Liposofast extruder

Liposomes were prepared from 100% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipid. Lipid powder was dissolved in chloroform to make 15–20 mg/mL stock solutions and stored at −20°C. Aliquots of this solution were dried under nitrogen stream and desiccated overnight. The resulting film was resuspended to approximately 4 mM in MOPS buffer (20mM MOPS, 147 mM NaCl, 2.7 mM KCl, pH 7.4) for at least one hour and vortexed. The resulting liposome solutions were used as is for the tubulation assays. For FCS measurements, large unilamellar vesicles (LUVs) were prepared by extruding this suspension 21 times through two stacked membranes of 50 nm pore size (Whatman) in a Liposofast extruder (Avestin). For EM, the suspension was extruded 13 times through 400 nm pore size membranes. LUVs are stored at room temperature for up to several days. Before analyses, all lipid concentrations were determined by assaying for total phosphate content²⁷ .

Large unilamellar vesicles (LUVs) were prepared using the film deposition technique. Lipid stocks in chloroform were added to a glass vial in the required proportions. The mixture was dried under nitrogen, and residual solvent was evaporated by exposing the sample to vacuum overnight. The lipid films were then hydrated in buffer, vortexed to create multilamellar vesicles (MLVs) and then extruded through a polycarbonate membrane with 100-nm pores (GE Healthcare BioSciences, Pittsburgh, PA) using a LiposoFast extruder (Avestin Inc., Ottawa ON) to make LUVs.
For SPR analysis, the molar composition of the liposomes was 90% POPC and 10% Chol, and the liposomes were hydrated in phosphate buffered saline (PBS) at a pH of 7.4. For membrane rigidity experiments, the molar composition of the liposomes was 79% POPC, 20% Chol, and 1% Laurdan. These laurdan-labeled LUVs were hydrated in liposome buffer (150 mM NaCl, 5 mM CaCl₂, 5 mM HEPES, 3 mM NaN₃, pH = 7.4)
Giant unilamellar vesicles (GUVs) were created by mixing POPC, Chol, and NBD-PE in a 79/20/1 molar ratio. The lipids, dissolved in 250 μL of chloroform and 30 μL of acetonitrile, were gently placed at the bottom of 3 mL of PBS (pH 7.4) in a round bottom flask. Using a rotary evaporator, the flask was rotated under vacuum in a 40 °C water bath for approximately 5 s to evaporate the solvents to form GUVs.

Liposomes were prepared using the lipid film technique^{28 (link)} from stock solutions of lipid and sterol in chloroform. For the ITC and CD experiments, the lipid films were hydrated with a phosphate buffer [10 mM PO₄ (pH 6.5)], and for the laurdan experiments, the lipid films were hydrated with a liposome buffer [150 mM NaCl, 5 mM CaCl₂, 5 mM HEPES, and 3 mM NaN₃ (pH 7.4)] to produce multilamellar vesicles (MLVs). The MLVs were then extruded through a 100 nm polycarbonate Whatman membrane (GE Healthcare BioSciences, Pittsburgh, PA) with a LiposoFast extruder (AVESTIN Inc., Ottawa, ON) to create large unilamellar vesicles (LUVs).²⁹

Cerasomes were prepared using a combined ethanol injection and extrusion method. First, 140 mM of CFL and 52 mM of cholesterol in acidic ethanol were incubated overnight at pH 3.0 and room temperature, permitting the hydrolysis of CFL. Stock solutions of DSPE-PEG (2000) Maleimide (MAL-DSPE-PEG) at a concentration of 6.8 mM were prepared in chloroform, and stock solutions of NBD-DPPE at a concentration of 0.5 mM were prepared in a 1:1 (v/v) acidic ethanol and chloroform mixture. The solutions were then mixed, leading to a lipid mixture containing CFL, cholesterol, NBD-DPPE, and MAL-DSPE-PEG at a 14.2:9.4:0.2:1 molar ratio. The lipid mixture was injected to degassed PBS at 70 °C and sonicated for 15 min in a bath-type sonicator, forming multilamellar cerasomes. The use of degassed PBS at 70 °C helped remove organic solvents, including chloroform with a boiling point 61.2 °C. To obtain unilamellar cerasomes, the multilamellar cerasomes were extruded through a 100 nm polycarbonate membrane mounted on the LiposoFast extruder (Avestin, Canada). The obtained cerasomes were allowed to further polymerize at room temperature for 1 hr, and then filtered using a 0.2 μm syringe filter and sterilized with ultraviolet (UV) exposure for 30 min.