Htb 37

Human epithelial colorectal adenocarcinoma cells (Caco-2, HTB-37) and human foreskin fibroblasts (HFF-1, SCRC-1041) were obtained from LGC Standards (Teddington, Middlesex, UK). Caco-2 cells were cultured according to manufacturer’s instruction in DMEM hg w/L-glu medium supplemented with 10% v/v FBS and penicillin-streptomycin solution at 37 °C in a humidified atmosphere of 5% CO₂. HFF-1 cells were cultured in DMEM hg w/L-glu/sp medium supplemented with 15% v/v FBS and penicillin-streptomycin solution at 37 °C in a humidified atmosphere of 5% CO₂.

Human colorectal adenocarcinoma cells (Caco-2 cell line, HTB-37) from ATCC/LGC Standards GmbH (Wesel, Germany) were grown in MEM medium (61100-87, Gibco by Life Technologies, Carlsbad, CA, USA) supplemented with 1.5 g/L of NaHCO₃, 1 mM of sodium pyruvate, 20% fetal bovine serum (10270-106, origin South America, Gibco, by Life Technologies, Carlsbad, CA, USA), and 1% antibiotic-antimycotic solution (A5955; Sigma-Aldrich, St. Louis, MO, USA). The cell culture was maintained in a humidified atmosphere at 37 °C and 5% CO₂. For treatment, the Caco-2 cells were seeded at a 5 × 10⁴ cells/mL density and allowed to adhere overnight. The nanoformulations (IONP:5-FU = 0.5:1, 1:1, and 1.5:1) and free components (IONPs and 5-FU) were added to the culture medium in various concentrations. Doses between 0.1–100 μg/mL IONPs and 5–250 μg/mL 5-FU were applied to the Caco-2 cells for 24 and 48 h for an MTT assay. For the other tests, a dose of 200 μg/mL of 5-FU was selected, and the corresponding doses of IONPs in the nanoformulation were 6 (0.5), 12 (1), and 18 μg/mL (1.5). Untreated Caco-2 cells were used as a negative control. Before treatment, the suspensions were sterilized under UV radiation for 1 h.

Caco-2 cells were obtained from the American Type Culture Collection (HTB-37, LGC Standards, Molsheim, France) and used at passages 32–40. Cells were grown in a culture medium (minimum essential medium containing 5.5 mM D-glucose, Earle’s salts, and 2 mM L-alanyl-glutamine (MEM GlutaMAX)), supplemented with 1% non-essential amino acids, 50 IU/mL penicillin, 50 μg/mL streptomycin, and 20% FCS, at 37 °C in an atmosphere containing 5% CO₂. Caco-2 cells were seeded at 5 × 10⁴ cells/cm² in 96-well plates for cytotoxicity and HCA, and in 6-well plates for microarrays and RT-qPCR assays. The day after, cells were exposed to toxins in FCS-free medium for 24 h. A vehicle control containing MeOH (5% for cytotoxicity and HCA, 0.62% for RT-qPCR, and 0.31% for microarrays) was included for each experiment. Four independent experiments were performed for cytotoxicity, HCA and RT-qPCR, and six independent experiments for microarrays were performed.