Biomacromolecules 2003, 4, 808-814
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Freeze-Fracture Electron Microscopy of Lipid Membranes on Colloidal Polyelectrolyte Multilayer Coated Supports S. Moya,†,§ W. Richter,‡ S. Leporatti,† H. Ba ¨ umler,$ and E. Donath*,† 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 freezefracture 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 glycocalyx. 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. Biotechnological 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 polyelectrolytelipid-polyelectrolyte composites in the colloidal dimension became only recently possible, when the layer-by-layer (LbL) technique5-9 was combined with the self-assembly of lipid * To whom correspondence should be addressed. E-mail: done@ medizin.uni-leipzig.de. † Leipzig University. § Present address: Colle ` ge de France, Chimie des Interactions Mole´culaires, 11 Place Marcelin Berthelot, F-75005 Paris, France. ‡ Friedrich-Schiller-University Jena. $ Humboldt University of Berlin.
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 evaporation 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 102 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 photobleaching10 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 oppositely charged ones. Charged lipids such as dipalmitoyl diphosphatidic acid (DPPA) formed bilayers, while zwitterionic 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
10.1021/bm034013r CCC: $25.00 © 2003 American Chemical Society Published on Web 04/15/2003
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might have been present in the form of adsorbed vesicles, which were then replaced by polyelectrolyte adsorption.12 The shape of lipid vesicles is rather smooth. The surface of a polyelectrolyte multilayer has a roughness on the order of a few nanometers. One of the intriguing questions is whether the lipid bilayer follows these surface inhomogeneities and, if so, to what extent. A better understanding of these problems would certainly contribute to improved assemblies. It can also be expected that the properties of the adsorbed lipid layer will largely depend on the degree of interaction between the lipid and polyelectrolyte phase. The use of lipid-coated capsules or lipid-coated colloidal particles allowed for the first time application of highresolution electron microscopy techniques to obtain a deeper insight into the topology of the lipid-polyelectrolyte interface. Freeze-fracture electron microscopy is especially suitable because it allows for a detailed two-dimensional view of the lipid membrane. In addition, this technique evidences the presence of the bilayer because the fracture plan runs along the hydrophobic interior (midplane) of the bilayer, splitting it into two leaflets. Materials and Methods Materials. The sodium poly(styrene sulfonate) (PSS) of MW 70.000 and 500.000 g/mol and the poly(allylamine hydrochloride) (PAH), MW 8000-11 000 g/mol were purchased from Aldrich. The PAH was used as received, whereas the PSS was dialyzed against Milli-Q water and lyophilized before use. Dipalmitoyl diphosphatidyl acid (DPPA) was obtained from Avanti polar lipids; dipalmitoyl diphosphatidylcholine (DPPC), labeled DPPC (L-R-phosphatidylcholine-(NBD-βaminohexanoyl)-γ-palmitoyl), and cholesterol were purchased from Sigma. Saturated lipids were chosen because they observe a phase transition in an experimentally suitable range of temperatures, which is currently under investigation by means of smallangle X-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS). Polystyrene (PS) latex particles of approximately 488 nm in diameter were synthesized following literature procedures.13 PS-latex particles with a diameter of approximately 21 µm were purchased from Microparticles GmbH, Berlin. The water used was prepared in a three-stage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18.2 MΩ cm. Freeze-Fracture Electron Microscopy. The colloidal dispersions were sandwiched between thin copper profiles and quick-frozen from room temperature by plunging them into a liquid propane/ethane mixture cooled in liquid nitrogen (cooling rate about 3-5.000 K s-1). Fracturing and replication were done with a BAF 400 T freeze-fracture device (Balzers/BAL-TE, Liechtenstein) using a double replica stage and electron gun evaporators. Pt(C) were deposited onto fracture faces under 35° angle to a controlled thickness of 2 nm. The replicas were placed on uncoated copper grids, cleaned in tetrahydrofurane, and examined in an EM 900 electron microscope (Zeiss, Germany). Height differences
