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

摘要

Cationic lipids are commonly used and relatively safe vectors for plasmid(pDNA) delivery. In search for an optimal lipid-based formulation for siRNAdelivery 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 complexedusing 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 muchhigher ratios (≥ 10) were still less efficient in complexing siRNA. pDNA complexeswere 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 mosteffectively.