Conductance and Capacitance of Polyelectrolyte and

Received January 14, 2000. In Final Form: May 23, 2000. Polyelectrolyte capsules were fabricated in aqueous media by stepwise adsorption of polyelectr...
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Langmuir 2000, 16, 7075-7081

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Conductance and Capacitance of Polyelectrolyte and Lipid-Polyelectrolyte Composite Capsules As Measured by Electrorotation R. Georgieva,†,‡,⊥ S. Moya,† S. Leporatti,† B. Neu,| H. Ba¨umler,| C. Reichle,§ E. Donath,*,† and H. Mo¨hwald† Max-Planck Institute of Colloids and Interfaces, Golm/Potsdam, D-14476, Germany; Department of Physics and Biophysics, Medical Faculty, Thracian University of Stara Zagora, Bulgaria; Department of Membrane Biology, Institute of Biology, Humboldt University, D-10098 Berlin, Germany; and Institute of Transfusion Medicine, Medical Faculty Charite´ , Humboldt University, Berlin, D-10098 Berlin, Germany Received January 14, 2000. In Final Form: May 23, 2000 Polyelectrolyte capsules were fabricated in aqueous media by stepwise adsorption of polyelectrolytes onto fixed erythrocytes and subsequent template dissolution. Lipid polyelectrolyte composite capsules were prepared assembling lipid layers on these polyelectrolyte capsules. Dipalmitoyl phosphatidyl acid (DPPA), dipalmitoyl phosphatidyl choline (DPPC), and a mixture of both were used. Confocal laser scanning microscopy showed that the lipids form a homogeneous coverage on the capsule surface. An electrorotation technique was used to study the electrical properties of polyelectrolyte and lipid-polyelectrolyte composite capsules. A conductivity of 1 S/m for polyelectrolyte capsule walls was found. Lipid-polyelectrolyte composite capsules yielded conductivities in the range from 10-4 to 10-1 mS/m and capacities of 2.7 µF/cm2 for DPPA and 0.5 µF/cm2 for DPPC. These conductivities of lipid-polyelectrolyte composite capsules were much higher than for black lipid membranes. They increased with the bulk electrolyte concentration, which was attributed to the presence of pores or defects in the lipid structures. The effective area of pores was estimated as 0.01% of the total capsule surface.

Introduction A novel method for the fabrication of polyelectrolytecoated colloids and hollow polyelectrolyte (PE) capsules was recently introduced.1-4 The method employs the electrostatic interaction between oppositely charged stepwise-adsorbed PE species.5 First, a PE film is formed on a colloidal template. Then the template is dissolved resulting in a hollow PE capsule. Besides melamine formaldehyde (MF) colloidal particles4 biological cells have recently also been used as templating cores.6,7 In the case of the biocolloids the core is removed by a treatment with an oxidizing NaOCl solution.6 After core dissolution, a PE capsule is obtained. A number of applications of these novel capsules have been outlined: Polymers have been synthesized within the capsules;8 organic and inorganic solids9 as well as organic solvents10 have been encapsulated.

For controlled and sustained release it is rather interesting to reduce the permeability for small polar species, which in most cases seem to easily diffuse through the polyelectrolyte capsule walls.11 In analogy to the barrier function of biological membranes, it was thus attempted to assemble lipids on the PE capsules to reduce the permeability for ions and neutral small molecules. The lipids form bilayer structures and in some cases also multilayers on the capsule surface.12 The ionic permeability and the integrity of the PE lipid composite wall is closely related to its conductance. The capacity of the lipid layers is a measure of its thickness. Therefore, the technique of electrorotation was applied to explore the electrical properties of composite capsules. Electrorotation is a dielectric spectroscopy technique for single particles. It is a useful tool for the investigation of electric and dielectric properties of colloidal particles and biological cells.13-20 In this method an external rotating

†

Max-Planck Institute of Colloids and Interfaces. Thracian University of Stara Zagora. § Department of Membrane Biology, Institute of Biology, Humboldt University. | Institute of Transfusion Medicine, Medical Faculty Charite ´, Humboldt University. ⊥ Present address: Institute of Transfusion Medicine, Medical Faculty Charite´, Humboldt University, Berlin, Germany. ‡

(1) Donath, E.; Walther, D.; Shilov, V. N.; Knippel, E.; Budde, A.; Lowack, K.; Helm, C. A.; Mo¨hwald, H. Langmuir 1997, 13, 5294. (2) Sukhorukov, G. B.; Donath, E.; Davis, S.; Lichtenfeld, H.; Caruso, F.; Popov, V. I.; Mo¨hwald, H. Polym. Adv. Technol. 1998, 9, 759. (3) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, E.; Knippel, M.; Mo¨hwald, H. Colloids Surf. A 1998, 137, 253. (4) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. Engl. 1998, 37, 2201. (5) Decher, G. Science 1997, 277, 1232. (6) Neu, B. Dissertation Math, Nat. Fakulta¨t, Humboldt University, Berlin, 1999. (7) Voigt, A.; Lichtenfeld, H.; Sukhorukov, G. B.; Zastrow, H.; Donath, E.; Ba¨umler, H.; Mo¨hwald, H. Ind. Eng. Chem. Res. 1999, 38, 4037.

