Hacat 300493

The in vitro cytocompatibility/cytotoxicity tests were developed on two types of healthy cell lines and on two tumor cell lines: immortalized human keratinocytes (HaCaT - 300493; CLS Cell Lines Service GmbH), primary epidermal melanocytes (HEMa - ATCC^® PCS-200-013™), human melanoma (A375 - ATCC^® CRL-1619™), and murine melanoma (B164A5 – 94042254; ECACC). HaCaT, A375, and B164A5 cells were cultured in specific culture medium - Dulbecco’s modified Eagle Medium high glucose supplemented with 10% fetal calf serum and 1% penicillin/streptomycin solution. HEMa cells culture required Dermal Cell Basal Medium supplemented with Adult Melanocyte growth kit, 1% penicillin/streptomycin mixture, and 1% FCS. Throughout the experiments, the cells were maintained in a humidified incubator in standard conditions (5% CO₂ at 37°C) and were passaged every other day. The cells were counted using Countess™ II Automated Cell Counter in the presence of Trypan blue.

Budesonide, methylcellulose (Methocel 90HG), and ethanol (96%) were purchased from Sigma Aldrich; poly(methacrylic acid-co-methyl methacrylate) 1:1 (Eudragit^® L100) from Evonik Röhm GmbH (Darmstadt, Germany); polyvinyl alcohol (PVA 22000) from Fluka Chemie AG (Germany); and Pluronic F127 from BASF (Ludwigshafen, Germany). Distilled water was prepared in the laboratory. For HPLC analysis, acetonitrile and methanol HPLC grades were used from Fisher Chemical (Thermo Fisher Scientific Inc., Waltham, MA, USA). Dulbecco’s Modified Eagle’s Medium, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), fetal bovine serum, and L-glutamine were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The Human immortalized keratinocyte cell line HaCaT (300493) was acquired from the CLS Cell Lines Service GmbH (CLS, Eppelheim, Germany).

HaCaT (300493) and A375 (CRL-1619™) cell lines were purchased as frozen vials from CLS Cell Lines Service GmbH (Eppelheim, Germany) and ATCC (American Type Cell Collection, Lomianki, Poland), respectively. The cells were cultured in their specific media—DMEM supplemented with 10% FCS and 1% penicillin (100 U/mL)-streptomycin (100 µg/mL) mixture. The cells were grown in an incubator at 37 °C, and 5% CO₂, presenting normal morphology and proliferation during all experiments.