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in the replica reveal themselves as bright shadows because behind an obstacle the platinum is not deposited causing a white (electron transparent) shadow. In front of the obstacle, naturally, a dark zone is observed indicating surplus accumulation of the evaporated Pt-C. From the length of the shadow, it is possible to calculate roughly the height of the obstacle because the evaporation was done under the angle of 35°. To avoid a large systematic error in height calculation, only those depressions or raises of the surface that were on a horizontal part of the fracture plane were considered. Confocal Laser Scanning Microscopy (CLSM). Confocal micrographs were taken with a Leica TCS NT inverted confocal system (Leica, Germany) equipped with a 100× oil immersion objective, numerical aperture 1.4. As a lipidfluorescent probe, 1% NBD-labeled DPPC was added to a mixture of 9:1 DPPC/DPPA. Scanning Force Microscopy (SFM). SFM images were recorded in air at room temperature (20-25 °C) using a Nanoscope III Multimode SFM (Digital Instrument Inc., Santa Barbara, CA). Silicon nitride (Si3N4) cantilevers with a force constant of 0.58 N/m (Digital Instruments) were used for contact SFM. Images were obtained on the surface of bare and coated PS-latex particles of 21 µm in diameter. Coating of Colloids with Polyelectrolyte. Latex particles were alternatively suspended in PAH solutions (1 mg/mL) in 0.5 M NaCl and PSS (1 mg/mL) in 0.5 M NaCl. After each adsorption of polyelectrolyte, the samples were washed three times with water. The coating and washing was performed in an Amicon filtration cell. The coating procedure has been detailed elsewhere.7,9 Coating with Vesicles. Vesicles were prepared by evaporating at 40 °C a 1 mg/mL chloroform solution of lipid in a rotavap. After solvent evaporation, water was added up to a lipid concentration of 1 mg/mL, and the solution was sonicated during 5 min in a Sonorex Super Digital. Lipid fractions correspond to mass percentages. For the latex coating, 100 µl of a 10% v/v solution of polyelectrolyte-coated latices was mixed with 500 µl of lipid dispersion. Excess lipids were removed by three repeated centrifugations in water. Flow Cytometry. Flow cytometry measurements were performed using commercial equipment FACScan Becton Dickinson device. As a lipid fluorescent probe, 10% NBDlabeled DPPC was added to the lipid mixtures. Results and Discussion Results. The successful deposition of lipids onto polyelectrolyte-coated particles is demonstrated by CLSM. One observes a homogeneous fluorescence all over the particle surface (see Figure 1). This proves within the limits of resolution of the CLSM technique the presence and homogeneous distribution of lipids on the particle surface. Because, however, the lateral resolution of the CLSM technique is limited to 150-200 nm (the vertical resolution is slightly less), one cannot draw conclusions about the topology of the lipid arrangement. Especially, it is not possible to find out whether the bilayer follows the local curvature and graininess of the polymer support. This is beyond the
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Figure 2. Flow cytometry data of erythrocytes coated with (PSS/ PAH)5 and (a) 80% DPPC, 10% NBD-DPPC, 10% DPPA and (b) 10% NBD-DPPC, 90% DPPA.
Figure 1. Confocal image of PS-latex particles coated with (PAH/ PSS)5PAH and DPPC/10% DPPA, 1% NBD-DPPC.
resolution of the technique. An arrangement of the lipids in the form of small and densely spaced patches could probably not be distinguished from a homogeneous bilayer coverage. Whether the lipids form a bilayer or a multilayer cannot be seen with CLSM alone because it is difficult to quantify the amount of fluorescence in absolute terms with CLSM. Even if the settings of the device are precisely kept, different orientations in the interface would result in changes of the brightness owing to the resolution dependency on orientation. Therefore, flow cytometry was applied to quantify the fluorescence of particles. This technique allows for simultaneous recording of light scattering and fluorescence on a single-particle level. In biological applications, the fluorescence intensity distribution is typically recorded over the particle population and correlated to the particle size. Subpopulations may be thus identified and eventually used for a subsequent sorting procedure. Below, we applied this technique to obtain information about the homogeneity of lipid coverage. A fluorescent lipid was used as a marker allowing us to quantify the lipid amount per particle by measuring the fluorescence histogram. It has to be stressed that the resolution of the technique is quite superb. It can easily distinguish between one and two polyelectrolytelabeled layers or identify an incomplete layer coverage on top of cells.14 It has been shown earlier that a variety of colloidal cores can be used as templates for the LbL coating technique. After a few layers are deposited (>8), the features of the particles become independent of the nature of the substrate. This allowed us to use glutaraldehyde-fixed red blood cells as templates for the LbL coverage followed by lipid adsorption. The advantage of this template is its superior size homogeneity. This is an essential advantage for flow-cytometry fluorescence quantification because the distribution of fluorescence would in the first instance depend on the particle surface area. Furthermore, the hydrodynamic features of the flow cytometry device are also optimized for particles of the size of biological cells. Thus fixed red blood cells were coated with 10 polyelectrolyte layers (PAH/PSS) followed by adsorption of lipids. Either a mixture of DPPC/DPPA or pure DPPA were employed together with 10% of NBD-
