Detecting Vulnerable Atherosclerotic Plaques by ⁶⁸Ga-Labeled Divalent Cystine Knot Peptide

Integrin α_vβ₃ has been considered as a promising biomarker for vulnerable atherosclerotic plaques, and it is highly expressed by those instability-associated factors, such as macrophages, vessel endothelial cells, and smooth muscle cells. Our previous study successfully showed that the ⁶⁴Cu-labeled divalent (containing two RGD motifs) cystine knot peptide, ⁶⁴Cu-NOTA-3-4A, had high binding affinity and specificity in targeting vulnerable carotid atherosclerotic plaques with increased α_vβ₃ levels. Therefore, considering that ⁶⁸Ga has excellent nuclear physical properties for positron emission tomography (PET), this study aimed to investigate the feasibility of using ⁶⁸Ga-NOTA-3-4A for PET study of vulnerable atherosclerotic plaques. The vulnerable carotid atherosclerotic plaques were induced and maintained in ApoE^-/- mice through carotid artery ligation and a high-fat diet. Divalent knottin peptide 3-4A was synthesized through solid-phase peptide synthesis chemistry and radiolabeled with ⁶⁸Ga after being conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The probe stability was analyzed in phosphate buffered saline (PBS) buffer and mouse serum. ApoE^-/- mice with atherosclerotic plaques ( n = 4) were imaged by PET/CT at 1 and 2 h after the tail vein injection of ⁶⁸Ga-NOTA-3-4A. The targeting specificity was determined by coinjection of ⁶⁸Ga-NOTA-3-4A and nonradioactive c(RGDyK) peptide. The carotid artery tissues were removed, and immunofluorescent staining was performed to evaluate α_vβ₃ integrin expression. It was found that ⁶⁸Ga-NOTA-3-4A displayed high stability in both PBS buffer and mouse serum. Small animal PET/CT images and quantification analysis indicated the quick and high plaque uptake of ⁶⁸Ga-NOTA-3-4A (6.67 ± 1.44 and 2.97 ± 0.46%ID/g at 1 and 2 h, respectively). The plaque-to-normal artery ratio was 15.88 and 9.90 at 1 and 2 h, respectively. Furthermore, the plaque accumulation of ⁶⁸Ga-NOTA-3-4A was significantly inhibited via coinjection of c(RGDyK). Finally, immunostaining identified integrin α_vβ₃ expressed by macrophages, vessel endothelial cells, and smooth muscle cells. In summary, ⁶⁸Ga-NOTA-3-4A has high potential to be a promising PET tracer for imaging vulnerable atherosclerotic plaques.