Serial molecular imaging of the receptor for advanced glycation end-products with multimodal nanoparticle-based targeted probe in preclinical models of hindlimb ischemia

170 Objectives In this study we assessed the feasibility of serial multimodal imaging of the receptor for advanced glycation end-products (RAGE) in preclinical models of hindlimb ischemia using targeted nanoparticle-based PET-optical probe. Methods For our studies, we used previously synthesized, optimized and chemically characterized G4-PAMAM dendrimer-based nanoparticle. The nanoparticle was functionalized with the specific RAGE ligand (Ne-carboxymethyl-lysine, CML), and labeled with both copper-64 (Cu-64) and high-yield fluorophore (AlexaFluor-647) for dual-modality PET-optical imaging. In vivo serial imaging was performed in C57BL6 mice (n=8) at 1, 3, 7 and 14 days after surgical ligation of right femoral artery to induce hindlimb ischemia (HI). To study RAGE expression in animal model of impaired collateral vessel development, we performed in vivo imaging of RAGE in diabetic (n=5) and non-diabetic (n=5) ApoE-/- mice at baseline and 1 week following hindlimb ischemia. All images were analyzed using CT-derived VOIs placed on PET images and specific uptake was expressed as SUV and ischemic-to-nonischemic (I/N) ratio. After last imaging session, mice were euthanized and selected tissues harvested for gamma well counting (GWC), Western blotting and immunofluorescence analysis using cell-specific antibodies for eNOS, CD31 and RAGE. Results GWC analysis demonstrated increased kidney, liver and lung retention paralleled by relative low uptake of RAGE-targeted nanoparticle in other critical organs. Analysis of PET-CT images and confirmed by GWC analysis in C57BL/6 mice subjected to HI demonstrated gradual increase of RAGE-targeted probe accumulation at 1 day (+9%), 3 days (+51%) and 7 days (+94%) followed by significant reduction in probe’s retention at 2 weeks post-HI (+34%). This observation was further supported by the immunohistochemical analysis of RAGE expression. Interestingly, the I/N ratio calculated for dimeric eNOS demonstrated highest expression as soon as 1 day post-HI (+292%) which gradually decreased to baseline levels at 2 weeks post-HI. Image analysis in ApoE-/- mice demonstrated that the I/N ratio was significantly higher in non-diabetic (+49%) vs. diabetic (-8%) at 1 week post-HI as compared to the baseline. However, the absolute activity of RAGE-targeted probe in non-ischemic muscles revealed increased baseline RAGE expression in diabetic ApoE-/- mice as compared to non-diabetic controls. These imaging- and molecular biology derived data suggest synergistic effect of both diabetes and apolipoprotein deficiency on RAGE expression, impaired collateral vessel development and possibly decreased peripheral perfusion recovery. Conclusions In this study we successfully assessed both temporal and animal model-specific changes in RAGE expression with PET-optical imaging using newly synthesized molecularly-targeted multimodal nanoparticle. This multimodal imaging strategy may allow optimization and monitoring of therapeutic RAGE-targeted interventions directed at the augmentation of functional recovery in diabetic patients with peripheral arterial disease. Studies were supported by the Ministry of Science and Higher Education Poland ("Mobility Plus" Program, AP), The Foundation for Polish Science (LK, WLD), and American Heart Association (WLD).