Liposome kit

A liposome kit purchased from Sigma-Aldrich (Catalog Number L4395, St. Louis, MO) was used. The user manual was followed to prepare multilamellar liposomes. Briefly, the lipid mixture was dissolved in CHCl₃/CH₃OH. The solution was dried under a stream of nitrogen. The vial containing the lipid film was placed under vacuum for 6 hrs to remove the traces of organic solvent. The dry lipid film was hydrated by adding PBS and vigorously shaking with a vortex mixer for >1 min. The MLV suspension were frozen and thawed for 6 cycles. A single freeze-thaw cycle consisted of freezing for 5 min at liquid nitrogen temperature (−196°C) and thawing for 5 min in a water bath at 65°C. Extrusion of the liposome suspension obtained was carried out with a filtration device (LiposoFast Liposome Factory, Sigma-Aldrich Chemicals) through a porous polycarbonate membrane with a pore size of 100 nm to obtain liposomes of uniform size. Size distribution of the liposomes prepared was measured by dynamic light scattering (DLS). Results are shown in Figure S1.

Liposomes were prepared using Liposome Kit (L4395, Sigma-Aldrich) containing powdered lipids (Total 90 μmoles/package; Cholesterol, 9 μmol/package, L-α-Phosphatidylcholine (egg yolk), 63 μmol/package, Stearylamine, 18 μmol/package). Powdered lipid was sequentially dissolved in 32 ml PBS, with or without 1000 μg of rNME1, and was incubated for 30 min with rotation at room temperature. The liposomes were put through three freeze–thaw cycles (− 196 °C for 4 min, 42 °C for 4 min) and then extruded 20 cycles through a 100 nm single clean track-etched polycarbonate membrane of NanoSizer MINI STERILE Extruder (TT-002S-010, T&T Scientific). Extruded liposomes were centrifuged (28,000 rpm, 30 min, 4 °C) for separating bound (pellet) and unbound (supernatant) rNME1. Efficiency of rNME1 encapsulation by the liposomes was assessed by Western blot, showing NME1 level at each step of liposome-rNME1 preparation.