In Vitro Antifungal Activity against Oral Candida Species Using a Denture Base Coated with Silver Nanoparticles

Although oral Candida easily adheres to denture base materials, many denture detergents are effective only against bacteria but not againstCandida. Silver nanoparticles (AgNPs), which are known to have potent antibacterial and antifungal activity, have been used in the prevention of oral candidiasis (OC). We evaluated the adherence of Candida albicans and Candida glabrata on a heat-cured Acron resin piece supported by AgNPs by low-vacuum scanning electron microscopy (SEM) andmeasuring colony-forming units. C. albicans and C. glabrata increasingly adhered to the resin surface of the control piece over time, but the adhesion AgNP of both Candida species to the AgNP-coated surface was significantly inhibited (P < 0.001). Low-vacuum SEM revealed that C. albicans and C. glabrata on the resin surface of control pieces appeared as oval colonies, with a major axis of 3-4 μm and a smooth cell wall, but those on the AgNP-coated resin surface were less abundant than the control and showed swollen yeast features, with a major axis of more than 5 μm and a corrugated cell wall. Our results suggest a way to prevent denture-associated OC by using denture base materials processed by AgNPs.


Introduction
Because oral care decreases the disease prevalence of respiratory tract infections of perioperative and elderly patients, as well as persons requiring nursing care [1], the importance of oral care is widely recognized.Progress in social aging has led to an increased population of denture-wearers, but denture cleaning is not easy for the elderly.Although the tedious care required renders denture wearing cumbersome, to such a patient or a person requiring social care, denture wearing is indispensable for food intake, as well as to maintain nutritional status.Candida, which easily adheres to denture base material, serves as a hotbed for mycotic stomatitis [2].However, there are very few commercially available denture-cleaning agents effective against Candida.Metal ions such as silver, copper, and zinc have bactericidal action.The antibacterial activities and safety of silver have become apparent in recent years, resulting in its growing use in the fields of food and medicine.Silver nanoparticles (AgNPs) have high antimicrobial activity and have shown potent inhibition of Candida, equivalent to that of amphotericin B [3][4][5].Studies on the targets and mechanisms of AgNPs have indicated that reactive oxygen species (ROS) and hydroxyl radicals produced by AgNPs disrupt the mitochondria and cell membrane of a fungus, leading to apoptosis [4,6].However, although AgNP-containing denture resin prevents the adhesion of Candida albicans to the resin [7], it does not influence the adhesion of other fungi or biofilm formation, despite antifungal activity [8].In order to demonstrate the anticandidiasis action of AgNPs, we evaluated the application of AgNP-processed denture base AgNP for the prevention of oral candidiasis (OC).

Preparation of Silver-Incorporated Heat-Cured Resin.
A heat-cured acrylic resin for denture base, Acron, was used in the study.A paraffin wax sheet (146 × 74 mm), with a 1.0 mm thickness (Bite Wax, Quest Corp., Japan), was embedded with Ag-coated Control (uncoated) Incubation time (h) Incubation time (h) plaster and washed with hot water after curing.A mixture of poly(methyl methacrylate) powder and solution (PMMA; Acron, GC Co. Ltd., Japan), at ratio of 100 g : 46 mL, was used to infill the gap in the plaster at 100 ∘ C and 15 MPa for 15 min.The plaster was carefully removed to obtain a heat-cured resin sheet with a 1.0 mm thickness, and the Acron piece was cut into 10 mm × 10 mm pieces to be used in the study.AgNPs were obtained by microwave irradiation of a solution mixture containing silver nitrate, polyethylene glycol, ethanol, water, and 24%/5% w/w aqueous ammonia in the presence of the Acron piece, yielding an Acron-AgNP composite [9].An Acron piece without AgNP coating was used as the control.

