Pre‐targeted Imaging of Protease Activity Via In Situ Assembly of Nanoparticles

The pre-targeted imaging of enzyme activity has not been reported, likely owing to the lack of a mechanism to retain the injected substrate in the first step for subsequent labeling. Herein, we report the use of two bioorthogonal reactions-the condensation reaction of aromatic nitriles and aminothiols and the inverse-electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO)-to develop a novel strategy for pre-targeted imaging of the activity of proteases. The substrate probe (TCO-C-SNAT4) can be selectively activated by an enzyme target (e.g. caspase-3/7), which triggers macrocyclization and subsequent in situ self-assembly into nanoaggregates retained at the target site. The tetrazine-imaging tag conjugate labels TCO in the nanoaggregates to generate selective signal retention for imaging in vitro, in cells, and in mice. Owing to the decoupling of enzyme activation and imaging tag immobilization, TCO-C-SNAT4 can be repeatedly injected to generate and accumulate more TCO-nanoaggregates for click labeling.