Abstract 425: Development of a Vascular Cell Adhesion Molecule-1 Targeted Esterase Nanofiber that Binds Activated Endothelial Cells

Abstract

Introduction: Novel therapies capable of directly targeting and regressing atherosclerotic plaques would provide tremendous benefit to patients with atherosclerotic cardiovascular disease. Molecular imaging studies have shown the feasibility of targeting vascular adhesion molecule (VCAM-1) for non-invasive atherosclerosis imaging. Histidine contains an imidazole ring with esterase activity that can convert cholesterol esters to free cholesterol. Thus, we hypothesize that a peptide amphiphile (PA) functionalized with a VCAM-1 binding peptide and histidine will spontaneously form a targeted nanofiber that binds activated endothelium and liberates free cholesterol from cholesterol esters. Methods: PAs were synthesized by solid phase methods and purified by reversed phase HPLC. Three different VCAM-1 binding peptide sequences were conjugated to the PAs (free N-terminus, free C-terminus, and cyclic). To assess 3-dimensional structure of the co-assembled PAs, cryogenic transmission electron microscopy (TEM) was used. To assess targeting of the VCAM-PAs (50μM), human umbilical endothelial cells (HUVECs) were stimulated with TNFα (10ng/ml). To assess the ability of the histidine-PA to liberate free cholesterol, a modified Amplex Red Cholesterol Assay was used. Results: Using TEM, we demonstrated that the co-assembled VCAM- and histidine-PAs formed characteristic nanofibers with diameters of 5-8nm and lengths ranging from 300nm to 1μm. TNFα-activated HUVECs were confirmed to express VCAM-1 protein by Western blot analysis. Confocal imaging revealed that the VCAM-1 binding peptide with a free N-terminus conjugated to the PA had the greatest binding affinity to the TNFα-activated HUVECs. Co-incubation of the histidine-PA with cholesterol esters demonstrated that free cholesterol was liberated from cholesterol esters in a concentration-dependent manner with a five-fold increase in free cholesterol release with 25μM of PA versus 1μM. Conclusion: We have designed and characterized a VCAM-1 targeted nanofiber that successfully binds activated endothelium and generates free cholesterol from cholesterol esters. This novel therapeutic nanotechnology has the capacity to target and regress atherosclerotic plaques.