Adhesion Assay and Analysis. Candidal adhesion to
Acron with (processed) or without (control) silver incorporation was assayed in sterile microplates (12 wells/plates).Each piece was placed in a well containing 1 mL of the yeast suspension (5 × 10 5 cells/mL), in order to cover the piece completely, and then incubated at 36 ∘ C for 1, 3, 8, and 12 h.Five processed pieces and 5 control pieces were used for each incubation time.Colony-forming capability, assessed by colony-forming units (CFU)/mL, was determined as follows: the test piece was removed from the cell suspension, rinsed 3 times with phosphate-buffered saline (PBS) to remove nonadherent Candida, and transmitted to a new sterile petri dish.
Adherent Candida was then removed by shaking for 10 min with 1 mL 0.1% TritonX-100 PBS solution.A 10 L aliquot of the solution was inoculated on CHROMagar Candida medium (Nippon Becton and Dickinson Company, Ltd., Japan) and incubated for 48 h to determine CFU/mL.Each piece was rinsed 3 times with PBS solution, fixed with 5% glutaraldehyde solution for 60 min, and incubated with 2% osmic acid for 30 min.The surface of the test pieces were observed with a low-vacuum scanning electron microscope (SEM, TM-1000, Hitachi, Japan) with magnification ratios of 1,000x and 10,000x.

Statistical Analysis.
The numbers of C. albicans and C. glabrata adherent to the surface of Acron pieces with and without silver coating were evaluated by two-way analysis of variance, followed by Tukey's test.A significance level of 0.05 was used for all analytical tests.Acron piece surface with a magnification ratio of 1,000x demonstrated that a similar number of C. albicans adhered to both AgNP-coated and control pieces after 1 h of incubation (Figure 3 (10,000x) of the Surface of the Acron Piece.Low-vacuum SEM with a magnification ratio of 10,000x was performed on uncoated and AgNPcoated Acron pieces.The Acron pieces were incubated for 3 h with C. albicans and demonstrated oval yeast cells with smooth cell walls and a major axis of 3-4 mm for the control, but on the AgNP-coated pieces, both yeasts showed deformed features, such as swollen cells with shriveled cell walls, a major axis of over 5 mm, and in some cases, leakage of cellular contents (Figure 4), indicating a loss of normal yeast cell function for adhering to the host.

Discussion
For the treatment of superficial mycoses such as OC and deep-seated mycosis, antifungal azoles, amphotericin B (AMPH-B), and micafungin are primarily used.These drugs are not prescribed prophylactically but administered following a definitive diagnosis of candidiasis.Oral care is recommended as a usual prophylactic measure, especially for elderly people, the population of whom has been increasing.
However, the daily practice of oral care, particularly cleaning of dentures, is difficult for elderly people.In a study by Dagistan et al. [2], 70% of stomatitis cases were denture induced, and in 68% of them, culture results were positive for fungi, with C. albicans being the most frequently isolated species.We have also demonstrated in a previous paper that Candida was detected in 78% of denture-wearing patients, who showed high pathogenic episodes of stomatitis and oral candidiasis, and ultrasonic cleaning of the dentures eliminated Candida [10].However, it is difficult for elderly patients to perform daily ultrasonic cleaning of their dentures.Several attempts to fabricate a denture base material to prevent the adhesion of Candida have been made.He et al. investigated in vitro adhesion of C. albicans, C. krusei, and C. dubliniensis to 4 different denture base materials and found that there was a significant difference in candidal adhesion among the types of denture base material, Candida species, and presence of absence of heat curing [11].Adherence of microorganisms varied with different surface textures of the denture base [12], with significantly greater adhesion of C. albicans on rough rather than smooth surfaces.Additionally, precoating the denture base materials with saliva reduced Candida adhesion [13].Treatment of a denture base with acrylic resin by glowdischarge plasma [14], ROS [15], or mannan [16] can also prevent Candida adhesion.Thus, several attempts to modify denture base materials have shown promising results but have not reached practical use in the clinical setting.Silver and its compounds have long been known to be effective against microorganisms and to demonstrate an antifungal activity equivalent to or more potent than AMPH-B and fluconazole (FLCZ) [3][4][5]17].A novel adduct of AgNPs to peptide hydrolysates from collagen, GX-95, was found to possess potent antifungal activities against C. albicans, including Ag-coated Control (uncoated) Incubation time (h) Incubation time (h) Ag-coated Control (uncoated) Incubation time (h) Incubation time (h) strains resistant to FLCZ, itraconazole, and flucytosine [18].In addition, the cytotoxicity of silver, especially of AgNPs, to human cells was found negligible in comparison with that to fungal cells [5,19].The antifungal effects by silver are attributable to the disruption of the structure of the fungal cell membrane due to the destruction of membrane integrity, resulting in leakage of intracellular ions and other materials and to the inhibition of the normal budding process by affecting the cell cycle at the G2/M phase [4].C. albicans exposed to AgNPs exhibited increased ROS and hydroxyl radical production, resulting in mitochondrial dysfunction and apoptosis [6].Because intracellular accumulation of ROS is accompanied by a mutation in CDC48 or by expression of mammalian bax, which are triggers of apoptosis, the generation of oxygen radicals is a key event in the ancestral apoptotic pathway of yeast [20].albicans and C. glabrata on the surface of the control Acron piece showed oval yeast cells with smooth cell walls and a major axis of 3-4 m, but they were remarkably deformed by the AgNP coating, as evidenced by swollen cells with shriveled cell walls, a major axis of over 5 m, and leakage of cellular components.The latter effect resulted in a loss in ability of yeast cells to adhere to the host.Thus, AgNPs-coated denture base material has several advantages in inhibiting candidal adhesion on the denture surface comparing AgNPscontained denture base material, such as easy preparation and high exposure of silver ions on the denture surface.
In conclusion, potent antifungal effects of AgNP coating of denture base material were demonstrated in vitro, as shown by inhibition of Candida adherence to the denture material surface and deformation of the normal morphology of Candida.Further attempts to apply AgNP-coated denture base materials for clinical use are expected.

