Photodynamic Therapy cytotoxicity using Photogem and LED on normal cells

Objective: The purpose of this in vitro study was to evaluate the cytotoxicity of a specific photodynamic therapy (PDT) after applying the photosensitizing agent Photogem associated with a blue light emitting diode (LED) to L929 fibroblasts and MDPC-23 odontoblast-like cell lines. Methods: After seeding the cells (30.000 cells/cm2) in 24-well dishes for 48 hours, they were incubated for 30 minutes with Photogem (0, 10, 25 and 50 mg/L) and irradiated or not with the LED (460 ± 3 nm; 25.5 and 37.5 J/cm2; 22 mW/cm2). It has been shown that this specific PDT is sufficient to inactivate Candida albicans in vitro. Subsequent to PDT, the cell metabolism was assessed at 0 h, 12 h and 24 h using the MTT assay and the cell morphology was evaluated by SEM. The flow cytometry technique was used to analyze the type of cell death (necrosis or apoptosis) as well as to measure intracellular ROS presence using a dihydrofluorescein diacetate technique. Results: The metabolism of those fibroblasts and odontoblast-like cells exposed to Photogem associated with LED irradiation (PDT) decreased 90% -98%, which was statistically lower than the negative control (no treatment). This reduction of the mitochondrial activity was not dose-dependent (Mann-Whitney, p<0.05). It was also shown that both cell types did not recover their viability at 24-hours after applying the PDT. Cells exposed only to Photogem or light-irradiation showed metabolic activity similar to the negative control. After PDT, it was observed plasma membrane disruption and cell swelling. The flow cytometry analysis demonstrated a strong PDT-induced cell necrosis. The photosensitizer increased the intracellular ROS level, which depended upon the concentration of Photogem and exposure to light. Conclusion: The association of Photogem with blue LED caused intense toxic effects to both cell lines evaluated, which were characterized by direct cellular necrosis.

Supported by FAPESP: 2007/04376-4