Fluoridated mouthrinse is indicated for individuals with high risk of caries. Chitosan (Chit) exhibits antibacterial properties, but little is known about its effects on enamel when combined with sodium fluoride (NaF) to form NaF/Chit supramolecular complexes. In our study, NaF/Chit supramolecular complexes structured as microparticles were synthetized and characterized, and their effects on human enamel were evaluated after cariogenic challenge simulating the daily mouthrinse use. Initially, NaF/Chit complex formation was investigated based on several titrations by measuring the zeta potential, electrical conductivity (κ), hydrodynamic diameter (D), viscosity (η) and heat flow (by isothermal titration calorimetry) against the molar ratio [NaF]/[Chit], which allowed us to identify the interactions between Chit-NaF with a stoichiometry of approximately 0.68. Spontaneous microparticle formation was observed. Samples of enamel blocks were prepared and divided into eight groups (n = 10/group): (i) 0.2% Chit; (ii) 0.2% NaF; (iii) 0.2% NaF/Chit suspension; (iv) 0.2% acetic acid; (v) 0.05% Chit; (vi) 0.05% NaF; (vii) 0.05% NaF/Chit suspension; and (viii) 0.05% acetic acid. Cariogenic challenge was performed in each sample by cycling in demineralization and remineralization solutions for 7 days. Before each demineralization cycle, the corresponding substances were passively applied daily for 90 s, even in groups with 0.02% concentration. After 7 days, samples were examined for Knoop hardness (KHN) measurements. The data were analyzed by repeated-measures ANOVA and Tukey tests (α = 0.05). The 0.2% NaF and 0.2% NaF/Chit groups showed higher KHN values than the other groups. The 0.2% NaF/Chit microparticle suspension reduced the enamel hardness loss after cariogenic challenge as effectively as the 0.2% NaF solution and demonstrated potential for use in a formulation with anti-caries effects.

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