Fig. 3: The bio-safety and mechanistic assessment of the selected leading AMPs.

a The hemolytic cytotoxicity of the selected AMPs was conducted by co-culturing with 4% (v/v) murine blood cells at 37 °C, 2 h, with distilled water and normal saline solution as positive control and negative control, respectively. The hemolytic cytotoxicity was quantified by experimentally testing OD₄₅₀ with three replicates, and all groups deducted the absorbance values of the negative control group. Standard formula was subsequently used to calculate the hemolysis rates. The data were presented as mean ± SD and the statistical significance was conducted by 2-sided Welch T test (p < 0.05, *, p < 0.01, **, p < 0.001, ***). All the experimental groups were conducted with three replicates. b–h The CCK8 cytotoxicity in vitro assessment of the selected AMPs by co-culturing with Caco-2 cells mimicking in vivo small intestinal epitheliums, at 37 °C, 12 h. The cell viability of those experimental groups with significant differences (One-way ANOVA-test (p* ≤ 0.05, p** ≤ 0.01, p*** ≤ 0.001) combined with Post-hoc multiple comparisons) compared to the control group were calculated by the standard formula. The box plots show the median (central line), the 25th percentile and 75th percentile, and interquartile range. All experimental groups were conducted with three independent replicates. AMP8, p-value = 0.9999, 0.9972, 1.0000, 0.7962, 0.9283; AMP13, p-value = 0.1765, 0.7083, 0.9806, 0.5713, and, 0.3634; AMP24, p-value = 0.6955, 0.5948, 1.0000, 0.6851, 1.0000; AMP28, p-value = 1.0000, 0.9884, 0.9975, 0.9982, and, 0.9998; AMP30, p-value = 0.1054, 0.9312, 0.0732, 0.0756, and, 0.0106; AMP31, p-value = 0.8062, 0.5982, 0.9620, 0.9373, and, 0.5814; AMP38, p-value = 0.0173, 0.0163, 0.0006, 0.0008, and, 0.00003. i The typical SEM morphologies of the destructed bacteria membranes by the representative AMPs (100 μM) co-culturing with different bacteria (1 × 10⁸ cfu/mL), at 37 °C, 24 h.