Controlling Ionic Conductivity in Lipid Polyelectrolyte Composite ...

J. Phys. Chem. B 2005, 109, 18025-18030

18025

Controlling Ionic Conductivity in Lipid Polyelectrolyte Composite Capsules by Cholesterol Radostina Georgieva,*,† Sergio E. Moya,‡ Hans Ba1 umler,§ Helmuth Mo1 hwald,† and Edwin Donath| Max Planck Institute of Colloids and Interfaces, Am Mu¨hlenberg 1, 14476 Golm/Potsdam, Germany, Center of Applied Chemistry InVestigations (CIQA), BouleVard Enrique Reyna No. 140, Saltillo, Coahuila, Mexico, Institute of Transfusion Medicine, Charite´ -UniVersita¨tsmedizin Berlin, 10098 Berlin, Germany, and Institute of Medical Physics and Biophysics, Faculty of Medicine, Leipzig UniVersity, Ha¨rtelstrasse 16/18, 04107 Leipzig, Germany ReceiVed: April 25, 2005; In Final Form: July 19, 2005

The effect of cholesterol on the formation and properties of bilayer lipid membranes deposited on polyelectrolyte multilayered capsules was studied. The permeability of lipid/cholesterol coated capsules for NaCl was derived from osmotic response experiments and ranged from 1.45 × 10-8 to 2.9 × 10-8 m‚s-1, which corresponds to a lipid layer conductivity of (0.7-1.4) × 10-8 S‚m-1. These conductivity values were in good agreement with the value of 0.8 × 10-8 S‚m-1 obtained by electrorotation and were by 3 orders of magnitude lower than those found earlier for lipid layers on polyelectrolyte capsules in the absence of cholesterol.

Introduction Engineering composite supramolecular structures with biological functions is an exciting research area, both because of its possible applications in medicine, drug delivery, and sensing, and from the viewpoint of scientific challenges met in this particular nano-mesoscale world.1-6 Recently a new type of supported membranes has been developed using polyelectrolyte capsules as templates.1,2 A polyelectrolyte capsule is a thin closed polyelectrolyte film with a void inside.1,7,8 Such capsules can be prepared by the consecutive assembly of oppositely charged polyelectrolyte molecules8,9 onto sacrificial colloidal templates such as melamine formaldehyde resin particles10 or erythrocytes,11,12 which after coating can be destroyed and washed out leading to the empty capsule. Polyelectrolyte multilayer capsule walls are generally permeable for small molecules and ions. To control the permeability is a challenging task. In analogy with cell membranes, phospholipid vesicles were adsorbed onto capsule walls. The vesicles spread due to electrostatic interaction with the support assembling a lipid membrane on top of the polyelectrolyte capsules. It closely follows the polyelectrolyte topology underneath all over the capsule surface and has a significant effect on capsule properties such as reducing the capsule permeability for small polar molecules.2 The conductivity and capacity of such supported membranes has been determined for different lipid compositions of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidyl acid (DPPA).13 To increase the complexity and selectivity of these bionanocomposites, so that they can perform more specific functions such as the selective retention of ions or the recognition of molecules, requires incorporation of more biological functional molecules such as proteins, DNA, or viruses.6 * Corresponding author. E-mail: radostina.georgieva@mpikg-golm. mpg.de. † Max Planck Institute of Colloids and Interfaces. ‡ CIQA. § Humboldt University. | Leipzig University.

The aim in this work was to further decrease the conductivity of the composites by incorporation of cholesterol into the phospholipid membranes in analogy to cell membranes. Cholesterol is a typical membrane lipid with a small polar OH headgroup, which can fill free space in the membrane, providing mechanical stability through its rigid structure and regulating the differences in temperature of phase transitions in mixtures of lipids. Many processes in membranes are controlled by local segregations or depletions of cholesterol, which have a significant influence in the local viscosity and consequently on membrane permeability and organization. The engineering of the capsule walls to reduce permeability, first by adding lipids and then by changing their formulation with cholesterol, is a necessary step before inclusion of channels in the structures, with which the nanocomposites could obtain ion-selective properties in a step forward to the mimicking of cell functions. Changes in ion conductivity for lipid polyelectrolyte capsules after incorporation of cholesterol have been studied by applying the electrorotation technique, while their permeability has been studied by osmotic response followed with confocal microscopy. Electrorotation is a dielectric spectroscopy technique14-16 extensively used to study electrical properties of cells. It has proved to be very useful in understanding electrical phenomena in polymer capsules and their dependency on layer architecture.13,17 The osmotic response is based on the fact that polyelectrolyte capsules shrink when a sudden osmotic pressure gradient is established through the capsule wall. The pronounced inward buckling of the capsule wall evidences this phenomenon.18 This was demonstrated also when capsules were suspended in polyelectrolyte solutions with concentrations varying from 2 to 12.5 wt % sodium poly(styrenesulfonate). From the polymer concentrations corresponding to the onset of buckling, the elasticity of capsules was calculated.19 The osmotic pressure is due to a difference in ion concentrations between capsule interior and exterior, and therefore kinetic aspects of buckling should yield quantitative information concerning ion permeation.20 This technique is complementary to electrorota-

