Fotossensibilizadores naturais e sintéticos aplicados à inativação fotodinâmica antimicrobiana: caracterização fotoquímica, espectroscópica e avaliação biológica

Abstract

Antimicrobial resistance and foodborne and cutaneous infections have intensified the search for alternative antimicrobial strategies. Antimicrobial photodynamic inactivation (aPDI) has emerged as a promising approach based on the generation of reactive oxygen species through the interaction of a photosensitizer, light, and molecular oxygen, exerting multifactorial effects on microbial cellular structures. This study investigated the potential of natural and synthetic photosensitizers for antimicrobial aPDI through phytochemical, spectroscopic, and photochemical characterization, assessment of antioxidant activity, and determination of antimicrobial efficacy against microorganisms of clinical and food-related relevance. In addition, a standardized aPDI protocol was developed and integrated into a custom-built LED illumination system designed for 96-well microplates. The system enabled irradiance control, wavelength selection, and homogeneous light distribution, allowing the simultaneous assessment of photosensitizer concentration and optical fluence with improved reproducibility and experimental throughput. The phenothiazinium dyes Azure A (AZA) and Toluidine Blue O (TBO) were evaluated as synthetic photosensitizers, whereas a hydroethanolic extract obtained from the flowers of Plumeria rubra L. (Apocynaceae) by ultrasound-assisted extraction was investigated as a natural photosensitizer. AZA and TBO exhibited absorption profiles in the visible region that were compatible with the red LED source used at 630 nm. Both dyes showed similar singlet oxygen generation rates and photodynamic saturation at concentrations above 34 µM. In the aPDI assays, both photosensitizers achieved complete inactivation of Cutibacterium acnes at the same concentrations, with minimum bactericidal concentrations (MBCs) ranging from 6.25 µM at 12 J/cm² to 0.78 µM at 50 J/cm². Against Staphylococcus epidermidis, AZA exhibited greater efficacy than TBO, with MBC values twofold lower at all evaluated fluences. Phytochemical analysis of the Plumeria rubra extract by UHPLC–MS/MS identified 21 compounds, with rutin (83.96 mg/g) and chlorogenic acid (13.38 mg/g) as the predominant constituents. The extract exhibited high antioxidant activity in the TBARS assay (EC₅₀ = 0.145 mg/mL) and broad spectral absorption between 400 and 630 nm, which was compatible with the white-light source used. In the absence of light, the extract showed no antibacterial activity under the tested conditions. Upon irradiation, however, it induced fluence-dependent photodynamic inactivation of all six Gram-positive strains evaluated, including methicillin-resistant Staphylococcus aureus (MRSA), with an MBC of 0.78 mg/mL at 18 J/cm². Lower efficacy was observed against Gram-negative bacteria.These findings demonstrate that AZA is an effective synthetic photosensitizer for antimicrobial photodynamic inactivation, with potential applications in the treatment of acne vulgaris. Furthermore, the Plumeria rubra extract represents a promising natural photosensitizer, combining a relevant phytochemical profile, photodynamic selectivity, and bactericidal activity against pathogens of clinical and food-related importance, including multidrug-resistant strains.