Figure 1 :
Figure 1: Macroscopic observation of (a) Candida albicans (C.albicans) and (b) Candida glabrata (C.glabrata) growth on CHROMagar Candida medium, in which the Acron pieces (AgNP-coated and uncoated) were rinsed with 10 mL PBS solution and incubated with the yeast suspension for 1, 3, 8, and 12 h.
(a)).Three hours after the start of incubation, C. albicans, in the form of yeast, began to aggregate and adhere to the control pieces, whereas very few C. albicans adhered to the AgNP-coated pieces in the form of yeast.The difference in the number of adherent C. albicans between AgNP-coated and control pieces became remarkably larger over time.A similar finding was observed for C. glabrata (Figure3(b)).

Figure 3 :
Figure 3: Low-vacuum scanning electron microscopic observation (1,000x) of the surface of an AgNP-coated and uncoated Acron piece.The Acron piece was incubated with (a) C. albicans or (b) C. glabrata for 1, 3, 8, or 12 h.(a) Although similar numbers of C. albicans adhered to both AgNP-coated and control pieces at 1 h, C. albicans began to aggregate and adhere to the control pieces in the form of yeast at 3 h, whereas only a few C. albicans adhered to AgNP-coated pieces in the form of yeast.The difference in the number of adherent C. albicans increased between the AgNP-coated and control pieces with incubation time.(b) A similar observation was observed with C. glabrata.

Figure 4 :
Figure 4: Low-vacuum scanning electron microscopy (10,000x) of the surface of Acron pieces coated with (Ag-coated) and without (uncoated) silver nanoparticles.Acron pieces were incubated with (a) C. albicans and (b) C. glabrata for 3 h.C. albicans and C. glabrata had oval yeast cells with smooth cell walls and a major axis of 3-4 mm in the control, but the Ag-coated piece showed swollen yeast cells with shriveled cell walls and a major axis of over 5 mm, with leakage of cellular contents in some cases.
[8]lication of silver, especially AgNPs, to denture base materials inhibits the adhesion of Candida to the denture surface and hence prevents OC.Acosta-Torres et al. reported that PMMA-AgNP discs significantly reduced the adherence of C. albicans in vitro, presenting a new biocompatible antifungal PMMA denture base material [7].Chladek et al. found in vitro antifungal efficacy of 16.3-52.5%bymodifyingthe soft lining of dentures with AgNPs [21].Wady et al. demonstrated that an AgNP solution with antifungal activity against C. albicans showed no effect on C. albicans adherence and biofilm formation after its incorporation into a denture base resin[8].Thus, potent antifungal activity of AgNPs has been commonly observed but inhibitory activity against Candida adhesion to a denture base has not.In the present study, adherence of C. albicans and C. glabrata to the surface of heat-cured Acron resin pieces modified by AgNPs was significantly lower than that of the control Acron piece, based on CFU.By SEM observation, C.