CC BY
3DOI 10.24412/cl-37136-2023-1-160-165
ANTIMICROBIAL PHOTODYNAMIC THERAPY IN THE TREATMENT OF ORAL
CANDIDIASIS
EWERTON MIMA1 AND ANA PAVARINA2
¡Department of Dental Materials and Prosthodontics, School of Dentistry, Araraquara, Sao Paulo State
University (Unesp), Brazil ewerton.mima@,unesp.br
ABSTRACT
The ageing of the global population is one of the most important medical and social demographic problem worldwide. According to World Health Organization, in 2020 there was 1 billion people aged 60 years and over and this population will double by 2050 [1]. In Brazil, elderly population corresponds to 14.7% in 2021 and they increased 39.8% in the last nine years [2]. In this scenario, illness and the well-being of elderly population became a global public health challenge. Especially in this population, edentulism (or teeth lost) is a healthcare problem associated with education and income status [3], leading to masticatory dificulties, speech alteration, impairment of systemic health, self-steem, and social life (4). Although the complete edentulism has decreased, partial edentualism has increased [5] and dental prostheses are required to restore the functions of the oral cavity. In addition to edentulism, elders may suffer from chronic diseases and debilitating conditions, such as Alzheimer, Parkinson, cancer, immunosuppression, etc., which affect their ability to selfcare, including oral hygiene [6]. Thus, elderly people may be more prone to oral diseases, such as caries, periodontal diseases, and also oppotunistic infections caused by fungi. Oral candidiasis is the most common oral fungal infection caused by species of Candida and associated with systemic and local predisposing factors, such as diabetes, antibiotic therapy, reduced salivary flow (xerostomia), poor hygiene, and, especially, immunosuppresion [7]. As opportunistic infections, yeasts thrive when the host's immune defences are debilitated. Among the manifestations of oral candidiasis, denture stomatitis is a chronic infection of oral mucosa beneath the denture characterized by mucosa inflammation ranging from to red dots to hyperplasia, usually without symptons, and with high prevalence in women [8]. An in vivo study with rats demonstrated that dentures with Candida albicans induce a shift in the oral microbiome from aerobic, health-associated species to anaerobic, inflammatory species [46]. Oral candidiasis may spread to oropharynge, causing oropharingeal candidiasis [9], and in immunosupprised patients the infection may reach the blood stream and cause candidemia, which have high mortality rates and it is a relevant nosocomial infection [10]. The genus Candida encompasses more than 150 species, but only a few are responsible by infections: C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. guilliermondii, C. dubliniensis, and C. lusitaneae [11]. C. albicans is the most prevalent and virulent species found as commensal in the human body, such as skin, oral and genital mucosae. Among the virulence factors shown by C. albicans, polymorphism is the most striking feature and corresponds to the ability of reversebly changing its round shape cell morphology of yeast to elongated filamentous form of hyphae (parallel sides without constiction) and pseudo-hyphae (eliptical cells with constrictions) (Figure 1) [12]. The filamentous form is pathogenic, able to invade the host's epithelium and promote infection [13]. Other species, despite less virulent, are isolated from infections alone or with C. albicans, they show intrinsic resistance to antifungal agents, such as C. glabrata and C. krusei [14], and C. tropicalis is the most virulent non-albicans species commonly verified in Latin America and Asia [15]. The treatment of ora candidiasis involves the use of antifungal agents, which may be topical or systemic. Different from the wide range of antibiotics, there are only three classes of antifungal agents available commercially: polyenes, azoles, and echinocandins [16]. Different from bacteria, fungal cells are eukariotic cells, similar to mammalian cells, and hence they share similar features, which hinder the development of antifungal agents against specific fungal targets without cytotoxicity to the host. Polyenes (nystatin and amphotericin B) were the first antifungal agents discovered in 1949 and clinically used [16,17], they bind to the ergosterol and disrupt the fungal cell membrane. Therefore, they are fungicides, but nystatin is only topical and amphotericin B, despite systemic, has nefrotoxic side effect. In the
