Toxicity, genotoxicity and adherence capacity of Candida albicans to oral keratinocytes after protocols for photodynamic inactivation mediated by Pelargonium sidoides

• Grant number: 21/02985-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2021
• End date: September 30, 2022
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Lívia Nordi Dovigo
• Grantee: Samuel Santana Malheiros
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Recently, international reports demonstrate an increase in the incidence of infections, mortality rates, and antifungal resistance of Candida albicans. Alternative treatments to combat this microorganism have been widely studied, such as photodynamic inactivation and the use of natural extracts with antimicrobial properties. EPs® 7630 is a commercial extract obtained from the roots of Pelargonium sidoides (P. sidoides), which has been showing potential use in combating bacteria, fungi, and viruses of human interest. Preliminary evidence obtained in our research group indicates that P. sidoides also has great potential as a photosensitizer in the photodynamic inactivation (PDI) of C. albicans. For this reason, this study aims to evaluate the parameters of cytotoxicity and genotoxicity of the use of P. sidoides in photodynamic inactivation and the ability of this therapy to influence cell migration and the adhesion of C. Albicans to epithelial cells. This is an experimental study, carried out in a laboratory. A standardized fungal suspension of the ATCC 90028 strain of C. albicans will be used. The human keratinocytes used in this study will be of the HaCaT lineage. To assess cell viability, keratinocytes will be exposed to P. sidoides for 15 minutes in the dark (pre-irradiation time) followed by lighting the samples for 18 minutes, obtaining a light dose of 50J/cm2 (LED ~ 450nm). After the proposed periods, a new culture medium containing the MTT solution will be added and an absorbance reading will be made on a spectrophotometer. Genotoxicity will be assessed by the micronucleus assay, in which the epithelial cells will be treated following the same protocols used in the previous assay. The samples will be analyzed under a fluorescence microscope, where the number of cells with micronucleus will be estimated. To assess cell migration, the in vitro healing test will be used, in which a "wound" will be made, followed by the application of the extract under the same protocols used in the previous tests. Images will be captured using an inverted light microscope and analyzed using the Image J. software. Migration will be determined by assessing the wound area and the percentage of wound closure (FF%) will be calculated. For the cell adhesion assay, a co-culture of keratinocytes and fungal cells previously treated will be performed following the PDI protocols previously described. The samples will be evaluated under fluorescence microscopy, where photomicrographs will be obtained for further analysis in ImageJ software. The percentage of adhesion in each culture will be determined as the ratio between the number of adherent cells of C. albicans on the entire surface of the monolayers and the number of cells of C. albicans inoculated. The experiments will be carried out on four separate occasions with three samples on each occasion. The effect of the experimental group on the different outcome variables will be investigated by descriptive analysis of the data, followed by one-way analysis of variance, if the assumptions of normal homoscedasticity are met. The level of significance adopted for the analyzes will be 5%. (AU)