Purpose: Near infrared fluorescence (NIRF) optical probes represent a promising means to assess enzymatic activity noninvasively in vivo. Quantification of enzyme activity is so far, however, hindered by the varying accumulation of the probes in tissues as well as errors resulting from differences in size and depth of tissues. Our aim was to develop a single NIRF probe which is able to measure Cathepsin B activity independent of probe accumulation or differences in tissue size and depth.
Methods and Materials: A single optical probe consisting of a Cy7-labeled cross-linked iron oxide (CLIO) core, derivatized with Cy5.5-labeled Cathepsin B sensitive peptide linkers, was synthesized. The probe was tested for in vitro activation with Trypsin and evaluated in phantom experiments for the ability to correct for size and depth. In vivo experiments were performed by injection of the probes into nude mice (n=4) bearing tumors of different size and depth and optical imaging in a whole mouse imaging chamber.
Results: Incubation with Trypsin increased the NIR fluorescence in the Cy5.5-channel 700% after 60 min., whereas the Cy7 attached to the CLIO produced a stable background fluorescence and was not activated by Trypsin, thus representing only the amount of probe accumulation. When liver samples of varying size from mice injected with the probe were imaged in phantoms, it was shown that the signal from the internal standard Cy7 reliably corrected for differences in Cy5.5 intensity caused by different organ sizes. Also, errors resulting from differences in depth could be corrected by division of the Cy5.5 signal through the Cy7 signal. Furthermore, the in vivo experiments showed that the Cy5.5/Cy7 ratio is able to reflect Cathepsin B activity independent of the size of tumors or their depth.
Conclusion: We present a second generation dual wavelength ratio imaging technique which uses a single probe with two NIR dyes, one of which is enzyme activated, whereas the second one serves as an internal standard. The technique enables non-invasive detection of cathepsin B activity that is corrected for differences in tissue size, depth, and probe accumulation.
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