Synthesis, Characterization, and Evaluation of the Antimicrobial Effects and Cytotoxicity of a Novel Nanocomposite Based on Polyamide 6 and Trimetaphosphate Nanoparticles Decorated with Silver Nanoparticles.

This study aimed to develop a polymeric matrix of polyamide-6 (P6) impregnated with trimetaphosphate (TMP) nanoparticles and silver nanoparticles (AgNPs), and to evaluate its antimicrobial activity, surface free energy, TMP and Ag+ release, and cytotoxicity for use as a support in dental tissue. The data were subjected to statistical analysis (p < 0.05). P6 can be incorporated into TMP without altering its properties. In the first three hours, Ag+ was released for all groups decorated with AgNPs, and for TMP, the release only occurred for the P6-TMP-5% and P6-TMP-10% groups. In the inhibition zones, the AgNPs showed activity against both microorganisms. The P6-TMP-2.5%-Ag and P6-TMP-5%-Ag groups with AgNPs showed a greater reduction in CFU for S. mutans. For C. albicans, all groups showed a reduction in CFU. The P6-TMP groups showed higher cell viability, regardless of time (p < 0.05). The developed P6 polymeric matrix impregnated with TMP and AgNPs demonstrated promising antimicrobial properties against the tested microorganisms, making it a potential material for applications in scaffolds in dental tissues.

Effect of nanometric ß-calcium glycerophosphate supplementation in conventional toothpaste on enamel demineralization: An in vitro study.

The aim of this study was to evaluate the effects of supplementing toothpastes containing 1100 ppm F with micrometric or nanometric [beta]-calcium glycerophosphate (ß-CaGPm/ß-CaGPn) on artificial enamel demineralization, using a pH cycling model. Bovine enamel blocks (4 mm × 4 mm, n = 120) selected using initial surface hardness were randomly allocated to ten toothpaste groups (n = 12): without fluoride or ß-CaGPm or ß-CaGPn (Negative control), 1100 ppm F (1100 F), and 1100 ppm F plus 0.125%, 0.25%, 0.5%, and 1.0% of ß-CaGPm or ß-CaGPn. Blocks were treated two times per day with toothpaste slurry and subjected to five pH cycles (demineralizing and remineralizing solutions) at 37 °C. The final surface hardness, percentage of surface hardness loss (%SH), cross-sectional hardness (ΔKHN), and profile analysis and lesion depth subsurface were analysed using polarized light microscopy (PLM). Fluoride (F), calcium (Ca), and phosphorus (P) concentrations were also measured. Data were analysed using ANOVA and Student-Newman-Keuls tests ([alpha] = 0.001). Blocks treated with 1100 F toothpaste containing 0.5%ß-CaGPm or 0.25%ß-CaGPn showed with reduced %SH values when compared with those treated with 1100 F alone (p < 0.001). Reduced lesion depths (ΔKHN and PLM) were observed for the slurry made up of 1100 F and 0.25%ß-CaGPn (p < 0.001). The addition of ß-CaGPm and ß-CaGPn did not influence the enamel F concentration, with the 1100 F/0.25%ß-CaGPn group exhibiting the highest Ca and P enamel concentrations (p < 0.001). Based on the findings of this in vitro study, we can conclude that the fluoride toothpaste produced a superior effect when combined at an appropriate ß-CaGP molar ratio. This effect was achieved with a lower proportion of ß-CaGP in the form of nanometric particles.

'Green' silver nanoparticles combined with tyrosol as potential oral antimicrobial therapy.

OBJECTIVES: This study aimed to evaluate silver nanoparticles (AgNPs) obtained by a 'green' route associated or not to tyrosol (TYR) against Streptococcus mutans and Candida albicans in planktonic and biofilms states. METHODS: AgNPs were obtained by a 'green' route using pomegranate extract. The minimum inhibitory concentration (MIC) against S. mutans and C. albicans was determined for AgNPs and TYR combined and alone, and fractional inhibitory concentration index (FICI) was calculated. Single biofilms of C. albicans and S. mutans were cultivated for 24 h and then treated with drugs alone or in combination for 24 h. RESULTS: AgNPs and TYR were effective against C. albicans and S. mutans considering planktonic cells alone and combined. The MIC values obtained for C. albicans was 312.5 µg/mL (AgNPs) and 50 mM (TYR) and for S. mutans was 78.1 µg/mL (AgNPs) and 90 mM (TYR). The combination of these antimicrobial agents was also effective against both microorganisms: 2.44 µg/mL/0.08 mM (AgNPs/TYR) for C. albicans and 39.05 µg/mL /1.25 mM (AgNPs/TYR) for S. mutans. However, synergism was observed only for C. albicans (FICI 0.008). When biofilm was evaluated, a reduction of 4.62 log10 was observed for S. mutans biofilm cells treated with AgNPs (p < 0.05, Tukey test). However, the addition of TYR to AgNPs did not improve their action against biofilm cells (p > 0.05). AgNPs combined with TYR demonstrated a synergistic effect against C. albicans biofilms. CONCLUSIONS: These findings suggest the potential use of AgNPs with or without TYR against C. albicans and S. mutans, important oral pathogens. CLINICAL SIGNIFICANCE: AgNPs obtained by a 'green' route combined or not with TYR can be an alternative to develop several types of oral antimicrobial therapies and biomaterials.

