Cellular Uptake of G4@IONPs

Cellular uptake of G₄@IONPs was evaluated by Prussian blue staining and measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) [38 (link),39 (link)]. Different concentrations of G₄@IONPs (500, 250, 100, 50, and 0 (control) μg/mL) were added to cell culture media. After 2 h incubation, cells were fixed with 4% formalin and incubated with 4% potassium ferrocyanide and 4% hydrochloric acid (50%, v/v) for 20 min. Finally, the G₄@IONPs cellular localization was observed by optical microscopy (Olympus, Tokyo, Japan). Furthermore, MC₄L₂ cells were trypsinized after 2 h incubation with G₄@IONPs (500, 250, 100, 50, and 0 (control) μg/mL) and lysed by 2 mL 65% nitric acid; the quantity of cellular uptake of G₄@IONPs was assessed using ICP-MS (Varian Inc, Palo Alto, CA, USA). To obtain the iron concentration per cell, the total iron concentration measured by ICP-MS was divided by the number of lysed cells.