10.1021/jp0521407 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/31/2005

18026 J. Phys. Chem. B, Vol. 109, No. 38, 2005 tion, because it depends on elasticity as an adjustable parameter, which can be measured independently. Experimental Section Chemicals. Sodium poly(styrenesulfonate) (PSS) of MW 70 000, poly(allylamine hydrochloride) (PAH) of MW 70 000, dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidyl acid (DPPA), and cholesterol were purchased from Aldrich/ Sigma, sodium hypochlorite solution 12% was purchased from Hedinger, and tetramethyl rhodamine ethyl ester, perchlorate (TMRE), was purchased from Molecular Probes. Weakly polymerized melamine formaldehyde (MF) resin particles (diameter 5 µm) were received from Microparticles GmbH, Adlershof, Germany. Erythrocytes were obtained from the donor center of Charite´, Berlin. Capsule Preparation. Capsules were fabricated by consecutive assembly of negatively charged PSS and positively charged PAH (layer-by-layer approach) on top of MF particles and fixed erythrocytes21,22 in the presence of 0.5 M NaCl. Eight layers on the melamine particles and five or nine layers on the cells were deposited. After assembling of the polyelectrolytes the melamine core and the interior of erythrocytes were destroyed by exposing the colloids to HCl solution of pH 1 and the cells to a 1.2% oxidizing solution of sodium hypochlorite for 20 min, respectively.11,23 The capsules templated on fixed erythrocytes were then covered with one layer of PAH to invert the surface charge from negative to positive. Lipid Membrane Assembly. Vesicles composed of a mixture of DPPC and DPPA with and without cholesterol respectively were prepared from chloroform/ethanol solutions. The organic solvents were evaporated in a rotavap. The deposited film was then dispersed in water to a final lipid concentration of 1 mg/ mL and sonicated above the phase transition temperature at 50 °C for 30 min. Vesicle adsorption and subsequent spreading was performed at 50 °C with a vesicle/capsule mixture under conditions of vesicle excess and resulted in the formation of a lipid layer on top of the polyelectrolyte multilayers. The nonadsorbed vesicles were then separated by centrifugation at 5000g. Finally, the lipid-coated capsules were washed three times in water. Confocal Microscopy. Confocal images were taken with a confocal laser scanning system TCS NT attached to an inverse microscope from Leica (Wetzlar, Germany), equipped with a 100× oil immersion objective with a numerical aperture of 1.4. The fluorescence labeling of the capsules was achieved by adding a minimal amount of TMRE, a membrane-permeable fluorescent dye, to the samples directly under the microscope. The micrographs were processed applying the TCLS software of Leica. The osmotic deformation was studied online, adjusting the salt concentration directly on the microscope glasses in order to define the critical concentration of osmotic response. Capsules templated on MF particles with nearly ideal spherical shapes were used, which allowed following the buckling process easily. For estimation of the critical permeation time, 100 µL of a 0.1 M NaCl stock solution was added under gentle stirring to a 100 µL capsule suspension in water using a 100 µL Hamilton syringe. The final salt concentration was 0.05 M. The flow rate of the stock solution was varied from 12 µL‚min-1 to 2 µL‚s-1. Then the deformation of the capsules was observed with the confocal microscope. Electrorotation Technique. Electrorotation of the capsules was performed in a microchamber with an electrode-electrode