labeled DPPC as the reporter molecule. Figure 2a,b shows that in each case a well-pronounced peak of fluorescence distribution was observed. The width of the peak is in each case less than 10% of its mean. In the case of DPPA, the width of the peak is about 40 arbitrary units for a mean value of 500 arbitrary units of fluorescence, while for DPPC, the width is also of 40 arbitrary units but for a mean value of 800. This is strong evidence for a homogeneous coverage of the particles. Had there been a significant amount of particles in the sample the surface of which, for example, is only covered by 50%, one would have observed a distribution with a width on the order of its mean. This is not the case. However, one can detect fluorescence attributed to remaining vesicles in the sample. In the case of DPPA, the vesicles are unilamellar and quite small, providing a fluorescence tail close to zero. The DPPC vesicles are multilamellar. They yield a wide tail of larger intensity located just below the comparatively narrow and intense peak related to the particles themselves. Next, we were interested to study the morphological details of the lipid coverage. Especially, we were interested to answer two fundamental questions: (I) what is the structure of the adsorbed lipid, and (II) to what extent are the lipids capable of following the local curvature of the polyelectrolyte support? SFM was employed in the past to study the roughness of polyelectrolyte multilayers on colloidal supports. Especially, polyelectrolyte multilayer capsules have been thoroughly studied by means of SFM investigations.15 The basic features of the PS-latex particle as the multilayer support are provided in Figure 3a. The particle surface is rather flat with irregularily spread invaginations of only about 2 nm in depth and a lateral extension of 40-70 nm. The typical height profile given below the image illustrates the almost perfect smoothness of the latex particle surface. The root mean square of the height variations (rms value) as obtained with the standard data evaluation protocol of the Nanoscope is less than 1 nm. The image in Figure 3b shows the PS-latex particle surface covered with nine polyelectrolyte layers prior to the lipid deposition. A considerable roughness is observed, evident also from a typical height profile given below the image. The rms value is about 7 nm. The patches of different height extend laterally over 30-50 nm. Height variations occur over relatively short distances. The slope between areas of different height is as much as 40°-50 °. These topological
Freeze-Fracture Electron Microscopy
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Figure 3. SFM image in contact mode of (a) part of a PS-latex particle surface, (b) the same surface covered with nine layers of polyelectrolytes (PAH/PSS/.../PAH), and (c) after adsorption of DPPC. Typical height profiles are given below the images. All three images are drawn in the same height scale.
features were attributed to changes brought about by drying caused by the random nature of the adsorption process of the individual polymers.15 The last image in Figure 3c was taken after lipid coverage. It is clearly obvious that the lipid coverage has considerably smoothed out the roughness of the polymeric support. The height variations are reduced by a factor of 2. Areas of different height change more gradually. The slope between areas of different height has decreased to 20°-30°, indicating that the lipid layer does not completely follow the topography of the support. Because SFM high-resolution imaging of soft objects requires a dry state, it was challenging to seek for a possibility to image the particle surface in the aqueous environment. A promising technique is freeze-fracture electron microscopy. This technique has been extensively used for the characterization of artificial and natural membranes.16 Samples are quick-frozen and then fractured. The fracture plane runs through the weakest part, which is especially the midplane of the lipid bilayer. A replica of the fracture plane is fabricated by means of evaporating platinum under a defined angle onto the surface. This allows the calculation of height differences in the fracture plane, and it will let us address the two originally raised questions. All micrographs are mounted with evaporation direction from bottom to top. Figure 4a shows the freeze-fracture image of bare PSlatex particles. The lattices did not fracture along the surface, but the fracture goes straight through the hydrophobic interior of the particle. Most of the particles show some additional plastic distortion indicating that their core is not completely solid and brittle at the low temperatures during freezefracturing. Figure 4b shows that PS-latex coated with (PAH/ PSS)2PAH behave in the same way as the bare lattices. The polyelectrolyte multilayer consisting of strongly interacting oppositely charged individual layers obviously does not permit the fracture plane to run along the surface. An interesting artifact can be observed in this particular image, as well as in some of the images given below. During the
Figure 4. Freeze-fracture electron microscopy images of (a) bare PS-latex particles and (b) PS-latex particles coated with (PAH/ PSS)2PAH. The inset shows the attached PE envelope.