Silver nanoparticles associated with a polyphosphate and fluoride enhance the prevention of enamel demineralization and impact on dual-biofilm adhesion.

OBJECTIVES: The aim of this study were to produce a multifunctional nanocomposite combining silver nanoaparticles (Ag), sodium trimetaphosphate (TMP) and fluoride (F), to investigate its effect on dental enamel demineralization and on biofilms of Streptococcus mutans and Candida albicans. METHODS: Bovine enamel blocks were submitted to five pH cycles and treated 2x/day with 100 ppm F, 225 ppm F, 100 ppm F + 0.2%TMP or 100 ppm F + 0.2%TMP+10% Ag (100F/TMP/Ag). Next, surface hardness loss (%SH), integrated loss of subsurface hardness (ΔKHN), enamel fluoride (F) and calcium (Ca) concentration were determined. Biofilms from single and dual species of S. mutans and C. albicans were treated with 100F/TMP/Ag, Ag or chlorhexidine gluconate for 24 h. The antibiofilm effect was evaluated by colony-forming unit counting and Scanning Electron Microscopy. RESULTS: The nanocomposite reduced 43.0% of %SH and was similar with samples treated with 225F, 100F/TMP and 100/TMP/Ag. The attribute of F and/or TMP in reducing ΔKHN in 5-20 µm was not affected by the addiction of Ag (110F = 225F = 100F/TMP = 100F/TMP/Ag > Negative Control). Further, 100F/TMP/Ag strongly reduced viable cells of S. mutans in dual biofilms (∼5 log10cm2) and structurally affected the biofilms. CONCLUSION: The 100F/TMP/F promoted a protective effect against enamel demineralization and was able to significantly inhibit the growth of biofilms of S. mutans and C. albicans. CLINICAL SIGNIFICANCE: The focus on prevention and non-invasive dental treatment is the most effective and least costly way to improve the population's oral health conditions. We present a nanocomposite for a multiple approach in prevention of caries.

Effects of nano-sized sodium hexametaphosphate on the viability, metabolism, matrix composition, and structure of dual-species biofilms of Streptococcus mutans and Candida albicans.

This study evaluated the effects of micrometric or nano-sized sodium hexametaphosphate (HMPnano), combined or not with fluoride (NaF, 1100 ppm), on dual-species biofilms of Streptococcus mutans and Candida albicans. Biofilms were treated with solutions containing the polyphosphates at 0.5% or 1.0%, with/without fluoride (F), in addition to positive and negative controls. Biofilms were analysed by colony-forming units (CFU) counting, metabolic activity, production of biomass, composition of extracellular matrix, and structure. 1% HMPnano + F led to the lowest S. mutans CFU, while C. albicans CFU counts were not affected by any solution. 1% HMPnano led to the lowest metabolic activity, except for 1% HMPnano + F. All solutions promoted reductions in biofilm biomass compared to controls. Also, 1% HMPnano + F promoted the lowest concentrations of carbohydrates in the biofilm matrix, besides substantially affecting biofilms' structure. In conclusion, HMPnano and F promoted higher antibiofilm effects compared with its micrometric counterpart for most of the parameters assessed.

Green and Chemical Silver Nanoparticles and Pomegranate Formulations to Heal Infected Wounds in Diabetic Rats.

