Comparison of the fungicidal efficacy of photodynamic therapy with methylene blue, silver nanoparticle, and their conjugation on oral Candida isolates using cell viability assay

Background and Purpose: This study aimed to evaluate the effect of common photodynamic therapy and photodynamic therapy by the silver nanoparticle, methylene blue, and their combination on biofilm and plankton cells of standard oral Candida isolates using cell viability assay. Materials and Methods: In this in vitro study, biofilm and plankton cells of Candida species(i.e. C .albicans and C. parapsilosis) and plankton cells of Candida glabrata were treated with methylene blue, silver nanoparticle, and their combination once alone and then with the irradiation of total dose of 1.92 J/cm² for 60 sec. The minimum inhibitory concentration and antifungal activity of each approach were evaluated using the XTT assay. Results: After photodynamic therapy, methylene blue showed antifungal effect only on Candida albicans, while the antifungal effect of silver nanoparticles was increased on all Candida species. On the other hand, photodynamic therapy with the combination of methylene blue and silver nanoparticles did not have any effect on C. albicans. However, it reduced the minimum inhibitory concentration value of C. parapsilosis, and the most antifungal effect was observed on C. glabrata. Conclusion: Photodynamic therapy with photosensitizers can serve as a treatment modality in Candida infections of the oral cavity. Antifungal effect of photodynamic therapy was strain- and photosensitizer-dependent.


Introduction
andida species are among the normal flora of the oral cavity and may become pathogenic, especially in patients with immunodeficiency [1][2][3]. Candida albicans is responsible for most oropharyngeal and fungal infections [4][5][6]. According to the reviewed literature, the prescribed systemic and local antifungal agents can cause the resistance of Candida species [7,8]. There are several factors related to resistance to antifungal medications, such as intrinsic resistance of the organism, exposure to antifungal agents for a long period, and the ability of Candida to form biofilm on the surfaces [9,10].
Candida species have produced biofilms on different surfaces, including mucosal cells, dentures, catheters, with C. albicans having the greatest potential to form biofilms [11]. Due to the density of the cells and the presence of the matrix, biofilms have more resistance against antifungal agents in comparison to planktonic cells [12].
Given the insensitivity of micro-organisms to conventional medications and their possible side effects, it is suggested to use new antimicrobial approaches. In photodynamic therapy, light with special wavelengths can produce hydroxyl free radical and singlet oxygen which can lyse the membrane and damage the cells [13,14]. Methylene blue is a potential photosensitizer thiazine dye and can transfer the absorbed energy to molecules to activate photodynamic therapy [15,16].
Given the antimicrobial properties of nanoparticles, silver nanoparticles have recently received special attention as an antimicrobial and antifungal agent [17,36 Curr Med Mycol, 2020, 6(4): 35-40 18]. Silver nanoparticles interact with the main parts of cells (membrane or cell wall, DNA, and microbial proteins) and produce intracellular reactive oxygen species. Accumulation of this intracellular production can regulate apoptosis [19]. Proper antifungal effect against C. albicans by gold nanoparticle-mediated photodynamic therapy was reported in the literature [20,21]. To the best of our knowledge, only few studies have addressed the antifungal effects of photodynamic therapy on silver nanoparticles.
In an in vitro study, gold nanoparticles enhanced the photodynamic therapy results with methylene blue for C. albicans biofilm inhibition [20]. In another similar assessment, gold nanoparticle-photosensitizer conjugate-based photodynamic therapy effectively killed both planktonic C. albicans cells and its hyphal form in biofilm [21].
Due to the antifungal prescription limitations and presence of non-pharmacological approaches, this study aimed to compare the effect of photodynamic therapy with methylene blue, silver nanoparticles, and their combination on standard oral Candida species using the Cell Proliferation Kit II (XTT)(2,3-bis-(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide) to introduce a new antifungal treatment with limited side effects.

Study sample
The present in vitro research was approved by the Ethics Committee of the Shiraz University of Medical Sciences (ethics code: IR.SUMS.REC.1396.S272).This study was conducted on standard species of C. albicans PTCC 50027(ATCC 10231), Candida parapsilosis (ATCC 22019), and Candida glabrata (ATCC 2001).

