Enhancing antifungal and antibacterial properties of denture resins with nystatin-coated silver nanoparticles | Scientific Reports

Scientific Reports volume 14, Article number: 23770 (2024) Cite this article

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Long-term oral health issues caused by fungi and bacteria are a primary concern for individuals who wear dentures. Denture stomatitis, primarily caused by Candida albicans (C. albicans), is a prevalent condition among denture users. Metal nanoparticles exhibit improved antimicrobial effectiveness and fewer adverse effects. This study aimed to evaluate the antifungal and antibacterial effects of nystatin-coated silver nanoparticles (Nys-coated AgNPs) embedded in acrylic resin as a more biocompatible material for denture resins. AgNPs and Nys-coated AgNPs were synthesized and characterized using UV-Vis, SEM, EDX, and DLS. Specimens of polymethyl methacrylate (PMMA) with three different concentrations of Nys, AgNPs, and Nys-coated AgNPs (0.1%, 1%, 10% w/w) were prepared. The water absorption properties of the disks and drug release were investigated for 14 days and 120 h, respectively. The hydrophilic and hydrophobic properties of the samples and their contact angles were evaluated using the sessile drop technique. The antifungal and antimicrobial activity of the prepared discs was studied against C. albicans and Streptococcus mutans, respectively. Adding Nys-coated AgNPs decreased the contact angle of discs from 67° to 49°. Furthermore, the water absorption rates of the different discs were not significantly different from those of the control groups. Results showed that Nys-coated AgNPs (10% w/w) in PMMA effectively inhibited C. albicans growth better than Nys composites (10% w/w). Additionally, Nys-coated AgNPs composites, as well as AgNPs-containing composites, showed considerable antibacterial activity against S. mutans. Nys-coated AgNPs (10% w/w) had no toxic effect on NIH3T3 cells. In conclusion, Nys-coated AgNPs could be considered a good candidate for incorporation into denture resins to address chronic oral diseases.

Denture bases can be made from various materials, such as heat-cured acrylic resins and metals. Metal denture bases have poor retention because of heavy dentures and limited aesthetic properties. Additionally, they can be more challenging to repair and modify when compared to acrylic resin in cases of highly resorbed alveolar ridges. Overall, while metal denture bases have certain drawbacks that make them less popular1, heat-cured acrylic resins have more favorable characteristics, such as acceptable aesthetics, biocompatibility, easy handling2, oral stability, and cost-effectiveness. However, it is not considered an optimal material because of its low mechanical and physical strength, including low thermal conductivity and a high coefficient of thermal expansion, which leads to internal stresses during the process3. Additionally, the denture base resins serve as a suitable surface for biofilm formation and the adhesion of microorganisms, which can lead to Candida-associated denture stomatitis (DS) and fungal infections, particularly in immunosuppressed and elderly patients4. Epidemiological studies indicate that the prevalence of denture stomatitis (DS) among denture wearers ranges from 15% to over 70%. In general, the incidence of DS is higher among women and elderly denture wearers. Despite its common occurrence, the exact cause of DS is not yet fully understood5. It has been demonstrated that DS can develop as a result of plaque accumulation on denture surfaces. One of the primary pathogenic microorganisms that causes DS is Candida albicans (C. albicans)6. Some factors appear to enhance the ability of C. albicans to colonize both oral mucosal and denture surfaces, including nighttime and continuous use of removable dentures, inadequate denture hygiene, accumulation of denture plaque, and yeast and bacterial contamination of the denture surface5. Mechanical cleaning is a successful method for removing biofilms; however, due to the challenges that elderly patients may face in keeping dentures clean7,8, chemical cleaning is recommended9. Nystatin (Nys) is a polyene macrolide commonly used to treat oral candidiasis in patients with DS or in patients who develop candidiasis after prolonged antibiotic therapy10. Nys also has an important function in preventing systemic and oral candidiasis in infants, premature newborns, and immunocompromised patients, such as organ transplant recipients, cancer patients, and those with AIDS10,11. The oral administration of Nys is not recommended primarily because it is not absorbed in the gastrointestinal tract12. Therefore, the topical application of Nys is the most common method of administration in dentistry, as systemic exposure is minimal. Oral forms of Nys should be administered multiple times a day due to the cleansing effect of saliva and the self-cleaning action promoted by the muscles. Additionally, it possesses a thick consistency, unappealing taste, and poor solubility. Furthermore, the oral suspension has a sweet flavor formulation, which can contribute to dental problems and may result in adverse reactions, particularly in diabetic patients13,14,15,16.

