2.5.2. Cell Treatment and LED Irradiation Protocol

To perform the in vitro experiments, Mel-Juso cells were seeded at a density of 5 × 104 cells/mL in 96 and 6-well plates and incubated at 37 °C, 5% CO₂. After adhesion, the cells were treated with, γ-Fe₂O₃ NPs_TPPS, free γ-Fe₂O₃ NPs and free porphyrin, diluted in complete culture medium until a final concentration of 0.05, 0.5, 2.75, 4.16, 5.5 and 11 μg/mL for NPs, 0.01, 0.1, 0.5, 0.75, 1 and 2 μg/mL for porphyrin and 0.05 + 0.01, 0.5 + 0.1, 2.75 + 0.5, 4.16 + 0.75, 5.5 + 1 and 11 + 2 μg/mL for NPs + TPPS in γ-Fe₂O₃ NPs_TPPS, respectively. Untreated cells were used as control. To allow the internalization of porphyrin and NPs, cell cultures were incubated for 24 h at 37 °C, 5% CO₂. Afterwards, the culture medium was replaced with a fresh one and the cells were irradiated or not for 0.5, 1, 2.5, and 5 min, and incubated at 37 °C. All experiments were performed at the interval of 24 h from the irradiation procedure. Before incubation with cells, the NPs and TPPS suspensions were sterilized using UVC radiation for 1 h. Verifications by absorption spectroscopy regarding possible photobleaching after UVC exposure were per-formed to be sure that the sterilization has no effect on suspensions. No changes of the spectra were noticed.

For cell exposure to UV light, a custom system of irradiation was developed (Figure 2). A 405 nm LED (Nikia, Japan, NVSU 233B) was used as a light source, with a radiant flux of 1400 mW. To control the intensity of LED and the time of irradiation, a Pulse Frequency Generator (PWM) and a Timer Relay Module were added to the pow-er supply circuit.

LED Irradiation experimental setup.

A Plano-Convex Lens, with the focal length of f = 100 mm, was placed between the LED and the plates, at a distance of 25 mm with respect to the light source to collect the entire radiation emitted by the diode (Figure 2), and 30 mm to the probe, to ir-radiate a large interest zone. The power density was measured at different duty cycles for the PWM, a calibration curve was drawn and used further in irradiation parameters selection.