M. A. Wasserman1,2,3, J. S. Rink1, C. S. Thaxton1,4, M. R. Kibbe1,2,3 1Simpson Querrey Institute For BioNanotechnology,Chicago, IL, USA 2Northwestern University,Department Of General Surgery,Chicago, IL, USA 3Northwestern University,Department Of Vascular Surgery,Chicago, IL, USA 4Northwestern University,Department Of Urology,Chicago, IL, USA
Introduction:
Current surgical and endovascular therapies for severe atherosclerotic disease often fail due to the development of neointimal hyperplasia with resultant arterial restenosis and occlusion. The objective of this study is to evaluate the targeting specificity, cellular binding, and internalization patterns of a novel synthetically engineered targeted delivery vehicle that can subsequently be designed to deliver a therapeutic agent to prevent arterial restenosis. Our hypothesis is that the targeted gold nanoparticle will bind with specificity to the site of arterial injury and demonstrate cellular binding and internalization to the cells that comprise the vascular wall.
Methods:
A citrate-stabilized 13 nm gold nanoparticle (AuNP) was surface functionalized with a molecular fluorophore and a collagen-binding peptide (CBP) or a scrambled peptide (SCR). In vivo assessment of targeting specificity was accomplished utilizing the rat carotid artery balloon injury model in 10-week-old male Sprague-Dawley rats. After balloon angioplasty of the left carotid artery, systemic injection of the AuNP into the inferior vena cava was performed with either the CBP-AuNP (15-60 nM, n=14) or the SCR-AuNP (30-60 nM, n=7). Arteries were harvested 20 minutes post injection. Binding was detected using fluorescent microscopy. The right carotid artery served as the control (n=21). In vitro determination of cellular binding and internalization was performed on rat aortic endothelial cells (EC), adventitial fibroblasts (AF), and smooth muscle cells (SMC) exposed to 50 nM of the AuNP. Fluorescent uptake was quantified at 4 and 24 hours using the Cytation 3 Cell Imaging Multi-Mode Reader. Statistical analysis was performed using ANOVA on Ranks.
Results:
In vivo, the collagen-targeted CBP-AuNP bound to the site of arterial injury and brightly fluoresced, with the most prominent signal at 45 nM. No binding was noted to the contralateral, uninjured right carotid artery. Rats injected with the SCR-AuNP expressed no fluorescent signal in either the right or left carotid arteries. In vitro, the AuNP bound to all three cell types and fluorescence significantly increased over time (EC p<0.001, AF p<0.001, SMC p<0.001), suggesting internalization within the cell. Among the three cell types, the greatest fluorescence was noted with EC (2.0-fold increase vs. 1.6-fold AF and 1.3-fold SMC at 24 hours).
Conclusion:
Our collagen-targeted AuNP binds with specificity to the site of vascular injury and demonstrates binding to and internalization within the cells that comprise the vascular wall. These studies serve as the foundation for further evaluation and optimization of our delivery vehicle for the vasculature. Ultimately, our goal is to systemically administer a therapeutic agent in a targeted manner to prevent neointimal hyperplasia and arterial restenosis following vascular interventions.