# In Vitro Antifungal Activity against OralCandida Species Using a Denture Base Coated with Silver Nanoparticles

Authors: Yoshiaki Kamikawa; Daisuke Hirabayashi; Tomohiro Nagayama; Jyunichi Fujisaki; Tomofumi Hamada; Ryoichi Sakamoto; Yasuko Kamikawa; Kazumasa Sugihara
Journal: Journal of Nanomaterials
 (2014)

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## Abstract

Although oralCandida easily adheres to denture base materials, many denture detergents are effective only against bacteria but not against Candida. 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) and measuring 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.


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## Body

## 1. 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 againstCandida. 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 ofCandida, equivalent to that of amphotericin B [3–5]. 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 ofCandida 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).


## 2. Materials and Methods

### 2.1. 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 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.


### 2.2. Preparation of Yeast Culture Solution

TheCandida standard strains used in the study wereC. albicans (ATCC18804) andC. glabrata (ATCC90030). The suspension solution of theCandida standard strains was prepared in RPMI 1640 culture medium adjusted to a McFarland factor of 0.5 (absorbance at 530 nm).


### 2.3. 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 × 105 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 non-adherentCandida, and transmitted to a new sterile petri dish. AdherentCandida was then removed by shaking for 10 min with 1 mL 0.1% TritonX-100 PBS solution. A 10 μL 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.


### 2.4. Statistical Analysis

The numbers ofC. albicans andC. 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.


## 2.1. 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 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.


## 2.2. Preparation of Yeast Culture Solution

TheCandida standard strains used in the study wereC. albicans (ATCC18804) andC. glabrata (ATCC90030). The suspension solution of theCandida standard strains was prepared in RPMI 1640 culture medium adjusted to a McFarland factor of 0.5 (absorbance at 530 nm).


## 2.3. 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 × 105 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 non-adherentCandida, and transmitted to a new sterile petri dish. AdherentCandida was then removed by shaking for 10 min with 1 mL 0.1% TritonX-100 PBS solution. A 10 μL 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.


## 2.4. Statistical Analysis

The numbers ofC. albicans andC. 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.


## 3. Results

### 3.1. Adhesion of Yeast to the Acrylic Denture Base Resin

As shown in Figure1, 48 h incubation of the rinse fluid on CHROMagar Candida medium exhibited time-dependent increases inC. albicans andC. glabrata colony formation. For the control uncoated piece,C. albicans increased to 7.50 × 104 CFU/mL with a 12 h incubation, whereas with the AgNP-coated piece,C. albicans increased to only 3.83 × 10 CFU/mL, showing statistical significance between the control piece and the AgNP-coated piece (P
<
0.001, Figure 2(a)). A similar result was obtained withC. glabrata, which increased to 6.99 × 105 CFU/mL with a 12 h incubation on the control pieces versus 2.18 × 102 CFU/mL on the AgNP-coated piece (P
<
0.001, Figure 2(b)).Macroscopic observation of (a)Candida albicans (C. albicans) and (b)Candida glabrata (C. glabrata) growth on CHROMagarCandida 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)
(b)Number of adherent cells on an Acron piece coated with AgNPs (Ag-coated,n
=
5) or without AgNPs (control, n
=
5), as measured by counting colony forming units (CFU) on CHROMagarCandida medium, where 10 mL of PBS rinse solution from the Acron pieces (Ag-coated or uncoated) were incubated with (a)C. albicans and (b)C. glabrata for 1, 3, 8, or 12 h. *Significantly different from control (P
<
0.001).
(a)
(b)


### 3.2. Low-Vacuum SEM Observation (1,000x) of the Surface of the Acron Piece

Low-vacuum SEM observation of the Acron piece surface with a magnification ratio of 1,000x demonstrated that a similar number ofC. albicans adhered to both AgNP-coated and control pieces after 1 h of incubation (Figure 3(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 fewC. albicans adhered to the AgNP-coated pieces in the form of yeast. The difference in the number of adherentC. albicans between AgNP-coated and control pieces became remarkably larger over time. A similar finding was observed forC. glabrata (Figure 3(b)).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 ofC. 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 fewC. albicans adhered to AgNP-coated pieces in the form of yeast. The difference in the number of adherentC. albicans increased between the AgNP-coated and control pieces with incubation time. (b) A similar observation was observed withC. glabrata.
(a)
(b)


### 3.3.  Low-Vacuum SEM Observation (10,000x) of the Surface of the Acron Piece

Low-vacuum SEM with a magnification ratio of 10,000x was performed on uncoated and AgNP-coated Acron pieces. The Acron pieces were incubated for 3 h withC. 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.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 andC. 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.
(a)
(b)
(c)
(d)