Compounds with antifungal effects can eradicate contamination of C. albicans and reduce DS symptoms, provided the dentures are disinfected and kept clean, otherwise, DS will recur when the treatment stops5. The matrix of the extracellular polymeric substance may limit the access of drugs to organisms located at a greater depth in the biofilm17. Therefore, numerous investigations have been focused on developing dental materials and cleaning techniques to prevent or reduce C. albicans adhesion18,19,20. Redding et al. applied a thin-film polymer coating that contained antifungal agents to decrease the formation of C. albicans biofilms21. Zhou et al. developed parylene coatings with a high antifungal activity which can reduce C. albicans adhesion on the surfaces of denture bases22. Also, Lazarin et al. used a photo-polymerized coating that included a hydrophilic monomer to reduce the adhesion of C. albicans23.

The addition of nanoparticles (NPs) to poly methyl methacrylate (PMMA) has recently gained attention due to the improvement of the resin’s physical and mechanical properties. NPs may also display remarkable antibacterial activity because of their chemical reactivity and high surface area24,25 leading to high efficiency even at small concentrations26. Among different NPs, silver NPs (AgNPs) have been investigated as one of the most popular materials due to their unique characteristics27. AgNPs have been shown to have effective antibacterial properties, providing a beneficial alternative to antibiotics in the treatment of several infections, and do not cause microbial resistance28,29,30,31,32. AgNPs can be added to the acrylic resin to prevent the growth of bacteria such as Escherichia coli, Staphylococcus aureus, and Streptococcus mutans (S. mutans)33. Also, it has been shown that acrylic resin incorporated with AgNPs inhibits the adhesion of C. albicans and provides antifungal activity against C. albicans34. AgNPs can also improve the mechanical features of acrylic resin. The effect of AgNPs on the mechanical properties depends on the AgNP concentration, the kind of acrylic resin, and the polar interactions between the AgNPs and PMMA chains24. AgNPs can increase the elastic modulus, compressive strength, thermal conductivity, and flexural strength of acryl resin35 and taste sensation36. Resin discoloration, limited application in esthetic areas, also inhibitory effect on tensile and flexural strengths are the disadvantages of acrylic resin incorporated with AgNPs37,38,39.

This study aimed to improve the composition of the PMMA by incorporating AgNPs and Nys-coated AgNPs to develop a new nanocomposite with better antifungal and antibacterial effects.

All materials, including polyvinyl (PVA) and silver nitrate (AgNO3), sodium borohydride (NaBH4), and Nys were provided from Sigma Aldrich (St. Louis, Missouri, United States) and used with no more purification. AgNPs were synthesized by chemical reduction40 of AgNO3 by using NaBH4. The reaction was completed in the presence of PVP to control the agglomeration and the growth of NPs41. 500 mg of silver nitrate AgNO3 was combined with 25 ml of distilled water. In addition, 766 mg of sodium borohydride (NaBH4) and 800 mg of PVP were dissolved in 75 mL of distilled water, and then the AgNO3 solution was slowly added to the second solution within 15 min. The reaction mixture was stirred for 30 min on a stirrer at room temperature to ensure completion of the reaction. Subsequently, it was kept at 4 °C for 2 days to allow for proper completion of the reaction. For the synthesis of Nys-coated AgNPs, 125 mg of Nys was dissolved in 25 mL of methanol. Following this, 25 mL of the prepared Nys and AgNO3 solution were combined and stirred. Then 25 mL of the prepared mixed solution was added dropwise to the 75 mL of the PVP and NaBH4(Fig. 1). Finally, the solution of AgNPs and Nys-coated AgNPs was dried using the freeze-drying method.

(A) Mixture of PVP and NaBH4 solution, (B) preparation of Nys-coated AgNPs.

Various methods have been used to characterize the prepared NPs. UV-vis analysis was performed at 25 °C using a Cecil UVVis spectrophotometer (Sydney, Australia) to investigate the presence of AgNPs, Nys, and Nys-coated AgNPs42. The size distribution and ζ-potential of AgNPs, Nys-coated AgNPs were analyzed using dynamic light scattering at pH 7.4 with a Nanotrac Wave (Microtrac, PA, USA)43. The morphology and elemental analysis of the prepared nanocomposites were studied by SEM and EDX. To perform SEM and EDX analyses, the NP samples were deposited onto thin glass slides and subsequently dried. Following this, a thin layer of gold was applied to the dried samples using a direct current sputter (Emitech K450X, UK). FTIR was utilized for structural analysis (Tensor 27, Bruker, Germany) with an attenuated total reflectance accessory44.

