Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Summary

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Abstract

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 µM, 40 µM, and 80 µM) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm²; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch's ANOVA and Games-Howell tests (α = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 µM or 80 µM curcuminoids. aPDT mediated by 80 µM curcuminoids promoted a 2.47 log₁₀ reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Introduction

Oral candidiasis (OC) is the main fungal infection of the oral cavity; it is caused by the overgrowth of Candida spp. Predisposing factors for OC include endocrine dysfunction, the use of broad-spectrum antibiotics, radio- and chemotherapy, nutritional deficiencies, xerostomia (low salivary flow), denture use, poor hygiene, and, especially, immunosuppression¹. Among Candida species, Candida albicans is the most prevalent and virulent one; it is found as a commensal species in the human body and as an opportunistic pathogen. C. albicans has the ability to change its morphology from commensal yeasts (blastopores) to pathogenic filaments (hyphae and pseudohyphae)². The filamentous forms, especially the hyphae, can invade the host epithelium by endocytosis or active penetration, causing infection³. Other virulence factors of C. albicans include adhesion, biofilm formation, and secretion of lipolytic and hydrolytic enzymes and toxins, such as lipases, phospholipases, proteinases, and candidalysin⁴.

OC treatments involve the use of antifungal agents, especially topical polyenes and azoles (nystatin and miconazole)⁵. However, they only show short-term efficacy, and recurrence is frequent. In addition, the overuse of antifungals has given rise to the problem of antifungal resistance development and spread⁶. Therefore, alternative therapies are needed, such as antimicrobial Photodynamic Therapy (aPDT), which combines a photosensitizer (PS) and light at an appropriate wavelength (the same as that of the PS absorption) in the presence of oxygen. PSs are bound to or taken up by cells and, when activated by light, produce reactive oxygen species (ROS) that are toxic to sensitized cells⁷.

In aPDT, one of the photosensitizers (PSs) employed is curcumin (CUR), a naturally occurring compound extracted from the rhizomes of the turmeric plant (Curcuma longa L.). Curcumin possesses numerous therapeutic attributes, including anti-inflammatory, antioxidant, anticancer, and antimicrobial capabilities^8,9. A prior investigation found that aPDT utilizing CUR effectively diminished C. albicans in a murine model of oral candidiasis without causing any harm to the host's tissues¹⁰. CUR is the main curcuminoid extracted from turmeric, but other polyphenols, such as demethoxycurcumin and bis-demethoxycurcumin, are also found in this plant. Curcuminoid-mediated aPDT demonstrated antibacterial activity against biofilms of Staphylococcus aureus grown in catheters¹¹. However, to the best of our knowledge, its antifungal activity against C. albicans remains unclear. Therefore, in this study, we evaluated aPDT mediated by a curcuminoid salt against C. albicans in a murine model of OC.

Protocol

The research protocol for the use of mice was approved by the Ethics Committee for Animal Use (case numbers 05/2008 and 09/2020) at the School of Dentistry, Araraquara, UNESP. C. albicans (ATCC 90028) was used as the reference strain. Six-week-old female Swiss mice (n = 45), with a body mass range of 20-30 g, were used for the present study. The animals were provided by São Paulo State University, UNESP, Botucatu.

1. Preparation of PS and selection of the light source for aPDT

1. Prepare the PS according to the specifications for the substance to be tested.
   NOTE: In this study, a water-soluble mixture of curcuminoids (CUR-based water-soluble salt mixture¹²) was used as the PS (see Table of Materials and Figure 1). Dilutions at 20 µM, 40 µM, and 80 µM (15.6, 29.2, and 58.4 mg/L, respectively) were prepared in ultra-pure water immediately before use and kept at 5 °C.
2. Select a light equipment with an appropriate wavelength (the same as that of the PS absorption) capable of illuminating the entire photosensitized target uniformly and simultaneously without heating the tissue.
   ​NOTE: In this study, a handpiece containing a light-emitting diode (LED, see Table of Materials), that was designed at the Instituto de Física de São Carlos (University of São Paulo, São Carlos, SP, Brazil), was used. The output power delivered by the light was 89.2 mW/cm² at a blue wavelength of approximately 455 nm.

