Tsai, Jeannette Ti-hua;
(1999)
Characterisation of non-viral DNA complexes for gene therapy.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Complexes formed between DNA and cationic polymers or lipids are attracting interest as gene delivery vehicles suitable for gene therapy and DNA vaccines. To better understand the structures formed by the interaction of DNA and polycations several techniques, which include the photon correlation spectrophotometry (PCS), zeta potenrial and fluorimetry, were used to characterise such DNA complexes. DNA and poly-L-lysine were normally found to form small particles of 100±20 nm. Aggregation, where particles were measured to have high polydispersity and large diameters of >1000 nm, was occasionally observed especially at charge ratio (+/-) close to 1. Mixing conditions were observed to be important and a syringe pump was designed where the DNA and polycation solutions were mixed under controlled conditions at the junction of a T-connector. The zeta potential of poly-L-lysine (PLL)/DNA complexes was found to be strongly negative (-40±5 nm) at low charge ratios (+/-) and positive at high charge ratios. Poly-L-lysine of 4 molecular weights (2900, 9600, 25900 and 99500) was used in this study and all expect the smallest one were able to condense DNA efficiently. Fluorimetry and gel exclusion studies further provided methods to assess the effectiveness various polycations interacted with DNA. Small molecules such as spermidine and PLL (m.w. 2900) and the cationic liposome DC-chol/DOPE (6:4) were found to be less efficient in forming complexes with DNA. Complexed DNA was also found to be resistant against shear and DNase degradation. The techniques developed here offer ways to characterise DNA complexes which will be crucial in the production and quality control of therapeutic materials for pharmaceutical use. Large-scale techniques were investigated for the production of poly-L-lysine condensed anionic liposome delivery system. A one-step process for producing liposomes did not appear feasible although scaleable homogenisation could be effective in producing small unilamellar liposomes (SUVs).
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Characterisation of non-viral DNA complexes for gene therapy |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10099818 |
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