## 3 Results and Discussion
### Toxicity  
The MTT cytotoxicity assay is based on the activity of mitochondrial dehydrogenase. Since this enzyme is present only in the active mitochondria, the reaction can occur exclusively in viable cells. Vero cells were treated with different concentrations of NPs for 24 h showing that there was no significant reduction in the viability compared to the untreated cells. More specifically, we could observe different cytotoxicity profiles for the differently prepared silver NPs (Figure 4). The nanoparticles prepared with 150 \(\mu\)L of N\({}_{2}\)H\({}_{4}\cdot\)H\({}_{2}\)O (AgNP1) were in fact revealed to be more toxic at higher concentrations (more than 50 \(\mu\)g/mL) and at longer time of exposure when compared to the nanoparticles prepared with 10 \(\mu\)L of N\({}_{2}\)H\({}_{4}\cdot\)H\({}_{2}\)O (AgNP2). A clear difference could also be observed between IndAgNPs and naked AgNPs, with a lower toxicity for the functionalized silver NPs, demonstrating an advantage of shedding the AgNPs direct external surface with a peptide coating. Overall, the observed toxicity was exerted at concentrations markedly higher than those required for antibacterial test. It should be noted that the peptide indolicidin on its own also showed to be toxic at high doses (Figure 5). \begin{table}
\begin{tabular}{l c c} \hline \hline
**Sample** & **Particle Size Distribution [nm]** & **\(\Delta\)max [nm]** \\ \hline Ag colloids prepared using 150 \(\mu\)L N\({}_{2}\)H\({}_{4}\cdot\)H\({}_{2}\)O in the presence of indolicidin & 12.5 \(\pm\) 6.2 & \(\sim\)454 \\ Ag colloids prepared using 150 \(\mu\)L N\({}_{2}\)H\({}_{4}\cdot\)H\({}_{2}\)O without & 32.6 \(\pm\) 11.4 & \(\sim\)427 \\ indolicidin & 5.9 \(\pm\) 2.9 & \(\sim\)462 \\ Ag colloids prepared using 10 \(\mu\)L N\({}_{2}\)H\({}_{4}\cdot\)H\({}_{2}\)O without & 21.4 \(\pm\) 11.4 & \(\sim\)444 \\ \hline \hline \end{tabular}  
* Particle size was evaluated using transmission electron microscopy; Amax was determined by UV-Vis spectroscopy.  
\end{table} 
Table 2: Characteristics of Ag colloids prepared with different amounts of N\({}_{2}\)H\({}_{4}\cdot\)H\({}_{2}\)O, in the presence indolicidin and without the peptide. Figure 4: Cytotoxicity evaluation of AgNPs via MTT assay. Vero cells were treated for 3, 10, and 24 h with different concentrations of NPs (\(\mu\)g/mL). However, the amount of the peptide on the functionalized NPs is lower than the concentration used in the MTT assay. Therefore, we can conclude that the NPs described herein do not raise safety concerns due to the low doses needed to produce an antimicrobial effect. Moreover, the less toxic nanoparticles is the IndAgNP2 produced in 10 \(\upmu\)L of N2H4-H2O pointing out that the method for production of silver NPs needs to be carefully considered since, at least partially, the toxicity could be due to the chemical used for production. Hemolysis assay was performed at the same concentrations used in the MTT assay. The hemolytic action of peptides or silver NPs is regarded as a negative function of antimicrobial compounds, posing a serious obstacle to their use for pharmaceutical application. Interestingly, in contrast to the hemolytic activity of indolicidin at high concentration (20 to 100 \(\upmu\)g/mL) (Table 3), all compounds were found to possess little hemolytic activity (less than 25%) at the highest concentrations tested (100 \(\upmu\)g/mL) (Table 4). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Name** & **Indolicidin (\(\upmu\)g/mL)** & **AgNPs (\(\upmu\)g/mL)** & **\% Hemolysis** \\ \hline \multirow{4}{*}{**IndAgNP1**} & 1.5 & 1 & 0 \\  & 3 & 5 & 0 \\  & 6 & 10 & 0 \\  & 12 & 20 & 0 \\  & 25 & 50 & 8 \\  & 50 & 100 & 21 \\ \hline \multirow{4}{*}{**AgNP1**} & - & 1 & 0 \\  & - & 5 & 0 \\ \cline{1-1}  & - & 10 & 0 \\ \cline{1-1}  & - & 20 & 0 \\ \cline{1-1}  & - & 50 & 12 \\ \cline{1-1}  & - & 100 & 24 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Hemolytic activity of nanosystems. Figure 5: Cytotoxicity evaluation of indolicidin via MTT assay.