Evaluating Cytotoxicity of Citric Acid-Modified MnFe2O4 Nanoparticles

Testing with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO, USA) assay was performed according to Pasupuleti et al.25 (link) RAW 264.7 murine macrophage-like cells (Rio de Janeiro Cell Bank, Rio de Janeiro, Brazil) were plated at a concentration of 1×10⁵ cells per well in supplemented Dulbecco’s Modified Eagle’s Medium (4 mM glutamine, 10% fetal calf serum, and 100 units/mL penicillin/streptomycin) and incubated with different concentrations of nanoparticles (0–750 µg/mL). After overnight incubation, 10 mL of the MTT solution (5 mg/mL in PBS) was added to each well. Plates were incubated for 4 hours in 5% CO₂ at 37°C. The generated blue formazan product was dissolved by the addition of 100 µL of 100% dimethyl sulfoxide (Mallinckrodt Chemical, Paris, KY, USA) per well. The absorbance was monitored at 575 nm in an enzyme-linked immunosorbent assay plate reader (BioTek, Winooski, VT, USA). The citric acid-modified MnFe₂O₄-NPs concentrations for the viability and proliferation assay were 250, 500, and 750 µg/mL.