Georgieva et al. distance of 250 µm.16 A computer-controlled generator (FOKUS Giesenhorst, Germany) provided four 90° phase shifted, symmetrical square-wave signals. The measurements were performed over the frequency range between 500 Hz and 32 MHz. The applied field strength was 20 kV/m. The electrorotation spectra of polyelectrolyte (PE) and lipid-PE composite capsules were measured as a function of the bulk conductivity in water and in NaCl solutions. The capsule rotation was recorded by means of a video microscopy system. Since rotational movement is effortlessly observed for ellipsoidal objects, we used capsules templated on erythrocytes, which, mimicking the shape of their template, have a biconcave discoid, slightly ellipsoidal shape. Theoretical electrorotation spectra were calculated, applying the algorithm of Pastushenko et al.24 Single shell and double shell spherical models17 were used for simulation of the spectra of the control and of the lipid-coated capsules, respectively. Although the capsules templated on erythrocytes have an ellipsoidal shape, the error introduced by modeling them as spheres is small since their eccentricity is not large.25 Results and Discussion Erythrocyte- and MF-templated polyelectrolyte capsules are highly permeable for salt ions. Previous electrorotation measurements on erythrocyte capsules of 11 layers showed a conductivity of 1 S‚m-1 of the polyelectrolyte wall.13 This would correspond to an ion permeability of approximately 7 × 10-2 m‚s-1 in 0.1 M electrolyte solution, as calculated from eq 4 assuming a layer thickness of 20 nm. In the case of MF-templated capsules the presence of salt in concentrations above 0.01 M caused an increase of permeability for macromolecules related to the softening of the multilayer26 but did not lead to buckling.27 Even upon further shockwise increase of the concentration of different salts up to 2 M, no osmotic response with deformation could be observed.28 This can be understood, because both erythrocyte- and MF-templated capsules undergo osmotic swelling during the procedure of core removal, which results in a rather porous structure of the polyelectrolyte film.10,12 When such capsules are suspended into salt solutions, an osmotic deformation cannot be induced, as we can see in Figure 1a for melamine-templated capsules. The small ions are free to permeate through the capsule wall, equilibrating very quickly the internal and external ion concentrations. The resulting osmotic pressure difference is too transient and probably too small to be capable of inducing buckling, which occurs on the time scale of plastic deformations. If, however, the capsule permeability for ions is reduced, an osmotic response can be induced. We have shown in previous papers that the assembly of lipids on top of polymer capsules reduces the permeability of the last for small organic ions, for example, 6-carboxyfluorescein,1 although the lipid bilayers still constitute a highly conductive structure (conductivity of 1.5 × 10-5 S‚m-1), as measured by electrorotation,13 and probably the difference in NaCl concentration will not be high enough to create a gradient of ion concentration and osmotic pressure. The coating of melamine capsules with a phospholipid membrane composed of 90% DPPC and 10% DPPA did not induce pronounced capsule deformation when 40 mM NaCl was added as shown in Figure 1b. One has also to consider the time constant of the osmotic response in relation to the time constant of plastic deformations. The faster the ions permeate and equilibrate with the external solution, the less pronounced the deformation should be. Assuming that conductivity or ion permeability is mostly resulting from a limited number of defects in the lipid

Ionic Conductivity in Polyelectrolyte Capsules

J. Phys. Chem. B, Vol. 109, No. 38, 2005 18027

Figure 1. Confocal micrographs showing the osmotic response of MF-templated PSS/PAH capsules with eight layers in 40 mM NaCl solution (a) without lipid coating, (b) coated with DPPC/DPPA (9:1), and (c) coated with DPPC/DPPA/Chol (6:1:3). The fluorescence label is TMRE. The size of all images is 25 µm.

Figure 2. Confocal micrographs of capsules coated with DPPC/DPPA/Chol at 50 mM NaCl. The final concentration was reached with different flow rates of a 100 mM NaCl stock solution to the bulk of 100 µL capsules suspension: (a) 12 µL‚min-1, image size 75 µm; (b) 24 µL‚min-1, image size 55 µm; (c) 36, 60, and 120 µL‚min-1, image size 75 µm.

