Whatman nuclepore track etched polycarbonate membranes

Liposomes (large unilamellar vesicles) were prepared as described earlier [19 (link)]. Briefly, lipid films were obtained by co-evaporation of aliquots of stock solutions of lipids in chloroform–methanol (2:1) in a round-bottom flask on a rotary evaporator, with subsequent drying for 45 min at 5 Pa. The resulting compositions (by mol) were ePC (PC); ePC–PI, 9:1 (10PI); ePC–GM₁, 9:1 (10GM₁); ePC–GM₁, 9.8:0.2 (2GM₁); ePC–CMG-PE, 9:1 (10CMG); ePC–CMG-PE, 9.8:0.2 (2CMG). If not stated otherwise, the lipid films were hydrated in phosphate buffered saline (PBS, pH 7.4), subjected to seven cycles of freezing/thawing (liquid nitrogen/+40 °C), and extruded 20 times through Whatman Nuclepore track-etched polycarbonate membranes (Cytiva, Marlborough, MA, USA) with a pore size of 100 nm on a mini-extruder (Avanti, Alabaster, AL, USA). Phospholipid concentrations in liposome dispersions were measured by the enzymatic colorimetric phosphatidylcholine assay (Sentinel Diagnostics, Milan, Italy). For anisotropy experiments, 0.025 mol% 1,3,5,7-tetramethyl-BODIPY-labeled phosphatidylcholine (TMB-PC) was added at the stage of lipid film formation.
Liposome dispersions were stored at 4 °C and used for experiments within 3 days.

Liposomes (large unilamellar vesicles) were prepared as described earlier [13 (link),26 (link),27 (link)]. Briefly, lipid films were obtained by co-evaporation of aliquots of stock solutions of lipids and MlphDG in chloroform–methanol (2:1) in round-bottom flask on a rotary evaporator, with subsequent drying for 45 min at 5 Pa. The resulting compositions were ePC; ePC–PI, 9:1; ePC–MlphDG, 9:1; ePC–MlphDG–PI, 8:1:1 (by mol). If not stated otherwise, the lipid films were hydrated in phosphate buffered saline (PBS, pH 7.4), subjected to seven cycles of freezing/thawing (liquid nitrogen/+40 °C), and extruded 20 times through Whatman Nuclepore track-etched polycarbonate membranes (Cytiva, Marlborough, MA, USA) with a pore size of 100 nm on a mini-extruder (Avanti, Alabaster, AL, USA). Phospholipid concentrations in liposome dispersions were measured by the colorimetric assay [39 (link)]. Prodrug concentrations were controlled by UV spectrophotometry after liposome disruption with ethanol (λ_max MlphDG 260 nm, ε 16,100 M⁻¹ cm⁻¹). For Förster resonance energy transfer (FRET) experiments, 1 mol% bis-cyclohexyl-BODIPY-labeled phosphatidylcholine (BCHB-PC) was added at the stage of lipid film formation.
Liposome dispersions were stored at 4 °C and used for experiments within 7 days.