Sustained Release of eDNA-Degrading Enzymes for Antibiofilm Dental Resins

The persistence of oral biofilms on teeth and dental restorative/reconstructive materials is promoted by their extracellular matrix (ECM), which stabilizes microbial communities and restricts antimicrobial penetration. Extracellular DNA (eDNA) is a conserved structural component of the ECM produced by multiple oral microorganisms and represents a promising target for biofilm control. NucB, a nonsequence-specific extracellular endonuclease, degrades eDNA and disperses diverse biofilms without direct bactericidal activity. In this study, we developed a sustained-release system for NucB using nonbiodegradable particles composed of 2-hydroxyethyl methacrylate and trimethylolpropane trimethacrylate (TMPT), and we evaluated the antibiofilm effects of NucB-loaded particles and dental resins incorporating these particles. NucB-loaded poly(2-hydroxyethyl methacrylate)/TMPT (polyHEMA/TMPT) particles released active enzyme for up to 28 d, with an initial burst followed by diffusion-controlled release. The released NucB retained DNA-digesting activity and significantly reduced Streptococcu mutans biofilm biomass and structural integrity without affecting bacterial growth or membrane integrity, consistent with ECM-targeted activity. Polymethyl methacrylate (PMMA) resins containing ≥10 wt% NucB-loaded polyHEMA/TMPT particles showed sustained NucB release for 28 d and inhibited S. mutans biofilm formation while maintaining flexural strength and modulus at loadings up to 30 wt%. Time-course analyses showed that NucB reduced eDNA accumulation from the earliest stages of biofilm development, thereby preventing dense microcolony assembly and maintaining sparse, discontinuous structures for at least 48 h. In a saliva-derived multispecies model, the PMMA resin containing 10 wt% NucB-loaded polyHEMA/TMPT particles significantly reduced biofilm biomass while preserving bacterial membrane integrity, demonstrating matrix-level activity in complex communities. In this study, we established a nonbiodegradable delivery system that enables sustained NucB release and persistent ECM disruption without compromising the mechanical properties of dental resins. This approach provides a promising strategy for developing biofilm-resistant dental materials to improve control of clinically relevant oral biofilms.