membranes, due to the interaction of the lipids with the polyelectrolyte support, a further reduction of the permeability hence requires a considerably more perfect lipid bilayer. Given the above-mentioned special features of cholesterol, such as space filling, we expected that its incorporation in the membrane could be a possible means for defect repair. Indeed, confocal micrographs of capsules coated with a mixture of DPPC/DPPA and cholesterol (30% w/w) show that in the presence of 40 mM NaCl the capsules observe buckling (Figure 1c). Further increasing the salt concentration results in complete shrinking of the capsules coated with phospholipids and cholesterol. Performing an initial set of experiments with adding various amounts of salt at once, we found that shape changes started to appear around a jump of ion concentration of 8 mM. Below this critical concentration step, the pressure, caused by the concentration gradient between the capsule interior and exterior, is not large enough to induce a significant degree of buckling. This salt concentration of 8 mM corresponds to a threshold osmotic pressure of approximately 40 kPa. Actually, the threshold pressure has been defined as the pressure necessary to induce buckling of half the capsules. Applying the relation between this threshold pressure and elastic modulus used in ref 19 with a wall thickness of 16 nm (approximately 4 nm per double layer of PSS/PAH) and capsules radius of 2.5 µm, one can calculate an elastic modulus of 240 MPa. This is half the value obtained by Gao et al.19 from osmotic response to PSS solutions and, probably, may be explained by slight variations in the preparation procedure or by the different nature of the capsule surface, in this work an extra lipid layer; in Gao’s work another PSS layer together with a polymer solution in the bulk was present. If a stock salt solution is added to a suspension of the cholesterol lipid coated polyelectrolyte capsules by different flow

rates as described in the Experimental Section, the shape changes occurring have to be related to the time necessary to reach the above-mentioned critical concentration. If the salt concentration equilibrates faster between the capsule interior and the bulk or with the same time constant as the increase of concentration in the bulk, the induced pressure difference will never be high enough to cause buckling. To find the time scale of permeation, the following experiment was conducted: We added stock salt solution with flow rates of 12, 24, 36, 60, and 120 µL‚min-1, corresponding to concentration changes of 0.19, 0.38, 0.55, 0.87, and 1.5 mM‚s-1, respectively, to capsule suspensions. Figure 2 illustrates the absence or presence of osmotic buckling with representative confocal micrographs at the final state. It is evident that, for the slowest applied increase of NaCl concentration, changes in the shape are yet starting to occur. A quite noticeable osmotic response is found when the NaCl addition is conducted with 0.38 mM‚s-1. The larger the rate of concentration increase, the more pronounced was the buckling of the capsules. Now, if we assume that the threshold concentration for collapsing is 8 mM, corresponding to a threshold pressure of πc ) 40 kPa, the time to reach this particular concentration is about 20 s, if a flow rate of 0.38 mM‚s-1 was applied. If salt addition occurs more slowly, the ions will be capable of at least partially equilibrating; consequently, deformations are largely absent, as observed in the experiment with a flow rate of 0.19 mM‚s-1. If the concentration increase is considerably faster, the collapse is complete because the water flux from the capsule interior to the bulk is much faster than salt equilibration. Taking 20 s as a lower limit for the characteristic time for NaCl permeation, we thus can estimate an upper limit for the permeability, P, of the capsule wall. It is natural to assume that

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Georgieva et al.

Figure 3. Electrorotation spectra of (a) (PSS/PAH)2PSS erythrocyte capsules, (b) (PSS/PAH)4PSS erythrocyte capsules, (c) (PSS/PAH)3 capsules coated with DPPC/DPPA/Chol, and (d) (PSS/PAH)5 capsules coated with DPPC/DPPA/Chol. Spectra were measured at bulk conductivities of 1 (squares), 7 (circles), and 21 mS‚m-1 (triangles). The curves correspond to theoretical calculations according to ref 23. Fitting parameters are given in Table 1.

the permeability is determined by the cholesterol-containing phospholipid layer providing the effective barrier for ion permeation. In the approximation of spherical capsules the following equation can be applied:29

P)

V r ‚ln 2 ) ln 2 Atc 3tc

(1)

V, A, tc, and r are the volume, the surface area, the characteristic time of permeation, and the capsule radius, respectively. The lower limit for the characteristic time was estimated as 20 s. On the other hand, it cannot be larger than 40 s. Hence, the calculated permeability ranges between 1.45 × 10-8 and 2.9 × 10-8 m‚s-1. The permeability obtained with eq 1 is generally controlled by both the ion concentration inside the membrane and the diffusion coefficient itself. In the case under consideration certainly the almost negligible ion content inside the lipid layer is responsible for the small permeability. The part of permeability, P*, related only to the ionic mobility and not to ion distribution would be

P* )

cb P cm

(2)

where cb and cm are the ionic concentrations outside and inside the lipid layer, respectively. P* is related to the diffusion coefficient by means of

