## 2 Results
### Toxicity Assessment  
After establishing the antibacterial and antibiofilm activities, we sought to test the toxicity of HRZN peptides by challenging defibrinated human red blood cells with the peptides for 1 h at 37 \({}^{\circ}\)C (Figure 3A). Defibrinated blood was chosen over EDTA-treated blood because we observed high hemolysis results when using the latter that did not correlate with the published results [69]. All peptides showed a high degree of hemolysis, except HRZN-13 and -14.  
Hemolysis was detected by the measurement of hemoglobin released (OD\({}_{540}\)). The percentage of hemolysis was calculated relative to the Triton X-100 control (100%) control. HRZN-13 and -14 showed negligible hemolysis (0.7% and 5.6%) with incubation at a 100 \(\upmu\)g/mL concentration of peptide. The hemolysis activity of these two peptides is less than LL-37, supporting our conclusion of little to no toxicity of the peptides. HRZN-15 and -16 completely lysed the human red blood cells at 100 \(\upmu\)g/mL, whereas HRZN-17 lysed 74% of the hRBCs. At 10 \(\upmu\)g/mL, HRZN-16 demonstrated 67% hemolysis, followed by HRZN-17 and HRZN-15 showing 18% and 8.6% hemolysis. These hemolysis values are higher than LL-37, demonstrating that HRZN-15, -16, and -17 have high levels of toxicity. All peptides presented little to no hemolysis at 1 \(\upmu\)g/mL. The human cathelicidin peptide LL-37 showed 5.4% and 22% hemolysis at 10 and 100 \(\upmu\)g/mL. A similar hemolysis of LL-37 has also been reported at 25 \(\upmu\)M (~112 \(\upmu\)g/mL) [70].  
The overall toxicity of the most active peptide HRZN-15 was then assessed in vivo in the waxworm _Galleria mellonella_ model. Although animal models are the most relevant for the assessment of novel therapeutics against MDR _A. baumannni_ infection [58; 59; 71], the waxworm has been developed as a rapid first assay against _A. baumannni_ infection [72; 73; 74; 75; 76; 77; 78; 79]. We first had to assess the toxicity of HRZN-15 in the waxworm. Waxworms were injectedwith 10 \(\upmu\)g of HRZN-15 or polymyxin B in 10 \(\upmu\)L of PBS, and survival was recorded every 24 h for 72 h. HRZN-15-treated worms resulted in only 40% survival of worms (\(p<0.05\)) (Figure 3B). This result is consistent with the HRZN-15 hemolysis results. Worms injected with polymyxin B showed 80% survival, and the result was not statistically different from the PBS-injected group (90% survival). Thus, HRZN-15 was toxic by these assays, and the peptide needs further development to reduce its toxicity. Because of this high level of toxicity of the peptide itself, we were unable to proceed to test the ability of HRZN-15 to eradicate an MDR _A. baumannii_ infection in the waxworm model.  
Figure 3: Toxicity assessment of HRZN peptides. (**A**) Hemolysis of HRZN peptides at peptide concentrations of 100, 10, and 1 \(\upmu\)g/mL on defibrinated human red blood cells (hRBCs) with LL-37 used as a control. (**B**) Toxicity assessment of HRZN-15 and polymyxin B using _Galleria mellonella_. Each worm was injected with 10 \(\upmu\)g of either HRZN-15 or polymyxin B in 10 \(\upmu\)L. Worms injected with 10 \(\upmu\)L of PBS were used as controls. The survival of worms was monitored for 3 days. The Mantel–Cox test was performed to calculate statistical significance (* \(p<0.05\), ns = not significant).