Ratio imaging of enzyme activity using dual wavelength optical reporters

The design of near-infrared fluorescent (NIRF) probes that are activated by specific proteases has, for the first time, allowed Enzyme activity to be imaged in vivo. In the current study, we report on a method of imaging Enzyme activity using two fluorescent probes that, together, provide improved quantitation of enzymatic activity. The method employs two chemically similar probes that differ in their degradability by Cathepsin B. One probe consists of the NIRF dye Cy5.5 attached to a particulate carrier, a crosslinked iron oxide nanoparticle (CLIO), through Cathepsin B cleavable L-arginyl Peptides. A second probe consists of Cy3.5 attached to a CLIO through proteolytically resistant D-arginyl Peptides. Using mixtures of the two probes, we have shown that the ratio of Cy5.5 to Cy3.5 fluorescence can be used to determine levels of Cathepsin B in the environment of nanoparticles with macrophages in suspension. After intravenous injection, tissue fluorescence from the nondegradable Cy3.5-D-arginyl probe reflected nanoparticle accumulation, while fluorescence of the Cy5.5-L-arginyl probe was dependent on both accumulation and activation by Cathepsin B. Dual wavelength ratio imaging can be used for the quantitative imaging of a variety of Enzymes in clinically important settings, while the magnetic properties of the probes allow their detection by MR imaging.