Avanços na terapia fotodinâmica: estudo comparativo dos fotossensibilizadores butil azul de toluidina, azul de toluidina e azul de metileno na viabilidade, proliferação e incorporação celular de fibroblastos – estudo in vitro

Abstract

Introduction: Antimicrobial photodynamic therapy has emerged as an adjunctive approach for infection control, employing photosensitizers that, when activated by light, generate reactive oxygen species capable of inducing microbial damage through non-specific mechanisms, with a low probability of resistance development. Although toluidine blue O and methylene blue are widely used, the butyl toluidine blue O derivative exhibits higher lipophilicity, lower cytotoxicity, and greater potential for singlet oxygen generation. This study compares the effects of these three photosensitizers on cell viability, proliferation, and cellular uptake in fibroblasts, under dark conditions and after photodynamic irradiation. Methodology: The concentration–response relationship of the photosensitizers BuTBO, TBO, and MB was previously determined to standardize the experimental conditions and identify concentrations capable of preserving cell viability and proliferation in the absence of light. L929 cells were exposed to the three dyes (0–100 µM) for 5 minutes, washed, and analyzed in the dark, defining 25 µM as the concentration for subsequent experiments. This concentration was then used to evaluate the response after irradiation (660 nm, 2.4 J; 125 s). Cell viability was quantified by the MTT assay and proliferation by the crystal violet method, under the same experimental conditions. Photosensitizer uptake (25 µM) was analyzed by fluorescence microscopy. Data were analyzed using two-way ANOVA (photosensitizer × concentration) and one-way ANOVA (photosensitizer), both followed by Bonferroni post hoc tests (p < 0.05). Results: A 5-minute exposure to the photosensitizers (0–100 µM) did not alter cell viability (MTT) or proliferation (crystal violet) at 0 h and 24 h (p > 0.05). After PDT, all groups showed a significant reduction in viability compared with the control (p < 0.05). Butyl toluidine blue O exhibited the lowest optical density (OD ≈ 0.63) in the MTT assay, indicating the highest cytotoxic effect under irradiation, whereas toluidine blue O and methylene blue showed intermediate values (OD ≈ 0.77 and 0.80). No significant differences in cell proliferation were observed among groups (p > 0.05). Fluorescence microscopy demonstrated cytoplasmic incorporation of BuTBO in L929 fibroblasts, with a diffuse pattern in the dark and a more granular distribution after irradiation, suggesting intracellular redistribution. Morphological changes occurred without loss of cell adhesion, consistent with the greater reduction in metabolic activity observed in the MTT assay, indicating a predominantly intracellular cytotoxic effect. Conclusions: After PDT, butyl toluidine blue O produced the greatest reduction in metabolic viability in L929 fibroblasts compared with the other photosensitizers, while preserving cell proliferation. Intracellular detection by fluorescence microscopy reinforces its superior photodynamic effect and its potential for future applications.