Infected cutaneous ulcers from diabetic rats with Candida albicans and Streptococcus aureus were treated with spray formulations containing green silver nanoparticles (GS), chemical silver nanoparticles (CS), or pomegranate peel extract (PS). After wound development and infection, the treatments were performed twice per day for 14 days. The wound healing was analyzed on days 2, 7, and 14 through the determination of CFUs, inflammatory infiltrate, angiogenesis, fibroplasia, myeloperoxidase, and collagen determination. Expressive improvement in wound healing was noted using both silver nanoparticles for 7 days. All the treatments were superior to controls and promoted significant S. aureus reduction after 14 days. CS presented better anti-inflammatory results, and GS and CS the highest number of fibroblasts. Despite the techniques' limitations, GS and CS demonstrated considerable potential for managing infected wounds, especially considering no early strategies prior to the drugs, such as the debridement of these wounds, were included.

Novel Colloidal Nanocarrier of Cetylpyridinium Chloride: Antifungal Activities on Candida Species and Cytotoxic Potential on Murine Fibroblasts.

Nanocarriers have been used as alternative tools to overcome the resistance of Candida species to conventional treatments. This study prepared a nanocarrier of cetylpyridinium chloride (CPC) using iron oxide nanoparticles (IONPs) conjugated with chitosan (CS), and assessed its antifungal and cytotoxic effects. CPC was immobilized on CS-coated IONPs, and the nanocarrier was physico-chemically characterized. Antifungal effects were determined on planktonic cells of Candida albicans and Candida glabrata (by minimum inhibitory concentration (MIC) assays) and on single- and dual-species biofilms of these strains (by quantification of cultivable cells, total biomass and metabolic activity). Murine fibroblasts were exposed to different concentrations of the nanocarrier, and the cytotoxic effect was evaluated by MTT reduction assay. Characterization methods confirmed the presence of a nanocarrier smaller than 313 nm. IONPs-CS-CPC and free CPC showed the same MIC values (0.78 µg mL-1). CPC-containing nanocarrier at 78 µg mL-1 significantly reduced the number of cultivable cells for all biofilms, surpassing the effect promoted by free CPC. For total biomass, metabolic activity, and cytotoxic effects, the nanocarrier and free CPC produced statistically similar outcomes. In conclusion, the IONPs-CS-CPC nanocarrier was more effective than CPC in reducing the cultivable cells of Candida biofilms without increasing the cytotoxic effects of CPC, and may be a useful tool for the treatment of oral fungal infections.

Novel nanocarrier of miconazole based on chitosan-coated iron oxide nanoparticles as a nanotherapy to fight Candida biofilms.

Overexposure of microorganisms to conventional drugs has led to resistant species that require new treatment strategies. This study prepared and characterized a nanocarrier of miconazole (MCZ) based on iron oxide nanoparticles (IONPs) functionalized with chitosan (CS), and tested its antifungal activity against biofilms of Candida albicans and Candida glabrata. IONPs-CS-MCZ nanocarrier was prepared by loading MCZ on CS-covered IONPs and characterized by physicochemical methods. Minimum inhibitory concentration (MIC) of the nanocarrier was determined by the microdilution method. Biofilms were developed (48 h) in microtiter plates and treated with MCZ-carrying nanocarrier at 31.2 and 78 µg/mL, in both the presence and absence of an external magnetic field (EMF). Biofilms were evaluated by total biomass, metabolic activity, cultivable cells (CFU), extracellular matrix components, scanning electron microscopy and confocal microscopy. Data were analyzed by two-way ANOVA and Holm-Sidak test (p < 0.05). A nanocarrier with diameter lower than 50 nm was obtained, presenting MIC values lower than those found for MCZ, and showing synergism for C. albicans and indifference for C. glabrata (fractional inhibitory concentration indexes of <0.12 and <0.53, respectively). IONPs-CS-MCZ did not affect total biomass and extracellular matrix. IONPs-CS-MCZ containing 78 µg/mL MCZ showed a superior antibiofilm effect to MCZ in reducing CFU and metabolism for single biofilms of C. albicans and dual-species biofilms. The EMF did not improve the nanocarrier effects. Microscopy confirmed the antibiofilm effect of the nanocarrier. In conclusion, IONPs-CS-MCZ was more effective than MCZ mainly against C. albicans planktonic cells and number of CFU and metabolism of the biofilms.

Antimicrobial Activity of Compounds Containing Silver Nanoparticles and Calcium Glycerophosphate in Combination with Tyrosol.

