentially important non-viral vectors and widely used for gene delivery system. More recently, liposomes carrying PEG on their exterior have ability to avoid uptake by reticuloendothelial system and can effectively stabilize lipid membranes. The purpose of my study is used PEG conjugate liposomes by metal ion affinity and investigated different types of modification affected gene transfer efficiency in Chinese hamster ovary cell.
The aim of this study, on one hand was synthesized novel polymer and its physical and chemical characterization of such metal affinity, therefore, we used NTA-chelator coupled to PEG; and on the other hand was grafting of single histidine functionality in molecular structure of different helper lipid, DSPE and DOPE, respectively. In the experiment, liposomes consisted of cationic lipid, DOTAP, in presence of DSPE, DOPE, DSPE-His, DOPE-His as helper lipid, respectively and the pEGFP-C1 as reported gene. PEG-NTA was linked to vector that employed two approaches to PEGylate liposomes by metal ion affinity. In addition, the different liposomal formulations were further characterized by the flow cytometry analysis and fluorescent microscopy.
Pre-PEGylation of liposomes by metal ion affinity was able to promote transfection efficiency, whereas Post-PEGylation decreased efficiency. Two types of PEGylation were reduced electric charge of liposomes and raised relative stable of particle size. Compared with unmodified DNA/liposomes complexes（lipoplexes）, DOTAP/DSPE and DOTAP/DOPE, grating of histidine onto helper lipid was found to increase zeta potential, DNA delivery and cellular viability, the DOTAP/DOPE-His lipoplexes increased the transfection activity about 1-fold.
This results showed that PEGylation of liposomes provide better stable of struction than unPEGylation, but Post-PEGylation of liposomes caused to inhibit nucleic acid escape into cytosol. The conjugation of histidine onto helper lipid enhances transgene expression contrast with unmodified liposomes.
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