Cationic lipid formaulation of short interference RNA (siRNA) for delivery to respiratory epithelial cells

Cationic lipids are commonly used and relatively safe vectors for plasmid<br/>(pDNA) delivery. In search for an optimal lipid-based formulation for siRNA<br/>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.<br/>Plasmid DNA encoding for luciferase and GAPDH siRNA were complexed<br/>using three model cationic lipid-based systems; DOTAP:DOPE,<br/>DOTAP:DOPE:DMPE-PEG5000, DOTAP:DOPE:protamine. Cationic lipid/pDNA<br/>(+/-) charge ratio of 2 or greater complexed pDNA most efficiently, while much<br/>higher ratios (≥ 10) were still less efficient in complexing siRNA. pDNA complexes<br/>were larger (up to 4 μm) and formed aggregates in physiological buffer, compared to water, whereas siRNA complexes remained small (&lt;300 nm). Gene silencing in bronchial and alveolar cell lines Calu-3 and A549 revealed a dependency on high lipid/siRNA (+/-) charge ratio (&gt; 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<br/>effectively.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2012

Author dc:creator

• Betkaoui, Radia

Advisors dc:contributor.advisor

• Forbes, Benjamin John
• Patel, Nilesh

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/9868d81f-3986-41d2-a3a4-a06f6b36a46b

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/9868d81f-3986-41d2-a3a4-a06f6b36a46b