Photodithazine^® (PDZ) is a chlorin e6 derivative (VETAGRAND, Co, Moscow, Russia), which has an absorption peak of 660 nm. The PDZ was prepared on the day of use from the stock solution (5,000 mg/L) at a concentration of 200 mg/L in natrosol gel (Farmácia Santa Paula, Araraquara, SP, Brazil) and was kept protected from light (Hidalgo et al., 2019). The bovine pancreas DNase I enzyme stock solution (AMPD1, Sigma-Aldrich, St. Louis, MO, USA) was prepared on the day of use in 0.1 M sodium acetate buffer (pH 5.5) at the concentration of 20 units/mL (Abreu-Pereira et al., 2022).

The red LED light device (LXHLPR09, Luxeon^® III Emitter, Lumileds Lighting, San Jose, California, USA) was used with an absorption band of 660 nm, and the light intensity at the end of the device (5 mm in diameter) was 44.6 mW/cm². Thus, a light dose of 50J/cm² (19 min) was applied to the tongues of animals infected with C. albicans.

The present study was approved by the Animal Ethics Committee of the School of Dentistry of Araraquara, UNESP (Case number: 09/2020). A total of 180 female mice of the Swiss strain (≅ 5 weeks old) were used from the vivarium of the School of Dentistry of Araraquara, UNESP. The animals were allocated in cages, with five animals per cage according to the study groups, and kept in a room with a controlled temperature (23 ± 2°C) with standard chow and water ad libitum (Carmello et al., 2016; Hidalgo et al., 2019).

Strains of C. albicans ATCC (American Type Culture Collection, Rockville, USA), susceptible to fluconazole (ATCC 90028; CaS) and resistant to fluconazole (ATCC 96901; CaR) were defrosted, reactivated on Agar Sabouraud Dextrose (SDA) medium (37°C, 48 h). Next, colonies were transferred and cultivated in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) buffered to a pH of 7.0 with 3-(N-Morpholino) propanesulfonic acid 4-Morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 16 h. Then, the cells were washed twice with sterile phosphate buffered saline (PBS) (50 mM), resuspended with 3 mL of RPMI 1640, and the cell suspension was standardized spectrophotometrically with an optical density (OD) of 540 nm (1.0 nm ± 0.08, corresponding to 10⁷ CFU/mL) (Carmello et al., 2016; Hidalgo et al., 2019).

For the induction of oral candidiasis, the methodology described before (Takakura et al., 2003; Carmello et al., 2016) was used with some modifications. Tetracycline (0.83 mg/mL) was administered in the water available to the animals during the experimental period. The animals were immunosuppressed with subcutaneous injections of prednisolone at a dose of 100 mg/kg of body mass on days 1, 5, 9, and 13. Inoculation with the strains was performed on day 2 of the experiment (Figure 1; Takakura et al., 2003; Carmello et al., 2016; Hidalgo et al., 2019). For this procedure, the animals were sedated with 0.1 mL of chlorpromazine hydrochloride (2 mg/mL), and sterile mini-swabs soaked in the CaS or CaR suspension were scrubbed across the dorsum of the animals’ tongues for 30 s.

On day 7, the presence of white patches or pseudomembranous lesions was verified, and the treatments were performed. The animals were anesthetized with an intraperitoneal injection of ketamine [100 mg/kg body weight (National Pharmaceutical Chemistry Union S/A, Embu, SP, Brazil)] and xylazine [10 mg/kg body weight (Veterinary JA Ltda., Sponsor Paulista, SP, Brazil)]. Then, the animals were placed in a supine position on the work table, the tongues were gently taken out of the oral cavity, and 50 μL of PDZ diluted in natrosol gel (200 mg/L) was applied with a pipette (Carmello et al., 2016; Hidalgo et al., 2019). The mice stayed in the dark for 20 min for a pre-incubation time. Then, each dorsum of the tongue was illuminated with LED for 19 min (50 J/cm²) (P + L+ group). The effect of the isolated application of PDZ (P + L-) and the LED (P-L+ group) was also evaluated. For DNase treatment, the tongue of the mice received 50 μL of DNase (20 units/mL) for 5 min. One group received the combined treatment with the enzyme and PDZ-aPDT (DNase+P + L+ group). After the treatments, neither DNase nor PDZ was removed from the mice’ tongue. The untreated control group (P-L- group) received no PDZ, light, or DNase. In addition, two additional negative infection control groups (NIC groups) with healthy animals were evaluated. In one of them, mice were immunosuppressed on days 1, 5, 9, and 13 (NIC+ group); in the other group, animals did not receive immunosuppression (NIC- group). Seven animals were evaluated for each experimental condition, except the group NIC+ (n = 3) and NIC- (n = 3). The therapies were performed once a day for five consecutive days (from day seven until day 11).

