# Use of Ferritin-Based Metal-Encapsulated Nanocarriers as Anticancer Agents
## 3 Results
### Cellular Uptake of Ferritin Nanocarriers
Figure 4 shows that HFt and HFt-Au do not affect cell cycle progression nor significantly modify the subG0-G1 apoptotic fraction, thus confirming our previous observations on the lack of cytotoxicity as assessed by the MTT method. Conversely, both HFt-cisPt and HFt-Ag caused a marked effect on cell cycle progression and/or cell viability. In particular, 10 uM HFt-Ag treatment induced a marked increase in the sub-G1 (hypodiploid) fraction of the total cell population, as compared with the control group, with percentages rising from about 8% to 28% at 24 h. By increasing the incubation time, most cells die upon treatment with 10 uM HFt-Ag (see Figure 3), thus impairing the analysis of the cell cycle. Treatment with 10 uM HFt-cisPt also induced a marked increase in the sub-G1 (hypodiploid) fraction of the total cell population, as compared with the control group, with percentages rising from about 9% at 24 h to 18% at 48 h and to 28% at 72 h. In the same timeframe, while the S fraction in the control group is in the 10%-12% range, it becomes about 16% at 24 h, 26% at 48 h and 23% at 72 h.  
Figure 3: Cell viability assay on SH-SY5 Ycells upon HFt-based nanocarriers exposure: (**A**): HFt; (**B**): HFt-Au; (**C**): HFt-cisPt and (**D**): HFt-Ag. For each type of nanocarrier, 24 h, 48 h and 72 h treatments with six different amounts of nanocarriers were assayed. Each series is formed by the following amounts of nanocarriers (expressed as metal concentration): control = no NCs; 0.3 uM; 1.0 uM; 3 uM; 10 uM; 30 uM. For each experimental condition, viability percentage with respect to control is shown. Each point represents the mean value of three different experiments, each of which is based on eight different points. Standard errors are indicated. Statistical significance assessed by _t_-test; *_p_ (_t_-test) < 0.05; **_p_ (_t_-test) < 0.01; ***_p_ (_t_-test) < 0.001. Figure 4: Cell cycle analysis by flow cytometry on SH-SY5 Ycells upon HFt-based nanocarriers exposure, after staining with propidium iodide dye to evaluate cell cycle progression and apoptosis. (**A**) 24 h treatment; (**B**) 48 h treatment; and (**C**) 72 h treatment. For each experiment, treatments with 10 \(\upmu\)M nanocarriers (NCs) (expressed as metal concentration) were assayed. The effects of no treatment (CTRL), treatment with HFt (CTRL), HFt-Ag, HFt-Au and HFt-cisPt are shown. Standard deviations are indicated. Statistical significance assessed by \(t\)-test; * \(p\) (\(t\)-test) \(<\) 0.05. Evaluation of Cellular Redox Homeostasis and Mitochondrial Transmembrane Potential in the Presence of Metal-Containing Ferritin Nanocarriers  
Previous work demonstrated that ferritin nanocarriers may induce oxidative stress-dependent damage to protein and DNA that may contribute to the toxic effect of the metal NCs [31]. The effect of the different metal-containing HFt-nanocarriers on the mitochondrial potential and on the intracellular glutathione (GSH) content of SH-SY5Y cells was then investigated. While HFt increases mitochondrial transmembrane potential after 24 and 48 h of incubation, neither treatment with HFt-cisPt nor with HFt-Au determines appreciable effects (Figure 5A). Administration of HFt-Ag dramatically destroys the mitochondrial potential, even after only 24 h of treatment. The changes in GSH levels of SH-SY5Y cells upon administration of nanocarriers corresponding to 10 \(\upmu\)M metal ions were also assessed (Figure 5B). HFt-Ag treatment determines fast and highly significant (\(p<0.001\)) reduction of GSH content, while HFt-cisPt administration determines a small, long-term (72 h) reduction of GSH content. Surprisingly, HFt-Au treatment elicits a 30% reduction (\(p<0.05\)) of GSH content within 4 h, and a remarkable long-term GSH increase of about 120% at 48 h and 72 h (\(p<0.01\)). Figure 5: Mitochondrial potential (**A**) and glutathione (GSH) intracellular levels (**B**) of SH-SY5Y cells upon treatment for the indicated time with 10 \(\upmu\)M nanocarriers, expressed as percentage of the control \(\pm\) SD or SEM.