Freeze-Fracture Electron Microscopy of Lipid Membranes on Colloidal Polyelectrolyte Multilayer Coated Supports S. Moya, ²,§ W. Richter, ‡ S. Leporatti, ² H. Ba ¨ umler, $ and E. Donath* ,² Institute of Medical Physics and Biophysics, Leipzig University, Liebigstrasse 27, D-04103 Leipzig, Germany, Institute of Ultrastructure Research, Friedrich-Schiller-University Jena, Ziegelmu ¨ hlenweg 1, D-07740 Jena, Germany, and Institute of Transfusion Medicine, Medical Faculty, Charite ´ , Humboldt University of Berlin, D-10098 Berlin, Germany Received January 10, 2003; Revised Manuscript Received February 17, 2003 Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze- fracture electron microscopy. Introduction Biological membranes are in most cases in close proximity with a network of proteins or carbohydrates or both forming, for example, the cytoskeleton, the cell wall, or the glyco- calyx. The bilayer membranes may be anchored to these structures via defined molecular sites such as the case in the cytoskeleton-plasma membrane interconnection. In a plant cell, the turgor pressure pushes the membrane toward the cell wall inducing the tight arrangement. Glycocalyx molecules quite often have a hydrophobic site being itself part of the plasma membrane. These hydrophilic surface (polymer) layers contribute in many ways to the properties and function of membranes. 1-4 Lipid vesicles have been widely used as model systems for the investigation of membrane properties. Biotechno- logical applications of vesicles as containers for transport have further contributed to the widespread study and subsequent use of these structures. Although the interaction of vesicles with macromolecules was for many years in the focus of interest, vesicular systems in which the lipid bilayer was intercalated between two polymeric supports had not been fabricated. Such systems would be an interesting model of biological cells. The assembly of these sandwich-like polyelectrolyte- lipid-polyelectrolyte composites in the colloidal dimension became only recently possible, when the layer-by-layer (LbL) technique 5-9 was combined with the self-assembly of lipid bilayers. The building process starts with the fabrication of an empty polyelectrolyte (PE) capsule on top of which a bilayer is assembled afterward. 10-12 This was conducted by vesicle adsorption and spreading. A reverse-phase evapora- tion protocol proved to be also quite suitable. Supplementary polyelectrolyte layers (and bilayers) can be conveniently adsorbed afterward in a similar fashion. This fabrication pathway allows for the build-up of containers as small as tens of nanometers to a few micrometers in diameter with quite organized yet complex walls with tuneable thicknesses on the order of 10 2 nm. The built-up thickness and regularity of these composites has been investigated by means of various fluorescence techniques. The permeability was assessed by fluorescence recovery after photobleaching 10 and dielectric spectroscopy. 11 Differential scanning calorimetry (DSC) data revealed a phase transition of lipids assembled on top of capsules. This is evidence for a bilayer structure, yet the phase-transition temperatures were in some cases shifted compared with free bilayers. 10 The polyelectrolyte multilayer assembly employs the electrostatic interaction between oppositely charged species. When lipids were adsorbed on top of the multilayer cushion, it was possible to assemble either neutral species or op- positely charged ones. Charged lipids such as dipalmitoyl diphosphatidic acid (DPPA) formed bilayers, while zwitter- ionic lipids such as dipalmitoyl diphosphatidic choline formed multilayers as was concluded from Fo ¨rster energy transfer and single-particle light-scattering measurements. 10 When subsequently polyelectrolytes were adsorbed on top of the lipid layer, some of the lipids came off. It remained unclear whether these lipids prior to adsorption formed patches on the bilayer. Another possibility was that they * To whom correspondence should be addressed. E-mail: done@ medizin.uni-leipzig.de. ² Leipzig University. § Present address: Colle `ge de France, Chimie des Interactions Mole ´cu- laires, 11 Place Marcelin Berthelot, F-75005 Paris, France. ‡ Friedrich-Schiller-University Jena. $ Humboldt University of Berlin. 808 Biomacromolecules 2003, 4, 808-814 10.1021/bm034013r CCC: $25.00 © 2003 American Chemical Society Published on Web 04/15/2003