AgNPs and Nys-coated AgNPs at three different concentrations (0.1%, 1%, and 10% (w/w)) were blended with heat-curing acrylic resin. The selection of the three different concentrations of Nys, AgNPs, and Nys-coated AgNPs for embedding in PMMA was based on preliminary experiments. These initial studies were conducted to evaluate the efficacy and compatibility of each concentration, ensuring optimal antifungal and antibacterial properties while maintaining the structural integrity of the PMMA matrix. These concentrations were determined based on a preliminary study, which indicated that adding less than 1% (w/w) of AgNPs to the acrylic resin powder does not exhibit antifungal and antibacterial activity. The Nys, AgNPs, and Nys-coated AgNPs were mixed using an ultrasonic machine (80 MPa for 5 min, Ultrasonic Homogenizers HD2070) with a resin monomer. The resulting powder and monomer liquid mixture were thoroughly blended and processed in accordance with the manufacturer’s instructions. After mixing, the prepared paste was filled into steel molds (Fig. 2) at the dough stage. The samples were placed between two glass slides before investing to achieve standardized and smooth specimens45,46. The samples were compressed using a hydraulic press (Delta, Vipi-Delta Maquinas Especiais, Pirassununga, Brazil). After applying 2 MPa pressure for 5 min, the flasks were separated, silicone patterns were removed, and excess material was trimmed. Subsequently, the mold was subjected to pressure for over 15 min. Following this, they were polymerized in a water bath until they reached the boiling point and then left to set for 30 min. The flasks were allowed to cool at room temperature, the specimens were deflasked and excess flash was removed using a sterile bur (Maxi-Cut; Lesfils de August Malleifer SA, Ballaigues, Switzerland)47.

The sample size was determined based on pilot study results (15 × 2-mm disks for contact angle and 5 × 2-mm disks for other tests). Thirty samples were given for each type of test, such as antifungal, antibacterial, contact angle, water sorption, drug release, and toxicity tests, making a total of 180 samples. Specimens for every test were subdivided into 10 groups as follows (Fig. 3): Group A: 3 specimens of pure acrylic resin were used as the control group.

Group B: 3 specimens of PMMA were mixed with 0.1% (w/w) of AgNPs composite. (AgNPs/PMMA composite). Group C: 3 specimens of PMMA were mixed with 1% (w/w) of AgNPs/ PMMA composite. Group D: 3 specimens of PMMA were mixed with 10% (w/w) of AgNPs/ PMMA composite. Group E: 3 specimens of PMMA were mixed with 0.1% (w/w) of Nys-coated AgNPs composite (Nys-coated AgNPs/PMMA composite). Group F: 3 specimens of PMMA were mixed with 1% (w/w) of Nys-coated AgNPs/PMMA composite. Group G: 3 specimens of PMMA were mixed with 10% (w/w) of Nys-coated AgNPs/PMMA composite.

Group H: 3 specimens of PMMA were mixed with 0.1% (w/w) of Nys composite. (Nys/PMMA composite). Group I: 3 specimens of PMMA were mixed with 1% (w/w) of Nys/ PMMA composite. Group J: 3 specimens of PMMA were mixed with 10% (w/w) of Nys/ PMMA composite.

Steel molds for (A) 15 × 2-mm disks (B) 5 × 2-mm disks and (C) sandwich steel molds ready for press.

Images of PMMA composite discs.

Water absorption was measured using distilled water at 37 °C. Firstly, the prepared disks were weighed (with ± 0.1 mg precision) using an “AND Weighing GR-200 Analytical” scale and after that, they were put in distilled water (10 mL) at 37 °C in individual jars. Disks were weighted daily for the 7 days. The extra water on the disks was removed before measuring the weight with filter paper. The water absorption (M∞) was calculated by the following formula:

M ∞ = m1 – m0/m0.

m1 is the saturated disk weight at equilibrium conditions, and m0 is the initial weight of dry samples before immersion48.

The hydrophobicity of the surface was assessed by measuring the water contact angle using the sessile drop technique. Each sample surface was air-dried, and then a small amount of distilled water (2 µL) was dispensed onto the surface using an auto-pipette. The contact angle measurements were carried out using an automated goniometer (Contact Angle Goniometer (CAG-20 PE)), which includes a CCD camera to capture the droplet image and proprietary image processing software to measure the contact angle. The measurements were performed in two different positions for each sample, and the average contact angle was calculated47,49.

To investigate the release of Nys from nanocomposites, prepared discs were placed in 10 mL of PBS and subjected to constant stirring at 37 ºC using a multipoint magnetic stirrer. At appropriate intervals, 1 mL of the medium was withdrawn and replaced with fresh PBS. The concentration of Nys in the withdrawn samples was measured three times using UV-vis spectrophotometry at 322 nm. Before the measurements, all samples were centrifuged for 5 min at 7,250 × g to obtain a clear sample50.

The antimicrobial properties of the prepared discs were assessed using agar diffusion assays on Muller-Hinton agar (for antibacterial activity) and Sabouraud Dextrose agar (for antifungal activity). To summarize, a microbial suspension with a 0.5 McFarland standard (1.5 × 108 CFU/mL) was cultured on Muller-Hinton Agar plates and allowed to dry for 10 min. Six-millimeter disks were placed on the agar surface, and the plates were then incubated at 35 °C for 24 h. After incubation, the zones of inhibition around the disks were measured51.

