In real time: Thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual‐emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage‐dependent spatial localization. The most advanced RACPP uses norleucine (Nle)‐TPRSFL as a linker that increases sensitivity to thrombin by about 90‐fold (see figure).

Activatable cell penetrating peptides (ACPPs) provide a general strategy for molecular targeting by exploiting the extracellular protease activities associated with disease. Previous work used a matrix metalloproteinase (MMP-2 and 9) cleavable sequence in the ACPP to target contrast agents for tumor imaging and fluorescence guided surgery. To improve specificity and sensitivity for MMP-2, an integrin αvβ3 binding domain, cyclic-RGD, was covalently linked to the ACPP. This co-targeting strategy relies on the interaction of MMP-2 with integrin αvβ3, which are known to associate via MMP-2’s hemopexin domain. In U87MG glioblastoma cells in culture, dual targeting greatly improved ACPP uptake compared to either MMP or integrin αvβ3 targeting alone. In vivo, dual-targeted ACPP treatment resulted in tumor contrast of 7.8±1.6, a 10 fold higher tumor fluorescence compared to the negative control peptide, and increased probe penetration into the core of MDA-MB-231 tumors. This platform also significantly improved efficacy of the chemotherapeutic monomethylauristatin E (MMAE) in both MDA-MB-231 orthotopic human and syngeneic Py230 murine breast tumors. Treatment with cyclic-RGD-PLGC(Me)AG-MMAE-ACPP resulted in complete tumor regression in one quarter of MDA-MB-231 tumor bearing mice, compared to no survival in the control groups. This rational mechanism for amplified delivery of imaging and potent chemotherapeutic agents avoids the use of antibodies and may be of considerable generality.

Extracellular proteases including thrombin are involved in numerous biological processes and play major roles in a variety of human diseases. The spatial and temporal patterns of activation of proteases in vivo control their biological role in diseases and amenability to therapeutic targeting. Previously we developed activatable cell-penetrating peptides (ACPPs) to monitor matrix metalloproteinase (MMP) and elastase activity in tumors. Later ACPPs detect thrombin activation in atherosclerosis and brain injury. We have now modified the thrombin ACPP in two independent ways, 1) to provide a FRET-dependent emission ratiometric readout and 2) to accelerate the kinetics of cleavage by thrombin. Emission ratioing improves kinetic detection of enzyme activity, because it reflects the ratio of cleaved versus uncleaved probe but cancels out total probe concentration, illumination intensity, detection sensitivity, and tissue thickness. Because pharmacokinetic washout of the uncleaved probe is not necessary, yet the cleavage converts a diffusible substrate into an immobilized product, thrombin activity can be imaged in real time with good spatial resolution. Meanwhile, placement of norleucine-threonine (Nle-Thr) at the P4-P3 substrate positions accelerates the kinetics of thrombin cleavage by 1-2 orders of magnitude, while preserving selectivity against related proteases. The new ratiometric ACPPs detect localized thrombin activation in rapidly forming blood clots minutes after probe injection, and the signal is inhibited by thrombin specific inhibitors.Thrombin is a serine protease and a key regulator of blood coagulation. It is responsible for the proteolytic cleavage and activation of multiple coagulation factors including Factor V,

Nerve degeneration after transection injury decreases intraoperative visibility under white light (WL), complicating surgical repair. We show here that the use of fluorescently labeled nerve binding probe (F-NP41) can improve intraoperative visualization of chronically (up to 9 months) denervated nerves. In a mouse model for the repair of chronically denervated facial nerves, the intraoperative use of fluorescent labeling decreased time to nerve identification by 40% compared to surgeries performed under WL alone. Cumulative functional post-operative recovery was also significantly improved in the fluorescence guided group as determined by quantitatively tracking of the recovery of whisker movement at time intervals for 6 weeks post-repair. To our knowledge, this is the first description of an injectable probe that increases visibility of chronically denervated nerves during surgical repair in live animals. Future translation of this probe may improve functional outcome for patients with chronic denervation undergoing surgical repair.