## 2 Results
### Cytotoxicity of Peptides In Vitro and In Vivo  
To determine whether the peptides induce a cytotoxic effect, we first analyzed the hemolytic activity of Pro9-3 and its analogs-Pro9-3D, Pro10-1, and Pro10-1D against sheep red blood cells (sRBCs) (Figure 2A). Incubation of sRBCs with 100 \(\upmu\)M Pro9-3 and Pro9-3D peptides induced 2% hemolysis; however, no hemolytic activity was observed with Pro10-1D and Pro10-1 peptides. In contrast, melittin exhibited 100% hemolysis at 25 \(\upmu\)M. As listed in Table 2, Pro10-1D showed a higher relative selective index followed by Pro9-3D, Pro10-1, and Pro9-3 (41.18, 31.82, 13.46, and 10.94, respectively) against gram-negative bacteria. Therefore, Pro10-1D exhibited greater bacterial selectivity than the other peptides.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Microorganisms**} & \multicolumn{5}{c}{**Minimal Inhibitory Concentration (MIC\({}_{100}\)) in \(\upmu\)M**} \\ \cline{2-6}  & **Pro9-3** & **Pro9-3D** & **Pro10-1** & **Pro10-1D** & **Melittin** \\ \hline Standard gram-negative & & & & & \\ bacteria & 16 & 8 & 16 & 8 & 8 \\ _E. Coli_ & 16 & 4 & 16 & 4 & 8 \\ _E. coli K1_ & & & & & \\ _A. baumannii_ & 16 & 4 & 8 & 2 & 4 \\ \hline MDR Gram-negative bacteria & & & & & \\ MDREC 1229 & 16 & 8 & 8 & 4 & 16 \\ MDREC 1238 & 32 & 4 & 32 & 4 & 32 \\ MDRAB 12010 & 16 & 8 & 8 & 4 & 8 \\ MDRAB 12220 & 16 & 8 & 16 & 8 & 8 \\ \({}^{\text{a}}\) GM & 18.29 & 6.29 & 14.86 & 4.86 & 12.0 \\ \({}^{\text{b}}\) HC\({}_{10}\) & 200 & 200 & 200 & 200 & 0.8 \\ \({}^{\text{c}}\) Relative selective index & 10.94 & 31.82 & 13.46 & 41.18 & 0.07 \\ \hline \hline \end{tabular}  
* The geometric means (GMs) are the mean minimum inhibitory concentration (MIC) values of all bacterial strains. The average MIC was calculated from three experimental measurements. \({}^{\text{b}}\) HC\({}_{10}\) is the degree of peptide concentration inducing 10% hemolysis of heparinized sheep red blood cells in vitro. When no detectable hemolysis was observed at 100 \(\upmu\)M, 200 \(\upmu\)M was used to calculate the relative selective index. \({}^{\text{c}}\) Relative selective index was calculated using GM of the MIC (\(\upmu\)M)/HC\({}_{10}\). The larger values indicate greater cell selectivity.  
\end{table}
Table 2: Activities of Pro9-3 and its analogs against microorganisms.  
Figure 2: Cytotoxic effects of Pro9-3 and its analogs: (**A**) dose–response curves for sheep red blood cell (sRBC) hemolysis induced by peptides and (**B**) dose-dependent (0–50 \(\upmu\)M) cytotoxicity of the peptides against mouse macrophage RAW264.7 cells for 24 h. Melittin was used as a control. The values are expressed as the mean \(\pm\) SEM of three independent experiments and are statistically significant at \({}^{\text{*}}\)\(p\)\(<\) 0.05, \({}^{\text{**}}\)\(p\)\(<\) 0.01, and \({}^{\text{***}}\)\(p\)\(<\) 0.001; ns, not significant (by _one-way ANOVA_, _[PERSON]’s post hoc test_).  
We next investigated the cytotoxicity of Pro-peptides against the murine macrophage cell line RAW264.7. Pro9-3 resulted in 95% and 94% survival rates of RAW264.7 cells when compared with the melittin control (12% and 11.2%) at concentrations of 25 and 50 \(\upmu\)M, respectively, whereas Pro9-3D showed a lethal effect with 68% and 51.8% survival rates, respectively (Figure 2B). In contrast, RAW264.7 cells treated with Pro10-1D, which showed higher antibacterial activity than the other peptides, exhibited 89% and 73% survival rates in response to 25 and 50 \(\upmu\)M treatments, respectively, when compared with Pro10-1 (90% and 87%). Notably, except for Pro9-3D, all other peptides exhibited an 85% survival rate at 25 \(\upmu\)M. Even though Pro9-3D, which has high hydrophobicity, showed high antibacterial activity against MDR gram-negative bacteria, it is very toxic to mammalian cells; thus, it cannot be an effective candidate peptide antibiotic. Therefore, we performed further experiments on the parent peptide, Pro9-3, as well as on newly designed peptides Pro10-1 and Pro10-1D.