*3.3. Hemolysis, Cytotoxicity and Immunomodulatory Activity*

Compared to the parent IDR1018 peptide, which appeared to be hemolytic against RBCs as well as toxic to PBMCs at high doses (up to 20% at 128 μg mL−1), neither of the conjugated derivatives exhibited any hemolysis or cytotoxicity at the highest concentration tested (Figure 2a,b). Consistent with other previous studies for other peptides [30,31,42,43], pegylation and glycosylation worked well in reducing HDP toxicity.

**Figure 2.** Hemolysis and Cytotoxicity of the peptide conjugates vs. IDR1018 itself. (**a**) displays the hemolysis analysis against RBCs while (**b**) depicts the cytotoxicity against PBMCs. Experiments were conducted in triplicate in three independent experiments and mean values ± standard deviation are presented.

The capacity of conjugated peptide derivatives to modulate the innate immune system was tested by assessing the induction and suppression of chemokines and cytokines from human PBMCs. These tested modulators including MCP1, TNFα, IL-1β, IL-1RA and IL-10, were selected based on previous studies where IDR1018 demonstrated an excellent ability

to modulate them in vitro and in vivo [17,19,24,38,44,45]. In addition, these immunity modulators are involved in multiple systemic mechanisms essential to suppress inflammations and indirectly defend against bacterial infections. For instance, MCP-1 attracts macrophages and enhances the recruitment of monocytes at the sites of infections, injuries and inflammations [46], while the anti-inflammatory cytokines, IL-1RA and IL-10 act as natural inhibitors of the harmful effect of pro-inflammatory immune mediators such as IL-1α and IL-1β [47]. Interestingly, in the absence of an LPS stimulant, PEG6-IDR1018 maintained a similar ability to modulate chemokine and cytokine expression to that observed for unmodified IDR1018. However, Glc-IDR1018 exhibited a significant increase in the expression of the immune mediators relative to unmodified IDR1018. Thus Glc-IDR1018 boosted the level of MCP1 and IL-1RA by 2–5 fold,1.5 ng mL−<sup>1</sup> and 0.8 ng mL<sup>−</sup>1, respectively, compared to only 0.7 and 0.15 ng mL−<sup>1</sup> induced by the unmodified IDR1018 (Figure 3a,b). These peptides did not induce TNFα (Figure 3c).

**Figure 3.** Enhanced effect of glycosylation on the immunomodulatory activities of IDR1018-derived peptides. (**a**–**c**) The effect of the peptides at 8 μg mL−<sup>1</sup> in inducing chemokine MCP1 and cytokines IL-1RA and TNFα production by PBMCs in the absence of LPS stimulation. (**d**) the effect of peptides on the immune mediator expression when co-treated with 20 ng mL−<sup>1</sup> LPS (% of fold changes relative to LPS-stimulated cells). Data are presented as mean ± standard deviation from three independent experiments (\* represents *p* < 0.05, based on ANOVA analysis with Tukey's correction for multiple testing).

When stimulated with LPS which amplified the secretion of immune mediators, substantial reductions in the proinflammatory TNFα were detected due to peptide action, with no significant difference between all three peptides. However, the expression of IL-1β was significantly suppressed when cells were treated with Glc-IDR1018 cf. the other peptides (Figure 3d). Moreover, remarkable peptide-induced increases in MCP1, IL-1RA and IL-10 induction levels were also detected with substantially higher expression values associated with Glc-IDR1018 treatment. In agreement with previous reports, the results indicated that pegylation had no marked effect on the immunomodulatory properties of the HDP IDR1018 [43,48]. However, this was the first report we have observed indicating that glycosylation markedly improves the immunomodulatory properties of HDPs. While the mechanistic effects of glycosylation on the immunomodulatory properties of IDR1018 as well as the underlying biophysical processes remain to be elucidated, the overall results showed exciting data with excellent outcomes for this peptide conjugate in modulating the immune system with efficacy exceeding that for previously reported HDPs [15,19].