1960s, the first azoles (imidazoles) were introduced in the market, such as ketoconazole, miconazole, clotrimazole; they inhibit an enzyme responsible for the synthesis of ergosterol, thus they are fungistatic and, most of them, topical [16,18]. In the 1980s, in parallel to the HIV pandemic and other immunosuppression conditions caused by the development of medicine, such as organ transplantations, opportunistic fungal infections rose and the triazole drugs (fluconazole, itraconazole, voriconazole) emerged and revolutionized the antifungal treatments of systemic fungemia due to their efficacy and safety [16,19]. However, the widespread and the miuse of azoles brought up the problem of antifungal resistance, which is currently a big challenge of public health and involves all classes of antifungal drugs [20]. In 2020s, a new class of antifungal drugs, echinocandins (caspofungin, micafungin, and anidulafungin), was introduced in the market, they inhibit the P-glucan of the fungal cell wall, they are fungicide against some specias and fungistatic against other yeasts and they are systemic only administered by intravenous route [16,21]. Although these antifungal agents have demonstrated efficacy against superficial and systemic fungal infections, some species has developed resistance against one or more classes of antifungal agents and new species has emerged with multidrug resistance, such as Candida auris [22]. Therefore, this current scenario has imposed the search of new therapeutic modalities. Antimicrobial Photodynamic Therapy (aPDT) has been empolyed as an alternative or adjuvant method against Candida spp. and candidiasis in in vitro and in vivo studies, including animal models and clinical trials [23-26]. Several photosensitizers (PS) have been used, from first to third generation, such as porphyrins, clorins, phthalocyanines, curcumin, etc. But studies have shown that planktonic (free-floating cells) are more susceptible to aPDT than biofilms [23,24], which are complex communities of microrganisms attached on a biotic or abiotic surface and encased in a selfproduced polymeric matrix [27]. The matrix acts as a barrier that protect microbial cells against external antimicrobial agents and the host's immunity cells. Therefore, cells in biofilms are tolerant to antimicrobial approaches and infections caused by them are difficult to treat. This is the reason why several studies have been shown that, although planktonic cultures have been suscessfully inactivated by aPDT, biofilms have shown only reduction in their viability. When Photodithazine (PDZ) was used as a PS in aPDT against strains of C. albicans, C. glabrata, and C. tropicalis, their planktonic cultures showed complete inactivation or reduction raging from 4 to 5 log10, while their biofilm cultures have their viability reduced by 60% to 70% [28]. However, clinical infections, including oral diseases, are not caused by monospecies biofilms, but instead they are characterized by multispecies biofilms, where the pathogens share a consortium of mutual benefits. When a multispecies biofilm formed by C. albicans, C. glabrata, and Streptococcus mutans (the main bacterium involved in dental caries) was submitted to PDZ-mediated aPDT, significant reduction in their viability was observed, but the biofilm biomass was not reduced, suggesting that aPDT inactivated the microbial cells, but was not effective in disrupting the biofilm structure [29]. Therefore, an effective therapy may require several applications of aPDT instead of a single one. Using the same multispecies biofilm, three applications of PDZ-mediated aPDT resulted in higher inactivation than a single application, reducing the biofilm viability and also its total biomass [30]. aPDT mediated by PDZ was also used in vivo for the treatment of oral candidiasis in mice. It reduced the fungal load on the tongue of mice without harm the tissue [31] and demonstrated similar results to nystatin [25]. Similar efficacy was observed in vivo against fluconazole-resistant strains of C. albicans [32]. The biofilm maturation also influences its susceptibility to photoinactivation. Thus, the multispecies biofilm grown at 24 and 48 hours was submitted to aPDT mediated by curcumin (CUR), and 24-h biofilm showed higher reduction of viability and total biomass than 48-h biofilm [33]. aPDT mediated by CUR also inhibited biofilms of C. dubliniensis [34], a species genetically similar to C. albicans discovered in oral candidiasis of a HIV patient. CUR is an interesting