After five consecutive days of treatment (day 11) and 7 days after the end of the treatment (day 18), C. albicans cells were recovered from the tongue of mice. For this procedure, the mini-swabs were swabbed on the dorsum of each tongue for 1 min. Then, they were transferred to tubes with 1 mL of saline solution and vortexed for 1 min to detach C. albicans cells. Then, serial dilutions were made (10⁻¹ a 10⁻³) and plated in duplicate in SDA culture medium containing 5 μg/mL of chloramphenicol. After 48 h of incubation at 37°C, the viable colonies were counted, and the values of CFU/mL were determined.

The white patches or pseudomembranous lesions in the tongues of mice were photographed before the beginning of the treatments, 24 h (day 12), and 7 days (day 18) after the last application. All photographs were standardized and obtained with the same digital camera (Sony Cyber-Shot DSCF717; Sony Corporation, Tokyo, Japan), by the same operator and under the same conditions (place, light, angle, and position of the animals), thus aiming to facilitate the reproducibility. The extension area of the lesion on the tongue in each photograph was evaluated using the ImageJ.exe program.¹ The percentage of the extension area of each lesion over the total area of the tongue was calculated using this software (Hidalgo et al., 2019).

Initially, mice were anesthetized with an intraperitoneal injection of ketamine and xylazine. Then, animals’ tongues were surgically removed and destinated for histopathological and fluorescence microscopic analyses. After the tongue excision, mice were euthanized by intramuscular injection of a lethal dose of ketamine (0.2 mL) and xylazine (0.4 mL) 24 h (day 12) and 7 days (day 18) after the last application of treatment (Carmello et al., 2016; Hidalgo et al., 2019).

The tongues were placed in plastic cassettes for the histopathological and fluorescence microscopic analyses. These cassettes were immersed in 10% paraformaldehyde (pH 7.2) (441.244, Sigma-Aldrich, St Louis, MO, USA). Then, the histological fixation process was made, and the blocks were fixed on wooden supports and placed in a rotating microtome. Sixteen serial histological sections of each block were obtained. These cuts were placed on glass slides and stained with hematoxylin–eosin (HE) stain to evaluate the histological events that occurred in each of the groups through light microscopy [Zeiss microscope LSM 700 (Carl Zeiss, Heidelberg, Germany)] at 100 and 200X magnification. A pathologist performed the histological analysis, and the following aspects were evaluated: the presence/absence of yeast and inflammatory infiltrate, epithelial tissue integrity, and adjacent connective tissue response. The material was classified into scores: 0—the absence of inflammation; 1—the presence of inflammatory infiltrate; 2—moderate inflammation; 3: severe inflammation; and 4: abscess formation (ISO 7405:1997). The evaluation was performed by a single examiner blinded to each experimental group at each evaluated time after treatment.

Initially, the samples were deparaffinated and hydrated in water. The antigenic retrieval was performed by heat. The sections were then immersed in 10 mM buffered sodium citrate, pH 6.0, and placed in the microwave twice for 5 min each (El-Habashi et al., 1995). Next, the slides were dried, and the sections were circled with a hydrophobic barrier pen (Sigma Advanced PAP Pen-Z377821) and 20 μL of the primary antibody (1 → 4)-β-mannan and galacto-(1 → 4)-β-mannan (400–4) (Table 1) diluted in 2% bovine serum albumin (BSA) and 0.1% Triton X100 (1:20 dilution) was pipetted on each section (Lobo et al., 2019). The slides were incubated overnight (4°C). After incubation, sections were carefully washed with 0.89% NaCl solution, and a blocking solution (3% BSA) was added, followed by incubation for 15 min (room temperature). Then, the sections were washed again with 0.89% NaCl solution, and the secondary antibody (20 μL) labeled with Alexa Fluor® 594 nm (1:500 dilution in 2% BSA) was added (Table 1), followed by incubation for 2 h (4°C). After the secondary antibody incubation time, the sections were washed with 0.89% NaCl and incubated with 20 μL of concavalin-A lectin conjugated with Alexa Fluor^® 488 nm (200 μg/mL) (Table 1) and Hoescht (6 μg/mL) (Table 1) for 30 min. Next, samples were washed with 0.89% NaCl. The mounting media [Fluoromount ^™ Aqueous Mounting Medium (F4680, Sigma-Aldrich, St Louis, MO, USA)] was added, and the slides were ready for image acquisition. Images were acquired using the Leica DM2500 LED microscope (Leica Microsystems, Wetzlar, Germany).

