Photoactivatable Cascade DNAzyme Nanomachines for Amplified Imaging of Tumor Response to STING-Activated Immunotherapy

Real-time monitoring of enzymatic cascades following Stimulator of Interferon Genes (STING) activation is vital for elucidating the mechanisms and efficacy of cancer immunotherapies. However, the concurrent visualization of multiple enzymatic targets in vivo remains restricted by insufficient specificity and sensitivity. Herein, we report a near-infrared (NIR) photoactivatable cascade DNAzyme nanomachine (PCDzNM) designed for the concurrent, amplified imaging of granzyme B (Gzm B) and cathepsin B (Cat B) activity during STING-activated immunotherapy. In this platform, DNAzymes are initially caged by protease-substrate peptides. Upon NIR irradiation, these peptides are cleaved by their target proteases, restoring DNAzyme activity and initiating a site-specific substrate cleavage that amplifies fluorescence signals in situ. Both in vitro and in vivo evaluations demonstrate that this spatiotemporally controlled PCDzNM enables precise, real-time visualization of enzyme dynamics. Notably, we observed divergent enzymatic profiles: immune-suppressed tumor microenvironments exhibit high Cat B but low Gzm B activity, whereas STING-activated tumors display the reverse pattern. This platform represents a robust tool for real-time monitoring immune-tumor interactions and holds potential for guiding personalized immunotherapy.