Targeted Optical Molecular Imaging of Atheroma Calcification Using Novel Aldendronate-based Probe

초록

Background/Objectives: Vascular spotty calcification is an actively regulated biological process resulting in plaque vulnerability. We investigated the feasibility of a novel alendronate-based near-infrared fluorescence (NIRF)-emitting probe to specifically target atherosclerotic calcification in a murine model in vivo using our customized high-resolution multichannel intravital molecular imaging system (IVFM). Methods/Results: We have fabricated a calcium-binding NIRF probe by chemically coupling alendronate, a specific targeting ligand, and NIRF-emitting Cy5.5 to the ends of azide-PEG-NHS ester (Al-Cy5.5). Prepared Al-Cy5.5 has high affinity for calcium phosphate-containing bone minerals. In vitro, Al-Cy5.5 specifically binds to RANKL-induced osteogenic-macrophages as compared to macrophages (p<0.01). On whole body fluorescence imaging to assess time-dependent excretion, NIRF signals remained visible up to 48 hrs. Then, in mice with calcified plaque induced by a combination diet of high-cholesterol and warfarin, Al-Cy5.5 (2.5 mg/kg) was intravenously injected. 48 hrs after administration, murine calcified atheroma was assessed using a customized high-resolution multichannel IVFM, which demonstrated highly enhanced NIRF signals in vivo in the calcified areas of murine carotid plaques (p<0.01, Figure). Ex vivo laser scanning fluorescence microscopic and immune-histological findings from the corresponding sister sections well corroborated the in vivo imaging results, which demonstrated the co-localization of NIRF signals with plaque calcifications (von-Kossa stain). Conclusions: Our novel calcification targeted probe, Al-Cy5.5, was able to selectively target atheroma calcification in vivo in a murine model as assessed by optical IVFM. This novel targetable strategy is expected to provide a promising theranostic basis for calcified high-risk plaques by integration with multimodal customized catheter imaging system.