64Cu-Labeled Divalent Cystine Knot Peptide for Imaging Carotid Atherosclerotic Plaques

The rupture of vulnerable atherosclerotic plaques that lead to stroke and myocardial infarction may be induced by macrophage infiltration and augmented by the expression of integrin alpha(v)beta(3). Indeed, atherosclerotic angiogenesis may be a promising marker of inflammation. In this study, an engineered integrin alpha(v)beta(3)-targeting PET probe, Cu-64-NOTA-3-4A, derived from a divalent knottin miniprotein was evaluated in a mouse model for carotid atherosclerotic plaques. Methods: Atherosclerotic plaques in BALB/C mice, maintained on a high-fat diet, were induced with streptozotocin injection and carotid artery ligation and verified by MR imaging. Knottin 3-4A was synthesized by solid-phase peptide synthesis chemistry and coupled to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) before radiolabeling with Cu-64. PET probe stability in mouse serum was evaluated. Mice with carotid atherosclerotic plaques were injected via the tail vein with Cu-64-NOTA-3-4A or F-18-FDG, followed by small-animal PET/CT imaging at different time points. Receptor targeting specificity of the probe was verified by coinjection of c(RGDyK) administered in molar excess. Subsequently, carotid artery dissection and immunofluorescence staining were performed to evaluate target expression. Results: Cu-64-NOTA-3-4A was synthesized in high radiochemical purity and yield and demonstrated molecular stability in both phosphate-buffered saline and mouse serum at 4 h. Small-animal PET/CT showed that Cu-64-NOTA-3-4A accumulated at significantly higher levels in the neovasculature of carotid atherosclerotic plaques (7.41 +/- 1.44 vs. 0.67 +/- 0.23 percentage injected dose/gram, P < 0.05) than healthy or normal vessels at 1 h after injection. F-18-FDG also accumulated in atherosclerotic lesions at 0.5 and 1 h after injection but at lower plaque-to-normal tissue ratios than Cu-64-NOTA-3-4A. For example, plaque-to-normal carotid artery ratios for F-18-FDG and Cu-64-NOTA-3-4A at 1 h after injection were 3.75 and 14.71 (P, 0.05), respectively. Furthermore, uptake of Cu-64-NOTA-3-4A in atherosclerotic plaques was effectively blocked (similar to 90% at 1 h after injection) by coinjection of c(RGDyK). Immunostaining confirmed integrin alpha(v)beta(3) expression in both the infiltrating macrophages and the neovasculature of atherosclerotic plaques. Conclusion: Cu-64-NOTA-3-4A demonstrates specific accumulation in carotid atherosclerotic plaques in which macrophage infiltration and angiogenesis are responsible for elevated integrin alpha(v)beta(3) levels. Therefore, Cu-64-NOTA-3-4A may demonstrate clinical utility as a PET probe for atherosclerosis imaging or for the evaluation of therapies used to treat atherosclerosis.