To determine the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of prepared discs including AgNPs incorporated in PMMA, nystatin-coated AgNPs incorporated in PMMA and plain PMMA overnight cultures of S. mutans were prepared in the respective broth media and adjusted to a concentration of 1 × 106 CFU/mL. The PMMA discs were immersed in sterile broth for 14 days to extract the nanoparticles and different dilutions were prepared. The inoculum was prepared by dilution in media to give a final organism density of approximately 5 × 105 CFU/ml in the well. In a 96-well microtiter plate, 100 µL of each extract was combined with 100 µL of the inoculum in each well, including control wells with only broth and inoculum. The plates were incubated at 37 °C for 24 h, and were coloured after the addition of 10 µl of resazurin (337.5 mg of resazurin powder in 50 ml of sterile distilled water, Serva, Germany) and incubated at 37 °C for 3 h for observation of the colour change, and the MIC was defined using ELx 800 microplate reader (BioTek, Winooski, VT, USA) at 570 nm as the lowest concentration exhibiting no visible growth. For the MBC determination, 10 µL samples from wells showing no growth were plated onto blood agar for S. mutans, followed by incubation at 37 °C for 24 h. The MBC was identified as the lowest concentration of extracts released from discs that resulted in a ≥ 99.9% reduction in CFU compared to the control. This comprehensive approach enabled the evaluation of the antimicrobial efficacy of prepared PMMA composite disc against the selected pathogens.

NIH3 cells were purchased from the Pasteur Institute of Iran. The toxicity of the prepared composites on NIH3 cells was evaluated by MTT assay. NIH3 cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 µg/mL) and then incubated in an incubator at 37 ˚C, 5% CO2, and 98% humidity. When the cell confluency reached 60-70%, the cells were detached using trypsin and then seeded in 96 well plates at a density of 10 × 10 3 cells/well and incubated for 24 h. After sterilization of the prepared composites, they were kept in media for 7 days at 4 °C. The media of the seeded NIH3 cells were replaced with the media incubated with the prepared composites. The untreated cells and 5% DMSO-treated cells were used as negative and positive controls, respectively in the MTT test. After 48 h, the previous media was removed and then fresh media containing 2 mg/ml MTT solution was added to each well and incubated for another 4 h. After that, to dissolve formazan crystal, DMSO was added to each well and the absorbance of each well as cell viability index was measured at 570 nm wavelength with the microplate reader.

The growth performance of the prepared composites (e.g., Nys/PMMA (10% w/w), AgNPs/PMMA (10% w/w), and Nys-coated AgNPs/PMMA (10% w/w)) on NIH3 cells was assessed through Resazurin reduction assay. After sterilization of the prepared composites, they were kept in media for 14 days at 4 °C. NIH3 cells were seeded in 96-well plate at a density of 10 × 103 cells per well and incubated for 24 h. The media of the cells were replaced with the media incubated with the prepared composites. Each well was supplemented with RPMI-1640 culture medium, and the plates were incubated for a duration of ten days, during which the treatment medium was replaced every two days. Upon reaching the specified time intervals, the viability of the cells was evaluated using the resazurin reduction assay. To perform the assay, first, high purity resazurin was dissolved in PBS (pH 7.4) to a concentration of 0.15 mg/ml and then filter-sterilized through a 0.2 μm filter. For the assay, the plates were incubated for the desired exposure period, after which 20 µl of the resazurin solution was added to each well. Following an additional incubation of 1 to 4 h at 37 °C, absorbance was measured at 570 nm wavelength with the microplate reader. The untreated cells were used as negative controls.

The synthesis of silver nanoparticles (AgNPs) through chemical techniques relies on three key factors: (a) reducing agents, (b) stabilizing agents, and (c) silver precursors. The shape and size of the NP are influenced by the concentrations of PVP and silver nitrate (Ag(NO3)). By carefully controlling these experimental parameters, the geometry of the NPs can be tailored. In this study, the size of the NPs was optimized, resulting in a size range of 5 nm to 100 nm52,53. UV-visible spectrophotometry was employed to qualitatively examine the UV absorbance profile of Nys, AgNPs, and Nys-coated AgNPs. Figure 4 illustrates that the maximum absorbance peak of the AgNPs solution was observed at 400 nm, while an aqueous solution of Nys exhibited absorption peaks in the range of 275 nm to 325 nm, which is characteristic of Nys. Additionally, the Nys-coated AgNPs solution displayed peaks in the range of 305 nm to 395 nm.

UV-vis of plain Nys, AgNPs, and Nys-coated AgNPs.

