Site-Specific [ ^99m Tc]-Labeled Nanobody Tracer for SPECT Imaging of P2X7 Expression in Atherosclerotic Plaques

Purinergic receptor P2X7 has been considered as a potential new target for detecting and treating high-risk plaque. Nanobodies are the smallest antibody fragments with high antigen binding ability and specificity, which are well-suited for radionuclide imaging. The present study aimed to develop a novel P2X7-targeted nanobody SPECT tracer and to investigate its potential for identification of atherosclerotic plaque (AP). The anti-P2X7 nanobody 1c81 was site-specifically conjugated with [^99mTc]Tc-HYNIC-GGGC via sortase A-mediated transpeptidation to prepare [^99mTc]Tc-1c81. Saturation binding experiments and cell-binding assays were performed to evaluate their affinity and specificity. Biodistribution studies in C57 mice were conducted at 0.5, 1, and 2 h postinjection (p.i.), and SPECT/CT imaging was performed in ApoE^-/- (high-fat diet) and C57 mice (normal diet) at 2 h p.i, respectively. Specific binding was validated by blocking studies (coinjection of [^99mTc]Tc-1c81 with excess unlabeled 1c81) in ApoE^-/- mice. Target-to-background ratio (TBR) was calculated for AP at aortic arch. The harvested aortas were analyzed by autoradiography, Oil Red O lipid staining, and immunofluorescence staining (CD68, P2X7) to correlate tracer uptake with AP characteristics. To further validate the clinical relevance, human coronary endarterectomy (CE) specimens were analyzed for P2X7 and CD68 expression using immunohistochemistry. [^99mTc]Tc-1c81 was synthesized with 53.77 ± 0.06% radiochemical yield, > 95% purity, 11.13 ± 2.78 MBq/nmol molar activity, and a binding dissociation constant of 6.38 nM. Biodistribution showed rapid clearance from the blood and normal organs except the kidneys. SPECT imaging at 2 h p.i. revealed clear aortic arch visualization, with significantly higher TBR in ApoE^-/- mice compared to both C57 and blocking groups (4.49 ± 1.88 vs 0.96 ± 0.64, <i>P</i> = 0.012; 4.49 ± 1.88 vs 1.40 ± 0.28, <i>P</i> = 0.017). Autoradiography further confirmed specific tracer accumulation in APs, colocalizing with Oil Red O-positive lipid-rich regions. Immunofluorescence and immunohistochemical staining validated high P2X7 receptor expression in both mouse AP aortic valve sections and human CE specimens, which was colocalized with CD68⁺ inflammatory cells, confirming the clinical relevance of P2X7 as an imaging target for inflammation of AP. [^99mTc]Tc-1c81 exhibited specific binding to the P2X7 receptor in AP <i>in vivo</i>. It may serve as a novel P2X7-targeted SPECT tracer to detect AP, with promising applications in clinical risk stratification and treatment response monitoring.