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Title: Synthesis and biophysical studies of cationic lipids as gene delivery vectors
Author: Ho, J. K. W.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2012
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Non–viral gene vectors exhibit several desirable properties, over their viral counterparts. This has generated vast interest in the development of efficient non–viral vectors for DNA in gene therapy. The first generation lipid/peptide/DNA (LID) vector was comprised of Lipofectin™, a 1:1 mixture of DOTMA and DOPE, together with a targeting peptide. The resulting LID vector exhibited significant advantages over cationic liposome vectors (lipoplex systems) for gene delivery, transfecting a range of cells with high transfection efficiencies. The LID vector is introduced, and its advantages are outlined. The results and discussion starts in Chapter 2, as we begin our investigation to understand the structural requirements that may lead to enhanced transfection efficiency. The lipid component in the LID vector is described, together with a systematic strategy to enhance transfection activity in the LID vector via new lipid design. The synthesis of a range of C14:1 cationic lipids is described. We have also synthesised asymmetric cationic lipids and lipids containing a short poly(ethylene glycol) (PEG) chain, outlined in Chapter 3. Furthermore, we have synthesised a series of lipids containing a reducible disulfide bridge attached to a PEG moiety, as our strategy is to enhance solubility and shielding during the early stages of cell entry. Once the complex is internalised into the cell, the protective PEG units are shedded, as described in Chapter 4. In addition, the cationic thiol lipid synthesised was coupled to a fluorescent label, to afford a fluorescently–labelled lipid, which has enabled us to track the movement of the lipoplex (lipid:DNA) during transfection. The synthesis towards a cyclic head group for the attachment of different lipid chains is described in Chapter 5. We have utilised a range of synthetic methodologies, including metathesis strategies for the formation of the cyclic lipid precursor. To grasp a greater understanding of how the lipids influence the packaging of DNA, we have studied the biophysical properties of the liposomes and lipoplexes generated at different lipid:DNA mixing ratios, using sizing and zeta potential measurements, given in Chapter 6. The mixing ratios are later used in the design of transfection experiments. Finally, the cationic lipids synthesised in the earlier chapters are formulated into LID complexes, and assessed in a range of different cell lines. The effect of the lipids on transfection efficiency is discussed in Chapter 7. The potential for future work in this area of research is discussed in Chapter 8. A formal description of the experimental procedures and analytical data is presented in Chapter 9.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available