Figure 5 depicts the dimensions of the synthesized AgNPs and Nys-coated AgNPs, which were measured using Dynamic Light Scattering (Nanotrac Wave II). The results indicate that the majority of AgNPs exhibited a size of 11.62 nm with a narrow distribution. As illustrated in Fig. 5b, this value increased with the addition of Nys. The average size of Nys-coated AgNPs was 12.18 nm. The zeta potential of AgNPs and Nys-coated AgNPs provides valuable insights into their stability and dispersion in solution. The zeta potential of bare AgNPs was influenced by the presence of PVP, which serves as a stabilizing agent. Our results indicated that bare AgNPs exhibited a zeta potential of -23 ± 2.1 mV, demonstrating sufficient stability; the negative charge helps prevent aggregation through electrostatic repulsion. The decoration of AgNPs with Nys altered the zeta potential due to the positive charge of the coating, resulting in a shift to a less negative value of -11 ± 1.4 mV for the Nys-coated AgNPs.

The size distribution of (A) AgNPs and (B) Nys-coated AgNPs.

Figure 6 presents the SEM image of the prepared composites. The control samples exhibited surface irregularities, while the AgNPs were observed as spherical and uniform particles. Additionally, the SEM findings verified a smoother surface with reduced vacuole formation. Furthermore, Fig. 7 illustrates the EDX findings regarding the ratios of silver (Ag), oxygen (O), and carbon (C) in AgNPs/PMMA, Nys-coated AgNPs/PMMA, Nys/PMMA composites, and control groups. The EDX spectrum demonstrated the presence of elements in a specific area of the SEM image and confirmed the successful preparation of various composites.

SEM and EDX images (A) control PMMA composite (B) Nys/PMMA composite (C) AgNPs/PMMA composite (D) Nys-coated AgNPs/PMMA composite. (E) AgNPs (F) Nys-coated AgNPs.

The EDX analysis was used to quantify the amounts of carbon (C), nitrogen (N), oxygen (O), and silver (Ag) in the control PMMA composite, Nys/PMMA composite, AgNPs/PMMA composite, and Nys-coated AgNPs/PMMA composite.

The FTIR spectrum of Nys-coated AgNPs showed no significant differences from AgNPs in terms of tension or stretching aspects. Additionally, the absence of peaks related to pure Nys in the FTIR spectrum of Nys-coated AgNPs indicates the complete involvement of Nys in the PVP coating around AgNPs during formulation (Fig. 8).

FTIR analysis data of plain Nys, AgNPs, and Nys-coated AgNPs (Nys/AgNPs).

The water absorption property was not significantly different between the prepared materials and the control group. The Ag/PMMA composites had the highest water absorption, which did not exceed 6%. Additionally, all nanocomposites and Nys/PMMA composites showed higher water absorption compared to the control groups (Fig. 9). The quantity of water absorption was influenced by the concentration of the added materials, with higher concentrations leading to increased water absorption and lower concentrations resulting in decreased water absorption. However, there was no significant difference in water absorption observed among the different disk samples.

Water absorption (%) of nanocomposites containing pure nystatin (Nys), AgNPs (Ag), and Nys-coated AgNPs (Nys/AgNPs) with different percentages of Nys and NPs.

The contact angle is used to evaluate the hydrophilicity and hydrophobicity of polymers. The hydrophilic polymers have low contact angles. Considering that AgNPs have hydrophilic properties, it is expected that the addition of these NPs to the polymer will decrease the contact angle of the polymer. As seen in Figs. 10 and 11, the study results indicate that AgNPs/PMMA composites exhibited the lowest contact angle and the best hydrophilic properties. Additionally, the contact angle of Nys-coated AgNPs/PMMA and Nys/PMMA composites decreased compared to the control group. Furthermore, Nys/PMMA composites were less hydrophilic than nanocomposites. The hydrophilicity of the polymer is shown to depend on the concentration of NPs and Nys in the composites. At high concentrations, the contact angle decreases, and the polymer’s hydrophilicity increases.

The contact angle of distilled water on different nanocomposite discs with different percentages of pure nystatin (Nys/PMMA), AgNPs (AgNPs/PMMA), and Nys-coated AgNPs (Nys/AgNPs/PMMA).

Quantitative results of contact angle of distilled water on different nanocomposite discs with different percentages of pure nystatin (Nys/PMMA), AgNPs (AgNPs/PMMA), and Nys-coated AgNPs (Nys/AgNPs/PMMA) at various.

Figure 12 depicts the Nys release profile from Nys-coated AgNPs/PMMA and Nys/PMMA composites over 120-hour. The release of Nys was faster and higher in the case of the Nys-coated AgNPs/PMMA composite compared to the Nys/PMMA composite. These findings are in line with the results of the contact angle analysis. As previously reported, Nys-coated AgNPs/PMMA composites exhibited greater water absorption compared to the Nys/PMMA composites and the control group, indicating the hydrophilic nature of the Nys-coated AgNPs/PMMA composites.

Cumulative release of Nys from Nys/PMMA (Nys; 0.1, 1 and 10% w/w) and Nys-coated AgNPs/PMMA (Nys-coated AgNPs; 0.1, 1 and 10% w/w) composites.

