Calcein leakage assay

For making calcein-encapsulated, unilamellar liposomes, a lipid film containing POPC/DOPS (85:15 or 70:30) was rehydrated with Tris buffer (10 mM, pH 7.4) containing 70 mM calcein. The liposome suspension was freeze-thawed for ten cycles and extruded ten times through polycarbonate filter (100-nm pore size). The free calcein was removed by dialyzing calcein-containing liposomes through a Slide-A-Lyzer 10K dialysis cassette (10,000 MWCO, 0.1–0.5 mL, ThermoFisher, MA).

To measure calcein leakage from the liposomes, a method in the Spectramax i3 (Molecular probes, CA) program was used with an excitation setting of 490 nm and an emission setting of 520 nm. Calcein leakage due to peptoid addition was monitored for 60 min. Maximal vesicle leakage was then measured by addition of the detergent Triton X-100 from a 10% stock solution to achieve a final concentration of 0.1% v/v concentration and by monitoring for a further 1.5 min period on the program.

Calculation of calcein leakage, % leakage, was done as follows:

In this equation, I is the fluorescence intensity 60 min after peptoid addition.

Io is the intensity of intact vesicle baseline acquisition, and is measured after the addition of Triton X-100.