# Use of Ferritin-Based Metal-Encapsulated Nanocarriers as Anticancer Agents
## 3 Results
### Cellular Uptake of Ferritin Nanocarriers  
The cellular uptake of rhodaminated iron-containing HFt by SH-SYSY cells was determined by fluorescence microscopy. Figure 2A shows images taken after 0, 2, 3 and 4 h of exposure to rhodaminated HFt. The images clearly show a time-dependent increase in the intracellular red fluorescence, along with a decrease of mean background fluorescence. Part of HFt-NCs form transient aggregates in the culture medium. Figure 2B shows the incorporation kinetics of rhodaminated HFt, where each timepoint represents mean rhodamine fluorescence intensity (AU) upon subtraction of the mean background fluorescence. The experiment shows that SH-SYSY cells efficiently incorporate the HFt. The fitting of the timecourse shows that 50% of the plateau is reached after about 2.5 h. A video showing the cellular uptake of rhodaminated HFt is included in Supplementary Materials. Figure 1: Summary of the synthesis of metal-containing human ferritin H chain (HFt)-based nanocarriers. Figure 2: (**A**) time-lapse experiment of rhodamine HFt uptake by neuroblastoma cells. SH-SYSY cells were seeded in 8-well-plates and treated with 0.03 mg/mL. rhodaminated HFt. Fluorescence images were acquired every 20 min for 4 h. Times: 0, 2, 3, 4 h (see also the Video S1 in Supplementary Materials). Scale bar: 10 \(\upmu\)m; (**B**) timecourse of the increase in fluorescence of SH-SYSY cells upon incubation of rhodaminated HFt. \(y\)-axis shows mean rhodamine fluorescence intensity (AU) measured for each cell, upon subtraction of the mean background fluorescence (average of eight cells). Evaluation of Cell Viability and Cell Cycle Progression in the Presence of Metal-Containing Ferritin Nanocarriers  
Investigations were carried out to determine the effect of the different metal-containing Hft nanocarriers on the viability of SH-SYSY cells. Figure 3 shows cellular viability in the presence of various HFT-metal concentrations, determined by the MTT assay up to 72 h. HFt (containing a small amount of iron, i.e., about 50 atoms per protein molecule) and HFt-Au (Figure 3A,B, respectively) did not show any apparent effect on cell viability up to 48 h of treatment. However, a significant proliferative effect was observed for HFt, and some effect for HFT-Au, for prolonged treatment (72 h). Conversely, HFT-cisPt and HFt-Ag (Figure 3C,D, respectively) showed a considerable cytotoxicity as compared to HFt. In the experiments, the cell viability was negatively correlated with metal concentration in a statistically significant manner. Data reveal that after 48 h or 72 h of exposure to HFT-cisPt (Figure 3C), cell viability was reduced by about 50% when exposed to 10 uM HFt-cisPt (\(p\) < 0.001 vs. control), and by 60% when HFt-cisPt were present at a final concentration of 30 uM (\(p\) < 0.001 vs. control). The IC50 value for HFT-cisPt was 10 uM after 48 h of treatment, and 8 uM after 72 h of treatment; these experiments indicated that the toxic effect of HFt-cisPt is mostly reached within 48 h-treatments. HFt-Ag (Figure 3D) had a dramatic effect on cell viability within 24 h of treatment even at very low concentrations, cell viability being lower than 30% upon incubation with 1 uM HFt-Ag. The IC50 value for HFt-Ag was 0.5 uM after 24 h of treatment; cell death is almost total after 48 h or 72 h treatments. Notably, HFt-Ag was found to be highly cytotoxic even for very short exposure periods, i.e., 2-4 h (data not shown), and the toxic effect of HFt-Ag was obtained within a shorter timespan (a few hours) with respect to HFt-cisPt. Conversely, treatment of SH-SYSY cells with free AgNO\({}_{3}\) elicits cytotoxic effects comparable to HFt-Ag only at higher concentrations (Supplementary Figure S1). Figure 3: \(Cont.\)  
The effect of HFt-metal exposure on SH-SY5 Ycells was also assayed by flow cytometry, after staining with propidium iodide dye to evaluate cell cycle progression and apoptosis.