PS because it is a natural compound with several therapeutic properties, such as antioxidant, anti-inflammatory, anticancer, and antimicrobial activities. However, CUR is not soluble in water (lipophilic) and unstable in solution, requiring toxic solvents, which hinder using CUR in vivo. Therefore, drug delivery systems have been employed to carry and solubilize CUR [35]. When CUR was encapsulated in polymeric nanoparticles and used as a PS against single and multispecies biofilms of C. albicans, S. mutans and methicillin-resistant Staphylococcus aureus (MRSA), the nanoparticles showed suitable chemico-physical properties and photoinactivation. Although cationic (positive charge) nanoparticles were more effective than anonic (negative charge) ones on biofilm photoinactivation, cationic nanoparticles were cytotoxic [36]. These nanoparticles were also used as PS in vivo in a murine model of oral candidiasis and significant
reduction of C. albicans from the tonghe of mice was verified without toxic effect on tissue, although the application of nystatin resulted in higher fungal inactivation [37]. However, in these studies the efficacy of CUR-loaded nanoparticles was lower than free CUR, which could be attributed to slow release of CUR from nanoparticles, which may be a shortcoming for topical use. Therefore, recently a smart nanosystem, a photoresponsive micelle was developed to load CUR and to be used as PS in aPDT. The photo-responsive micelle showed nano features similar to convencional micelles, improved the CUR release, and demonstrated photoinactivation of biofilms of C. albicans, MRSA, and Pseudomonas aeruginosa similar to CUR loaded in conventional micelles [38]. When nanotechnology is employed against microbial cells and biofilms, ideally the nanosystem should be cationic, i.e., it should have positive superficial charge. The superficial charge of microbial cell wall and of biofilm matrix is negative (anionic) due to their composition of polyssaccharide and also extracellular DNA in the matrix. Thus, the attraction between the different charges improves the antimicrobial activity [39]. Another PS, a phthalocyanine loaded in cationic nanoemulsion, demonstrated photoinactivation of a multispecies biofilm of C. albicans, C. glabrata, and S. mutans, although the total biomass was not reduced [40]. When used in vivo, aPDT mediated by phthalocyanine in cationic nanoemulsion also reduced the viability of C. albicans recovered from oral candidiasis in mice without adverse effect on the tongue's tissue [41] and reduced the virulence factors of adhesion and biofilm formation of C. albicans [42]. Other strategies have been also used to improve the photoinactivation of biofilms. The combination of aPDT with nystatin in a in vivo model of oral candidiasis resulted in reduction of C. albicans resistant to fluconazole and remission of the lesions on the mice's tongue [43]. The association of aPDT with Sonodynamic Therapy (SDT) demonstrated increased efficacy against biofilms of C. albicans compared with each therapy used alone, reducing both the fungal viability and the biofilm biomass [44]. Some drugs are able to reverse antimicrobial resistance by inhibiting the efflux pumps, which are transporters on microbial membrane that carry antimicrobial agents out of the cell. CUR acted as a efflux pump inhibitor of C. albicans, increasing its susceptibility to fluconazole and showing synergism with this antifungal agent. Although CUR-mediated aPDT reduced the viability fluconazole-resistant C. albicans in vitro, in vivo model using systemic infection of the larvae Galleria mellonella demonstrated that aPDT mediated by CUR did not increase the larvae survival neither reduced the recovery of C. albicans from larvae. Moreover, verapamil, a calcium blocker, was more effective as efflux pump inhibitor, incresing the survival of larvae infected with C. albicans resistant to fluconazole [45]. A clinical trial demonstrated that aPDT mediated by PDZ was effective as nystatin in the treatment of denture stomatitis, despite recurrent of lesions after both treatments [26]. All these studies have demonstrated that biofilms are still a challenge to be combated, despite all the technologies and recent advanced of aPDT. The combination of therapies aimed to target the biofilm matrix and also (but not only) the microbial cells demands further investigations.
Figure 1: Morphological forms of Candida albicans: yeast (A), hyphae (B), and pseudo-hyphae (C)
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