cleaning of the sample with tetrahydrofurane, the polystyrenelatex particle used as the template for layer build-up is dissolved. The adsorbed polyelectrolyte layer, however, is not and remains sometimes attached to the replica. The subsequent observation in the TEM device reveals these remaining polyelectrolyte layers as “capsules” often in close neighborhood of particles (Figure 4b, inset). In some cases the core particle was not completely removed. These (few) remains appear as electron-dense dark spheres or parts of them because of their high electron absorption (Figure 6a). When a composite layer consisting of polyelectrolytes and one or more complete lipid layers was fabricated, regardless of the particular lipid composition used, in all cases the fracture plane runs along the particle surface. This is proper evidence for lipid bilayer coverage. The fracture planes reveal a wealth of details. Much in contrast to commonly fractured lipid vesicles, the lipid layers on solid latex particle supports
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Figure 6. Freeze-fracture electron microscopy images of (a) (PAH/ PSS)2PAH-coated latex particles + DPPC (10%)/DPPA (30%)/ cholesterol (dark, not completely dissolved latex sphere) and (b) (PAH/PSS)2PAH-coated latex particles + DPPC (10%)/DPPA (30%)/ cholesterol + PAH/PSS/PAH. Figure 5. Freeze-fracture electron microscopy images of (a) (PAH/ PSS)2PAH-coated latex particles + DPPA, (b) PE-coated latex particles + DPPA + PAH/PSS/PAH, (c) (PAH/PSS)2PAH-coated latex particles + DPPC (10% DPPA), and (d) (PAH/PSS)2PAH-coated latex particles + DPPC (10% DPPA) + PAH/PSS/PAH.
show a remarkable roughness, which we attribute to the original roughness of the polyelectrolyte layer-coated latex particle surface. One can also observe areas where the fracture plane suddenly changes in height with regard to the surface normal. Below these details of the fracture planes are quantitatively analyzed. This allows drawing of conclusions about the properties of the lipid coverage, as well as about the interaction between the lipid layer and the adjacent polyelectrolyte layers. Figure 5 displays freeze-fracture images of latices coated with PE multilayers and DPPA or DPPC/DPPA mixtures as the top layer or underneath three supplementary polyelectrolyte layers. The micrograph in Figure 5a displays the situation in which DPPA forms the top layer. Figure 5a clearly shows a rough texture of the particle surface. The whole fracture plane of the particle surface is composed of numerous small wartlike patches of slightly different height. This is consistent with a lipid layer following closely the inhomogeneities of the underlying support. The quantitative description of the surface roughness provided for the average height variance a value of 1.1 nm, while the average diameter of the patches is 27 nm with a variance of about 8 nm. Next, three additional polyelectrolyte layers (PAH/PSS/ PAH) were adsorbed on top of the DPPA layer (Figure 5b). The topology of the fracture plane changed remarkably compared with the case of DPPA being the top layer. Islands are identified where the fracture plan suddenly changed its distance from the particle surface. Instead of running continuously at the same level, sudden jumps into the direction of the particle surface and back to the original fracture level are observed. The depth of these islands is about 8 nm. The size of the islands is 84 nm with a variance of 55 nm. The latter value indicates that these islands are rather inhomogeneous in shape and lateral extension. The
surface of these islands has a different roughness compared with the lipid-layer fracture plane. Small, more or less uniform, round particle-like patches with a diameter of 7 ( 1 nm and an estimated height of 3 ( 1 nm are found. The contour of these structures is well defined. Because they are described in the concave fracture face, they have to be related to the fracture plane running beyond the DPPA bilayer. DPPC (10% DPPA)-coated latices show concave (Figure 5c, upper part), as well as convex, fracture faces (Figure 5c, lower part). The surface is again not flat, seemingly composed of numerous wartlike patches. The contour of these “warts” is not well defined. They extend over about 20 nm with a variance of 3 nm. The estimated height of these patches (6 ( 3 nm) is somewhat larger than that of the corresponding structures found in the case of DPPA as the lipid layer. Jumps of the fracture plane resulting in islandlike areas are not observed. When, as shown in Figure 5d, three supplementary