Influence of Polyelectrolyte Chemical Structure on their Interaction with Lipid Membrane of Zwitterionic Liposomes Francois Quemeneur, † Marguerite Rinaudo, ‡ and Brigitte Pe ´ pin-Donat* ,† Laboratoire Electronique Mole ´ culaire Organique et Hybride/UMR 5819 SPrAM (CEA-CNRS-UJF)/INAC/ CEA-Grenoble, 38054 Grenoble Cedex 9, France, and Centre de Recherches sur les Macromole ´ cules Ve ´ge ´ tales (CERMAV-CNRS), affiliated with Joseph Fourier University, BP53, 38041 Grenoble Cedex 9, France Received April 14, 2008; Revised Manuscript Received May 26, 2008 In this paper we extend our previous experimental work on interaction between polyelectrolytes and liposomes. First, the adsorption of chitosan and alkylated chitosan (cationic polyelectrolytes) with different alkyl chain lengths on lipid membranes of liposomes is examined. The amount of both chitosans adsorbed remains the same even if more alkylated polysaccharide has to be added to get saturation if compared with unmodified chitosan. It is demonstrated that alkyl chains do not specifically interact with the lipid bilayer and that electrostatic interaction mechanism governs the chitosan adsorption. The difference observed between unmodified and alkylated chitosans behavior to reach the plateau can be interpreted in terms of a competition between electrostatic polyelectrolyte adsorption on lipid bilayer and hydrophobic autoassociation in solution (which depends on the alkyl chain length). Second, interaction of liposomes with hyaluronan (HA) and alkylated hyaluronan (anionic polyelectrolytes) is analyzed. The same types of results as discussed for chitosan are obtained, but in this case, autoassociation of alkylated HA only occurs in the presence of salt excess. Finally, a first positive layer of chitosan is adsorbed on the lipid membrane, followed by a second negative layer of HA at three different pHs. This kind of multilayer decoration allows the control of the net charge of the composite vesicles. A general conclusion is that whatever the pH and, consequently, the initial charge of the liposomes, chitosan adsorption gives positively charged composite systems, which upon addition of hyaluronan, give rise to negatively charged composite vesicles. I. Introduction Adsorption of polyelectrolytes on charged surfaces plays an important role in materials science 1–5 and biomedical applica- tions. 6–9 In particular, interactions between polyelectrolytes and charged lipid bilayers have been extensively investigated under theoretical 10–16 and experimental approaches. 17–22 Electrostatic coupling of polyelectrolytes to lipid bilayers produces materials with specific properties and enhanced stabilities when used in various devices, such as chemical sensors 23 or drug carriers. Many factors can affect polyelectrolyte adsorption on a lipid bilayer: (i) the initial bilayer properties (chemical structure of the lipids, autoassociated configuration), (ii) polyelectrolyte properties (chemical structure, charge density, molecular weight (Mw), solution concentration), and (iii) equilibrium solution properties (pH and ionic strength). 24,25 The combined effects of these factors control polymer-polymer or polymer-lipid bilayer interactions. Liposomes consist of self-closed phospholipid bilayers (or multilayers). 26 Their diameters range between a few nanometers and hundred micrometers. Three main kinds of liposomes are distinguished: small unilamellar vesicles (SUVs; 20-100 nm), large unilamellar vesicles (LUVs; 100-500 nm), both usually used as protective capsules, 27–29 and giant unilamellar vesicles (GUVs; 0.5-100 µm), generally studied as oversimplified models of biological cells. 30 Interaction of liposomes with macromolecules is of relevance to simulate intercellular, polymer-cell, 31–34 and liposome-cell interactions 35–39 and is of interest in the domain of soft-matter physics. 40 Furthermore, liposomes decorated with polyelectrolytes are extensively used as drug carriers 41,42 and it is demonstrated that coating liposomes with polyelectrolytes enhance their therapeutic activity 43–46 and circulation lifetime 47,48 in an intravenous environment. For example, complexes of liposomes with DNA have recently received much interest as nonviral gene delivery vehicles 49–52 for a variety of biomedical applications. In our previous works 53,54 we have quantified chitosan (a linear biocompatible cationic polyelectrolyte) adsorption on both large and giant vesicles. We have demonstrated that chitosan-lipid bilayer interaction enhances vesicles stability with regard to various stresses (for example, pH and salt shocks). However, it seems of interest to further study the changes in liposome structural and physical properties resulting from their interaction with polyions relaying with the structure of the polyelectrolytes. Actually, it is reported that polyelectrolyte adsorption affects membrane behavior: permeability, 55–57 fu- sion, 58 phase transformations, 59–61 and stabilization. 62,63 It is the reason why, in the present paper, we first focus on the effect of alkyl chain lengths grafted on chitosan on the mechanism of its adsorption. Then, we show that hyaluronan (HA, an anionic polyelectrolyte) and a HA-alkylated derivative can also be adsorbed on the same DOPC lipid bilayer. The two polysac- charides investigated (HA and chitosan) were chosen for their good biocompatibility recognized for biomedical applications. 64 Finally, we report the formation of the chitosan/hyaluronan complexes on the lipid bilayers. The advantage of the use of HA as external layer is to confer better biocompatibility to the decorated vesicles. * To whom correspondence should be addressed. Tel.: 00 33 4 38 78 38 06. Fax: 00 33 4 38 78 51 13. E-mail: brigitte.pepin-donat@cea.fr. † Laboratoire Electronique Mole ´culaire Organique et Hybride/UMR 5819 SPrAM (CEA-CNRS-UJF)/INAC/CEA-Grenoble. ‡ Centre de Recherches sur les Macromole ´cules Ve ´ge ´tales. Biomacromolecules 2008, 9, 2237–2243 2237 10.1021/bm800400y CCC: $40.75  2008 American Chemical Society Published on Web 07/01/2008