Liposome dye leakage

The liposome dye leakage assay was performed as we reported previously [67] to determine whether the peptides had any membrane‐damaging properties. Lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA), and carboxyfluorescein was purchased from Sigma‐Aldrich. Carboxyfluorescein‐encapsulated liposome vesicles were created and tested as previously described [67]. Briefly, a 5 mg mixture of 3 : 1 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC, a zwitterionic lipid)/1‐Palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐(phospho‐rac‐(1‐glycerol)) (POPG, a negatively charged lipid) was weighed and dissolved in 2 mL chloroform; the chloroform was evaporated using nitrogen, leaving a thin lipid film which was dried in a vacuum desiccator overnight. After addition of 500 µL of 30 mm carboxyfluorescein dye in sodium phosphate buffer (pH 7.5), the tubes were vortexed and incubated for 1 h at RT. Vesicles were formed by five successive freeze–thaw cycles in liquid nitrogen. This solution was then extruded through a polycarbonate filter (pore size 100 nm) 21 times using a mini‐extruder from Avanti Polar Lipids (fitted with two 0.5‐mL Hamilton gastight syringes). Nonencapsulated dye was removed from the vesicles using a Sephadex G50 gel exclusion column. Stock solutions of peptides (70 μm) were prepared in PBS (pH 7.4) with 5% DMSO; controls also contained 5% DMSO/PBS, which was predetermined not to cause dye leakage. Final peptide concentration in the wells also containing PBS and vesicles was 20 µm. After a 10‐min period, fluorescence values of the samples in 96‐well plates were measured by a spectrofluorometer (filter set to 485 nm excitation and 528 nm emission). Triton X‐100 detergent (10% v/v in PBS) was used as the positive control for determination of 100% leakage, while the negative control was 5% DMSO in PBS.

Dye leakage was calculated by the equation below where F _solvent is the fluorescence of the negative control (no peptide). Values reported are the average of triplicate runs.