## 3 Results
### Combined Effects of Fe\({}_{3}\)O\({}_{4}\) NPs and Chemotherapeutic Agents on Cell Viability  
Next, we investigated the inhibitory effects of treatment with rapamycin or carboplatin alone or with each chemical agent combined with Fe\({}_{3}\)O\({}_{4}\) NPs on prostate cancer cell growth using alamarBlue assays. Treatment with rapamycin induced a concentration-dependent decrease in viability in both cell lines (\(p\) < 0.01 at 1, 10 and 20 nM in PC-3 cells; \(p\) < 0.05 at 1 nM, \(p\) < 0.01 at 10 and 20 nM in DU145 cells; Figure 4A). Treatment with carboplatin also induced a concentration-dependent decrease in viability in both cell lines (\(p\) < 0.01 at 10, 20 and 30 nM; Figure 4B). The selection of the dose for chemotherapeutic agents was based on a dose-seeking study showing approximately 25% inhibition in cell growth (Figure 4A,B). Interestingly, Alamar Blue assays showed that 10 nM rapamycin combined with Fe\({}_{3}\)O\({}_{4}\) NPs induced a concentration-dependent decrease in viability in DU145 cells (\(p\) < 0.01 at 1, 10, and 100 \(\mathrm{\SIUnitSymbolMicro g/mL}\) Fe\({}_{3}\)O\({}_{4}\) NPs) and in PC-3 cells (\(p\) < 0.01; 100 \(\mathrm{\SIUnitSymbolMicro g/mL}\) Fe\({}_{3}\)O\({}_{4}\) NPs; Figure 4C). Moreover, Alamar Blue assays showed that 10 nM carboplatin combined with Fe\({}_{3}\)O\({}_{4}\) NPs produced a concentration-dependent reduction in cell viability in DU145 cells (\(p\) < 0.05 at 10 \(\mathrm{\SIUnitSymbolMicro g/mL}\), and \(p\) < 0.01 at 100 \(\mathrm{\SIUnitSymbolMicro g/mL}\) Fe\({}_{3}\)O\({}_{4}\) NPs) and in PC-3 cells at 100 \(\mathrm{\SIUnitSymbolMicro g/mL}\) Fe\({}_{3}\)O\({}_{4}\) NPs (\(p\) < 0.01; Figure 4D).  
Figure 4: Effects of chemotherapeutic agents alone and in combination with Fe\({}_{3}\)O\({}_{4}\) NPs on cell viability. (**A**) Effects of rapamycin on PC-3 or DU145 cell viability for 24 h; (**B**) Effects of carboplatin on PC-3 or DU145 cell viability for 24 h; (**C**) DU145 or PC-3 cells were treated with rapamycin alone or in combination for 24 h; and (**D**) DU145 or PC-3 cells were treated with carboplatin alone or in combination for 24 h. * Significantly different from the group with chemotherapeutic agent alone at \(p\) < 0.01; ** significantly different from the group with chemotherapeutic agent alone at \(p\) < 0.05.  
\begin{table}
\begin{tabular}{c c c c c} \hline
**PC-3** & **Sub-G\({}_{1}\)** & **Co/G\({}_{1}\)** & **S** & **G\({}_{2}\)** \\ \hline Control & 3.3 \(\pm\) 0.1 & 66.1 \(\pm\) 0.3 & 8.0 \(\pm\) 0.2 & 22.7 \(\pm\) 0.3 \\ Fe\({}_{3}\)O\({}_{4}\) NPs (1 g/mL) & 4.5 \(\pm\) 0.2 & 68.2 \(\pm\) 0.5 & 8.3 \(\pm\) 0.1 & 19.0 \(\pm\) 0.7 \\ Fe\({}_{3}\)O\({}_{4}\) NPs (10 g/mL) & 5.1 \(\pm\) 0.2 & 67.9 \(\pm\) 0.5 & 8.4 \(\pm\) 0.1 & 18.5 \(\pm\) 0.6 \\ Fe\({}_{3}\)O\({}_{4}\) NPs (100 g/mL) & 5.5 \(\pm\) 0.1 & 68.7 \(\pm\) 0.3 & 8.9 \(\pm\) 0.2 & 16.9 \(\pm\) 0.3 \\ \hline
**DU145** & **Sub-G\({}_{1}\)** & **Go/G\({}_{1}\)** & **S** & **G\({}_{2}\)** \\ \hline Control & 0.5 \(\pm\) 0.1 & 53.7 \(\pm\) 0.3 & 5.1 \(\pm\) 0.1 & 40.8 \(\pm\) 0.4 \\ Fe\({}_{3}\)O\({}_{4}\) NPs (1 g/mL) & 0.5 \(\pm\) 0.1 & 52.3 \(\pm\) 0.4 & 5.7 \(\pm\) 0.1 & 41.4 \(\pm\) 0.4 \\ Fe\({}_{3}\)O\({}_{4}\) NPs (10 g/mL) & 0.5 \(\pm\) 0.1 & 53.0 \(\pm\) 0.2 & 5.8 \(\pm\) 0.1 & 40.8 \(\pm\) 0.3 \\ Fe\({}_{3}\)O\({}_{4}\) NPs (100 g/mL) & 0.6 \(\pm\) 0.1 & 50.9 \(\pm\) 0.7 & 8.5 \(\pm\) 0.4 & 40.0 \(\pm\) 1.1 \\ \hline \end{tabular}
\end{table}
Table 1: Effect of Fe\({}_{3}\)O\({}_{4}\) NPs on cell cycle of prostate cancer cells.