Assessing Cytotoxicity, Proteolytic Stability, and Selectivity of Antimicrobial Peptides: Implications for Orthopedic Applications

Abstract

In orthopedics, the use of anti-infective biomaterials is considered the most promising strategy to contrast the bacterial contamination of implant surfaces and reduce the infection rate. KSL, KSL-W, and Dadapin-1 are three antimicrobial peptides (AMPs) that possess significant antibacterial properties, making them promising candidates for producing anti-infective biomaterials not based on antibiotics. To fully assess their true potential, this study explores in detail their cytocompatibility on human osteoblast-like MG63 cells, murine fibroblastoid L929 cells, and hMSCs. To this end, the cytotoxicity of the AMPs in terms of IC₅₀ was tested over a range of concentrations of 450-0.22 µg/mL using the ATP bioluminescence assay. The tests were performed both in the presence and absence of bovine serum to assess the effects of serum components on peptide stability. IC₅₀ values obtained under the most stringent conditions were used to extrapolate the selectivity index (S.I.) toward salient bacterial species. In medium containing serum, all AMPs exhibited minimal to no cytotoxicity, with IC₅₀ values exceeding 100 µg/mL. Dadapin-1 was the peptide that exhibited the lowest cytotoxicity, KSL-W exhibited the highest stability, and KSL exhibited the highest selectivity. Overall, these findings highlight the potential of these AMPs for the future production of anti-infective materials.

Keywords: AMPs; L929; MG63; antimicrobial peptides; cytocompatibility; cytotoxicity; hMSCs; implant orthopedic infections; proteolytic stability; selectivity index.

Figure 1

The bar graphs show the effects of Dadapin-1 on the cellular metabolism of (a) L929 cells and (b) hMSCs in the absence and presence of fetal bovine serum (FBS). Bioluminescence readings in relative luminescence units (RLUs) were normalized to the mean of the reference control set to 100%. Bars represent the mean ± S.D. of three independent experiments (N = 9), except for the assay performed with L929 without FBS (four independent experiments, N = 12). The statistically significant difference with respect to the reference control: * p < 0.05; ** p < 0.01; *** p < 0.001. Detailed information on the statistical analysis performed and exact p-values can be found in Tables S2 and S3.

Figure 2

The bar graphs show the effects of KSL on the cellular metabolism of (a) MG63 cells, (b) L929 cells, and (c) hMSCs in the absence and presence of FBS. Bioluminescence readings in relative luminescence units (RLUs) were normalized to the mean of the reference control set to 100%. Bars in the graph represent the mean ± S.D. of three independent experiments (N = 9), except for the assay performed with MG63 and hMSCs without FBS (four independent experiments, N = 12). The statistically significant difference with respect to the Reference control: * p < 0.05; ** p < 0.01; *** p < 0.001. Detailed information on the statistical analysis performed and exact p-values can be found in Tables S2 and S3.

Figure 3

The bar graphs show the effects of KSL-W on the cellular metabolism of (a) MG63 cells, (b) L929 cells, and (c) hMSCs in the absence and presence of FBS. Bioluminescence readings in relative luminescence units (RLUs) were normalized to the mean of the reference control set to 100%. Bars in the graph represent the mean ± S.D. of three independent experiments (N = 9), except for the assay performed with MG63 and hMSCs without FBS (four independent experiments, N = 12). The statistically significant difference with respect to the Reference control: * p < 0.05; ** p < 0.01; *** p < 0.001. Detailed information on the statistical analysis performed and exact p-values can be found in Tables S2 and S3.

Figure 4

The bar graph reports the calculated IC₅₀ values of Dadapin-1 when tested on MG63 cells, L929 cells, and hMSCs, in the presence (blue bars) and absence of FBS (red bars). Plotted values for MG63 cells refer to our previously published work [18]. Conversely, the values for L929 cells and hMSCs are newly introduced by the present investigation. It may be noticed that Dadapin-1 exhibits no or very low cytotoxicity. For both MG63 cells and hMSCs, the level of cytotoxicity increased in the absence of serum.

Figure 5

The bar graph presents the calculated IC₅₀ values of (a) KSL and (b) KSL-W when tested on MG63 cells, L929 cells, and hMSCs, in the presence (blue bars) and absence of FBS (red bars). Confidence intervals for IC₅₀ values can be found in Table S4.

Figure 6

Representative images of MG63 cells treated for 24 h with the indicated substances. The nuclei staining (Hoechst 333432) is shown in blue, and the actin cytoskeleton staining (Phalloidin-FITC) is shown in green. Bottom scale bars: 50 µm.