## 3.1. Adhesion of Yeast to the Acrylic Denture Base Resin

As shown in Figure1, 48 h incubation of the rinse fluid on CHROMagar Candida medium exhibited time-dependent increases inC. albicans andC. glabrata colony formation. For the control uncoated piece,C. albicans increased to 7.50 × 104 CFU/mL with a 12 h incubation, whereas with the AgNP-coated piece,C. albicans increased to only 3.83 × 10 CFU/mL, showing statistical significance between the control piece and the AgNP-coated piece (P
<
0.001, Figure 2(a)). A similar result was obtained withC. glabrata, which increased to 6.99 × 105 CFU/mL with a 12 h incubation on the control pieces versus 2.18 × 102 CFU/mL on the AgNP-coated piece (P
<
0.001, Figure 2(b)).Macroscopic observation of (a)Candida albicans (C. albicans) and (b)Candida glabrata (C. glabrata) growth on CHROMagarCandida 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)
(b)Number of adherent cells on an Acron piece coated with AgNPs (Ag-coated,n
=
5) or without AgNPs (control, n
=
5), as measured by counting colony forming units (CFU) on CHROMagarCandida medium, where 10 mL of PBS rinse solution from the Acron pieces (Ag-coated or uncoated) were incubated with (a)C. albicans and (b)C. glabrata for 1, 3, 8, or 12 h. *Significantly different from control (P
<
0.001).
(a)
(b)


## 3.2. Low-Vacuum SEM Observation (1,000x) of the Surface of the Acron Piece

Low-vacuum SEM observation of the Acron piece surface with a magnification ratio of 1,000x demonstrated that a similar number ofC. albicans adhered to both AgNP-coated and control pieces after 1 h of incubation (Figure 3(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 fewC. albicans adhered to the AgNP-coated pieces in the form of yeast. The difference in the number of adherentC. albicans between AgNP-coated and control pieces became remarkably larger over time. A similar finding was observed forC. glabrata (Figure 3(b)).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 ofC. 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 fewC. albicans adhered to AgNP-coated pieces in the form of yeast. The difference in the number of adherentC. albicans increased between the AgNP-coated and control pieces with incubation time. (b) A similar observation was observed withC. glabrata.
(a)
(b)


## 3.3.  Low-Vacuum SEM Observation (10,000x) of the Surface of the Acron Piece

Low-vacuum SEM with a magnification ratio of 10,000x was performed on uncoated and AgNP-coated Acron pieces. The Acron pieces were incubated for 3 h withC. 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.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 andC. 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.
(a)
(b)
(c)
(d)


## 4. 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 Dağistan et al. [2], 70% of stomatitis cases were denture induced, and in 68% of them, culture results were positive for fungi, withC. albicans being the most frequently isolated species. We have also demonstrated in a previous paper thatCandida was detected in 78% of denture-wearing patients, who showed high pathogenic episodes of stomatitis and oral candidiasis, and ultrasonic cleaning of the dentures eliminatedCandida [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 ofCandida have been made. He et al. investigated in vitro adhesion ofC. albicans,C. krusei, andC. 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 ofC. albicans on rough rather than smooth surfaces. Additionally, precoating the denture base materials with saliva reducedCandida adhesion [13]. Treatment of a denture base with acrylic resin by glow-discharge plasma [14], ROS [15], or mannan [16] can also preventCandida 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) [3–5, 17]. A novel adduct of AgNPs to peptide hydrolysates from collagen, GX-95, was found to possess potent antifungal activities againstC. albicans, including 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]. Application of silver, especially AgNPs, to denture base materials inhibits the adhesion ofCandida to the denture surface and hence prevents OC. Acosta-Torres et al. reported that PMMA-AgNP discs significantly reduced the adherence ofC. 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% by modifying the soft lining of dentures with AgNPs [21]. Wady et al. demonstrated that an AgNP solution with antifungal activity againstC. albicans showed no effect onC. 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 againstCandida adhesion to a denture base has not. In the present study, adherence ofC. albicans andC. 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. albicans andC. glabrata on the surface of the control Acron piece showed oval yeast cells with smooth cell walls and a major axis of 3-4 μm, but they were remarkably deformed by the AgNP coating, as evidenced by swollen cells with shriveled cell walls, a major axis of over 5 μm, 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 AgNPs-contained 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 ofCandida adherence to the denture material surface and deformation of the normal morphology ofCandida. Further attempts to apply AgNP-coated denture base materials for clinical use are expected.



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*Source: 780410-2014-04-30.xml*