2. Preparation of C. albicans inoculum

1. Keep the reference strain C. albicans (ATCC 90028) in yeast-peptone-glucose (YEPD, see Table of Materials) with 50% glycerol at -80 °C until use.
2. Thaw the frozen strain at room temperature, spread a 100 µL aliquot on a Petri dish with Sabouraud dextrose agar (SDA) with chloramphenicol (see Table of Materials), and incubate at 37 °C for 48 h.
3. Transfer five colonies into 10 mL of yeast nitrogen broth with 2% glucose (YNBg) medium and grow aerobically at 37 °C for 16 h.
4. Dilute the yeast culture in fresh YNBg (1:10) and incubate aerobically at 37 °C until the optical density reaches the mid-log phase of growth.
   NOTE: Standardize the microbial growth curve for each laboratory previously. In the current research, cultures were allowed to incubate for around 8 h, until they reached the mid-log phase of growth as indicated by the optical density at 540 nm (OD₅₄₀), with a mean value of 0.536 ± 0.062 arbitrary units (mean ± standard deviation [SD]).
5. Centrifuge the culture at 5,000 x g at room temperature for 5 min, discard the supernatant and wash the pellet twice with the same volume of sterile phosphate-buffered saline (PBS; 0.136 M NaCl, 2 mM KCl, 1 mM KH₂PO₄, 10 mM Na₂HPO₄, pH 7.4).
6. Use 100 µL aliquots for performing four serial 10-fold dilutions in PBS, spread dilutions on SDA plates, and incubate at 37 °C for 48 h for colony count. As a standard, fungal suspensions are used at concentrations of approximately 4.5 × 10⁷ colony forming units per milliliter (CFU/mL)].

3. Induction of OC in mice

NOTE: The following methodology was previously described by Takakura et al.¹³ and reproduced by our group^10,14, with some modifications.

1. Randomly distribute mice in nine groups (n = 5), corresponding to the same number of treatments (Supplementary File 1).
   NOTE: The oral cavity of mice should be negative for Candida growth. This needs to be checked by swabbing the oral cavity and plating the sample on SDA.
2. Keep the animals in propylene boxes¹⁰(5 mice per box max.), with wood shavings for floor covering, in a temperature-controlled vivarium at 23 ± 2 °C under a 12/12 h light/dark cycle. No specific enrichment needs to be provided.
3. Maintain mice on extruded mouse diet and filtered water ad libitum.
4. Subcutaneously inject the immunosuppressive drug, prednisolone (100 mg/kg body weight, see Table of Materials), for the first time on day one to all the members of groups C+L+ 20, C+L+ 40, C+L+ 80, C+L- 20 µM, C+L- 40, C+L- 80, C-L+, and C-L- (Supplementary File 1).
   NOTE: Keep mice from the same group in the same box and monitor them daily for any sign of stress and weight loss. Seek veterinary advice for analgesic or altered food/water if stress or weight loss are noted..
5. Starting on day one and until the end of the experiment, provide tetracycline hydrochloride in drinking water at 0.83 mg/mL¹³.
6. Inoculate mice tongues on day two, including groups C+L+ 20, C+L+ 40, C+L+ 80, C+L- 20, C+L- 40, C+L- 80, C-L+, and C-L- (Supplementary File 1), with the C. albicans inoculum. Do not inoculate those mice from the negative control group (NCtrl).
   1. Sedate animals by intramuscular injection of 10 mg/kg chlorpromazine chloride (see Table of Materials) on each thigh muscle before carrying out inoculation.
   2. Perform intraoral inoculation of the mice by soaking a sterile swab (one per animal) into a freshly prepared (i.e., prepared immediately before inoculation) standardized suspension of C. albicans.
   3. Place sedated mice in a supine position. Gently pull their tongues out of their mouths using a sterile forceps. Rub each mouse's tongue with a soaked swab for 30 s. Monitor mice until they recover from sedation.
7. On day five, administer a complementary subcutaneous injection of prednisolone (100 mg/kg body weight) to maintain the immunosuppression.
   ​NOTE: This protocol is adequate to keep the infection for 7 days after intraoral inoculation. The protocol timeline is shown in Figure 2.

