Cell viability assays

Cell culture. Immortalized human keratinocytes (HaCaT) were chosen due to their good proliferative capacity in culture and their use in previous in vitro cytotoxicity studies of local anesthetics and drug delivery systems [24,25]. The HaCaT cells (BCRJ 0100, Rio de Janeiro Cell Bank, Rio de Janeiro, RJ, Brazil) were grown in 75 cm² TPP^® bottles (Techno Plastic Products AG, Trasadingen, Switzerland) containing Dulbecco’s Modified Eagle Medium (DMEM, Vitrocell, Campinas, SP, Brazil) supplemented with 10% (v/v) fetal bovine serum (Vitrocell), 10,000 U/mL penicillin (Vitrocell), and 100 μg/mL streptomycin (Vitrocell), at 37°C, in a humid atmosphere with 5% CO₂ [24,25]_.

MTT colorimetric assay. Semi-confluent cells were seeded into 96-well tissue plates, at approximately 1x10⁴ cells/well, followed by incubation for 48 h, at 37°C, in a humid atmosphere with 5% CO₂. Subsequently, the cells were exposed to ATC, ATC_epi, ATC_nano, and ATC_nanoepi, at concentrations in the range from 0.06% to 1%, for 1 h or 24 h. The half-maximal inhibitory concentration (IC₅₀) was determined for all formulations and exposure periods.

Cell viability was evaluated by incubation with 0.3 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) for 3 h at 37°C. The cell viability values were obtained by conversion of the absorbance readings (A_{570 nm}) into percentages of viable cells. The values obtained for DMEM were used as reference controls (100% cell viability).

Cell viability by fluorescence imaging. The fluorescence imaging analysis of cell viability was performed as described previously, using the LIVE/DEAD^® assay (Invitrogen, Carlsbad, CA, USA) [25]. Briefly, approximately 1x10⁵ cells were inoculated into 24-well plates and were incubated for 48 h, at 37°C, under a humid atmosphere with 5% CO₂. Subsequently, the cells were exposed to ATC, ATC_epi, ATC_nano, and ATC_nanoepi, at the ATC IC₅₀ obtained in the MTT assay. Methanol (70%) was used as the cell death positive control. After exposure to the formulations for 1 h or 24 h, the cells were washed twice with phosphate-buffered saline (PBS), followed by addition of 300 μL of the LIVE/DEAD^® solution to each well containing HaCaT cells. The plates were then incubated at room temperature for 30–45 min. Fluorescence images were acquired using an inverted microscope (Axiovert 40 CFL, Carl Zeiss, Germany) coupled to an MEC camera (AxioCam, Carl Zeiss, Germany). Calcein/AM was detected using wavelengths of 450–490 nm (excitation) and 515–565 nm (emission). Cells labeled with EthD-1 were detected using wavelengths of 528–546 nm (excitation) and 590–617 nm (emission) [25].