polyelectrolyte layers were deposited on top of the PC/DPPA layer, one can again observe jumps of the fracture plane producing defined islands in the surface. These islands have extensions of tens of nanometers with a height of about 11 nm. Rather similar to the case of supplementary polyelectrolyte layers assembled on DPPA, also small well-defined circular particles such as patches with a diameter of about 6.5 nm and height of about 5 nm are characteristic for the surface of the islands. Because these structures represent elevations in the concave fracture plane, they have to be related to a fracture plane beyond the lipid layer. The roughness of the fracture plane running through the midplane of the lipid layer is very similar to the cases described above. Figure 6 shows freeze-fracture images of polyelectrolytecoated latices and covered with a mixture of DPPC (70%), cholesterol (20%), and DPPA (10%) without (Figure 6a) and with three supplementary (PAH/PSS/PAH) polyelectrolyte layers on top of the lipid layer (Figure 6b). In both cases, the fracture plane is rather homogeneous. No islands as the result of fracture jumps have been observed. However, a certain roughness is clearly observable. Small wartlike areas
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of 28 ( 7 nm extension can be identified. Both micrographs, Figure 6, parts a and b, show additionally some lipid vesicles. We assume they represent remains from the coating procedure. Probably they were adsorbed to the particles and could be thus not be completely removed during the washing steps. Occasionally the multilayer structure of these vesicles is revealed by a jump of the fracture plane (Figure 6a, arrowhead). The roughness of the lipid layer on the solid support is clearly evident when comparing it to the rather smooth fracture along the bilayer of the lipid vesicle surface. The thickness of the lipid membrane of the vesicles can be roughly estimated from fractures of multilayer vesicles. We found a value of about 5 nm. When, as shown in Figure 6b, three supplementary polyelectrolyte layers were adsorbed, uniform small particlelike almost circular defects are observed in the concave fracture face. The fracture plane runs toward the colloidal core surface thus probably indicating a particle-like defect in the bilayer. These defects have a depth of 5-8 nm and a diameter of about 12 nm. Discussion. Both confocal microscopy and flow cytometry studies proved the presence of lipids on top of polyelectrolyte-coated latices. Confocal microscopy investigations assured the continuity of the coating over the latex surfaces and flow cytometry allowed for quantification of the coating, thus proving that the lipid coverage was homogeneous over the particle population. But neither of these techniques provided a deeper insight into the local topology of the lipid coverage on top of the polyelectrolyte support. Thus many interesting questions remained unanswered. For example, it is quite interesting to look for pores or defects in the lipid layer. If they were present, they would have a large impact on the permeability properties of these composite layers. Another question often raised concerns the significance of the lipid-polyelectrolyte interaction for the topology of the lipid membrane. If the interaction was strong enough, one would expect the lipid layer to follow closely the profile of the underlying polyelectrolyte cushion. If the interaction was weak, one would expect a bilayer attached point-wise to the polyelectrolyte support but relatively free between these attachment sites. Naturally, the physicochemical properties of the bilayer should depend to a large extent on this interaction. One of the techniques to study the local topology of the lipid coat is SFM. This technique has been employed to study the surface of the polyelectrolyte multilayers. It was found that such layers generally observe a grainy texture. After coating with lipids, it was found that the roughness and texture of the capsule surface changed considerably. The surface appeared smoother than the original polyelectrolyte multiplayer support, but was remarkably rougher than a free lipid bilayer. Defects, such as holes, could not be found with the available resolution of the SFM. From these findings, it was concluded that lipids form a continuous regular layer on top of the polyelectrolyte matrix. Lipid multilayer patches were not seen. The disadvantage of the SFM technique is, however, that for high resolution drying was required. This may have affected the obtained images in the dry state.