4. Antimicrobial Photodynamic Therapy and recovery of C. albicans from oral lesions

1. On day seven, prior to treatment, anesthetize the animals using an intraperitoneal injection of ketamine hydrochloride (100 mg/kg body weight) combined with xylazine (10 mg/kg body weight) (see Table of Materials).
2. Place each animal in a supine position on a pad device^14,15equipped with stainless steel wires that can be looped around the incisors to keep the mouth open.
   NOTE: Isothermal pads are recommended for warming the mice while they are sedated, preventing hypothermia at room temperature and avoiding mortality related to anesthesia¹⁵. Each anesthetized animal should be monitored by the lack of a withdrawal reflex when toes are pinched to confirm the anesthesia depth. One maintenance dose of ketamine at 1/3 the original dose (without xylazine) may be administered if needed.
3. With the mandible and cheeks retracted, gently place the tongue outside the mouth.
4. For mice from C+L+ groups, pipette 70 µL of the PS on the dorsum of the tongue (at one of the tested concentrations). Maintain the mice in a dark environment for a duration of 20 min as a pre-irradiation period. Ensure that during this time, the tongue of each animal remains positioned inside the oral cavity to prevent the photosensitizer (PS) from being ingested.
5. After the photosensitization period, pull the tongue out of the mouth for illumination. Place the LED device onto the dorsum of the tongue and illuminate at 37.5 J/cm² (7 min with the present device) (Figure 3).
6. For animals from the C+L- groups, pipette 70 µL of the PS at one of the tested concentrations on the dorsum of the tongue, and keep these mice in the dark for 27 min.
7. For animals from the C-L+ group (treated with light without any previous photosensitization), pipette 70 µL of sterile saline solution on the dorsum of the tongue. Keep the mice in the dark for 20 min and then illuminate their tongues at 37.5 J/cm² (7 min with the present device).
8. For mice from the C-L- group (neither treated with PS nor with light), pipette 70 µL of sterile saline solution on the dorsum of the tongue and keep them in the dark for 27 min.
9. Recover C. albicans from the tongues of all mice immediately after treatment. Swab the dorsum of the tongue for 1 min with a sterile cotton swab.
10. Place each sample swab in a tube containing 1 mL of sterile saline and vortex for 1 min to resuspend the microbial cells.
11. Immediately perform 10-fold serial dilutions in PBS and plate 25 µL aliquots over SDA plates in duplicate. Incubate the plates aerobically at 37 °C for 48 h.
12. After 48 h, use a digital colony counter (see Table of Materials) to determine yeast colony counts. Calculate C. albicans load (CFU/mL) for each animal.
13. Transform CFU/mL values into log₁₀ and analyze properly according to the design of the experiment and data assumptions.
    NOTE: In the present investigation, Welch's one-way ANOVA and Games-Howell post-hoc test (α = 0.05)¹⁶ was used.

5. Histopathological analyses

1. On day eight, anesthetize mice with ketamine at 100 mg/kg combined with xylazine at 10 mg/kg via intraperitonial injection and euthanize them with an overdose of ketamine (200 mg/kg administered via intraperiotonial). Confirm death by checking for the absence of respiratory movement (apnea), and absence of heartbeat (asystole).
2. Carefully pinch the tongue and pull it out of the animal's mouth. Using a manual scalpel, make an incision before the circumvallate papillae to surgically remove the entire tongue without causing any injuries to the anterior and central regions.
3. Fix the tongue in 10% buffered formalin (pH 7.2-7.4) for 24 h and wash it in running water for 2 h.
4. Proceed with the inclusion in the paraffin process: dehydration, clarification, and impregnation^10,14.
5. Cut serial sections (5 µm thick) using a microtome, then affix the sections onto glass slides. Proceed to stain these sections using periodic acid-Schiff and hematoxylin (PAS-H) for histopathological examination and identifying fungi through observation under a light microscope.
6. Ask a pathologist, preferably blind to the groups studied, to examine the tissue reaction due to C. albicans infection.
7. Describe the histological characteristics of the tissue (epithelium and connective tissue), especially local inflammatory responses of varying intensities.

