Development of an Experimental Protocol for Combined Electroporation and Phototherapy Treatment Against Melanoma Cells

Melanoma is the most common cutaneous tumor with significant challenges for successful pharmacological treatment. The aim of this study was to determine the optimal treatment conditions using a combined therapy based on electroporation and the photoactivation of acridine orange (AO) with a blue LED lamp, designed by Biopulse srl. The effect of combined therapy was investigated by MTT assay and optical microscopy observations. Drug uptake and apoptosis induction were analyzed by flow cytometry. The best treatment schedule was obtained using electric pulses (sequences of eight biphasic pulses lasting 50 + 50 μs with 300 μs interpulse, at a voltage of 300 V), an AO concentration of 0.01 μg/mL, and LED exposure for 30 s. The combined treatment was more effective against melanoma cells than human dermal fibroblast-adult cells. The results demonstrated that electroporation increased AO accumulation, allowing for reduced drug concentrations and exposure times. This combination induced programmed cell death consistent with apoptotic features in melanoma cells, as shown by cell cycle analysis. These preliminary results demonstrated the efficacy of combination therapy based on electroporation and phototherapy with AO on melanoma cells, and they encourage the possibility of testing efficacy on melanoma models in veterinary oncology.