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 a…

Representative 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

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