Nanocomposites containing antimicrobial agents and calcium phosphates have been developed. Thus, this study assessed the effects of two compounds containing silver nanoparticles (AgNPs) and ß-calcium glycerophosphate (CaGP), associated or not with tyrosol (TYR), against planktonic cells and biofilms of Candida albicans and Streptococcus mutans. The nanocompounds were synthesized through chemical and 'green' processes and characterized by scanning electron microscopy. The minimum and fractional inhibitory concentrations of each compound were determined for planktonic cells. Next, 24-h single biofilms of C. albicans and S. mutans were treated for 24 h with the nanocompounds alone or in combination with TYR, and the antibiofilm effect was assessed through enumeration of colony forming units. Biofilm data were statistically examined using one-way ANOVA and the Kruskal-Wallis test (α = 0.05). The chemically synthesized nanocompound in combination with TYR demonstrated a synergistic effect against planktonic cells of C. albicans and S. mutans. For the nanocompound obtained through the 'green' route associated with TYR, a synergistic effect was observed only against C. albicans. For biofilms, only the combination obtained through the 'green' route + TYR demonstrated a synergistic effect against C. albicans. Our results may contribute to the development of oral care products containing AgNPs-CaGP and TYR to combat oral infections.

Antibiofilm effect of chlorhexidine-carrier nanosystem based on iron oxide magnetic nanoparticles and chitosan.

This study synthesized and characterized a chlorhexidine (CHX)-carrier nanosystem based on iron oxide magnetic nanoparticles (IONPs) and chitosan (CS), and evaluated its antimicrobial effect on mono- and dual-species biofilms of Candida albicans and Streptococcus mutans. CHX was directly solubilized in CS-coated IONPs and maintained under magnetic stirring for obtaining the IONPs-CS-CHX nanosystem. Antimicrobial susceptibility testing for planktonic cells was performed by determining the minimum inhibitory concentration (MIC) of the nanosystem and controls. The effects of the IONPs-CS-CHX nanosystem on the formation of mono- and dual-species biofilms, as well as on pre-formed biofilms were assessed by quantification of total biomass, metabolic activity and colony-forming units. Data were analyzed by the Kruskal-Wallis' test or one-way analysis of variance, followed by the Student-Newman-Keuls' or Holm-Sidak's tests (α = 0.05), respectively. Physico-chemical results confirmed the formation of a nanosystem with a size smaller than 40 nm. The IONPs-CS-CHX nanosystem and free CHX showed similar MIC values for both species analyzed. In general, biofilm quantification assays revealed that the CHX nanosystem at 78 µg/mL promoted similar or superior antibiofilm effects compared to its counterpart at 39 µg/mL and free CHX at 78 µg/mL. These findings highlight the potential of CS-coated IONPs as preventive or therapeutic agents carrying CHX to fight biofilm-associated oral diseases.

In situ effect of fluoride toothpaste supplemented with nano-sized sodium trimetaphosphate on enamel demineralization prevention and biofilm composition.

OBJECTIVE: To evaluate the effect of a fluoride toothpaste containing nano-sized sodium trimetaphosphate (TMPnano) on enamel demineralization in situ and composition of the biofilm. DESIGN: This crossover double-blind study consisted of four phases (seven days each) and 12 volunteers who wore oral appliances containing four enamel bovine blocks. The cariogenic challenge was performed by 30% sucrose solution (6x/day). The toothpaste treatments (3x/day) were as follows: no F/TMP/TMPnano (Placebo), 1100 ppm F (1100F), 1100F plus 3% micrometric or nano-sized TMP (1100F/TMP; 1100F/TMPnano). Percentage of surface hardness loss (%SH), and integrated loss of subsurface hardness (ΔKHN), as well as enamel calcium (Ca), phosphorus (P), and fluoride (F) were determined. Moreover, biofilm formed on the blocks were analyzed for F, Ca, P, and insoluble extracellular polysaccharide (EPS) concentrations. Data were analyzed using one-way ANOVA, repeated measures followed by Fisher LSD test (p < 0.001). RESULTS: 1100F/TMPnano promoted the lowest %SH and ΔKHN among all groups (p < 0.001). Regarding the F concentrations in the enamel and in the biofilm, there were no significant differences between 1100 F and 1100 F/TMPnano, but significantly increased enamel Ca concentrations (p < 0.001). 1100F/TMPnano showed lower values of EPS concentration when compared with 1100F (∼80%) (p < 0.001). CONCLUSION: 1100F/TMPnano promoted a greater protective effect against enamel demineralization and significantly affected the composition of biofilm formed in situ when compared to 1100F toothpaste.

Antimicrobial Potential and Cytotoxicity of Silver Nanoparticles Phytosynthesized by Pomegranate Peel Extract.

