Polycarbonate track etched membrane

POPC was dissolved in chloroform, distributed into 5 mg aliquots and dried first under a nitrogen stream, then under vacuum for 2 h. The desiccated lipid aliquots were stored under a nitrogen atmosphere at −20 °C until used. Liposomes were prepared by first rehydrating the above-mentioned aliquots in phosphate-buffered saline (PBS), typically at concentrations of 5 mg/mL. The lipid suspensions were then passed 11 times through a fully assembled Mini-Extruder (Avanti Polar Lipids), fitted with a polycarbonate track-etched membrane (Whatman) featuring either 100 nm or 200 nm pore sizes, sandwiched between four extruder drain discs (i.e. two on each side of the membrane). The fluorescent labelling of POPC liposomes was achieved by first dissolving Texas Red (TR)-modified lipids in a 1:1 (v/v) mixture of chloroform and methanol (0.5 mg/mL) and adding 100 μL of it to a 5 mg POPC aliquot (i.e. 1% (w/w)) prior to performing the drying and extrusion steps described above.

Large unilamellar vesicles with ∼1 μm diameter were prepared by extrusion. First, 45 μl of a lipid solution in chloroform (4 mg ml⁻¹) was deposited into a brown glass vial and dried to a film under vacuum. The solution included either TX-DHPE or OG-DHPE at 0.8 mol% to allow fluorescence imaging of the vesicles and to distinguish between vesicle mixtures. Second, the lipid film was rehydrated with 300 μl of water (MilliQ) and vortexed for 5 min. After transferring the sample to an Eppendorf tube, four freeze/thaw cycles were carried out in liquid nitrogen followed by water at 30 °C to facilitate break up and aid in forming a homogeneous large unilamellar vesicle suspension after extrusion. The sample was then extruded using a mini-extruder (Avanti Polar Lipids, Alabaster) with a polycarbonate track-etched membrane with 1.0 μm pores (Whatman). A typical size distribution for the vesicles formed via this method is provided in Supplementary Fig. 5. The initial concentration of vesicles in a sample is approximately 1% by volume.

SUVs were prepared by lipid film hydration and subsequent extrusion. Appropriate amounts of DOPC, DOPE, and PE-CF stock solutions were added to a round bottom flask (DOPC: DOPE: PE-CF = 700:300:4, molar ratio). Chloroform was evaporated with a gentle stream of nitrogen while simultaneously spreading the lipids to form a thin film. Remaining trace of chloroform was removed by keeping the flask under vacuum for at least 2 h in a desiccator. The obtained thin film was hydrated with a solution of 4 mM dextran and 15% v/v glycerol, to a final lipid concentration of 20 mg/mL. The hydration was facilitated by incubating at 37 °C while shaking for at least 30 min till the entire film was dispersed in the solution. The dispersed film was then sonicated for 30 min in an ultrasonic bath in order to break large aggregates and thus ease the subsequent extrusion step. A mini-extruder (Avanti Polar Lipids) was assembled and set on a heating block at 70 °C^{43 (link)}. The lipid suspension was then sequentially passed through, first, a 100 nm and then a 30 nm polycarbonate track-etched membrane (Whatman) 21 times each. The extruded SUVs were then stored at 4 °C for use.