Biofilm preparation
Candida species were cultured on sabouraud dextrose agar (manufactured by Merck, Germany) at room temperature for24-48 h. To increase the fungal cells, they were suspended in 20 ml yeast peptone dextrose and incubated overnight in an orbital shaker (60 rpm) at 30 °C. The tubes were centrifuged at 3000 g for 5 min at 4°C.The harvested cells were washed with 20 ml ice-cold sterile phosphate buffer saline (PBS) (manufactured by Gibco, USA) in triplicate.
Afterward, the sediment was suspended in 10 ml prewarmed (37 °C) RPMI 1640 medium (manufactured by Sigma, UK)without sodium bicarbonate and with Lglutamine which buffered to pH 7.0 with 0.165 M morpholinepropane sulfonic acid (manufactured by Sigma, Germany).The suspension was diluted 1:1000 and adjusted to the 10 6 cells/mL in RPMI 1640) with a hematocytometer Neubauer chamber. Biofilms were produced on commercially 96 microtiterflat-bottom cell and tissue culture Plates (Nantong Renon Laboratory Equipment Co., Ltd, Jiangsu, China). Subsequently, 100 μL of a standard Candida suspension was transferred into each well, and the plates were incubated for two h at 37°C in a shaker at 75 rpm to allow them to adhere to the surface of the wells. After the adhesion phase, the supernatant was aspirated and each well was washed twice with 150 μL of PBS to remove the loosely adherent cells. A total of 100 μL of RPMI 1640 was added to each washed wells, and the plates were incubated at 37°C in a shaker at 75 rpm. The biofilms were allowed to develop for 24 h and used in different assays. It should be mentioned that all assays were repeated three times. The C. glabrata cannot produce biofilms; accordingly, the experiments were performed on the plankton cells.

Minimum inhibitory concentration values of Candida species
Minimum inhibitory concentrations (MIC) of methylene blue, silver nanoparticle, and combination were assessed based on the CLSI M27-A3 standard procedure [22]. A dilution equal to 0.5 McFarland was prepared in normal saline for each Candida species and diluted to 1:1000 with RPMI.Afterward, 100 µL of each photosensitizer dilution and 100 µL of the Candida species suspensions were added to each well. Serial dilutions were prepared ranging from 100 to 0.195 µl/ml from methylene blue, silver nanoparticles, and combination. In each series, the 11th and 12th wells were considered as positive and negative wells without photosensitizer and yeast suspension, respectively. The plates were incubated at 37°C and their MIC was recorded after 24 h. Furthermore, similar plates were processed and treated with laser. The MIC is defined as the lowest concentration of photosensitizer with a 50% decrease in growth, compared with the positive control group. The mean values recorded in three different experiments are reported as MIC values.

Photosensitizer
The combination included methylene blue as photosensitizer and silver nanoparticles. To obtain a uniform suspension of silver nanoparticles, 200 mg of silver nanoparticles powder (manufactured by Sigma Aldrich, the USA) [18,23,24] with particles sized <100 nm and 100 ml normal saline was poured into the tube and sonicated at 200 W for two min in a sonicator device(manufactured by Hielscher, Germany). The concentration of 200 mg/ml of methylene blue was made in phosphate-buffered saline(20 Mm phosphate buffer, 2.7 Mm potassium chloride, 137 Mm sodium chloride; pH 7.4) [14,16,25].
A combination of silver nanoparticles and methylene blue with a concentration of 200 mg/ml was prepared. In addition, 10-fold serial dilutions were prepared ranging from 100 to 0.195 µl/ml from methylene blue, silver nanoparticles, and their combination. Biofilms were treated with 100 µl of each concentration for 30 min in darkness (under a laminar flow hood) under two conditions, i.e. with and without photodynamic therapy. Those that were not exposed to laser light or photosensitizer were considered as the positive control group (n=10). Finally, the vitality of Curr Med Mycol, 2020, 6(4): 35-40 37 Candida biofilms was assessed using XTT.

Light exposure
In the pilot study, we examined the efficacy of photodynamic therapy on the yeasts in durations of 1-5 min since it was difficult for patients to tolerate the procedure for more than five min. There was no difference between the applications of laser for different durations in terms of inducing a reduction in viable Candida species; therefore, one-min irradiation was applied in this study. Moreover, the light source gallium-aluminum-arsenide laser (manufactured by Easy-Laser, Clean Line, Brazil) was employed. The total output power was 0.025 W, and the continuous mode of the device with 660 nm was used. The crosssection of the laser probe was 0.78 cm², and all Candida preparations were irradiated with a total dose of 1.92 J/cm² for 60 sec.

