*3.2. In Vitro Peptide(s) Aggregation*

Aggregation is typically a limitation of HDPs since it not only impacts biological activity but also biocompatibility and immunogenicity [39,40]. In previous studies, the HDP IDR1018 was found to aggregate in various in vitro solvents as well as in vivo under certain conditions and as a consequence, a reduction in the immunomodulatory activity was observed under conditions that promote aggregation [19,33]. Here, we examined the aggregation tendency of peptide conjugates in multiple solutions, including water, saline (0.9% NaCl), 5% dextrose, and 10% RPMI tissue culture medium with 1% fetal bovine serum (FBS). While no aggregation (measured by turbidity) was observed in water, saline, or 5% dextrose for any sample, including IDR1018 (Figure 1a), a substantial increase in aggregation was detected in tissue culture medium for parent peptide IDR1018 but not for the pegylated and glycosylated derivatives.

When the peptides were prepared in sodium salts of abundant polyatomic anions, such as citrate and phosphate (Figure 1b,c), a substantial increase in turbidity was observed with IDR1018 at solute concentrations as low as 10 mM. In contrast, no turbidity increase was detected with PEG6-IDR1018 or Glc-IDR1018 at higher solute concentrations, suggesting a significant reduction in the salt-induced peptide aggregation. When incubated with PBMCs in tissue culture, neither peptide conjugate, PEG6-IDR1018, and Glc-IDR1018 showed any obvious sign of aggregation, in strong contrast to IDR1018, which showed amorphous aggregates (Figure 1d). These results, indeed, were consistent with earlier reports, that demonstrated the utility of pegylation and glycosylation in increasing solubility and diminishing aggregation for other antimicrobial peptides [31,41].
