Fluoridated silver nanocomposites for caries management: an in-vitro assessment of the cytological and antibacterial profiles

Abstract

Background: Silver nanoparticles (AgNPs) have antibacterial properties with potential applications in managing dental caries. Functionalization with fluoride may further enhance AgNPs' antibacterial efficacy. This study evaluated the impact of fluoridated AgNPs coated with various surface moieties on their safety profile and antibacterial effects against cariogenic bacteria as a potential anti-cariogenic treatment.

Methods: AgNP synthesis followed citrate and gallic acid reduction methods with polyethylene glycol (PEG) and polyvinylpyrrolidone coating. Functionalizing AgNPs with sodium fluoride (NaF) proceeded. Testing the safety of synthesized compounds was done on human gingival fibroblasts and oral epithelial cells. Meanwhile, minimum inhibitory concentration (MIC) determination against Streptococcus mutans was executed to verify antibacterial activity.

Results: Gallic-reduced AgNPs revealed higher yielding capacity than citrate-AgNPs. Cytologically, PEGylation reinforced citrate-AgNPs stability and improved IC50 range up to ∼ 4.2 × 10¹⁶ µg/mL and 64.3 µg/mL on fibroblastic and epithelial lineages. PEGylated AgNPs counteracted the cytotoxicity of free NaF with antagonistic combinational effect of NaF@PEG gallic-AgNPs on gingival fibroblasts. Microbiologically, AgNPs recorded an enhanced antimicrobial activity of ∼ 5.3 ± 2.3 µg/mL averaged MIC against Streptococcus mutans. Furthermore, fluoridation of PEG gallic-AgNPs depicted an additive antimicrobial propensity.

Conclusions: This dual action nanoplatform successfully integrates fluoride and silver components, reducing fluoride concentrations to safety range while maximizing silver's antibacterial properties. Engineered NaF@PEGylated nanosilver formulation represents promising anti-cariogenic strategy that optimizes therapeutic efficacy while maintaining biological safety.

Keywords: Streptococcus mutans; Cytotoxicity; Gallic acid; Minimum inhibitory concentration; Nano silver; Polyethylene glycol.

Fig. 1

Physio-optical properties of different coated gallic acid-reduced AgNPs. (a) UV-Vis spectrophotometer reveals the smooth mono-peaked nanopopulation synthesized by the green reduction method. PEG and PVP capping agents improve the stability of the bared gallic acid-reduced AgNPs, which increases with time. (b) DLS displays the non-homogeneity in the nano sizes with multi-peaked curves. (c) TEM photomicrographs reveal the pronounced nanoparticle aggregation (clouds) disclosed from the uncapped gallic-AgNPs while moderated by PVP capping

Fig. 2

Characterization of the various capped citrate-reduced AgNPs. (a) Optically, the uncoated chemically synthesized AgNPs display a broader peak at a lower absorbance value than the capped AgNPs, where PVP and PEG are of added value regarding nano stability and concentration. (b) PEGylation of citrate-AgNPs reduces the nanosize by DLS to the smallest size in the same range of the PDI (0.5) as PVP and uncapped nanoparticles. (c) TEM visualization indicates the synthesis of monodispersed, widely distributed spherical PVP- and PEG-stabilized AgNPs of more miniature sizes than bared citrate-AgNPs

Fig. 3

Panel i demonstrates the safety profile of the AgNPs on HGFs and OEC lineages depicted in the non-linear regression curves. (a) The uncapped citrate AgNPs reveal higher cytotoxicity towards primary cell lines than the green synthesized AgNPs. (b and c) PEGylation of the gallic-AgNPs (b) and the citrate-AgNPs (c) boost their safety profile, while PVP capping reveals a toxic influence on epithelial cells. Panel ii shows the high sensitization of HGFs and OEC lineages to NaF with an acute drop in cell viability on the dose-dependent curves at very low doses of NaF. The calculated IC50 are the mean of three independent experiments, each of at least triplicates. In AgNP panels, Kruskal-Wallis test reveals insignificance of p > 0.05 For HGFs. Meanwhile, one-way ANOVA followed by Tukey multiple comparison test reveals significance of p < 0.05 between OEC-treated groups tagged with similar symbols ^† and ^‡. In NaF panel, Mann Whitney test reveals insignificance of p > 0.05

Fig. 4

Assessment of mitochondria stress in PEGylated AgNPs treated primary cells versus NaF. (a) Representative MitoTracker Red-stained confocal microscopic images (scale bar = 25 μm) show the intense signals in the stressed NaF-treated cells against the mild fluorescence retrieved from cellular emolliated PEGylated AgNP panels. (b) The bar graphs for corrected log fluorescence intensities, where*** of p < 0.0001 marks the significant results of one-way ANOVA followed by multiple comparison test. Data are the mean of three random microscopic fields captured per each of the three wells for each group

Fig. 5

The cytological combinational index (CI) of NaF@PEG gallic-AgNPs on primary cell lines. (a) In gingival fibroblasts, the constant 1:1 (NaF: PEG gallic-AgNPs) combination ratio exhibits an antagonistic impact at all doses. Meanwhile, the epithelial cells (b) are highly sensitized to the combinational nanoplatform, inducing a significant synergistic impairment of cell viability than the treatment alone. CompuSyn (0.1) results of CI > 1 indicate an antagonistic effect, CI = 1 points out additive impact, while CI < 1 denotes synergistic effects. In the line graph, *points out the significance (p < 0.001) of combined nanoplatform with NaF, while # marks the significant relation (p < 0.001) with PEG gallic-AgNPs. Viability inhibition (%) data are the mean of three independent experiments, each of triplicates analyzed by tow-way ANOVA followed by Tukey multiple comparison test

Fig. 6

The combinational index (CI) of the NaF@PEG citrate-AgNPs on HGFs and OEC cells. (a) In HGFs, the combination of NaF in a constant ratio of 1:1 with PEG citrate-AgNPs induces a significant inhibitory effect on cell viability, fluctuating between additive and synergistic influences. (b) In OEC, the nanocomposite reveals a consistently synergistic effect along all doses. CompuSyn (0.1) results of CI > 1 indicate an antagonistic effect, CI = 1 points out additive impact, while CI < 1 denotes synergistic effects. In the line graph, * points out the significance (p < 0.001) of combined nanoplatform with NaF, while # marks the significant relation (p < 0.001) with PEG citrate-AgNPs. Viability inhibition (%) data are the mean of three independent experiments, each of triplicates analyzed by tow-way ANOVA followed by Tukey multiple comparison test