Optimization of a Protease Activated Probe for Optical Surgical Navigation

Molecularly
targeted optical contrast agents have the potential
to enable surgeons to visualize specific molecular markers that can
help improve surgical precision and thus outcomes. Fluorescently quenched
substrates can be used to highlight tumor lesions by targeting proteases
that are highly abundant in the tumor microenvironment. However, the
majority of these and other molecularly targeted optical contrast
agents are labeled with reporter dyes that are not ideally matched
to the properties of clinical camera systems, which are typically
optimized for detection of indocyanine-green (ICG). While a wide range
of near-infrared (NIR) dyes are suitable for use with highly sensitive
and highly tunable research-focused small animal imaging systems,
most have not been evaluated for use with commonly used clinical imaging
systems. Here we report the optimization of a small molecule fluorescently
quenched protease substrate probe 6QC-ICG, which uses the indocyanine
green (ICG) dye as its optical reporter. We evaluated dosing and kinetic
parameters of this molecule in tumor-bearing mice and observed optimal
tumor over background signals in as little as 90 min with a dose of
2.3 mg/kg. Importantly, the fluorescence intensity of the probe signal
in tumors did not linearly scale with dose, suggesting the importance
of detailed dosing studies. Furthermore, when imaged using the FDA
approved da Vinci Si surgical system with Firefly
detection, signals were significantly higher for the ICG probe compared
to a corresponding probe containing a dye with similar quantum yield
but with a slightly shifted excitation and emission profile. The increased
signal intensity generated by the optimal dye and dose of the ICG
labeled probe enabled detection of small, flat lesions that were less
than 5 mm in diameter. Therefore, 6QC-ICG is a highly sensitive probe
that performs optimally with clinical imaging systems and has great
potential for applications in optical surgical navigation.