The phytosynthesis of metal nanoparticles is nowadays attracting the increased attention of researchers and is much needed given the worldwide matter related to environmental contamination. The antimicrobial activity of colloidal and spray formulation of silver nanoparticles (AgNPs) synthesized by pomegranate peel extract against Candida albicans and Staphylococcus aureus, and their cytotoxicity in mammalian cells were tested in the present study. Dry matter, pH, total phenolics, and ellagic acid in the extract were determined. Then, AgNPs were phytosynthesized and characterized by X-ray diffraction, electron transmission microscopy, dynamic light scattering, zeta potential, and Ag⁺ dosage. Spray formulations and respective chemical-AgNP controls were prepared and tested. The peel extract reduced more than 99% of Ag⁺, and produced nanoparticles with irregular forms and an 89-nm mean size. All AgNP presented antimicrobial activity, and the spray formulation of green-AgNP increased by 255 and 4 times the effectiveness against S. aureus and C. albicans, respectively. The cytotoxicity of colloidal and spray green-AgNP was expressively lower than the respective chemical controls. Pomegranate peel extract produced stable AgNP with antimicrobial action and low cytotoxicity, stimulating its use in the biomedical field.

Nanosynthesis of Silver-Calcium Glycerophosphate: Promising Association against Oral Pathogens.

Nanobiomaterials combining remineralization and antimicrobial abilities would bring important benefits to control dental caries. This study aimed to produce nanocompounds containing calcium glycerophosphate (CaGP) and silver nanoparticles (AgNP) by varying the reducing agent of silver nitrate (sodium borohydride (B) or sodium citrate (C)), the concentration of silver (1% or 10%), and the CaGP forms (nano or commercial), and analyze its characterization and antimicrobial activity against ATCC Candida albicans (10231) and Streptococcus mutans (25175) by the microdilution method. Controls of AgNP were produced and silver ions (Ag⁺) were quantified in all of the samples. X-ray diffraction, UV-Vis, and scanning electron microscopy (SEM) analysis demonstrated AgNP associated with CaGP. Ag⁺ ions were considerably higher in AgCaGP/C. C. albicans was susceptible to nanocompounds produced with both reducing agents, regardless of Ag concentration and CaGP form, being Ag10%CaGP-N/C the most effective compound (19.5⁻39.0 µg Ag mL−1). While for S. mutans, the effectiveness was observed only for AgCaGP reduced by citrate, also presenting Ag10%CaGP-N the highest effectiveness (156.2⁻312.5 µg Ag mL−1). Notably, CaGP enhanced the silver antimicrobial potential in about two- and eight-fold against C. albicans and S. mutans when compared with the AgNP controls (from 7.8 to 3.9 and from 250 to 31.2 µg Ag mL−1, respectively). The synthesis that was used in this study promoted the formation of AgNP associated with CaGP, and although the use of sodium borohydride (B) resulted in a pronounced reduction of Ag⁺, the composite AgCaGP/B was less effective against the microorganisms that were tested.

Effect of fluoride toothpaste with nano-sized trimetaphosphate on enamel demineralization: An in vitro study.

OBJECTIVE: This study evaluated the effect of toothpastes containing 1100ppm F associated or not with micrometric or nano-sized sodium trimetaphosphate (TMP) on enamel demineralization in vitro, using a pH cycling model. DESIGN: Bovine enamel blocks (4mm×4mm, n=96) were randomly allocated into eight groups (n=12), according to the test toothpastes: Placebo (without fluoride or TMP); 1100ppm F (1100F); 1100F plus micrometric TMP at concentrations of 1%, 3% or 6%; and 1100F plus nanosized TMP at 1%, 3% or 6%. Blocks were treated 2×/day with slurries of toothpastes and submitted to a pH cycling regimen for five days. Next, final surface hardness (SHf), integrated hardness loss (IHL), differential profile of integrated hardness loss (ΔIHL) and enamel fluoride (F) concentrations were determined. Data were analyzed by ANOVA and Student-Newman-Keuls' test (p<0.05). RESULTS: The use of 1100F/3%TMPnano led to SHf 30% higher (p<0.001) and IHL∼80% lower (p<0.001) when compared to 1100F. This toothpaste also resulted in ∼64% reduction of mineral loss (ΔIHL) when compared to 1100F. Moreover, the addition of nano-sized TMP promoted increases in enamel F uptake of 90%, 160% and 100%, respectively for the concentrations of 1%, 3% and 6%, when compared to 1100F (p<0.001). CONCLUSION: The addition of nano-sized TMP at 3% to a conventional toothpaste significantly decreased enamel demineralization when compared to its counterparts without TMP or supplemented with micrometric TMP.