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Biophysical characterisation of plasmid formulation

Sarkar, Supti; (2005) Biophysical characterisation of plasmid formulation. Doctoral thesis , UCL (University College London). Green open access

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Abstract

DNA vaccination and gene therapy offer significant advantages in the treatment of many intractable diseases but many technical challenges must be overcome before the potential of these techniques are realised. A major challenge for non-viral vector particles is their physical instability in physiological conditions. The delivery vector investigated was a Lipid-Peptide-DNA (LPD) complex. The initial system was composed of lipofectin, peptide 6 and a 6.9 kb plasmid DNA. The aim of this work was to study parameters with a view of optimising the biophysical characteristics of the system, namely with regard to storage and transportation. The effects of ionic strength and pH on colloidal stability and structure of the gene delivery vector were investigated using dynamic and static light scattering. Results showed increased levels of aggregation at physiological concentrations (150mM NaCl), although a more stable system was observed in distilled water and at extreme sah conditions of IM. The rates of aggregation were found to be related to the zeta potential of the system and could be predicted using Monte Carlo simulations. Fractal dimension of complexes (where higher values correspond to more compact structures) showed values of 1.6 for 150 mM NaCl and 2.4 for IM NaCl. Lipids, peptides and plasmid DNA were investigated to see the effect of chemistry and physical structure on the aggregation properties of the system. Lipid studies assessing the release of fluorescent calcein from a variety of lipids, over a range of pH found Cl4 Unsaturated + DOPE had the greatest level of release when observed at pH 5.0. The effect of increasing plasmid DNA size (number of base pairs 5.7-72) was also investigated, larger plasmid sizes showed greater stability although further investigation is required to confirm this.

Type: Thesis (Doctoral)
Title: Biophysical characterisation of plasmid formulation
Identifier: PQ ETD:602804
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
UCL classification: UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering
URI: http://discovery.ucl.ac.uk/id/eprint/1446862
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