Persistence of internalized DMSA-SPION. (A) MCF-7 cells incubated with nanoparticles for 24 h, stained with Prussian blue reaction after different post-incubation times and visualized by bright field microscopy. (a) Untreated control cells. (b-d) Cells incubated for 24 h and stained 24, 48 and 72 h after incubation, respectively. Scale bar represents 10 μm. (B) Study of persistence by electron microscopy: (a) Cells were incubated with DMSA-SPION for 24 h. The cells were further incubated in medium without particles for additional (b) 24 h, (c) 48 h and (d) 72 h. Insets show larger magnification details of the endosomes. Scale bars represent 5 μm in overall areas and 500 nm in larger magnification insets, respectively. (C) Intracellular iron content quantification by ferrozine assay (expressed as weight of iron per cell) in control (non-treated) cells (c), and immediately (0) or 48 h after incubation with DMSA-SPION.