Figures 13 and 14 display the results of the agar diffusion test conducted after introducing the samples into Sabouraud dextrose agar containing C. albicans. There was a notable contrast in the antifungal properties of the different groups. The inhibition zone was not observed at low concentrations (0.1% and 1% (w/w)) in all groups, while 10% (w/w) of Nys-coated AgNPs/PMMA exhibited the maximum inhibition zone around the discs, similar to the 10% (w/w) of Nys/PMMA (24 mm) (the diameter of all discs is 5 mm). Discs containing AgNPs also displayed antifungal activity (13 mm) but not as much as Nys/PPMA composite (16 mm).

The diameter of the inhibitory zone for the 10% (w/w) AgNPs/PMMA composite was 15 mm, whereas for the 10% (w/w) Nys-coated AgNPs/PMMA, it was 13 mm (the diameter of all discs was 5 mm). The MIC for AgNPs/PMMA ranged from 220 to 323 µl/ml, while the MIC for Nys-coated AgNPs/PMMA varied between 62 and 189 µl/ml (Table 1). Notably, the Nys-coated AgNPs/PMMA (10% w/w) exhibited a significantly lower MIC (P < 0.01) against S. mutans compared to other composites. Additionally, the results of the MIC and MBC assays corroborated the findings from agar diffusion tests. These results confirm that the Nys-coated AgNPs/PMMA composite exhibits effective antibacterial activity, particularly in comparison to the AgNPs/PMMA composite.

(A) Antibacterial and (B) antifungal activity of different PMMA composites samples containing 0.1, 1, and 10% w/w of plain nystatin (Nys/PMMA), AgNPs/PMMA (AgNPs) and Nys-coated AgNPs/PMMA (AgNPs/Nys).

Quantitative result of (A) antibacterial and (B) antifungal activity of samples with different PMMA composites containing 0.1, 1, and 10% (w/w) of plain nystatin (Nys/PMMA), AgNPs/PMMA, and Nys-coated AgNPs/PMMA.

The MTT assay was used to investigate the cytotoxicity of the prepared discs. The findings indicated that the release media of neither the 0.1% nor the 1% (w/w) composites differed significantly from the negative control, suggesting no significant toxicity effects. However, the AgNPs composites (10% w/w) exhibited toxic effects, unlike the composites containing Nys (10% w/w). It seems that the toxic effects of AgNPs are diminished as a result of their association with Nys in composites containing Nys-coated AgNPs (Fig. 15A). Furthermore, the positive control (cells treated with DMSO (5% v/v)) led to a 75% inhibition of healthy NIH3 cells (Fig. 15C).

The proliferation of cells in the presence of media released from the composites was evaluated using healthy NIH3 cells (10 × 103 cells per well) over periods of 5, 7 and 10 days, utilizing the Resazurin reduction assay (Fig. 15B). After five days, the release media of both the Nys-coated AgNPs/PMMA (10% w/w) and Nys/PMMA (10% w/w) exhibited a slight increase in cell proliferation compared to the control sample (PMMA). In contrast, at the 7-day and 10-day marks, the Nys-coated AgNPs/PMMA (10% w/w) and Nys/PMMA (10% w/w) demonstrated significantly greater cell proliferation than the control. Specifically, after 7 days, the release media of Nys-coated AgNPs/PMMA (10% w/w) and Nys/PMMA (10% w/w) were recorded as 1.32 and 1.21 times, respectively. After 10 days, these rates increased to 1.36 and 1.27 times, respectively. However, cell proliferation assay confirms the results obtained from cell crypticity assay which imply the toxicity of AgNPs/PMMA (10% w/w).

(A) exhibits cytocompatibility assessment of different prepared nanocomposites on NIH3 cells. (B) shows cell proliferation performance of Nys/PMMA (10% w/w), AgNPs/PMMA (10% w/w), and Nys-coated AgNPs/PMMA (10% w/w). (C) shows cytocompatibility of plain PMMA. DMSO (5% v/v) was used as positive control.

In this study, AgNPs were synthesized using a chemical reduction technique and subsequently characterized. The UV-vis analysis of the AgNPs revealed a peak absorption near 400 nm, confirming the successful synthesis of the AgNPs31,41,54,55,56,57. The AgNPs-coated Nys exhibited a distinct peak absorption for AgNPs at approximately 400 nm, as well as peaks at around 280–330 nm for Nys58. Therefore, the presence of both of these peaks confirms the successful coating of AgNPs with Nys. The particle size was found to be in the range of 11–12 nm. It has been shown that smaller NPs exhibit a stronger antimicrobial effect, attributed to their larger surface area for interaction with various microorganisms59,60. The EDX-SEM analysis revealed that the nanocomposites primarily consist of Ag and C, representing the doping material and polymer, respectively61. The FTIR analysis data indicated that Nys was effectively involved in PVP56,62.

Inorganic NPs have been successfully utilized in the development of innovative nanodevices for various physical, pharmaceutical, biomedical, and biological applications63,64. AgNPs are particularly known for their effective antimicrobial properties, as they exhibit high toxicity against most microorganisms, with very limited occurrence of silver-resistant strains65,66, while silver-resistant strains are very limited67. Some researchers have observed the inhibitory effect of AgNPs/PMMA composites on the growth of S. mutans31. In the case of silver/polymer composites, the AgNPs can be freely dispersed in the target area and exert their cytotoxic activity. Additionally, the nanometer size of AgNPs provides a larger surface area for interaction, resulting in stronger bactericidal activity compared to micrometer-sized particles59,68. It has been demonstrated that metallic silver releases silver ions upon contact with water. The concentration of ions released from PMMA was very low and according to the toxicity assay results exhibited non-toxicity towards human cells. These ions function as a broad-spectrum biocide, effective even at minimal concentrations. In water, silver ion concentrations exceeding 0.1 µg/L are sufficient to eradicate bacteria. Additionally, disruption of Candida albicans occurs even at very low concentrations. Compared to other metal salts, silver’s toxicity to human cells is relatively low; concentrations above 10 mg/L are required to induce toxic effects. Therefore, the concentrations of silver ions remain well below the toxic threshold29,69. The mechanism of action of AgNPs on microorganisms has been studied, revealing that AgNPs can alter bacterial structure by incorporating into the bacterial membrane, increasing membrane permeability, and ultimately leading to cell death. Additionally, AgNPs have been found to bind to phosphorus-containing elements such as DNA due to silver’s strong tendency to react with such elements54,59,60,70,71. Asimuddin et al. reported the concentration-dependent functionality of AgNPs in mediating bacterial death, and our results also demonstrated a concentration-dependent effect of these nanoparticles72. Therefore, the synthesized AgNPs/PMMA composite exhibits significant contact antibacterial activity compared to other samples. The impact of AgNPs on C. albicans and their antifungal activity remains incompletely understood57,73,74,75. Similar to our findings, previous studies have shown that AgNPs exhibit antifungal effects on C. albicans57,74, due to membrane disruption and depolarization, enhanced release of trehalose and glucose levels, damage to the envelope structure, and inhibition of the normal budding process74. Upon adding AgNPs to the PMMA, no effect was observed on C. albicans at low concentrations. Therefore, the absence of an antifungal effect of the AgNPs/PMMA and Nys-coated AgNPs/PMMA composites observed in this study could be attributed to the low release of AgNPs or silver ions from the samples. These findings may also be linked to the polymer network structure. The denture base acrylic resin VIPI Wave contains a cross-linking agent (ethylene glycol dimethacrylate) in the liquid. Hence, it is possible that the polymeric network of the samples may have trapped AgNPs or silver ions and limited their diffusion in the test environment47. However, a strong antifungal effect was observed at high concentrations of AgNPs and Nys-coated AgNPs. These findings demonstrated the antifungal impact of AgNPs and their ability to compromise membrane integrity, as well as disrupt organelle and cellular structure, which are crucial for fungal survival and growth76. Interestingly, Nys-coated AgNPs/PMMA composites exhibited superior antifungal effects compared to Nys and AgNPs alone. The exact antimicrobial mechanism of AgNPs remains incompletely understood. As previously mentioned, their antimicrobial effects depend on multiple factors, including interactions with microbial cell walls, the release of toxic ions, cell penetration, the generation of free radicals and ROS, and ultimately, DNA damage77. These studies have demonstrated that the conjugation of various drugs to AgNPs enhances their antimicrobial effects, likely due to AgNPs high drug-loading capacity and small size. Nys-AgNPs have shown greater efficacy against specific microorganisms compared to Nys alone78. Furthermore, AgNPs not only exhibit significant antibacterial potential but also interact synergistically with conventional antibiotics, thereby enhancing their antibacterial activities79. This synergistic effect may also extend to antifungal activity. Nys disrupts the yeast cell membrane, leading to increased permeability of the cell membrane and other cellular constituents, while AgNPs act through multiple mechanisms80. The disruption of the cell membrane by Nys may facilitate the uptake of AgNPs into the microbial cytoplasm, allowing them to bind to specific targets81. Furthermore, the rate at which Nys is released from the Nys-coated AgNPs/PMMA composite could impact the antifungal activity of the composites. Due to the hydrophilic nature of Nys, the release of Nys from discs in the Nys-coated AgNPs/PMMA composite is faster than in the Nys/PMMA composites. As a result, the higher antifungal activity of the Nys-coated AgNPs/PMMA composite is expected. Conversely, the Nys-coated AgNPs/PMMA composite may exhibit greater porosity compared to the Nys/PMMA composite, leading to an inevitable increase in the release speed of Nys. This observation was reported by Kamaly et al. in 201682.

