Whatman nuclepore membrane filters

Liposomes (large unilamellar vesicles) were prepared by lipid film hydration followed by extrusion as described earlier [37 (link),52 (link)]. Mixtures of ePC, PI, SiaLe^X-conjugate and MlphDG in the required molar ratios (Table 1) in chloroform–methanol (2:1) were dried by rotary evaporation in round-bottomed tubes and then in vacuum at 7 Pa for at least 1.5 h. The lipid films were hydrated with PBS (unless otherwise specified) under shaking at room temperature for 2 h. Then, the suspensions were subjected to 5–7 cycles of freezing/thawing (N₂ liquid/+40 °C) and extruded 20 times through polycarbonate Whatman Nuclepore membrane filters (Cytiva, Marlborough, MA, USA) with a pore size of 100 nm using a Mini-Extruder setup (Avanti Polar Lipids, Alabaster, AL, USA). The resulting dispersions were stored at 4 °C and used for experiments within 3 days.
Phospholipid concentrations in liposome dispersions were measured by the enzymatic colorimetric phosphatidylcholine assay (Sentinel Diagnostics, Milan, Italy), as described in [52 (link)]. Prodrug concentrations were measured by UV spectrophotometry after liposome disruption with ethanol (λ_max MlphDG 260 nm, ε 16,100 M⁻¹cm⁻¹).
To obtain fluorescently labeled liposomes for microfluidic experiments, 0.5 mol % TMB-PC was added at the stage of lipid film formation.

Liposomes (large unilamellar vesicles) were prepared by lipid film hydration followed by extrusion. Mixtures of ePC, Chol, and POPG in the required molar ratios (see Table 1 for liposome compositions) were co-evaporated from solutions in chloroform in round-bottom tubes on a rotary evaporator. The lipid films were further dried on an Iney-4 (Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Russia) freeze dryer at 7 Pa and hydrated with PBS (unless otherwise indicated) at room temperature for 2 h with stirring. Then, the mixtures were subjected to 5–7 cycles of freezing (N₂ liquid)–thawing (+40 °C) and extruded through Whatman Nuclepore membrane filters (Cytiva, Marlborough, MA, USA) with calibrated pore size of 100 nm 20 times using an Avanti Polar Lipids (USA) mini-extruder. The resulting dispersions were stored at +4 °C and were used for experiments within 3 days.
To obtain fluorescently labeled liposomes for cell internalization experiments, 1 mol% TMB-PC was added at the stage of lipid film formation.

Liposomes (large unilamellar vesicles) were prepared by lipid film hydration followed by extrusion [16 (link),36 (link)]. Mixtures of ePC, and POPG, and the aC-PC phospholipid prodrug in the required molar ratios were co-evaporated from solutions in chloroform in round-bottom tubes on a rotary evaporator. The lipid films were further dried on an Iney-4 (Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Russia) freeze dryer at 7 Pa and hydrated with PBS (unless otherwise indicated) at room temperature for 2 h with stirring. Then the mixtures were subjected to 5–7 cycles of freezing (N₂ liquid)–thawing (+40 °C) and extruded through Whatman Nuclepore membrane filters (Cytiva, Marlborough, MA, USA) with calibrated pore size of 100 nm 20 times using an Avanti Polar Lipids (USA) mini-extruder. The resulting dispersions were stored at 4 °C and used for experiments within 3 days. To study the stability of the dispersions, they were stored at +4 °C for up to three weeks.