Hybrid in vivo FMT-CT imaging of protease activity in atherosclerosis with customized nanosensors

OBJECTIVE-: Proteases are emerging biomarkers of inflammatory diseases. In atherosclerosis, these enzymes are often secreted by inflammatory macrophages, digest the extracellular matrix of the fibrous cap, and destabilize atheromata. Protease function can be monitored with protease activatable imaging probes and quantitated in vivo by fluorescence molecular tomography (FMT). To address 2 major constraints currently associated with imaging of murine atherosclerosis (lack of highly sensitive probes and absence of anatomic information), we compared protease sensors (PS) of variable size and pharmacokinetics and coregistered FMT datasets with computed tomography (FMT-CT). METHODS AND RESULTS-: Coregistration of FMT and CT was achieved with a multimodal imaging cartridge containing fiducial markers detectable by both modalities. A high-resolution CT angiography protocol accurately localized fluorescence to the aortic root of atherosclerotic apoE mice. To identify suitable sensors, we first modeled signal kinetics in-silico and then compared 3 probes with oligo-l-lysine cleavage sequences: PS-5, 5 nm in diameter containing 2 fluorochromes, PS-25, a 25-nm version with an elongated lysine chain and PS-40, a polymeric nanoparticle. Serial FMT-CT showed fastest kinetics for PS-5 but, surprisingly, highest fluorescence in lesions of the aortic root for PS-40. PS-40 robustly reported therapeutic effects of atorvastatin, corroborated by ex vivo imaging and qPCR for the model protease cathepsin B. CONCLUSIONS-: FMT-CT is a robust and observer-independent tool for noninvasive assessment of inflammatory murine atherosclerosis. Reporter-containing nanomaterials may have unique advantages over small molecule agents for in vivo imaging.