2.4. Liposome Leakage Assay

The ability of MNT₁ to provide liposome leakage was demonstrated on unilamellar phosphatidylcholine (Sigma-Aldrich, Burlington, MA, USA) liposomes loaded with the fluorescent dye calcein (Fluka, Seelze, Germany). Unilamellar liposomes were loaded with fluorescent dye calcein to the concentration of 100 mM which is self-quenching. To do this fresh lipid suspension in liposome buffer containing HEPES (20 mM), MES (20 mM), citrate (20 mM), and sodium chloride (150 mM), pH 7.4 was sonicated until clear, using a W-181-T sonicator (Ulta Sonic Finland LTD, Lanti, Finland; 40 kHz, 90 W, 0 °C, 30 min), Then the resulting liposomes were passed 10 times through 0.22 µm Durapore filters (Millipore, Burlington, MA, USA) for size standardization. The liposomes were stored at 4 °C under an argon atmosphere. Liposomes were purified on PD-10 prior to experiment, then the same day incubated for 30 min with 100 nM MNT₁ in liposome buffer at the different pHs at room temperature in triplicate. After that, samples were diluted tenfold in liposome buffer pH 7.5, and the fluorescence of free calcein (leaked from liposomes) was measured at 520 nm (excitation at 490 nm). As a positive control (100% calcein leakage), a 0.5% Triton X-100-containing sample was used. Parallel probes where MNT₁ was omitted served to assess background calcein leakage.