Breaking biofilm barriers and reprogramming immunity: NIR-activated protease–copper nanozymes drive cuproptosis-like killing and accelerate infected wound repair

Protease-armed GCP breaks EPS barriers for deep biofilm penetration. • NIR-boosted copper overload drives cuproptosis-like bacterial killing. • GCP amplifies ROS generation through peroxidase-like nanozyme activity. • GCP eradicates mature biofilms and prevents new biofilm formation. • GCP promotes early M1 defense and late M2 repair in infected wounds. Biofilm infections are notoriously difficult to eradicate because the EPS matrix limits drug penetration and the local immune environment often fails to clear pathogens, leading to chronic inflammation and delayed healing. Here, we present a protease-armed copper nanozyme–antibiotic platform, Gen@CuS@BSA/protease (GCP), designed to integrate biofilm disruption, copper-overload–mediated killing, and stage-adaptive immunoregulation under near-infrared (NIR) activation. GCP incorporates alkaline protease to degrade EPS and create diffusion channels, CuS-based photothermal heating to enhance copper uptake and induce copper-overload-associated, cuproptosis-like bacterial killing, and peroxidase-like activity to amplify oxidative stress in the presence of H₂O₂, thereby boosting antibacterial efficacy. In vitro , GCP robustly eradicated established P. aeruginosa PAO1 biofilms and inhibited biofilm formation, consistent with improved intrabiofilm penetration. Transcriptomic analysis and RT–qPCR revealed suppression of central carbon metabolism and virulence- and biofilm-associated pathways, supporting a copper-driven lethal mechanism. In an infected wound model, GCP with NIR accelerated bacterial clearance and wound closure. Notably, GCP outperformed the protease-free counterpart (GC) in modulating macrophage dynamics—promoting early M1 activation for antibacterial defense while facilitating a subsequent M2 shift to resolve inflammation and support tissue remodeling. This study provides a unified strategy for treating refractory biofilm infections by synchronizing deep penetration, cuproptosis-like killing, and immuno-regenerative healing. Download: Download high-res image (336KB) Download: Download full-size image