Effect of composition, hydrogenation of phospholipids and lyophilization on the characteristics of eugenol-loaded liposomes prepared by ethanol injection method

The effect of composition, hydrogenation of phospholipids and lyophilization on liposomes characteristics was investigated. Liposomes were produced by ethanol injection method, employing hydrogenated (Phospholipon 80H, Phospholipon 90H) or non-hydrogenated soybean phosphatidylcholine (Lipoid S100) with cholesterol. Using Phospholipon 80H, cholesterol addition was crucial for liposomal homogeneity. Phospholipids concentration, stirring rate, and ethanol/water volume ratio were the most determinant factors controlling the vesicles size. Phospholipid assays showed that saturated phospholipids resulted in higher incorporation in vesicles than unsaturated ones. Smaller vesicles were obtained with Phospholipon 80H, due to lower content of phosphatidylcholine and to the presence of phosphatidylethanolamine which might increase repulsion between phospholipids, resulting in an increase in Zeta-potential and a decrease in vesicle size. TEM images revealed nanometric sized and spherical shaped oligo-lamellar vesicles. Although Lipoid S100 liposomes possessed the highest encapsulation efficiency of eugenol (86.6%), they were not stable in aqueous suspension at 4 °C after 2 months of storage, limiting their use in freeze-drying. Among several cryoprotectants, hydroxylpropyl-β-cyclodextrin protected eugenol-loaded Phospholipon 90H liposomes during freeze-drying as the mean vesicle size, polydispersity index, Zeta potential and encapsulation efficiency of eugenol were similar before and after lyophilization. This study demonstrated the possibility of maintaining a volatile essential oil in a suitable lyophilized liposomal formulation despite the very low applied pressures.