D ) hP*

(3)

where D is the diffusion coefficient inside a membrane of thickness h. Applying the relationship between ionic diffusion and conductivity, the permeability of 2.9 × 10-8 m‚s-1 can be converted into a conductivity σ ) 1.4 × 10-8 S‚m-1 with eq 4:

2F2z2cmhP* 2F2z2cmhP(cb/cm) 2F2z2cbhP ) ) (4) σ) RT RT RT

with h ) 8 nm. F is Faraday’s number, z2 ) 1 for NaCl, R is the gas constant, and T is the absolute temperature. The lower limit of the permeability of 1.45 × 10-8 m‚s-1 corresponds thus to 0.7 × 10-8 S‚m-1. In eq 4 it was assumed that anions and cations contribute equally to the conductivity. Actually, this must be, in contrast to the case of diffusion, where the coupling of anion and cation fluxes is required by electroneutrality, not necessarily the case. Hence, the calculated value can only be considered as an estimate of the conductivity. Nevertheless, this provides an opportunity to compare the calculated values from the osmotic response experiments with electrorotation measurements. For this purpose we performed lipid/cholesterol coating of capsules of five and nine layers of PSS/PAH templated on fixed erythrocytes. The electrorotation behavior of capsules templated on erythrocytes was described and analyzed in previous papers.13,17 The spectra of the polymer support, in this case capsules with five or nine polymer layers, are shown in Figure 3a,b. Polyelectrolyte capsules show in water a positive, co-field electrorotation in the megahertz region, which generally indicates that the capsules are more conducting than the surrounding electrolyte solution. This was attributed to the wall conductance as a result of its polyelectrolyte nature. With increasing conductivity of the bulk solution, the rotation speed decreases progressively accompanied by a shift of the rotation maximum to higher frequencies. Finally, the direction of the movement changes to negative, antifield rotation as soon as the bulk conductivity exceeds the conductivity of the polyelectrolyte layer. It was found that capsules with nine layers were more conducting than those made by depositing only five layers. This can be seen from the position of the rotation maxima at equal bulk conductivities, which appear at higher frequencies for more conductive particles. For example, we found in water a maximum rotation for capsules of five and nine layers at approximately 0.5 and 4 MHz, respectively. With increasing bulk conductivity this difference decreases slowly but remains in the range of some megahertz.

Ionic Conductivity in Polyelectrolyte Capsules

J. Phys. Chem. B, Vol. 109, No. 38, 2005 18029

TABLE 1: Parameter Values for Calculation of Theoretical Curves in Figure 3a sample

radius, µm

PE layer capacity, µF‚cm-2

PE layer conductivity, S‚m-1

(PSS/PAH)2PSS (PSS/PAH)2PSS, DPPC/DPPA/Chol coated (PSS/PAH)4PSS (PSS/PAH)4PSS, DPPC/DPPA/Chol coated

3 3 3.5 3.5

1.7 1.7 0.85 0.85

0.75 0.1 1 0.15

lipid layer capacity, µF‚cm-2

lipid layer conductivity, S‚m-1

0.16

0.8 × 10-8

0.16

0.8 × 10-8

a

Polyelectrolyte capsules are simulated using a single shell model. Those coated with a lipid layer were explored with the double shell model. The conductivity of the capsule interior was set equal to the conductivity of the bulk for the polyelectrolyte capsules. For capsules coated with a lipid layer, a constant conductivity of the interior of 1 mS‚m-1 was assumed.

