br Activatable optical imaging probes Optical fluorescence i
Activatable optical imaging probes Optical fluorescence imaging, which directly detects photons emitted from fluorescent probes, has become as a powerful analytical method to study biological processes both in vitro and in vivo. It is characterized by high sensitivity, is free of radioactive irradiation, and has the capacity for real-time imaging to obtain quantitative information about molecular targets . Activatable fluorescence imaging probes that amplify the fluorescent signal upon interaction with a certain enzyme have several advantages for in vivo applications, including low background signal and high specificity to detect enzyme activity . Moreover, activatable fluorescence imaging probes can also detect and monitor enzyme activity in real time. There are several principles to consider when designing activatable fluorescence probes for in vivo imaging. First, the probes should be designed to have fluorescence emission in the near-infrared (NIR) range (700–900nm) to guarantee high tissue-penetration capacity and low autofluorescence from tissues. Second, the probes should have high specificity to a target enzyme, that is, the fluorescence signal emission should only occur when the probe interacts with the enzyme of interest to ensure accurate detection. Third, the probe should exhibit minimal inhibition of enzyme activity upon recognition, allowing a single target enzyme to activate many molecular probes, thus amplifying the fluorescence emission and generating high SBR at the target site. Furthermore, upon activation, the probes should prolong accumulation at the target site with a continuous fluorescence signal, enabling long-term monitoring of enzyme activity. A number of chemical strategies have been developed to design activatable optical imaging probes and have been summarized in several excellent reviews 11., 12., 13., 14., 15.. Fluorogenic enzyme substrates are one of the most explored activatable probe types for enzyme detection. These probes contain a fluorophore that is capped with an enzyme recognition substrate to quench their fluorescence, and after enzymatic removal of the capping substrate, the fluorescence is recovered. Extremely high turn on ratios are commonly observed for these probes 16., 17.. Pre-quenched probes are another type of widely used activatable optical imaging probes, in which a fluorophore and a quencher are linked together via an enzyme-cleavable peptide substrate. The initial fluorescence of the fluorophore is quenched due to Förster resonance nuclear receptor transfer (FRET), and upon cleavage by a specific enzyme, the quencher is released to eliminate the FRET quenching effect, resulting in strong fluorescence recovery 8., 18.. Pre-quenched probes with other quenching mechanisms involving fluorophore H-dimer formation  and photon-induced electron transfer (PeT) have also been reported. Pre-quenched probes have found successful applications for in vivo imaging of various enzymes, such as cathepsins , MMPs , caspases , and β-lactamase . Enzyme-triggered self-assembly of small molecules into nanostructures has recently been exploited as a novel approach to build activatable probes for molecular imaging 24., 25.. The approach initially uses small molecules, which upon interaction with a target enzyme, form small probes that are converted into nanostructures through self-assembly. The probes are trapped and accumulated at the target site, producing a strong fluorescence signal. In a pioneering work, Rao and co-workers  employed a first-order bioorthogonal cyclization reaction between a D-cysteine residue and a 2-cyano-6-hydroxyquinoline (CHQ) moiety to construct a caspase-3/7 sensitive self-assembling fluorescent probe (C-SNAF) to image caspase-3/7 activity in living animals  (Fig. 2). Caspase-3/7 has been recognized as an “executioner” for cell apoptosis, and imaging its activity can provide invaluable predictive information regarding therapeutic efficacy and anti-cancer drug screening. C-SNAF is designed to have (1) D-cysteine and CHQ moieties linked to an amino luciferin scaffold, (2) a DEVD capping sequence with an ethyl disulfide, and (3) an NIR fluorophore Cy5.5 that is amenable to in vivo imaging (Fig. 2a). Compared to saline treatment, significantly brighter images were obtained in response to chemotherapy for C-SNAF in nude mice bearing subcutaneous HeLa tumors that received three rounds of doxorubicin (DOX) through i.v. administration (Fig. 2b). The brighter images were associated with caspase-3/7 activity in the tumors undergoing chemotherapy. Importantly, the degree of tumor size decrease during the course of chemotherapy directly correlated with the intensity of fluorescence in the tumors enhanced by C-SNAF, suggesting the potential of C-SNAF for early monitoring of tumor therapeutic efficacy (Fig. 2c). Moreover, fluorescence quenching/dequenching was also integrated with the self-assembly approach to construct pre-quenched probes, which could not only turn on fluorescence upon caspase-3/7 recognition but also show enhanced retention in apoptotic tumor cells. The self-assembly approach has also been extended to design a 18F-labeled positron emission tomography (PET) tracer 28., 29. and a Gd-based MRI contrast agent 30., 31., 32. for caspase-3/7, enabling in vivo imaging of tumor cell apoptosis with high sensitivity and high spatial resolution.