2.2.7. In Vitro Cellular Studies

The in vitro cytotoxic activity of rutin-loaded ethosomes (NV-A₄) was evaluated on keratinocyte cells (NCTC2544) using an MTT assay, and the results were expressed in terms of cell viability. The cells were seeded at a density of 5 × 10³ cells/well in 96-well plates. After 24 h of incubation, the cells were treated with 100 µL of the fresh medium added with empty ethosomes^® or DMSO, used as negative controls, at several concentrations referred to loaded ethosomes^® and free active solution tested during the evaluation of antioxidant effect. Hence, 10 µL of MTT solution (5 mg/mL dissolved in PBS solution) was added to each well. After 3 h of incubation, precipitated formazan salts were dissolved with 100 µL of ethanol–DMSO solution (1:1 v/v), and the plate was shaken for 20 min at 230 rpm (IKA^® KS 130 Control, IKA^® WERKE GMBH & Co., Staufen, Germany).

The cell viability (%) was determined by the use of microplate spectrophotometer (Multiskan MS 6.0, Labsystems, Vantaa, Finland) at a wavelength range between 540 nm and 690 nm using the following equation:

where Abs_t represents the absorbance of treated cells and Abs_c represents the absorbance of untreated ones.

For antioxidant efficacy, NCTC 2544 cells were seeded in a 96-well plate at a density of 7.5 × 10³ cells/well. After 24 h of incubation, the cells were treated with free active solution or with rutin-loaded ethosomes (NV-A₄) at the concentrations of 0.1, 1, 10, 50, and 100 µM. After 24 h of treatments, each well was treated with 700 µM of H₂O₂ for 1 h, and then the MTT assay was used to obtain cell viability values in order to investigate the cytoprotective effect of rutin. The results were reported as the mean value of three independent experiments ± standard deviation.