## 3 Results and Discussion
### Antibacterial Activity  
The antibacterial potential of the different NPs containing or not containing indolicidin was analyzed by MIC against both Gram-positive and Gram-negative bacteria. Determination of MIC values of indolicidin and AgNPs alone (Table 5) and their conjugates (Table 6) was carried out using three model bacteria strains: _E. coli_, _P. aeruginosa_, and _S. aureus_. The results showed that the effective doses were in a similar range (5-12.5 \(\upmu\)g/mL) for the two nanoparticles containing indolicidin, regardless of the amount of N\({}_{2}\)H\({}_{4}\)-H\({}_{2}\)O that was used for their preparation. Therefore, hydrazine has a negative effect on toxicity, but a non-discernible effect on the antibacterial activity. The inhibitory activity was moderately increased with the nanoparticles containing indolicidin compared to AgNP1 and AgNP2, suggesting that the addition of indolicidin is of great importance for the general mechanism and probably for the shape and size of the nanoparticles. In fact, both IndAgNp1 and IndAgNP2 are considerably smaller than their counterparts without indolicidin, and the antibacterial activity has been often correlated to the size of nanoparticles used. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & _E. coli_ & _P. aeruginosa_ & _S. aureus_ \\ \hline
**Name** & \(\upmu\)g/mL & \(\upmu\)g/mL & \(\upmu\)g/mL \\ \hline
**IndAgNP1** & 5 & 12.5 & 12.5 \\
**IndAgNP2** & 5 & 7.5 & 10 \\ \hline \end{tabular} Values are expressed as mean \(\pm\) SD.  
\end{table} 
Table 6: Antibacterial activity of AgNPs conjugated to the peptide indolicidin against Gram-positive and negative bacteria. The antibacterial activity is expressed as MIC values. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Name** & **Indolicidin (\(\upmu\)g/mL)** & **AgNPs (\(\upmu\)g/mL)** & **\% Hemolysis** \\ \hline  & 1.5 & 1 & 0 \\  & 3 & 5 & 0 \\
**IndAgNP2** & 6 & 10 & 0 \\  & 12 & 20 & 0 \\  & 25 & 50 & 0 \\  & 50 & 100 & 15 \\ \hline  & - & 1 & 0 \\  & - & 5 & 0 \\
**AgNP2** & - & 10 & 0 \\  & - & 20 & 0 \\  & - & 50 & 7 \\  & - & 100 & 18 \\ \hline \end{tabular} Values are expressed as mean \(\pm\) SD.  
\end{table} 
Table 4: _Cont._ \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{2}{c}{_E. coli_} & \multicolumn{2}{c}{_P. aeruginosa_} & \multicolumn{2}{c}{_S. aureus_} \\ \hline
**Name** & \begin{tabular}{c} **Indolicidin** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} & \begin{tabular}{c} **AgNPs** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} & \begin{tabular}{c} **Indolicidin** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} & \begin{tabular}{c} **AgNPs** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} & \
\begin{tabular}{c} **Indolicidin** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} &
\begin{tabular}{c} **AgNPs** \\ **(\(\upmu\)g/mL)** \\ \end{tabular} \\ \hline
**Indolicidin** & 30 & - & 30 & - & 20 & - \\
**AgNP1** & - & 15 & - & 15 & - & 25 \\
**AgNP2** & - & 10 & - & 10 & - & 15 \\ \hline \end{tabular} Values are expressed as mean \(\pm\) SD.  
\end{table} 
Table 5: Antibacterial activity of indolicidin and AgNPs alone against Gram-positive and negative bacteria. The antibacterial activity is expressed as MIC values. Indeed, it has been reported that smaller particles have greater surface area compared to larger particles [55; 56; 57; 58; 59]. Since metal ions are released from the surface, the greater the surface area, the greater the release of ions. Presumably the strong antibacterial potential of smaller nanoparticles derives from the release of a wider number of ions than the larger particles per unit mass. Furthermore, it is well known that AgNPs are generally more active against Gram-negative compared to Gram-positive bacteria, and our results are constant with this observation. It has been attributed to the differences in the bacterial cell wall and the resulting interaction with the nanoparticles. Gram-positive and Gram-negative bacteria are both characterized by a negative charge due to the presence of teichoic acids in the former and lipopolysaccharidesaccharides in the latter.