The Cell Viability Reduction assay
The XTT, which is a colorimetric assay system, was used to reassess the susceptibilities of the Candida species to photosensitizer since the viability of the cells was not clear by spectrophotometric readings. The XTT solution was prepared as a saturated solution at 0.5 mg/ml in sterile ringer lactate and covered with aluminum foil during preparation. After filtering it with a 0.22 µm pore size filter, the XTT solution was aliquoted into 10 ml working volume and stored at -70°C. Afterward, 10 Mm stock solution of menadione in 100% acetone was prepared, liquated into smaller volumes (about 50 µm), and stored at -70°C. Moreover, tubes containing 10 ml of the XTT solution were thawed (one per plate) as required for the experimental design.
Subsequently, 1µl of the stock solution of menadione was added to each tube to achieve a final menadione concentration of 1 µM. Besides, 100 µl XTT/menadione solution was added to each well, and the plate was covered with aluminum foil and incubated under the dark condition at 37°C on a rotating shaker. After one h, the plates were uncovered, and 75-80 µl of the colored supernatant of each well was transferred to a new plate. The plates were measured at 475 and 660 nm with a spectrophotometer, and the wells without biofilms were considered as a blank.
Absorbance of each sample was evaluated using the following formula according to the XTT assay kit: Specific Absorbance=A475nm (Test) -A475nm (Blank) -A660nm (Test)

Results
Sensitivity results of the photosensitizer (i.e., methylene blue, silver nanoparticles, and their combination) were different in various Candida species. As shown in Table 1, the reported MICs (plankton cells) for methylene blue were the same before and after irradiation (100 µg/ml) in C. parapsilosis, while the silver nanoparticles and combination MICs decreased after illumination. The MIC values of both silver nanoparticles and combination in C. parapsilosis were 50 and 25 before and after irradiation, respectively. Viability of biofilm formation (by XTT test) of C. parapsilosis in silver nanoparticles and combination decreased after irradiation by 30% and 50%, respectively. Methylene blue showed no anti-C. parapsilosis effects in the biofilm and plankton cells before and after irradiation.
The MICs of Methylene blue and the combination for C. albicans (100 µg/ml) and silver nanoparticles (50 µg/ml) were the same before irradiation (Table 1). However, after photodynamic therapy, the MIC values of Methylene blue and silver nanoparticle underwent a decrease. In vitro viability evaluation of C. albicans by the XTT method revealed 15%, 50%,and 10% viability reduction after photodynamic therapy in the yeasts treated with Methylene blue, silver nanoparticle, and the combination, respectively. Lowest MIC value of C. albicans, (25 µg/ml) was observed when yeasts were treated with silver nanoparticle and photodynamic therapy. Methylene blue and the combination that treated C. albicans did not show a significant colonyforming unit (CFU) reduction viability in comparison with silver nanoparticles alone.
Since C. glabrata does not have pseudohypha, the anti-biofilm activity could not be evaluated and results for plankton form were reported. The MIC values of Methylene blue for C. glabrata before and after photodynamic therapy were the same, while the MICs of silver nanoparticles and combination treatments decreased after photodynamic therapy (50 to 25 and 25 to 12.5). According to the XTT assay, 20% and 50% viability reduction was seen in silver nanoparticle and  Overall, photodynamic therapy with silver nanoparticles was the most effective treatment for C. albicans and C. parapsilosis biofilms. Moreover, photodynamic therapy with the combination was the most effective treatment for the inhibition of C. glabrata plankton cells. Figure 1 shows the laboratory assessments of XTT assay and MIC determination.

