pubs.acs.org/JAFC Published on Web 03/22/2010 © 2010 American Chemical Society J. Agric. Food Chem. 2010, 58, 5679–5684 5679 DOI:10.1021/jf100133b Effects of Chitosan and Rosmarinate Esters on the Physical and Oxidative Stability of Liposomes ATIKORN PANYA, † MICKAEL LAGUERRE, § JEROME LECOMTE, § PIERRE VILLENEUVE, § JOCHEN WEISS, # D. JULIAN MCCLEMENTS, † AND ERIC A. DECKER* ,† † Department of Food Science, Chenoweth Laboratory, University of Massachusetts, 100 Holdsworth Way, Amherst, Massachusetts 01003, § CIRAD, Dept PERSYST, UMR IATE, F-34398 Montpellier 5, France, and # Department of Food Science and Biotechnology, University of Hohenheim, D-70599 Stuttgart, Germany Liposomes have substantial potential to deliver bioactive compounds in foods. However, the oxidative degradation and physical instability of liposomes limit their utilization. This research evaluated the ability of chitosan and rosmarinic acid and its esters to increase the physical and oxidative stability of liposomes. Particle size analysis studies showed that the physical stability of liposomes was enhanced by depositing a layer of cationic chitosan onto the negatively charged liposomes. The combination of octadecyl rosmarinate (40 μM) and chitosan coating resulted in significantly greater inhibition of lipid oxidation in the liposomes compared to chitoson or octadecyl rosmarinate alone. Increasing the concentrations of octadecyl rosmarinate to a concentration of 40 μM in the chitosan-coated liposomes decreased lipid oxidation. Only butyl rosmarinate exhibited stronger antioxidant activity than free rosmarinic acid. Eicosyl rosmarinate (20 carbons) had lower antioxidant activity than all other rosmarinic acid derivatives. These results suggest that by combining the inclusion of appropriate antioxidants such as rosmarinic acid and the deposition of a chitosan coating onto the surface of liposomes may significantly increase the oxidative and physical stability of liposomes. KEYWORDS: Chitosan; rosmarinic acid; liposomes; lipid oxidation; antioxidant; lipid delivery system INTRODUCTION Liposomes are spherical, single- or multiple-layer vesicles that are spontaneously formed when phospholipids are dispersed in water. In recent years, liposomal encapsulation technologies have been extensively investigated in the food and agricultural indus- tries as delivery systems to entrap and protect functional and unstable components such as antimicrobials, flavors, antioxi- dants, and bioactive ingredients. Liposomes can entrap both hydrophobic and hydrophilic compounds within their structure, protect entrapped compounds from decomposition, and release the entrapped compounds at designated targets ( 1 , 2 ). Commer- cially available phospholipid preparations, commonly referred to as lecithin, are isolated from natural sources such as chicken egg yolk and soybeans ( 3 ) and are composed of mixtures of a variety of individual phospholipids. In the food industry, lecithins are generally recognized as safe (GRAS) food ingredients that are biocompatible, biodegradable, and nontoxic. They are used as both emulsifiers and texture modifiers ( 2 , 4 , 5 ). Phosphatidylcho- line (PC) is the major phospholipid found in most lecithins ( 6 ). One of the problem with liposomes in practical applications is their insufficient physical and chemical stability, leading to changes in particle size distribution, turbidity, and ability to contain the encapsulated compounds. Aggregation, rupture, and coalescence of liposomes will change their size distribution. This destabilization is particularly prevalent when surface charges are reduced at low pH conditions and at high ionic strengths ( 7 ). The chemical stability of liposomes may also be problematic due to oxidation or hydrolysis of the fatty acids ( 8 , 9 ). Many lecithins are susceptible to lipid oxidation because the phospholipids in the lecithin may contain fatty acids that are highly unsaturated. Transition metals such as iron can accelerate the oxidation of liposomes by interacting with residual lipid hydroperoxides in the phospholipids to produce free radicals that promote oxidation ( 10 , 11 ). In addition, the overall surface charge of liposomes manufactured from commercial lecithins is generally negative, resulting in electrostatic attraction of transition metals and thereby increasing metal-lipid interactions and further promoting oxidation ( 12 ). To minimize oxidative degradation of liposomes, several strategies have been reported including selecting high-quality lecithins with low levels of hydroperoxides and transition metals ( 13 ), using phospholipids that are high in saturated fatty acids (e.g., hydrogenated phospholipids; 14 ), adding antioxidants ( 15 ), and modifying the liposomal surface charges ( 11 , 16 , 17 ). Chitosan has been used successfully as a secondary layer on phospholipid-stabilized oil-in-water emulsion droplets to increase physical stability ( 18 -20 ). Modification of liposome surfaces by coating with chitosan has been demonstrated to enhance the physical stability of liposomes against aggregation for up to 45 days ( 21 ). Electrostatic deposition of chitosan onto phospholipid-stabilized