A dual fluorochrome probe for imaging proteases

Near-infrared fluorescence (NIRF) optical probes have been able to provide a noninvasive assessment of enzyme activity for a number of different enzymes and types of pathology. Here we describe a dual fluorochrome enzyme-activatable probe featuring one NIRF fluorochrome that is activated by protease activity and a second fluorochrome that is protease resistant and serves as an internal standard. The probe was prepared by attaching Cy7 directly to an amino-CLIO, an amine functional cross-linked iron oxide (CLIO) nanoparticle carrier, in a protease resistant manner. Cy5.5 was attached to a protease sensitive polyarginine peptide spacer, also attached to amino-CLIO. In vitro and in vivo the ratio of the Cy5.5 to Cy7 fluorescence was increased by protease, reflecting the increase in Cy5.5 fluorescence by protease in the vicinity of the probe. In vitro and in vivo the absolute values of the Cy5.5 and Cy7 fluorescence reflected lesion size and the distance of lesions from the surface, while the ratio of Cy5.5 to Cy7 fluorescence obtained was constant and independent of lesion size and depth. The dual fluorochrome probe, and related dual wavelength imaging method, represents a novel approach for imaging protease activity in vivo.