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The freeze-fracture electron microscopy technique is an elegant means for proving the success of the coating and for studying details under hydration conditions. First, we observed that both polystyrene latices and PE-coated latices are cross-fractured right through the particle and not along their surface. This behavior points to the hydrophilic nature of the particle surface. Furthermore, the interaction between the individual polyelectrolyte layers is sufficiently strong to resist any fracturing between them. When, however, lipids are adsorbed, the fracture plane runs along the lipidic surface because the weakest part of the system, which is preferentially the middle of the bilayer, breaks at low temperatures when the sample is subjected to the external fracturing force. It is worth emphasizing that this “surface” fracture is thus an unambiguous proof of the presence of the lipids being organized as a bilayer. There were, however, few cases when the fracture went partly through the particle itself regardless of the adsorbed lipid bilayer. This was only the case when after the bilayer formation supplementary polyelectrolyte layers were adsorbed on top of it. Such behavior of the fracture plane could be interpreted as a partial removal of the lipid coverage by the adsorbing polyelectrolyte layers. The freeze-fracture images provide additional information on the topology of lipid layer interacting with PE. It is worth comparing, for example, the smooth surface of DPPC/DPPA/ cholesterol vesicles with the roughness of the same lipid mixture on top of the PE support (Figure 6a,b). In the same cases, vesicles are still attached to the latex particles and the change in roughness can be observed within the same object. Regardless of the employed lipid composition, all covered particles showed a very similar lateral texture of the lipid-layer fracture plane. It is characterized by relatively flat but clearly distinguishable height changes with a lateral extension of 20-30 nm. The size of these local lipid curvature variations is smaller, being about one-third, than that of the grains of the polyelectrolyte support as revealed by SFM in a dried state. There, grains on the order of about 102 nm in diameter have been observed.15 It was interpreted as being at least partly a result of the drying procedure, which may have induced additional lateral clustering of the constituents of the multilayer. On the other hand, AFM measurements conducted on planar PSS/PAH films in water also demonstrated the presence of small patches ranging from 30 to 80 nm.17 The size of individual PSS molecules of a molecular weight of about 70.000 kD was found to be 27 nm with SFM on gold substrates.18 All of these findings underline that indeed the patches observed by SFM in multilayers represent most likely complexes of a few polyelectrolyte molecules, maybe in some cases also single molecules. Remarkably, the single-molecule size of 27 nm18 is also very close to the size of the patches observed in this work by means of freeze-fracturing. Although here the layer immediately beneath the lipid layer consisted of PAH of 70.000 kD instead of PSS, the size of them should be similar to that of PSS.19 Hence, we conclude that the appearance of the lipid layer reflects the topology of the underneath polyelectrolyte surface. The interaction between the lipid layer and the support is obviously stronger than the resistance of the bilayer to bend. Although the general appearance of
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the roughness was very similar, there were nevertheless small variations among the different lipid compositions. Pure DPPA formed a less wavy layer than was the case for the different DPPC mixtures. This could indicate differences in bending behavior. When cholesterol was present, it may have been used to compensate topological constraints induced by the mismatch of the inherent flat bilayer and the locally rough support. The coating with supplementary layers of PE changed neither the size nor the height of the local variations in lipid curvature but caused in the case of the cholesterol-free samples the appearance of islands where the fracture plane suddenly jumped below the level of the lipid layer. The height of these islands was remarkably larger (about 8 nm) than the thickness of a lipid bilayer. The islands themselves showed a clearly visible graininess on their surface. Rather small particle-like patches of a characteristic height of about 5 nm cover their surface. Such behavior would not have been observed if there was a second bilayer underneath. Therefore, we assume that these islands represent areas where the fracture plane jumped down into the polyelectrolyte multilayer region. It could have been that some lipids were locally removed from the particle surface after supplementary PE adsorption. This may have caused these irregular jumps of the fracture plane. This conclusion is further supported by the fact that occasionally latex particles were cross-fractured. Another explanation could be that the supplementary PE layers on top of the lipid layer disturbed the continuity of the bilayer thus hindering smooth fracturing along the midplane. This could have been induced by a small fraction of polyelectrolyte molecules extending through the lipid layer connecting via its strong interaction both adjacent polyelectrolyte layers together. Small holes indicating the presence of such molecules can be identified as described in Figure 6. The fact that samples with cholesterol showed no islands might be consistent with a less undisturbed lipid structure brought about by the space filling potential of cholesterol. Contrary to what has been originally expected was the obvious absence of multilayer fracturing in lipid layers on the PE-coated colloids. From single-particle light scattering (SPLS) and energy-transfer measurements, it was supposed that DPPA forms bilayers while DPPC is organized in multilayers. The presence of a single fracture plane confirms this hypothesis for DPPA but is in contradiction to what has been expected for DPPC. A possible explanation could be that the interaction of lipids with the PE underneath induced a more favorable fracture through the layer directly assembled on top of the PE. Another explanation, probably more likely, could be that DPPC forms also a single bilayer but with a vesicle attached, which had not completely spread upon interaction. Neither SPLS nor flow cytometry would be able to discriminate between these two cases if the number of vesicles is not too different.
