Supporting data for On-Demand Nanozymes for Biofilm Eradication

Discovery of a copper oxide-ascorbic acid nanozyme system: We designed an innovative system based on copper oxide (CuO) nanoparticles as the nanozyme platform, combined with biocompatible ascorbic acid as the substrate to enhance functionality . Our design incorporates an external-trigger mechanism (visible light) to precisely control nanozyme activity, enabling rapid microbial eradication while minimizing resistance risks and host-cell damage.We first demonstrated the efficacy of the light-activated CuO-AA nanozyme system against both planktonic bacteria and monospecies biofilms. We then asked if the CuO-AA nanozyme system can eradicate three species of biofilms relevant to root canal infections. The antimicrobial performance of CuO-AA was compared to conventional disinfectants using viability assays and confocal microscopy. While we found promising results, the tested biofilm model is oversimplified and lacks clinical relevance, which might potentially overestimate the antimicrobial effectiveness.Development of an endodontically-relevant multispecies biofilm for antimicrobial testing: Our observation that a single parameter (biofilm age) significantly impacts the outcome highlights the absence of a validated, standardized testing platform in endodontics. To address this gap, we developed a clinically representative biofilm model through systematic optimization of multiple parameters. This sophisticated model has the potential to serve as a robust platform for comparative disinfectant assessment.This 10-species biofilm model was characterized for its architecture, microbial, extracellular matrix composition, and inflammatory potential. We then evaluated our CuO-AA nanozyme system against this biofilm using a series of complementary approaches, including microbial viability, spatial architecture, and extracellular matrix composition.This body of work developed a CuO-AA nanozyme system with excellent antibiofilm activity. We also systematically developed and characterized a highly reproducible polymicrobial biofilm model and validated the efficacy of the CuO-AA system against this biofilm. Taken together, this work provides both a novel biofilm-targeting strategy and an advanced testing platform for endodontic therapeutics.