Analyses were performed using the IBM SPSS Statistics for Windows Version 27; IBM Corp., Armonk, NY, USA. Data from each strain was evaluated separately. The normality and homoscedasticity of the data from CFU converted in base-10 logarithms for each strain were assessed using the Shapiro–Wilk and Levene’s tests, respectively. The data were normal and heteroscedastic, so they were analyzed by a two-way ANOVA test, considering two treatment evaluation periods (immediate and 7 days after). Games-Howell post-hoc analysis was performed for multiple comparisons (α = 5%). The percentage values of tongue’s lesions assumed normality and were homoscedastic for the data evaluated in both periods (24 h and 7 days after the treatments) for CaS and CaR. Thus, they were analyzed by one-way ANOVA, followed by Tukey’s post-hoc (α = 5%). Descriptive analyses were performed for the images obtained for the histopathological and fluorescence microscopy evaluations.

The results of viability from CaS immediately after the treatments demonstrated that the animals treated with DNase followed by PDZ-aPDT (DNase+P + L+ group) showed the highest log₁₀ reduction value (CFU/mL), compared to the negative control group (P-L-) equivalent to 4.26 log₁₀ (Figure 2) and different from the other groups and the control (p ≤ 0.0001). The P + L+ group (PDZ-aPDT) showed a reduction of approximately 2.50 log₁₀ compared to the control (P-L- group) (Figure 2). The other groups showed statistically similar values with the control (P-L-) (p ≥ 0.05) (Figure 2).

The results of CaR showed that DNase+P + L+ group immediately after the treatments was statistically different from the other groups and exhibited the highest log₁₀ reduction value (CFU/mL), compared to the P-L-group (p ≤ 0.0001), equivalent to 2.89 log₁₀ (Figure 3). The group treated only with PDZ-aPDT (P + L+ group) showed a reduction of 0.34 log₁₀ compared to the P-L- group. The P + L-, P-L+, P + L+, DNase and P-L-groups presented statistically similar effects (Figure 3).

The results of 7 days after the end of the treatments demonstrated that the DNase+P + L+ group exhibited the greatest reduction in the viable colonies of CaS when compared to the negative control group (P-L-) (p ≤ 0.0001); this reduction was approximately 1.97 log₁₀ (Figure 2). The P + L+ group was similar to DNase+P + L+ group and showed a statistically different value from the other groups, with a reduction of 1.18 log₁₀ compared to the P-L- group (p ≤ 0.0001). The other experimental groups (P + L-, P-L+, DNase) showed statistically similar effects among themselves and with the negative control (P-L-) (p ≥ 0.05) (Figure 3).

For CaR after 7 days of the end of the treatments, the DNase+P + L+ group showed the greatest reduction in viable colonies when compared to the P-L- group (p ≤ 0.0001), with a reduction of approximately 1.27 log₁₀ (Figure 3). The P + L+ group showed a reduction of 0.22 log₁₀ compared to the P-L- group. The other experimental groups (P + L-, P-L+, P + L+, and DNase) showed statistically similar values among themselves and the P-L- group (p ≥ 0.05) (Figure 3).

The results after 24 h of treatment (Figure 4) for mice inoculated with the CaS showed that the DNase+P + L+ and P + L+ groups significantly reduced oral lesions by 98.92 and 97.71%, respectively, when compared to the P-L- group (p ≤ 0.0001). The results for 7 days after the treatments showed a reduction in oral lesions of 83.31% for DNase+P + L+ group, compared to the P-L- group (p ≤ 0.0001). The DNase+P + L+ group was statistically different from P + L+ group that reduced the oral lesions by around 63.81% compared to the P-L- group (p ≤ 0.0001). The other groups evaluated immediately and after 7 days (Figure 4) showed a statistically similar effect to the P-L- control (p ≥ 0.05). The images presented in Figure 5 illustrate the presence of white patches or pseudomembranous plaques on the dorsum of the animals’ tongues inoculated with CaS for each group 24 h and 7 days after the treatments.

