Effect of Oleic Acid, Cholesterol, and Octadecylamine on Membrane Stability of Freeze-Dried Liposomes Encapsulating Natural Antimicrobials

Abstract

Liposomes have been broadly studied as delivery systems for bioactive compounds, although its relatively low stability remains a limitation for commercial application. In this study, phosphatidylcholine (PC) liposomes were prepared entrapping a mixture of garlic extract (GE) and nisin (Nis) using cholesterol (CHO), oleic acid (OA), or octadecylamine (ODA) as membrane stabilizers to evaluate their physical, chemical, bioactive, and stability properties, in fully hydrated state and after freeze-drying. GE/Nis-loaded liposomes presented hydrodynamic diameter below 200 nm and polydispersity index below 0.30, typical for small unilamellar vesicles produced by thin film method. Under induced oxidation, the PC-OA-GE/Nis liposomes presented 91% less lipid peroxidation compared with the unloaded PC liposomes. The Fourier transform infrared spectroscopy (FTIR) analysis revealed a high level of hydrogen bonds in the polar head group of PC after addition of GE/Nis in all liposome formulations, in agreement to the high values of water activity and hygroscopicity found in the samples after freeze-drying. During 5 months storage at 4 °C, fully hydrated and lyophilized liposomes showed an increment in their average size and polydispersity index, but these values were reduced by the trehalose addition as lyoprotector. All liposome preparations maintained 100% activity against Listeria monocytogenes; nevertheless, a gradual reduction of activity against Salmonella enterica serovar Enteritidis was observed, suggesting a partial loss of GE active compounds. Despite some physical modifications, freeze-dried liposomes containing OA as stabilizer showed best antimicrobial properties and high lipid oxidation resistance, constituting a promising approach to stabilize GE/Nis for long-term storage.