Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.628115
Title: Cationic lipid formaulation of short interference RNA (siRNA) for delivery to respiratory epithelial cells
Author: Betkaoui, Radia
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2012
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Abstract:
Cationic lipids are commonly used and relatively safe vectors for plasmid (pDNA) delivery. In search for an optimal lipid-based formulation for siRNA delivery to the lungs, cationic lipid-based carriers were investigated for the delivery of siRNA in comparison with pDNA delivery. The aim was to determine whether the factors that influence cationic-lipid mediated pDNA delivery would similarly affect siRNA delivery. Plasmid DNA encoding for luciferase and GAPDH siRNA were complexed using three model cationic lipid-based systems; DOTAP:DOPE, DOTAP:DOPE:DMPE-PEG5000, DOTAP:DOPE:protamine. Cationic lipid/pDNA (+/-) charge ratio of 2 or greater complexed pDNA most efficiently, while much higher ratios (≥ 10) were still less efficient in complexing siRNA. pDNA complexes were larger (up to 4 μm) and formed aggregates in physiological buffer, compared to water, whereas siRNA complexes remained small (<300 nm). Gene silencing in bronchial and alveolar cell lines Calu-3 and A549 revealed a dependency on high lipid/siRNA (+/-) charge ratio (> 8) compared to the delivery of pDNA complexes (0.5-1). Confocal microscopy and endocytic inhibitors studies indicated that the cellular uptake of siRNA/lipid complexes was via a temperature-dependent pathway, which lead to the vesicular localisation of siRNA in the peri-nuclear region. Gene silencing activity was not dependent on the endocytosis-mediated uptake of the complexes. In conclusion, important differences in the factors that affect of siRNA versus pDNA delivery were revealed: the optimal properties of gene silencing were different depending on the nucleic acid and the lipid used. DOTAP:DOPE:DMPE PEG5000 and DOTAP:DOPE:protamine at charge ratios 8-10 delivered siRNA most effectively.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.628115  DOI: Not available
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