The calculated conductivity of the capsule wall was 0.75 and 1 S‚m-1, respectively. The assembly of DPPC/DPPA on top of the capsules caused a negative second peak in the kilohertz range accompanied by a reduction of the rotation speed in the megahertz region.13 Increasing ionic strength results in the progressive decrease of the positive peak and in the shift of the negative one toward higher frequencies. A conductivity of 1.5 × 10-5 S‚m-1 was obtained for the supported lipid layer in this case by the fitting of electrorotation spectra. The spectra of capsules coated with DPPC/DPPA and cholesterol, shown in Figure 3c,d, are very similar to those of pure DPPC/DPPA in water, showing the two previously mentioned peaks, a negative one in the kilohertz region and a positive one in the megahertz region. However, when the ionic strength is raised to 7 mS‚m-1, the behavior becomes quantitatively different, resulting in a change of the sign of the peak from positive to negative in the case of fivelayer polyelectrolyte support and in a vanishing co-field peak in the case of a nine-layer support. Further increases in the ionic strength induced more negative electrorotation values, together with a shift toward higher frequencies, which corresponds to an increase of the difference between the conductivity of the capsules and that of the bulk. The explanation for this behavior is certainly related to the considerably lower permeability for NaCl of the capsules coated with a lipid/cholesterol mixture. The speed of electrorotation and its direction depend on the difference in conductivity between media and the particle rather than on their absolute values. If the permeability for NaCl is sufficiently small in the presence of cholesterol, a concentration gradient between the capsule interior and the bulk is produced when the bulk electrolyte concentration is increased. Assuming a conductivity corresponding to water in the internal volume, we obtain a conductivity value for the phospholipid/cholesterol layer of about 0.8 × 10-8 S‚m-1, which is in rather good agreement with the values obtained from the osmotic experiment for the melamine capsules regardless of the striking conceptual difference in the principles of measurements. Although the value of conductivity obtained in the presence of cholesterol is 3 orders of magnitude lower than the values found earlier for DPPC/DPPA coated capsules without cholesterol, it is still 3-5 orders of magnitude higher compared with biological membranes and black lipid membranes, where values for the area specific resistance on the order of 107-109 Ω‚cm2 corresponding to conductivities of 5 × 10-12 to 5 × 10-14 S‚m-1 assuming a membrane thickness of 5 nm are typical.30,31 However, for solid supported lipid membranes, resistances ranging from 103 to 106 Ω‚cm2 and conductivities from 5 × 10-8 to 5 × 10-11 S‚m-1, respectively, have been reported.32-34 Both osmotic and dielectric experiments showed that lipid membranes supported on capsules have lower ion permeability when cholesterol is incorporated in the membrane. The assembly of phospholipids on a polyelectrolyte matrix results very likely in the presence of defects and free space in the membrane. This

is quite understandable since the lipids are charged and can interact with the polyelectrolytes underneath and this interaction may be locally stronger than the cohesion force in the membrane. Even DPPC, which is zwitterionic, has to be regarded as charged with two charges, positive and negative, capable of interacting electrostatically with the polymer substrate. Phospholipid membranes closely follow the polymer film underneath. This creates a region of significant bending and possibly “free” space in the layer. At least, large distortions from optimal packing can be expected. Consequently, to reduce the electrostatic interactions with the polyelectrolytes, the headgroups should be amphiphilic but uncharged. This is the case for lipids with OH- groups, e.g., for poly(ethylene oxide)s and cholesterol. Cholesterol gives more stability and density to the membranes, occupying free interstitial space. This was also supported by freeze fraction electron microscopy of lipid membranes containing cholesterol on top of polyelectrolyte coated latex particles.35 Probably the incorporation of cholesterol in the lipid membrane will give more bending elasticity to the membrane, and this as a whole will be less affected by interactions of the polyelectrolyte support with lipids at the molecular level. Cholesterol could segregate in areas where through the lipid polyelectrolyte interaction free space could be induced, sealing the membrane. In a way, we can say that cholesterol smoothes the lipid membranes. The low values of permeability found by the two techniques foresees the incorporation of channels and proteins in the lipid structure and the study of selective permeability by either electrorotation or osmotic response. Conclusions By including cholesterol in the formulation of phospholipid membranes on top of polyelectrolyte capsules, we have been able to decrease the capsule permeability as could be proved with the electrorotation technique and the osmotic response technique. This can be ascribed to the fact that cholesterol reduces the coupling to the polyelectrolyte, increases the mechanical strength, and seals holes possibly created via the preparation or via a too strong binding of the phospholipids to the polyelectrolytes. These low conductivities enable future studies of specific ion permeation through membrane integral pumps and channels. The conductivity determined by two independent techniques was derived on the order of 10-8 S‚m-1. This value is 3 orders of magnitude lower than the one found for lipid layers without cholesterol assembled on polyelectrolytes. Acknowledgment. We acknowledge financial support from the German Ministry of Education and Research for Project Nos. 0312011B-C and 0313316A. References and Notes (1) Donath, E.; Moya, S.; Neu, B.; Sukhorukov, G. B.; Georgieva, R.; Voigt, A.; Ba¨umler, H.; Kiesewetter, H.; Mo¨hwald, H. Chem.sEur. J. 2002, 8, 5481.

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