Caco 2 cells

Human intestinal epithelial Caco-2 cells were from the European Collection of Authenticated Cell Cultures (ECACC) (Merck, Milan, Italy). Cells were routinely grown in vented TC-treated 75 cm² flasks (Star-lab, Milano, Italy) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂, using DMEM supplemented with 10% inactivated fetal bovine serum, 2mM L-glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. All reagents were purchased from Sigma-Aldrich (Milan, Italy).

Caco-2 cells, obtained from American Type Culture Collection (Manassas, Virginia), were cultivated as described previously in an atmosphere of 90% air and 10% CO₂ (12 (link)). Briefly, Caco-2 cells (passage 95 to 105) were seeded on permeable polycarbonate filter supports (0.45 μm pore size, 12-mm diameter; Transwell Costar, Sigma-Aldrich) at a density of 44,000 cells/cm² in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 1% minimum essential medium nonessential amino acids, penicillin (100 U/mL), and streptomycin (100 μg/mL). Monolayers were used for experiments between day 21 and 26 after seeding.

HEK293T cells as well as Caco-2 cells were from Sigma Aldrich (St. Louis, MO, USA). HEK293T cells and ADAM10/ADAM17 double-deficient HEK293 cells [30 (link)] were grown in DMEM (High glucose; Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Pen/Strep). Caco-2 cells were cultured in RPMI (Thermo Fisher Scientific) with 10% FCS and 1% Pen/Strep.

Caki-2 cells were cultured in McCoy’s 5A (modified) medium supplemented with 10% FCS, Calu-3 cells were cultured in MEM with 10% FCS and 1x non-essential amino acids and both obtained from ATCC (Manassas, VA, USA). HEK293T cells were cultured in DMEM supplemented with GlutaMAX, 10% heat-inactivated FCS. Caco-2 cells were from Sigma Aldrich (Schnelldorf, Germany) and were cultured in EMEM medium supplemented with 10% FCS, L-glutamine and non-essential amino acids (M7145, Sigma Aldrich, Schnelldorf, Germany). HepG2 was obtained from Sigma Aldrich (Schnelldorf, Germany) and cultured in DMEM supplemented with GlutaMAX and 10% heat-inactivated FCS. All media contained 1% penicillin/streptomycin and the cells were cultured at 37 °C in a 5% CO₂ atmosphere. Silvestrol was dissolved in DMSO and further diluted in media (c_stock = 6 mM, maximal DMSO concentration during experiments 0.1% v/v). EGTA, famotidine and carbamazepine were from Sigma Aldrich (Schnellendorf, Germany). Lucifer Yellow (sc-215269) was obtained from Santa Cruz. Celecoxib was synthesized by WITEGA Laboratorien Berlin-Adlershof GmbH (Berlin, Germany). Forskolin and ionomycin were obtained from Sigma Aldrich (Schnelldorf, Germany). Silvestrol was provided by the Sarawak Biodiversity Centre (Kuching, North-Borneo, Malaysia; purity > 99%) and zotatifin was bought from MedChemExpress (USA, purity: 98%).

Caco-2 cells and mucus-secreting HT29-MTX cells, both from human colon carcinoma, were purchased from Sigma-Aldrich (Villefranche-sur-Saône, France). These two cell lines were routinely cultivated in 75 cm² flasks (Sarstedt, Nümbrecht, Germany), under 5% CO₂, at 37 °C, in DMEM (PAN Biotech, Aidencach, Germany) supplemented with penicillin/streptomycin (100 U·mL⁻¹), 2 mM L-glutamine, and 10% heat-inactivated FBS.

The safety of using Mel-PLGA NPs was examined, in vitro, before being used in vivo. Caco2 cells (Sigma Aldrich, USA) were cultured, at 37 °C in 5% CO2 and relative humidity of 95%, in Dulbecco’s modified Eagle medium (DMEM) supplemented with NaHCO₃ (2.2 g/l), d-glucose (4.5 g/l), 1% non-essential amino acids, 10% fetal bovine serum, 100 IU/ml penicillin and 0.1 mg/ml streptomycin (all materials used in the culture process were purchased from Sigma Aldrich, USA). In vitro cytotoxicity assay were accomplished according to Alaa et al.^{28 (link)}. 100 µl/well of 10⁵ Caco2 cells in tissue culture plates were incubated at 37 °C for 24 h to allow for the development of cell monolayers. After medium decantation, a washing media was used to wash the monolayers. Graded concentrations of Mel-PLGA NPs were produced by combining NPs with RPMI medium. The produced NPs dilution was diluted to 0.1 ml, added to the wells, and then left to sit for another 24 h. The wells received 20 µl of MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) at a concentration of 5 mg/ml. Plates were shaken for five min to ensure MTT mixing, then incubated for four h at 37°C with 5% CO₂. To dissolve the developed formazan, 200 µl of dimethyl sulfoxide (DMSO) were applied to the plates. At 560 nm, the absorbance (which was directly linked to formazan) was measured.

The study was conducted by Cyprotex (Macclesfield, United Kingdom). CACO 2 cells (Sigma-Aldrich, catalogue: 86010202) were seeded on Millipore Millicell plates and formed a confluent monolayer over 20 days before the experiment. On day 20, the tested compounds at a final concentration of 10 μM were added to the apical side of the membrane. The transport of the compound across the monolayer was monitored over a 2 h period by liquid chromatography (LC)–MS/MS quantification. The permeability coefficient (Papp) was calculated from the following equation: , where dQ/dt is the rate of permeation of the drug across the cells, C₀ the donor compartment concentration at time zero and A the area of the cell monolayer. C₀ was obtained from analysis of the dosing solution at the start of the experiment.