Antibacterial effects and remineralization potential of silver nanoparticles in a novel nanosilver fluoride formulation

Silver-based therapies are effective in preventing caries but often cause undesirable tooth staining. This study aimed to develop a dual-action nanosilver-fluoride (NSF) formulation that arrests caries while minimizing discolouration. Silver nanoparticles (AgNPs) were synthesized via chemical reduction and characterized using transmission electron microscopy and dynamic light scattering. Various AgNP concentrations (0–150 µg/mL) were evaluated for their ability to inhibit Streptococcus mutans biofilm formation using a colorimetric assay, while bacterial viability within established biofilms was further assessed at AgNP concentrations of 5, 15, 50, 150, and 300 µg/mL through colony-forming unit (CFU) counts and live/dead staining analysed via confocal laser scanning microscopy. Brain Heart Infusion (BHI) medium and 1% NaOCl served as negative and positive controls, respectively. Next, the optimal AgNP concentration was combined with three fluoride concentrations (1,000 ppm, 20,000 ppm, and 40,000 ppm) to assess synergistic antibacterial effects compared with fluoride alone. Although all three AgNP–fluoride combinations inhibited biofilm formation, only those containing 20,000 ppm and 40,000 ppm fluoride exhibited significantly enhanced bactericidal activity. Therefore, two NSF formulations were developed for further analysis: NSFlow (300 µg/mL AgNPs + 20,000 ppm fluoride) and NSFhigh (300 µg/mL AgNPs + 40,000 ppm fluoride). These formulations were compared with silver diamine fluoride (SDF), fluoride varnish (Fvarnish), and untreated controls in terms of: (1) antibacterial activity (biofilm inhibition and bacterial susceptibility within biofilms), (2) fluoride release (measured using an ion-selective electrode; n = 6 per group), and (3) remineralization potential of artificial carious lesions (evaluated via microcomputed tomography; n = 10 per group). All experiments were conducted in triplicate, and statistical analyses were performed accordingly. The synthesized AgNPs were spherical with an average diameter of 2.87 ± 0.96 nm and exhibited biofilm inhibition beginning at 2.34 µg/mL. At 300 µg/mL, AgNPs significantly reduced CFU and increased the percentage of dead bacteria compared with BHI, showing results comparable to 1% NaOCl. When 300 µg/mL AgNPs were combined with fluoride, all concentrations (1,000, 20,000, and 40,000 ppm) produced over 80% biofilm inhibition, similar to fluoride alone. However, the AgNP-fluoride combinations demonstrated greater bactericidal activity overall, showing lower CFU and higher percentages of dead bacteria than fluoride alone. Except the 1,000ppm fluoride combination, the percentage of dead bacteria was similar to that observed with fluoride alone. Accordingly, only NSFlow and NSFhigh were selected for further evaluation. Both NSF formulations, along with SDF and Fvarnish, significantly inhibited biofilm formation and decreased bacterial viability compared with the untreated control, with no statistically significant differences among the treatment groups. Fluoride release from NSF and SDF occurred rapidly within the first two days, after which concentrations dropped below the detection limit, whereas the release of Fvarnish was gradual and sustained over 7 days. Microcomputed tomography analysis revealed that only NSFhigh achieved remineralization levels comparable to those observed with SDF and Fvarnish. NSFhigh, combining 300 µg/mL AgNPs with 40,000 ppm fluoride, demonstrated strong antibacterial efficacy and remineralization potential equivalent to established treatments. This formulation shows promise as an effective, stain-reducing topical therapy for caries arrest.