Abstract 4957: Robust Transfection of Vascular Endothelium for siRNA Knockdown of Vascular Cell Adhesion Molecule-1 Through a Novel Nanoparticle-Lipid Raft Pathway
Controlling adhesion molecule expression as an antiatherosclerotic therapy is a heretofore untested concept that could modify disease progression and/or interdict acute coronary syndromes.
Methods: To design a synthetic vehicle to deliver vascular cell adhesion molecule-1 (VCAM-1) siRNA to endothelial cells, perfluorocarbon nanoparticles (PFC-NP) were loaded with the cationic lipid 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) in the lipid monolayer to form transfection complexes (10 pM PFC-NP + 5 nM siRNA) with VCAM-1 specific siRNA. Mouse 2F2B endothelial cells were treated with TNFα (10 ng/ml) to induce VCAM-1 expression and after 48h, mRNA and protein levels were measured. For endocytosis colabeling studies, transfection complexes were produced with fluorescently labled siRNA (red) and incubated with endothelial cells in the presence of fluorescently labeled pathway specific markers (green, macropinocytosis: dextran, clathrin: transferrin, lipid raft: cholera toxin-B).
Results: PFC-NP mediated siRNA transfection led to a significant decrease in both VCAM-1 mRNA and protein levels(A) . siRNA exhibited little intracellular colocalization with common markers of both macropinocytosis (B) and clathrin mediated endocytosis (C), while abundant membrane colocalization with lipid rafts was observed(D).
Conclusion: Because endothelial cells harbor an excess of lipid rafts in their plasma membranes, the development of an effective targeted nanoparticle carrier for siRNA that utilizes lipid raft transport rather than traditional endocytotic pathways could represent a valuable strategy for controlling vascular inflammation in atherosclerosis.
This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).