Moya et al.
Conclusions It was shown that by means of AFM and freeze-fracture electron microscopy it is possible to study the lipid coating on latices covered with polyelectrolyte multilayers with a resolution down to the range of a few nanometers. The change in the fracture pattern from cross-fractured particles to a surface fracture after lipid coating under hydration conditions provides an unambiguous proof for the existence of a lipid bilayer. Freeze-fracture electron microscopy imaging revealed a wealth of details concerning the arrangement of lipids on top of the PE cushions. The lipids closely follow the surface texture of the PE support. Zwitterionic lipids form more pronounced patches than charged ones, probably because the latter may have a larger resistance toward bending because of their charges, which would become more compressed as a result of bending. The absorption of extra PE layers on top of the lipid-coated latices produced different fracture patterns. Maybe this indicates a reorganization of the lipid layer underneath or a partial removal of the lipids by the incoming polyelectrolytes. Acknowledgment. This study was partly supported by grants from BMBF (Grant 0312011C) and from VW foundation (Grant I/78168). References and Notes (1) Shen, W. W.; Boxer, S. G.; Knoll, W.; Frank, C. W. Biomacromolecules 2001, 2, 70. (2) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58. (3) Sackmann, E. Science 1996, 271, 43. (4) Wong, J. Y.; Park, Ch. K.; Seitz, M.; Israelachvili, J. Biophys. J. 1999, 77, 1458. (5) Decher, G. Science 1997, 227, 1232. (6) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S.; Mo¨hwald, H. Angew. Chem. 1998, 110, 2323. (7) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, E.; Knippel, M.; Mo¨hwald, H. Colloids Surf. A 1998, 137, 253. (8) Sukhorukov, G. B.; Donath, E.; Davis, S.; Lichtenfeld, H.; Caruso, F.; Popov, V. I.; Mo¨hwald, H. Polym. AdV. Technol. 1998, 9, 759. (9) Voigt, A.; Lichtenfeld, H.; Sukhorukov, G. B.; Zastrow, H.; Donath, E.; Ba¨umler, H.; Mo¨hwald, H. Ind. Eng. Chem. Res. 1999, 38, 4037. (10) Moya, S.; Donath, E.; Sukhorukov, G. B.; Auch, M.; Lichtenfeld, H.; Ba¨umler, H.; Mo¨hwald H. Macromolecules 2000, 33, 4538 (11) Georgieva, R.; Moya, S.; Leporatti, S.; Neu, B.; Ba¨umler, H.; Reichle, C.; Donath, E.; Mo¨hwald, H. Langmuir 2000, 16, 7075. (12) Fery, A.; Moya, S.; Puech, P. H.; Brochard-Wyart, F.; Mo¨hwald, H. C. R. Acad. Sci., submitted for publication. (13) Furusawa, K.; Norde, W.; Lyklema, J. Kolloid Z. Z. Polym. 1972, 250, 908. (14) Neu, B.; Voigt, A.; Mitlo¨hner, R.; Leporatti, S.; Gao, CY.; Kisewetter, H.; Meiselman, H. J.; Ba¨umler, H. J. Microencapsulation 2001, 18, 385. (15) Leporatti, S.; Voigt, A.; Mitlo¨hner, R.; Sukhorukov, G. B.; Donath, E.; Mo¨hwald, H. Langmuir 2000, 16, 4059. (16) Meyer, H. W.; Richter, W. Micron 2001, 32, 615. (17) Lavalle, Ph.; Gergely, C.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C.; Picart, C. Macromolecules 2002, 35, 4458. (18) Zhu, M.; Schneider, M.; Papastavrou, G.; Akari, S.; Mo¨hwald, H. Nano Lett. 2001, 1, 569. (19) Donath, E.; Walter, B.; Shilov, V. N.; Knippel, E.; Budde, A.; Lowack, K.; Helm, C. A.; Mo¨hwald, H. Langmuir 1997, 13, 5294.
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