Results

The murine model of OC showed typical white patches and pseudomembranes on the tongue of all infected mice (Figure 4A). C. albicans recovered from C-L- animals confirmed tissue colonization by this microorganism (values ranged from 1.62 x 10⁴ to 4.80 x 10⁵ CFU/mL). As expected, animals from the NCtr group did not show any tissue alterations or colony growth after sampling (Figure 4B).

aPDT decreased the ...

Discussion

C. albicans has been associated with oral and esophageal infections in individuals with an immunocompromised state, diabetes mellitus, prolonged use of antibiotics, and poor oral hygiene^1,3. The study of human infectious diseases requires both in vitro and in vivo investigations before clinical trials can be safely and accurately designed. The present study describes a method for establishing a murine model of OC, which can be used to e...

Materials

Name	Company	Catalog Number	Comments
C. albicans	ATCC (Rockville, Md, USA)	90028	Used to prepare the Candida inoculum
Centrifuge	Eppendorf Centrifuge 5804/5804R,B. Braun, Melsungen, Hesse, Germany	022628146 (NA)	Used to prepare the Candida inoculum
Chlorpromazine chloride 2 mg/mL	Compounding pharmacy, Araraquara, SP, Brazil	-	Used to sedate animals during candida inoculation
Curcumin-based water-soluble salt	PDTPharma, Cravinhos, Brazil	-	Consisting of 53.4% of natural curcumin, and  46.6% of other curcuminoids (demethoxycurcumin and bis-demethoxycurcumin). Prepared in water and N-MethylD-Glucamine (final average molecular weight of 730.32 g.mol−1)
Digital colony counter	CP 600 Plus, Phoenix Ind Com Equipamentos Científicos Ltda, Araraquara, SP, Brazil	-	Used to count colonies on agar plates
Extruded mouse chow	Benelab food, Industry Qualy Animal Nutrition and Commerce Ltda., Lindóia, São Paulo State, Brazil.	-	Used for the feeding of the mice
Ketamine Hydrochloride 10%	Ketamina Agener, União Química Farmacêutica Nacional S/A, Embu-Guaçu, SP, Brazil	-	Used to anesthetize animals before  treatments and for euthanasia
Light-emitting diode handpiece (prototype)	Instituto de Física de São Carlos, University of São Paulo, São Carlos, SP, Brazil	-	Fabricated with LXHL-PR09, Luxeon III Emitter, Lumileds Lighting, San Jose, California, USA
Methylprednisolone acetate 40 mg	DEPO-MEDROL, Pfizer, New York	-	Used as an immunosuppressant
Microtome	Leica Microsystems, Bannockburn, IL, USA	SM2500	Used to cut the serial sections of the tongues
Propylene boxes (cages housing) H13 x L20 x D30 cm	Bonther Equipaments, Ribeirão Preto, SP, Brazil	-	Used to keep the animals throughout  the experimental period
Sabouraud Dextrose Agar with Chloramphenicol	HiMedia, Mumbai, India	MM1067-500G	Culture medium for yeast growth (agar)
Spectrophotometer	Spectrophotometer Kasvi K37-VIS , São José dos Pinhais, PR, Brazil	K37-VIS	Used to standardize the inoculum concentration
Tetracycline hydrochloride	Compounding pharmacy, Araraquara, SP, Brazil	-	Antibiotic given to induce oral dysbiosis
Wood shavings	J.R. Wood Shavings, Comerce of Sawdust Ltda., Conchal, São Paulo State, Brazil	-	Used for floor covering inside the housing boxes
Xylazine 2%	Calmiun, União Química Farmacêutica Nacional S/A, Embu-Guaçu, SP, Brazil	-	Used in combination with ketamine for anesthesia
Yeast Nitrogen Broth	Difco, InterLab, Detroit, MI, USA	DF0919-07-3	Culture medium for yeast growth (broth)
Yeast Peptone Dextrose Broth	NutriSelect Basic, Sigma Aldrich	Y1375	Culture medium for maintaining the strains at -80°C and grow