Assessment of Ultrasmall Nanocluster for Early and Accurate Detection of Atherosclerosis Using Positron Emission Tomography/Computed Tomography

Introduction: Researches have indicated the potential of chemokine receptor 2 (CCR2) as a valuable target for atherosclerosis imaging and therapy. Ultrasmall nanocluster has attracted significant interest for biomedical applications due to the favorable pharmacokinetics and accurate targeting. To improve the targeting efficiency and imaging contrast for CCR2 imaging, we prepared CCR2 targeted nanocluster to assess the initiation, progression, and regression of atherosclerosis in ApoE^-/- mouse models.

Methods: ApoE^-/- mice fed on high fat diet (HFD) were used as progressive atherosclerosis model. The regression model was established by transplanting aortic arches from ApoE^-/- mice to syngeneic C57BL/6 wildtype (B6 WT) recipients fed on normal chow. Gold nanoclusters intrinsically radiolabeled with ⁶⁴Cu and conjugated with CCR2 targeting peptide ECL1i were synthesized for CCR2 PET imaging in the atherosclerosis models. Histopathology and real-time PCR (RT-PCR) assay were carried out on the aortic arteries. Results: ⁶⁴Cu-AuNCs-ECL1i in WT mice showed rapid blood and renal clearance, as well as low liver and spleen uptake due to ultrasmall size. At 4 weeks post HFD in ApoE^-/- mice, PET/CT image showed a strong signal at aortic arch (SUV=0.62±0.06, n=4), which gradually increased overtime to SUV=1.32±0.13 (n=4) at 35 weeks post HFD, significantly higher than those acquired at 4-, 12-, 17-, and 21-weeks post HFD. By contrast, non-targeted ⁶⁴Cu-AuNCs in ApoE^-/- mice and ⁶⁴Cu-AuNCs-ECL1i in WT mice both showed minimal tracer uptake, indicating ⁶⁴Cu-AuNCs-ECL1i targeting specificity to detect the progression of plaques. Furthermore, the ApoE^-/-/WT PET uptake ratios showed correlation with ApoE^-/-/WT CCR2 RT-PCR ratios determined from aorta arches, suggesting the CCR2 targeting specificity of as-developed nanoclusters along the progression of plaques. After aorta arch transplantation from ApoE^-/- mice to B6 WT recipient, the tracer uptake in the aortic arch graft decreased from SUV=1.08±0.14 (n=4) to SUV=0.69±0.08 (p<0.001, n=4) at 2 months and SUV=0.46±0.08 (p< 0.01, n=4) at 4 months post transplantation. By contrast, minimal uptake was determined in neither the native aortic arch nor the aortic arch graft in the B6→B6 transplantation model, suggesting little contribution to the tracer uptake from the surgical procedures, which further confirmed the sensitivity of ⁶⁴Cu-AuNCs-ECL1i tracking the regression of atherosclerosis. Histopathological characterization demonstrated the upregulation of CCR2 in atherosclerotic plaques and association with CD68 macrophages. Conclusion: We developed a CCR2 targeted ⁶⁴Cu-AuNCs-ECL1i with rapid in vivo pharmacokinetics and clearance. PET/CT in ApoE^-/- mice showed sensitive and specific detection of CCR2 along the dynamic variation of plaques during the progression and regression of atherosclerosis. The results demonstrated its promising potential for translational imaging of human atherosclerosis. Acknowledgements: This work was supported by 1R35HL145212 from the National Heart, Lung, and Blood Institute of the National Institutes of Health.