Liposomes (large unilamellar vesicles) were prepared by lipid film hydration followed by extrusion as described earlier [37 (link),52 (link)]. Mixtures of ePC, PI, SiaLeX-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 (λmax 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.
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 (λmax 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.
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