Fetal bovine serum (fbs)

MDA-MB-231 human breast adenocarcinoma cell lines (ATCC, HTB-26) were cultured to investigate the cytotoxic effects of vitamin C on triple-negative breast cancer (ATCC, Manassas, VA, USA). These cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM). This contained 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate (ATCC), and was supplemented with 10% fetal bovine serum (FBS, ATCC) and 1% penicillin/streptomycin (100 U/mL penicillin, 200 µg/mL streptomycin) (Sigma Aldrich, St. Louis, MO, USA). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂.
To compare vitamin C’s effects on different breast cancer subtypes, MCF-7 cells (ATCC, HTB 22), which represent estrogen and progesterone receptor-positive, HER2/neu-negative breast cancer, were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC) under similar conditions, with identical FBS and penicillin/streptomycin supplementation.
For non-cancerous controls, human epithelial kidney (HEK 293, ATCC CRL-11268) and lung fibroblast (ATCC, CCL 205) cell lines were cultured in DMEM and EMEM, respectively, in the same conditions. Most of the cell passages ranged between 5 and 12. Additionally, rat spleen cells, provided by Dr. W. Thierfelder from Union University’s Biology Department, were prepared for cell viability assays to further evaluate the selectivity of VC’s cytotoxic effects.

Cytotoxicity studies were conducted on nanoparticle dispersions containing: SPION@OA_T80, conjugated with CBD (SPION@OA_T80_CBD), or CBG (SPION@OA_T80_CBG), and their combination with epirubicin (SPION@OA_T80_CBD_epi, and SPION@OA_T80_CBG_epi). Pure epirubicin was also tested. Nanoparticle concentrations ranged from 0.5 to 4 µg/mL corresponding to 0.05–0.4 µg/mL of cannabinoids and Epi (10% w/w). SKOV-3 cells were purchased from the American Type Tissue Culture Collection (ATCC, Rockville, MD, USA) and cultured in McCoy’s 5A medium supplemented with 10% FBS and 1% penicillin–streptomycin (all from Beth Haemek, Israel). SKOV-3 cells were seeded into 96-well plates at a density of 2.5 × 10³ cells per well and incubated at 37 °C in a humidified environment with 5% CO₂. After 24 h, the cells were washed with PBS, and various concentrations of SPIONs/CBD/CBG/epi compounds were added. The treated cells were then incubated for an additional 24, 48, and 72 h. Cytotoxicity was assessed using the MTS assay with the CellTiter-96^® AQueous One Solution Cell Proliferation Assay kit (Promega Corporation, Madison, WI, USA). The absorbance of the formazan product was measured at 490 nm using a microplate reader (Berthold Technologies, Bad Wildbad, Germany). Results are expressed as the percentage of viable cells relative to untreated control cells.