*2.3. Hemolysis Test*

Red blood cells (RBCs) were isolated and used to test the hemolytic activity of the peptide conjugates according to documented procedures [27,34] with some modifications. The blood samples were collected from healthy donors according to the University of British Columbia ethics certification and guidelines. Informed consent was obtained from all donors involved in the study. A 0.5% (*v*/*v*) RBC suspension in PBS was transferred into 96 well plates, where pre-diluted peptides series were incubated. The samples were placed at 37 ◦C for 1 h prior to reading the hemoglobin release at 414 nm and 546 nm. Triton X-100 (1% final concentration) was used as a positive control (100% hemolysis). The % hemolysis was then determined relative to the negative control (untreated samples).

### *2.4. Cytotoxicity and Immunomodulatory Assays*

The cytotoxicity of the conjugates was assessed in vitro against PBMCs as described earlier using the lactate dehydrogenase (LDH)-based assay [35]. Blood samples were collected from healthy donors with ethics approval and PBMCs were isolated following our previously reported protocol [35]. The cells were resuspended in RPMI medium supplemented with 10% FBS and seeded in tissue culture-treated plates at a final density of <sup>1</sup> × 105 cells well−1. Subsequently, the cells were treated with the conjugates or IDR1018 and incubated for 24 h at 37 ◦C in 5% CO2, to determine the LDH release. Triton X-100 (1% final concentration) was used as a toxic (positive) control.

To evaluate the capacity of the conjugates to modulate the immune system, suspended PBMCs (a final density of 1 × <sup>10</sup><sup>5</sup> cells well−1) were treated with the conjugates at 8 μg mL−<sup>1</sup> in the presence or absence of 20 ng mL−<sup>1</sup> *P. aeruginosa* LPS, incubated for 24 h, centrifuged, and then the supernatants were collected to quantifying the levels of monocyte chemoattractant protein 1 (MCP-1), the tumor necrosis factor-alpha (TNFα), and interleukins (IL) IL-1β, IL-10, and the anti-inflammatory IL-1 receptor antagonist (IL-1RA) using specific ELISA assays (ELISA kits, eBioscience, Carlsbad, CA, USA). All experiments were conducted in triplicate in three independent experiments and average values were presented.

**Figure 1.** In vitro aggregation experiments of IDR1018 and its conjugates as tested in various solvents under different conditions. The aggregations of the peptides are indicated by the level of turbidity when dissolved in water, saline, 5% dextrose, or RPMI medium (**a**), or when dissolved in various concentrations of sodium citrate (**b**), and sodium phosphate (**c**). Microscopic images show PBMCs after treatment with IDR1018 or its conjugates, indicating aggregation by IDR1018 cf. the conjugates (**d**). Black arrows point to clusters that formed when PBMCs were treated with IDR1018 in a tissue culture medium. Data were described as means ± standard deviations, and significant values (\*\*) were determined using the analysis of variance (ANOVA) test; ns in (**a**) indicates no significance. The scale bar is 500 μm.
