Hypoxia‐instructed pro‐protein therapy assisted with self‐catalyzed nanozymogen

The success of intracellular protein therapy demands potent intracellular delivery efficiency and selective protein activity in diseased cells. To fulfil these requirements, a cascaded nanozymogen incorporating hypoxia-instructed pro-protein engineering and hypoxia-strengthened intracellular protein delivery was developed. RPAB, a pro-protein of RNase A reversibly caged with hypoxia-cleavable azobenzene yet deprived of the enzymatic activity, was designed and co-delivered with glucose oxidase (GOx) using hypoxia-dissociable nanocomplexes (NCs) consisting of azobenzene-cross-linked oligoethylenimine (AOEI) and hyaluronic acid (HA). Upon NCs-mediated tumor targeting, cancer cell internalization, and endolysosomal escape, GOx catalyzed intracellular glucose decomposition and aggravated tumoral hypoxia, which drove the recovery of RPAB back to the hydrolytically active RNase and concurrently expedited the degradation of AOEI to release more protein cargoes. Thus, the catalytic reaction of the nanozymogen was self-accelerated and self-cycled, ultimately leading to cooperative anti-cancer efficacies between GOx-mediated starvation therapy and RNase-mediated pro-apoptotic therapy. This study represents the first example of hypoxia-instructed pro-protein engineering, which expands the available chemical tools for the conditional control over protein functions. The self-catalyzed nanozymogen design also provides an effective strategy to mediate intracellular protein delivery and highly sensitive and selective intracellular pro-protein activation.