Calcein Leakage Assay

A calcein leakage assay was performed according to established protocols (Kim et al., 2018). Briefly, lipid mixtures consisting of 7:3 weight ratios of the phosphatidylcholine (POPC) and phosphatidylglycerol (POPG) were prepared from stock lipid solution in 100% methanol. The organic solvents were evaporated, and the resulting lipid film was lyophilized for 1 day. The dried lipid film was resuspended in 1 ml of dye buffer solution (70 mM calcein, 10 mM Tris–HCl, 0.1 mM EDTA, 150 mM NaCl, pH 7.4). Liposomes were prepared through 10 freeze-thaw cycles in liquid nitrogen, followed by an incubation in a 50°C water bath. Then, the suspensions were extruded through 100 nm pore-polycarbonate membranes 20 times. After extrusion, the calcein-entrapped large unilamellar vesicles (LUVs) were separated using gel filtration through a Sephadex G50 column and eluted with a buffer solution (10 mM Tris–HCl, 0.1 mM EDTA, 150 mM NaCl, pH 7.4). Calcein leakage from the LUVs was measured at 20°C by monitoring the fluorescence intensity at an excitation wavelength of 490 nm and an emission wavelength of 520 nm on a spectrophotometer (Cary 5000 UV-Vis-NIR). The peptide and lipid molar ratios ranged from 1:1 to 1:128. The liposome without the peptide was used as a negative control, and the Triton X-100 was used as a positive control. Each run of the experiment contained three replicates, and the entire experiment was run in entirely twice. The percentage of the calcein leakage was calculated according to the following formula: