Liposome leakage assay

Following earlier work²⁶, lipids DOPC and DOPG were dissolved in chloroform at a concentration of 25 mg.mL⁻¹, mixed in a molar ratio of 7:3, and subsequently diluted further to 10 mg.mL⁻¹ using chloroform. The chloroform was evaporated using rotary evaporation (350 mbar, 313 K, 100 rpm). The lipid film was then dried in vacuum for at least 2 h after which the lipids were re-suspended in calcein-containing buffer (70 mM calcein in 10 mM Tris-HCl, pH 7.5) to get a 30 mM lipid suspension by hydration for 1 h in a rotary evaporator (no vacuum, 323 K, 100 rpm). Multilamellar vesicles thus formed were freeze-thawed four times using liquid nitrogen and a 310 K water bath to get unilamellar vesicles. A mini-extruder (Avanti Lipids) equipped with a 200 nm pore size polycarbonate membrane was used to perform 21 extrusions to homogenize the size of the lipid vesicles. Vesicles were separated from free calcein on a gravity driven Sephadex G-50 size exclusion column and eluted with a 10 mM Tris-HCl buffer containing 100 mM NaCl (pH = 7.5). The vesicles were characterized using dynamic light scattering, for which an ALV instrument equipped with an ALV5000/60 × 0 external correlator and a 300 mW Cobolt Samba-300 DPSS laser operating at a wavelength (λ) of 532 nm was applied. A cumulant analysis showed the vesicles to be monodisperse, and to have an average diameter of 190 ± 5 nm. The degree of dilution of the vesicles during size exclusion chromatography was estimated by comparing the intensity of scattered light (count rate) before and after size-exclusion chromatography. For this scattering angles θ ranging from 20° to 140° were used with steps of 5°, five measurements of 30 s were recorded for each scattering angle and the average was taken over all measurements. A fluorescence filter was applied that only transmits the laser light, to prevent the fluorescence light emitted by calcein from disturbing the measurements. Vesicles used for the leakage assay were diluted with a 10 mM Tris-HCl buffer containing 100 mM NaCl (pH = 7.5) to a final lipid concentration of 50 μM. Peptides were dissolved in the same buffer, at a concentration of 6.4 mg.mL⁻¹. The calcein leakage caused by the AMPs was measured by following the fluorescence intensity of the liposome solutions over time in a Cary Eclipse cuvette fluorimeter using an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The widths of the excitation and emission slits were 2.5 nm. The fluorescence measurements were started with 588 μL of the vesicle solution without AMPs. After two minutes the fluorescence intensity signal was stable and 12 μL of 6.4 mg.mL⁻¹ AMP solution was added, to achieve a final peptide concentration of 128 μg.mL⁻¹, equal to the highest peptide concentration used in the MIC assays. In total, the measurements lasted 10 minutes. At the end of the experiments, 10 μL of a 10% (v/v) Triton X-100 solution was added, which is a surfactant that disrupts the liposomes. The signal after Triton addition was taken to correspond to 100% liposome leakage. For all peptides, the liposome leakage assays was repeated four times with freshly prepared liposomes and results were found to be reproducible.