Furthermore, it is well established that the water absorption of the carrier matrix has a significant impact on the drug release profile83,84. As a result, drug release is accelerated in specimens with a higher water absorption ratio due to the increased mobility of the resin chains85. Complete antimicrobial action was observed over extended time scales due to prolonged surface contact, attributed to the controlled and slow release of silver ions from the polymer86. The release of silver ions from these polymers was limited; in fact, only about 4% of the total silver content was released after 2 days of immersion in water. This minimal release does not significantly alter the dimensions or shape of the polymers. Such long-term antimicrobial action can effectively prevent the formation and adhesion of microbial colonies87. We believe this novel approach holds promising applications for prosthetic dentures, as it provides sustained antimicrobial effects. Water absorption has two primary effects on the resin: it reduces mechanical properties and causes expansion due to the pressure of water molecules on the resin polymers. Limited water absorption over an extended period of use can result in resin expansion, which, in some cases, helps to counteract the shrinkage resulting from polymerization. However, high water absorption can be detrimental. Additionally, increased water absorption may reduce internal polymerization stresses88. The limited water absorption of the discs prepared in this study means that only the positive impact of water absorption is considered. Additionally, the influence of adding AgNPs and Nys on the hydrophobicity of the composites was examined. Research has shown that the hydrophobicity of the polymer surface is linked to the adhesion of Candida species89,90,91. Yoshijima et al. also found that the adhesion of C. albicans hyphae was reduced by hydrophilic coatings on acrylic denture bases92. Regardless of the concentration of added substances, the different PMMA composites led to a significant decrease in the hydrophobicity of all compounds. Given the hydrophobic nature of C. albicans hyphae, these findings suggest a substantial reduction in the formation and adherence of biofilm on the surface of the prepared discs. The findings obtained indicate that AgNPs embedded in poly methyl methacrylate denture base material have significant antifungal properties, which warrant further investigation for clinical applications.

In this study, we have demonstrated the enhanced antifungal and antibacterial properties of denture resins modified with nystatin-coated silver nanoparticles Nys-coated AgNPs through in vitro experiments. However, a significant limitation of our research is the absence of in vivo experiments. While our findings provide promising insights into the antimicrobial efficacy of these nanocomposites, the true translational potential of Nys-coated AgNPs can only be fully realized through in vivo investigations. Such studies are crucial for assessing the safety, efficacy, and long-term durability of these materials in biological systems, particularly in the context of real-world applications involving C. albicans and S. mutans infections. Future research should prioritize in vivo evaluations, including animal studies and clinical trials, to explore various administration routes and optimize the use of Nys-coated AgNPs as a drug delivery system. This will not only enhance our understanding of their biological interactions but also pave the way for developing effective treatment strategies for oral infections, addressing the pressing need for improved therapeutic options in dental care.

This article discusses the synthesis, characterization, and biological efficacy of Nys-coated AgNPs embedded in polymethyl methacrylate (PMMA) denture base material. Denture stomatitis, caused by C. albicans, is a common condition among denture users. The study aimed to evaluate the antifungal and antibacterial effects of Nys-coated AgNPs in denture resins. The Nys-coated AgNPs/PMMA nanocomposites exhibited a greater inhibitory effect on the growth of C. albicans compared to both AgNPs and plain Nys composites. Additionally, the antibacterial properties of the Nys-coated AgNPs/PMMA composite were found to be similar to those of AgNP-containing discs. The NPs did not have any toxic effects on cells and did not significantly alter the water absorption properties of the denture base material. Given their long-term and sustainable effectiveness, lower toxicity, and ease of handling, Nys-coated AgNPs could be considered a favorable option for incorporation into denture resins to address chronic oral diseases caused by fungi and bacteria.

Data is provided within the manuscript or supplementary information files.

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The authors are grateful for the financial support [grant number 70714] provided by Tabriz University of Medical Sciences, Tabriz, Iran.

Department of Prosthodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran

Elaheh Salehi Abar & Ali Torab

Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran

Elaheh Salehi Abar & Morteza Eskandani

Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Somayeh Vandghanooni

Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Mohammad Yousef Memar

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Elaheh Salehi Abar: Investigation, Data analyses, Writing – original draft. Mohammad Yousef Memar: Investigation, Writing – review & editing. Somayeh Vandghanooni: Formal analysis, Writing – review & editing. Morteza Eskandani: Conceptualization, Supervision, Writing – review & editing. Ali Torab: Supervision, Writing – review & editing.

Correspondence to Morteza Eskandani or Ali Torab.

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Abar, E.S., Vandghanooni, S., Memar, M.Y. et al. Enhancing antifungal and antibacterial properties of denture resins with nystatin-coated silver nanoparticles. Sci Rep 14, 23770 (2024). https://doi.org/10.1038/s41598-024-74465-7

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DOI: https://doi.org/10.1038/s41598-024-74465-7

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