Bacterial biofilm-associated infections, particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA), present a major challenge in clinical wound management due to their intrinsic resistance to conventional antibiotics and their capacity to severely impair tissue repair and wound healing. To address the limitations of current therapeutic approaches in combating biofilm-associated infections, we developed a multifunctional bimetal-doped nanozyme system, Cu,Fe,S,N-GQDs@Ru-NO, which synergistically integrates chemodynamic therapy (CDT), photothermal therapy (PTT), photodynamic therapy (PDT), and nitric oxide (NO) gas therapy to achieve enhanced antibiofilm efficacy through multiple complementary mechanisms. The nanozyme was synthesized via a hydrothermal method, during which Cu and Fe co-doped graphene quantum dots (Cu,Fe,S,N-GQDs) were covalently conjugated to an NO-releasing prodrug (Ru-NO) through amide bond formation, enabling precise functionalization. This nanozyme architecture confers multiple catalytic and therapeutic functionalities: the bimetallic doping significantly enhances the catalytic generation of reactive oxygen species (ROS), including hydroxyl radicals (•OH) and singlet oxygen (O₂); concurrently, the nanozyme demonstrates efficient photothermal conversion under 808 nm near-infrared (NIR) irradiation and facilitates controlled release of NO. These multimodal actions act synergistically to disrupt bacterial membranes, oxidize NADH (Nicotinamide adenine dinucleotide), and deplete ATP, leading to effective biofilm disintegration and achieving over 99 % bacterial eradication along with complete biofilm clearance in vitro. In a murine model of MRSA-infected wounds, treatment with the nanozyme markedly accelerated wound closure, characterized by attenuated inflammatory responses (reduced IL-6 levels) and enhanced angiogenesis (upregulated VEGF and α-SMA expression), with no detectable cytotoxicity or systemic adverse effects. This study presents a multimodal nanozyme platform that not only achieves potent eradication of multidrug-resistant biofilms but also actively promotes tissue regeneration, offering a promising paradigm for the development of next-generation antimicrobial therapeutics. 1
