Dipalmitoylphosphatidylethanolamine

In this work, pH-sensitive liposomes were prepared. Different lipid formulations were studied using the lipids dioleoylphosphatidylethanolamine (DOPE, from Avanti Polar Lipids, Birmingham, AL, USA), dipalmitoylphosphatidylethanolamine (DPPE, from Sigma-Aldrich, St. Louis, MO, USA), cholesterol (Ch, from Sigma-Aldrich, St. Louis, MO, USA) and cholesteryl hemisuccinate (CHEMS, from Sigma-Aldrich, St. Louis, MO, USA) at the molar ratios DOPE:CHEMS (7:3), DOPE:Ch:CHEMS (45:45:10) and DPPE:Ch:CHEMS (45:45:10). Liposomes composed of these formulations were synthetized following the ethanolic injection method [40 (link)], where an ethanolic lipid solution (1 × 10⁻³ M) was initially prepared according to the desired proportion. The volume of the lipid solution equivalent to this concentration was evaporated under an ultrapure nitrogen flow and subsequently redissolved in absolute ethanol in the same volume. Finally, the ethanolic solution was injected, drop by drop and under vortexing, to 3 mL of ultrapure water, inducing the formation of liposomes. The injection was performed while ensuring that the aqueous medium was at a higher temperature than the main phospholipid phase transition temperature (−16 °C for DOPE and 63 °C for DPPE) [41 (link)].

The analysis was performed essentially as described earlier [33 (link)] with modifications. The following lipids were used: semisynthetic bovine brain sphingomyelin (SM), C16-ceramide, cholesterol, dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) (all from Sigma). The lipids were dissolved in chloroform:methanol:H₂O (1:1:0.3) and 1 μl of the solution containing 25–500 pmoles of lipid was spotted onto a 0.45-μm nitrocellulose membrane. The membrane was pressed with a hot block at 60°C for 5 s according to Taki and Ishikawa [34 (link)], blocked with 1% gelatin and 1% polyvinylpyrrolidone and incubated for 45 min at 20°C with 1 μg/ml GST-PFO in PBS buffer containing 0.03% Tween-20. After washing, the membrane was exposed for 45 min to chicken anti-GST IgY-peroxidase (Rockland). Immunoreactive spots were visualized with the SuperSignal West Pico chemiluminescence substrate (Pierce).