Screening of the nanoparticles for cytotoxicity

The culture environment was maintained at 37 °C in humidified, concentrated CO₂ (5%) atmosphere. Cells (MCF-7, MDA-MB231, and Vero) were grown in DMEM media, supplemented with 10% serum (FBS), and incubated in the culture environment.

Trypsinization was performed when the cells reached approximately 90% confluency. Aliquots of 2 mL of warm (37 °C) trypsin–EDTA solution were added for 2 min to detach the cells. Then trypsin–EDTA was neutralized by adding equal amounts of complete medium¹⁵.

The cell viability was determined by using trypan blue staining solution, and cell concentration was counted by an automatic cell counter (Invitrogen). The cell suspension of 1 × 10⁵ cells/mL in aliquots of 100 µl was seeded in a 96-well plate, after which 100 µl growth medium was added to each well, followed by incubation at 37 °C in humidified 5% CO₂ atmosphere for 24 h.

Following a 24-h incubation period, medium was aspirated, and the cells were treated with 100 µl of a range of dilutions (100–0,001 µg/mL) of nanoparticles and other control samples, in triplicates. Aliquots of 100 µl of media were added to make a final volume of 200 µl. The plates were incubated for 48 h. Cell growth and metabolic activity were measured using the MTT assay as described by Mossman et al. 1983. Excel and Prism Graph Pad 8 were used to analyze growth inhibition activity.