An Endocellulase‐Triggered NO Targeted‐Release Enzyme‐Prodrug Therapy System and Its Application in Ischemia Injury

Abstract Nitric oxide (NO) is a crucial gaseous signaling molecules in regulating cardiovascular, immune, and nervous systems. Controlled and targeted NO delivery is imperative for treating cancer, inflammation, and cardiovascular diseases. Despite various enzyme‐prodrug therapy (EPT) systems facilitating controlled NO release, their clinical utility is hindered by nonspecific NO release and undesired metabolic consequence. In this study, a novel EPT system is presented utilizing a cellobioside‐diazeniumdiolate (Cel2‐NO) prodrug, activated by an endocellulase (Cel5A‐h38) derived from the rumen uncultured bacterium of Hu sheep. This system demonstrates nearly complete orthogonality, wherein Cel2‐NO prodrug maintains excellent stability under endogenous enzymes. Importantly, Cel5A‐h38 efficiently processes the prodrug without recognizing endogenous glycosides. The targeted drug release capability of the system is vividly illustrated through an in vivo near‐infrared imaging assay. The precise NO release by this EPT system exhibits significant therapeutic potential in a mouse hindlimb ischemia model, showcasing reductions in ischemic damage, ambulatory impairment, and modulation of inflammatory responses. Concurrently, the system enhances tissue repair and promotes function recovery efficacy. The novel EPT system holds broad applicability for the controlled and targeted delivery of essential drug molecules, providing a potent tool for treating cardiovascular diseases, tumors, and inflammation‐related disorders.