The results obtained for CaR (Figure 6) demonstrated that the DNase+P + L+ and P + L+ groups immediately after the treatments exhibited reductions of oral lesions by 96.07 and 50.41%, respectively, when compared to the P-L- group (p ≤ 0.0001). Immediately after the treatments, the other groups showed a statistically similar effect to the P-L- group (p ≥ 0.05). In addition, 7 days after the treatments, the DNase+P + L+ group showed a significant reduction in oral lesions by 75.24% when compared to the P-L- group (p ≤ 0.0001). The other groups showed statistically similar values to the P-L- control (p ≥ 0.05) (Figure 6).

The images in Figure 7 illustrate the presence of white patches or pseudomembranous plaques on the dorsum of the tongues 24 h and 7 days after the treatments performed on animals inoculated with CaR (Figure 7).

The histopathological evaluation demonstrated that the sections from tongues contaminated with CaS exhibited mild inflammatory infiltrates for groups DNase+P + L+ and P + L+ 24 h after the treatment (Figure 8). These groups presented histopathological characteristics similar to those observed in the NIC- and NIC+ groups. The stratified epithelium exhibited normal and healthy features, with lingual papillae covered by a fine keratin layer. The other groups (P-L-, P-L+, P + L- and DNase) presented similar histopathological characteristics with moderate inflammatory infiltration and the presence of numerous hyphae/pseudohyphae on the keratin layer and some hyphae/pseudohyphae invading the epithelial tissue of the tongues (Figure 8). Regarding the histological sections evaluated at 7 days after the treatment for CaS, the morphological characteristics remained relatively unchanged, with the exception of the P-L-, P + L-, P-L+, and DNase groups, which showed moderately degraded muscle tissue (Figure 8).

For the animals inoculated with CaR (Figure 9), the group treated with DNase+P + L+ presented histopathological characteristics similar to those observed in the NIC- and NIC+ groups (Figure 8) after 24 h of the treatments. The stratified epithelium exhibited normal and healthy features, with lingual papillae covered by a fine keratin layer (Figure 9). The group treated with P + L+ showed a greater number of hyphae and pseudohyphae within the keratin epithelial layer. The animals in the P-L-, P + L-, P-L+, and DNase groups presented extensive amounts of C. albicans covering the epithelial tissue, and there was a loss of the papillae (Figure 9). This epithelium showed intense inflammation with the existence of mononuclear cells inside the dilated blood vessels caused by the inflammation. The underlying connective tissue was formed by muscle fibers with normal characteristics.

The histological findings 7 days after treatments (Figure 9) for the P-L-, P + L-, P-L+, and DNase groups contaminated with CaR were similar to those observed 24 h after treatments associated with damage in the muscular tissue. In the P + L+ and DNase+P + L+ groups, many hyphae and pseudohyphae were observed in the epithelium keratin layer, but the epithelial tissue remained with normal characteristics (Figure 9).

The representative images from animals for P-L- group inoculated with CaS and CaR 24 h after the treatment (Figures 10, 11, respectively) showed a thick layer of biofilm (green) surrounded by polysaccharides (1–4 β-mannan and galacto) (red) produced by C. albicans. The images of DNase+P + L+ group presented similarity with NIC group once the polysaccharides (red) and fungal cells (green) were not labeled. The P + L+ group showed a small amount of biofilm (green) and polysaccharides (red) (Figures 10, 11).

After 7 days of the treatment for CaS and CaR (Figures 12, 13), the P-L- group was labeled with polysaccharides produced by C. albicans (red), with higher intensity of label in fungal cells (green) (Figures 12, 13). The DNase+P + L+ group of animals contaminated with CaS presented a small layer of C. albicans biofilm and polysaccharides (red color) (Figure 12). The P + L+ group demonstrated a slight presence of fungal biofilm (green) and polysaccharides (red color) (Figure 12). For CaR, the group DNase+P + L+ presented less biofilm and polysaccharides than the group treated only with PDZ-aPDT (P + L+ group) once there was a thick layer of CaR biofilm (green) surrounded by polysaccharides (red) (Figure 13).