br Activatable photoacoustic probes Photoacoustic PA imaging
Activatable photoacoustic probes Photoacoustic (PA) imaging is a recently developed technique . The imaging approach uses probes that absorb a pulsed laser beam and convert light energy into acoustic signals. It combines the advantages of the high sensitivity offered by optical imaging and the high spatial resolution offered by ultrasonic imaging. PA also enables high-throughput and real-time imaging and has wide applications for the in vivo molecular imaging of biological process 48., 49., 50., 51.. Currently, great attention has been paid to the development of activatable PA imaging probes for in vivo applications; however, only a few enzyme-activatable PA imaging probes have been reported due to the lack of an efficient approach to probe design. One of the first demonstrated concepts is based on enzyme-triggered in situ self-assembly to form nanomaterials, causing assembly-induced retention (AIR) to produce enhanced PA Phusion high-fidelity DNA polymerase at the target site . The first enzyme-activated PA imaging probe was reported by Gambhir and co-workers  based on a biocompatible reaction-mediated probe self-assembly strategy. The probe showed a 7.1-fold PA signal in furin-positive MDA-MB-231 tumors than in furin-deficient LoVo tumors in mice. Encouraged by this result, Wang and co-workers  recently reported another work, wherein αvβ3 integrin-targeted probe delivery and the gelatinase-triggered formation of nanofibers significantly amplified the PA signal in a U87 tumor xenografted mouse model. Compound 1 was designed to include the NIR dye purpurin18 (P18) as a light absorber, a peptide sequence of PLGVRG as a selective substrate for gelatinase, and RGD as the targeting ligand (Fig. 4a). When the probe was injected into the vasculature, its small size and hydrophilic nature assisted in the diffusion, extravasation and targeting to the αvβ3 integrin that was overexpressed on the tumor cell membranes. The active gelatinase in the tumor microenvironment then cut the peptide to form hydrophobic P18-PLG molecules, which could self-assemble into nanofibers, exhibiting an AIR effect in tumors and resulting in an enhanced PA signal. A comparable study with compound 1, gelatinase-inert control compound 2 (P18-PMGMRGRGD) and compound 3 (P18-PLGVRGRDG), which is gelatinase sensitive but lacks the RGD moiety for targeting the αvß3 integrin, showed that the PA signal of a tumor treated with 1 was 16-fold that of 2 at 8h post-injection and nearly twofold that of 3 (Fig. 4b, c). These results indicated that both αvβ3 integrin-mediated probe delivery and gelatinase-triggered probe activation and retention contributed to the enhancement of the PA signal. In contrast to the activatable PA imaging probes based on the AIR effect, a new study using NIR-absorbing CuS nanoparticles and a red light-absorbing BHQ3 organic quencher to build an MMP-activatable PA imaging nanoprobe for ratiometric imaging of MMP activity in vivo was recently reported by Liu and co-authors . This probe may represent an attractive strategy for the design of other enzyme-activatable PA imaging probes.
Summary and perspectives In recent decades, enormous advances have been achieved in the development of enzyme-activatable imaging probes, enabling the accurate detection of enzyme activity in vivo to better understand the biological function of enzymes in disease processes. Activatable probes are characterized by high sensitivity and specificity, which are superior to “always on” probes because the switch from the “off” to “on” state upon interaction with a specific enzyme at the target site allows signal amplification to detect enzyme activity in real time. However, the majority of the currently reported enzyme-activatable probes are only responsive to the hydrolase family (e.g., protease 52., 55., kinase ). The design of new activatable imaging probes for the detection of other classes of enzymes remains a challenge. However, the continuously emerging strategies (e.g., RIME) will pave the way to design activatable probes for in vivo imaging of protein disulfide isomerase  and oxidoreductase 57., 58..