Immobilized enzymes in inorganic nanoparticles for cariogenic biofilm eradication

Abstract

Dental caries is a sugar-mediated microbial dysbiosis in which extracellular polymeric substances (EPS) play a crucial role in biofilm formation and virulence. Mutans streptococci, a member of the oral microbiome, produce water-soluble (dextran) and insoluble (mutan) glucans from dietary sugars such as sucrose. These glucans form an extracellular matrix that promotes biofilm accumulation and acidification favoring enamel demineralization. Although mutanase and dextranase, enzymes that hydrolyze mutan and dextran, can disrupt the biofilm structure, they have sparsed use in oral care products, due to the difficulty of preserving enzymatic activity in formulations. Conversely, enzyme immobilization on surfaces, which can enhance stability while maintaining its catalytic activity, may provide an alternative to overcome this challenge. Therefore, the objective is to evaluate the combined effect of mutanase, and dextranase immobilized in inorganic nanoparticles on EPS matrix degradation as well as biofilm prevention and removal. For prevention, in addition to regular biofilm treatment, the acquired pellicle on the substrate will be treated previously to biofilm formation. An in vitro polymicrobial biofilm model containing S. mutans UA159 (ATCC 700610), well-characterized cariogenic streptococci, and S. oralis ATCC 35037, commensal streptococci will grow for 21 h and then be submitted to the following treatments for 2 min, twice daily: a) Negative control: sodium acetate buffer (0.2 M, pH 5.5); b) Positive control: chlorhexidine digluconate 0.12%; c) Free dextranase and mutanase at a final activity ratio of 5:1 (Dex: Mut); d) immobilized dextranase and mutanase at a final activity ratio of 5:1 (Dex: Mut). Biofilm disruption will be analyzed by quantitative and imaging assays, including the total number of viable cells, dry weight, the content of dextran/mutan, biofilm 3-D architecture, and in situ pH measurements combined with enamel surface analysis and transcriptomics via RNA-seq. This approach may be relevant to targeting a specific virulence factor without disturbing the ecological diversity of the host microbiota.