Desenvolvimento e validação preliminar de um sistema de irradiação por LED para aplicação em terapia fotodinâmica e ensaios in vitro

Abstract

Photodynamic Therapy (PDT) is based on the light activation of a photosensitizer in the presence of molecular oxygen, resulting in the generation of reactive species with localized cytotoxic effects. The reproducibility of PDT assays depends on accurate control of the light dose, irradiation homogeneity, and light source stability, which are often limited in conventional systems. Therefore, this study aimed to develop and validate an LED-based irradiation system with controlled light intensity and exposure time for in vitro PDT applications. The system was developed using an Arduino Nano V3 microcontroller coupled to a Metal-Oxide-Semiconductor Field-Effect Transistor (MOSFET), an LED array emitting in the 460–465 nm range, and a 3D-printed support structure that enables precise alignment with 96-well plates. Calibration of the light source was performed by chemical actinometry using potassium ferrioxalate, allowing the determination of radiant intensity, photon dose, and energy density. Actinometric results demonstrated a dose-dependent increase in light intensity, with values ranging from 5.47 × 10−9 to 1.79 × 10−8 einstein·s−1 at intensities from 20 to 100%. The corresponding energy densities ranged from 0.85 to 16.63 J·cm−2, depending on the combination of light intensity and irradiation time (10 min, 30 min, and 1 h). Photodegradation assays using sodium fluorescein showed a progressive decrease in absorbance and fluorescence with increasing light intensity and exposure time, confirming the reproducible photochemical response of the system. Biological evaluation included light-induced phototoxicity assays on L929 (murine fibroblasts) and A549 (human lung adenocarcinoma) cell lines, in which the MTT colorimetric assay revealed differences in cellular sensitivity, possibly associated with tolerance to thermal stress. Additionally, the system proved applicable to PDT through in vitro photoactivation of the Ru(phen)2(4Bzpy)NO2 complex, resulting in antimicrobial activity against Staphylococcus epidermidis ATCC 12228, with a minimum inhibitory concentration (MIC) of 64 μg·mL−1 and a minimum bactericidal concentration (MBC) of 128 μg·mL−1 under a light dose of 6.28 J·cm−2. In conclusion, the developed system provides reproducible control of light dose, homogeneous irradiation, and promising performance for in vitro photodynamic therapy applications.