4.8. Preparation of Liposomes

Conventional liposomes were obtained by using standard thin film hydration. Egg lecithin (10% in 1.6 mL) and cholesterol (69.9 mg/mL) solutions were prepared in chloroform–methanol 9:1 (v/v) and mixed. The mixture was precipitated as a film on the wall of a glass test tube by evaporating the solvent with a vacuum-line. The test tubes were then placed under a vacuum for at least 2 h to remove residual solvent. The thin film of the lipid was hydrated with 1 mL H₂O, using the sonication, and then characterized. An extruder with membranes of 0.4 µm was used for obtaining more homogeneous samples.

CRG/Ech-containing liposomes were produced by performing the next steps. At first, the complex CRG/Ech (10:1 w/w) was prepared as described above. Then 1 mL of this solution was added to the dried lipid film. The mixture was sonicated three times for 15 min in an ultrasonic bath, and then 0.1 mL suspension was added to 1.4 mL H₂O in different tubes and then centrifuged for 15 min at 15,000× g. The supernatant was removed, and the pellet was suspended in 1 mL of water and again centrifuged. The centrifugation step was carried out to remove the CRG/Ech complex not included in the liposomes. Washing was performed twice. An extruder was used to reduce the heterogeneity of the liposomes. The suspension of liposomes was passed through 0.4-micron membranes 10 times, on an extruder, according to the instructions.