Discussion
In the present study, standard species of C. albicans, C. parapsilosis, and C. glabrata were assessed since Candida albicans is the major cause of oropharyngeal candidiasis in immunocompromised patients. Furthermore, according to the literature, due to prolonged use of fluconazole in prophylaxis and treatment of the patients, non-albicans Candida species like C. parapsilosis and C. glabrata which are isolated from oral candidiasis are resistant to antifungal agents and non-wild type species [26,27]. In this regard, the use of safe and effective therapy for the treatment of patients is necessary.
According to the results, photodynamic therapy with methylene blue, silver nanoparticle, and their combination as photosensitizers were effective in reducing the MIC values and viability of some Candida species based on the XTT assay. During photodynamic therapy, the cells are damaged by necrosis and apoptosis since the interaction between light and the biological cells produces highly cytotoxic singlet oxygen and other reactive oxygen species [28]. The role of photosensitizers is to transfer photon energy to the cells and create interaction between a chemical constituent of photosensitizers and a specific wavelength of light [29][30][31].
In this study, methylene blue, silver nanoparticles, and their combination were used as the photosensitizers. Methylene blue promotes cell death by the production of cytotoxic singlet oxygen and other reactive oxygen species [14].Silver nanoparticle damages the cell membrane by attaching to the sulfur-containing proteins of the cell membrane which decreases the intracellular adenosine 5'-triphosphate level and destroys the microorganism DNA. Besides, the silver nanoparticle has a significant role in mitochondrial dysfunctional apoptosis due to the generated hydroxyl radicals [32]. Kim et al. found that silver nanoparticle has considerable antifungal activity on C. albicans by the pit formation on the cell membrane and ion leakage was confirmed by the transmission electron microscopy image [18].
Usage of photodynamic therapy alone and with different photosensitizers must be synergistic; however, in the present study, different results were observed based on the yeast type. Based on the results of a study performed by Sousaet al., the MIC reduction was significant (P˂0.05) for C. albicans during five-min incubation with laser with a concentration of 150 μg/mL of methylene blue [32]. In this study, a one-min photodynamic therapy treatment was selected with the concentration of 100 μg/mL of methylene blue since in higher concentrations the dark color of methylene blue influences the spectrophotometric reading.
Based on the results of a study carried out by Souza [33], the MIC values of C. albicans for methylene blue decline dafter irradiation and methylene blue was found to be an effective photosensitizer in antifungal photodynamic therapy. These findings are in line with those of the present study. According to the related literature, the antimicrobial properties of methylene blue mediated photodynamic therapy [34,35] and also photosensitizer alone had a non-fungicidal effect on C. albicans [36,37].
In this study, methylene blue did not have any antifungal effects on C. glabrata and C. parapsilosis plankton cells before and after the irradiation. Moreover, it was found that silver nanoparticles were effective on all Candida species and after photodynamic therapy, the MICs of the yeasts decreased. Antifungal effect of silver nanoparticles on C. albicans was reported in the literature [38,39]; however, there are limited data about its effect on nonalbicans Candida species. In the present study, the antifungal effect of the combination on non-albicans Candida species (C. glabrata and C. parapsilosis) increased after photodynamic therapy which is important in the clinic since these species are more resistant to antifungal agents, compared to C. albicans [1,2]. In biofilm formation, microorganisms can attach to a surface and grow on by secreting extracellular polysaccharides. Biofilms are more resistant to antibiotics than planktonic cells and have a significant role in public health. Bacteria are the major microorganisms that make biofilms; however, the fungi are becoming an increasingly common organism, especially C. albicans [14]. Evaluation of susceptibility of biofilms to antimicrobial agents cannot be determined by standard microdilution testing which is used for the planktonic cells since photons or antibacterial agents affect the surface of the biofilms and cannot penetrate the depth of the biofilm.
In the present study, the viability of C. albicans biofilm declined about 15%after irradiation with methylene blue as a photosensitizer; however, it was not effective on C. glabrata and C. parapsilosis with or without irradiation. In the study carried out by Sousa et al., no reduction in CFU/mL of C. albicans was reported after the use of methylene blue in the absence of irradiation, which was consistent with the results of this study [32].
Silver nanoparticles were effective on the viability of all Candida species in this study and the viability of the yeasts decreased after photodynamic therapy. Addition of silver nanoparticles to methylene blue in photodynamic therapy induced more potent antifungal activity on the biofilm of these species. Dovigo et al. observed a significant decrease in the viability of C. albicans and C. glabrata biofilm after photodynamic therapy [37]. Mima et al. reported that photodynamic therapy resulted in the reduction of CFU/mL of C. albicans, C. glabrata, C. tropicalis, C. dubliniensis, and C. krusei [40]. Moreover, inhibition of C. albicans biofilm formation by silver nanoparticles in the surgical sites was reported in the literature [41].
There are some limitations for in vivo application of photosensitizers in teeth, lips, buccal mucosa, tongue, and removable or fixed prosthetics due to staining; therefore, non-dye photosensitizers may be more appropriate for intraoral use of photodynamic therapy.

Conclusion
Based on the results, photodynamic therapy with the photosensitizers was effective in reducing the number of Candida species. In addition, the fungicidal effect of photodynamic therapy was strain-and photosensitizer-dependent. Methylene blue, silver nanoparticle, and their combination were effective photosensitizers with antifungal property against Candida species.