The thin film method used to prepare pH-responsive liposomes has been described previously [22 (link)]. In brief, the DOPE, CHEMS, and DSPE-PEG750 or DSPE-PEG2000 (molar ratio of 6:4 and 6:4:0.1, respectively) were dissolved in a 50:50 methanol:chloroform (v/v) solvent mixture in a round bottom flask. The solvents were removed under reduced pressure on a Büchi Rotavapor R-114 with vacuum pumpV-500 (Büchi Labortechnik, Flawil, Switzerland) at 45 °C and 60 rpm to obtain a thin lipid film that was left to completely dry at 50 mbar for 1 h. The dry lipid film was hydrated with a buffered solution of 80 mM calcein for the calcein containing liposomes or PBS (pH 7.4) for the non-calcein containing liposomes. The liposomes were stepwise hand extruded through Nucleopore® polycarbonate membranes (0.8, 0.4, 0.2, and 0.1 µm pore sizes, three cycles for each size).
For calcein containing liposomes, the extravesicular calcein was removed by ultracentrifugation at 300,000× g for 1 h at 10 °C (L8-70M Ultracentrifuge; Beckman Instruments, Palo Alto, CA, USA). The supernatant was carefully removed before the pellet was gently resuspended in PBS using a bench vortex mixer and centrifuged for an additional 1 h. The supernatant was then removed and the pellet gently resuspended in PBS by vortexing to obtain the final liposomal formulations.
For calcein containing liposomes, the extravesicular calcein was removed by ultracentrifugation at 300,000× g for 1 h at 10 °C (L8-70M Ultracentrifuge; Beckman Instruments, Palo Alto, CA, USA). The supernatant was carefully removed before the pellet was gently resuspended in PBS using a bench vortex mixer and centrifuged for an additional 1 h. The supernatant was then removed and the pellet gently resuspended in PBS by vortexing to obtain the final liposomal formulations.
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