Precise Delivery of Nitric Oxide Controlled by Bioorthogonal Endocellulase Ameliorates Hindlimb Ischemia

Peripheral artery disease (PAD) remains a great threat to the health of older people globally. Nitric oxide (NO), as an important signaling molecule, is integral to processes such as angiogenesis, inflammation, and tissue regeneration, making it a potential candidate for PAD treatment. Nevertheless, NO-based therapies are frequently limited in clinical utility, primarily due to the lack of effective strategies for fine-tuning the release of exogenous NO. In this study, we developed an enzyme-prodrug pair based on endocellulase (Cel5A-h38), which ensured complete bioorthogonality, thus avoiding interference with endogenous enzymes and eliciting an inflammatory response. This delivery system enables localized and controlled NO release, thus preventing side effects induced by systemic exposure. The therapeutic efficacy of the NO delivery system was systematically evaluated in a porcine model of hindlimb ischemia. Our results confirmed the benefits of targeted NO delivery in hindlimb ischemia, which include enhanced neovascularization and tissue perfusion, reduced inflammation, and alleviated muscle fibrosis, demonstrating its optimal translational potential.