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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628115 |
| Date | January 2012 |
| Creators | Betkaoui, Radia |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://kclpure.kcl.ac.uk/portal/en/theses/cationic-lipid-formaulation-of-short-interference-rna-sirna-for-delivery-to-respiratory-epithelial-cells(9868d81f-3986-41d2-a3a4-a06f6b36a46b).html |
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