Measurement of iron concentration in cells

To examine the release of ferrous or ferric iron from IONPs, we checked the intracellular iron concentration using a Spectroquant reagent (code 1.1476, 1.0001; EMD Millipore, Billerica, MA, USA). Cells (2×10⁴ cells/mL) were plated on a gelatin-coated 100 cm² culture dish and incubated until ~70% confluence. The medium was replaced with fresh medium containing 0–50 μg Fe/mL MPS-IONPs or ferucarbotran, and incubation was continued for 24, 48, and 72 hours. Subsequently, 10⁶ cells from each sample were collected by scraping, washed three times with PBS, and intracellular iron concentration was measured according to the manufacturer’s protocol. Briefly, cells were lysed in 100 μL of iron assay buffer and centrifuged at 16,000 g for 10 minutes to remove insoluble materials. Then, 5 μL iron reducer was added to each sample to reduce iron (III) to iron (II) in a 96-well plate. The sample mixture was incubated at 25°C for 30 minutes, and 100 μL iron probe was added to each well. The plate was incubated in the dark at 25°C for 60 minutes. Colorimetric changes were measured by a Multiskan microplate reader (Thermo Fisher Scientific) at 593 nm. To determine iron concentration, the iron standard provided by the manufacturer was used to prepare a standard curve.