Efeitos de formulações líquidas e em gel na terapia fotodinâmica: avaliação da atividade antimicrobiana e da biocompatibilidade celular

Abstract

Staphylococcus aureus is a Gram-positive pathogen associated with severe infections, and the increasing antimicrobial resistance highlights antimicrobial photodynamic therapy (aPDT) as a promising alternative due to its effective action and lack of resistance induction. The aim of this study was to evaluate, in vitro, an optimized aPDT protocol for the decolonization of S. aureus, comparing methylene blue in liquid solution (0.005% and 0.01%) and gel formulation (0.01%), under different pre- irradiation times (3 and 5 min), as well as to investigate the cytotoxicity and biocompatibility of the liquid formulations in L929 fibroblasts. To ensure standardization of the bacterial load used in the assays, the time point of highest bacterial growth activity was established, ensuring experimental reproducibility. Experiments were performed to assess aPDT activity in planktonic cultures, with groups organized as follows: (i) control, without treatment; (ii) laser, subjected only to irradiation; (iii) MB, incubated with 0.005% or 0.01% methylene blue, in liquid or gel formulation, with pre- irradiation times of 3 or 5 minutes; and (iv) aPDT, combining methylene blue (0.005% or 0.01%, liquid or gel) and laser after 3 or 5 minutes of pre-irradiation. Irradiations were performed using a laser prototype (400 mW, 120 J per point, 660 nm). After treatment, samples were diluted, plated on BHI agar, and incubated for 24 h for CFU/mL determination. Biocompatibility was assessed in murine L929 fibroblasts using the same experimental groups, except for the MB gel. Cells were treated and analyzed after 24 and 48 h using MTT, Crystal Violet, and Griess assays. Curve standardization at the exponential growth phase (mid-log) was achieved at approximately 5 h, with OD600 ≈ 0.7 (2 × 10⁸ CFU/mL). Neither laser irradiation alone nor methylene blue alone promoted CFU/mL reduction, whereas the combination of laser and liquid methylene blue at both concentrations resulted in partial inactivation after 3 minutes of pre-irradiation and complete inactivation after 5 minutes. The 0.01% methylene blue gel formulation did not reduce bacterial growth, regardless of the pre-irradiation time evaluated (3 or 5 minutes). In L929 fibroblasts, control, laser-only, and methylene blue- only groups preserved morphology, monolayer integrity, and metabolic viability, whereas groups subjected to aPDT (laser + methylene blue at 0.005% and 0.01%) exhibited marked loss of adherent cells, low Crystal Violet retention, and reduced viability in the MTT assay. Nitrite levels at 24 and 48 h did not increase among the groups. It can be concluded that aPDT using liquid methylene blue was effective in the inactivation of S. aureus, particularly after 5 minutes of pre-irradiation at both concentrations, while the gel formulation showed limited performance. However, the combination of methylene blue and laser exhibited cytotoxicity in murine L929 fibroblasts.