(8) Donath, E.; Ba¨umler, H.; Neu, B.; Caruso, F.; Sukhorukov, G. B.; Moya, S.; Mo¨hwald, H. German Patent Application 1999, DPA-Nr. 19907552. (9) Sukhorukov, G. B.; Da¨hne, L.; Donath, E.; Mo¨hwald, H. Adv. Mater. 2000, 12, 112. (10) Moya, S.; Sukhorukov, G. B.; Auch, M.; Donath, E.; Mo¨hwald, H. J. Colloid Interface Sci. 1999, 216, 297. (11) Sukhorukov, G. B.; Brumen, H.; Donath, E.; Mo¨hwald, H. J. Phys. Chem. B 1999, 103, 6434. (12) Moya, S.; Donath, E.; Sukhorukov, G. B.; Auch, M.; Lichtenfeld, H.; Ba¨umler, H.; Mo¨hwald H. Macromolecules 2000, 33, 4538. (13) Arnold, W. M.; Zimmermann, U. Z. Naturforsch. 1982, 37c, 908. (14) Arnold, W. M.; Schwan, H. P.; Zimmermann, U. J. Phys. Chem. 1987, 91, 5093. (15) Arnold, W. M.; Zimmermann, U. J. Electrost. 1988, 21, 151. (16) Glaser, R.; Fuhr, G.; Gimsa, J. Stud. Biophys. 1983, 96, 11. (17) Egger, M.; Donath, E. Biophys. J. 1995, 68, 364. (18) Hu, X.; Arnold, W. M.; Zimmermann, U. Biochim. Biophys. Acta 1990, 1021, 191. (19) Sukhorukov, V. L.; Zimmermann, U. J. Membr. Biol. 1996, 153, 161.

10.1021/la0000421 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/29/2000

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field is applied to a dispersion of particles. A phase difference and, hence, an angle between the electric field vector and the field induced particle polarization is created as a result of a dispersion process. This produces a torque acting upon the particles causing particle rotation in the frequency range between 101 and 108 Hz. The rotation speed of the particle is measured as a function of the frequency of the external field. An electrorotation spectrum is thus obtained. The polarization vector and the phase difference depend on the particle structure, the conductivity and the dielectric constant, on the conductivity of the external solution, and on the field frequency. As a rule, antifield rotation (against the external field) occurs if, on average, the particle is less conductive than the bulk electrolyte. In this case a minimum in the electrorotation spectrum can be observed. If the particle is more conducting than the surrounding electrolyte it rotates in cofield direction and a maximum of rotation is observed. The frequencies at which the torque has an extremum are defined as characteristic frequencies. If the particle is inhomogeneous a spectrum with more than one minimum and/or maximum may be observed. Each of them corresponds to a specific dispersion process. The advantage of electrorotation compared with other dielectric spectroscopy techniques is that dielectric properties of single particles can be studied. Specifically, in this paper electrorotation was applied to obtain information about the conductivities and capacities of PE and lipid layers of composite capsules. Materials and Methods Materials. The sources of chemicals were as follows: poly(styrenesulfonate, sodium salt) (PSS), Mw 70 000, Aldrich; poly(allylamine hydrochloride), (PAH), Mw 8 000-11 000, Aldrich; poly(diallyldimethylammonium chloride); DPPA (dipalmitoyl phosphatidyl acid), Avanti polar lipids; Sodium hypochlorite solution, ∼12% chlorine, Hedinger; DPPC (dipalmitoyl phosphatidyl choline), and fluorescently labeled DPPC (L-R-phosphatidylcholine (NBD-β-aminohexanoyl)-γ-palmitoyl), were purchased from Sigma. PAH was used as received, whereas PSS was dialyzed against Milli-Q-water (cut off 14 000) and lyophilized before use. The water used in all experiments was prepared in a threestage Millipore Milli-Q Plus 185 purification system and had a resistivity higher than 18.2 M Ω cm Capsule Preparation. Erythrocytes obtained from fresh human blood anticoagulated with EDTA (ethylenediamine tetraacetate) were washed twice in buffered NaCl solution (5.8 mM phosphate buffer, 5.6 mM KCl, 150 mM NaCl, pH ) 7.4), then treated with glutardialdehyde (Grade I, Sigma-Aldrich, Sternheim, Germany) at a final concentration of 2% for 60 min at 20 °C.21 After fixation the cells were washed four times with distilled water. Then PSS and PAH were adsorbed on the cells applying the step-wise assembling protocol.6 After each absorption step the samples were centrifuged in an Eppendorf rotor (Hettich Universal 30F) at 2000 rpm, and washed three times in water. For the preparation of the microcapsules, erythrocytes with 10 layers (PAH/PSS)5 were suspended in a solution of 140 mM NaCl and 1.2% NaOCl. After 20 min of incubation at 20 °C the cellular template was dissolved.6 Polyelectrolyte hollow capsules were obtained. Afterward the sample was washed three times with a 10-2 M NaCl solution. Lipid Assembly. Two different protocols have been applied to adsorb lipids onto PE capsules. In the first protocol the electrostatic attraction between charged phospholipid vesicles and the oppositely charged capsules is used to adsorb the lipids onto the polyelectrolyte multilayer. Lipid vesicles were mixed with a capsule dispersion and incubated for (20) Wang, X.-B.; Huang, Y.; Ho¨lzel, R.; Burt, J. P. H.; Pething, R. J. Phys. D: Appl. Phys. 1993, 26, 312. (21) Ba¨umler, H.; Djenev, I.; Iovchev, S.; Petrova, R.; Lerche, D. Stud. Biophys. 1987, 125, 45.

Georgieva et al. 20 min for adsorption to occur. Then the samples were centrifuged for 20 min at 30 000g and the supernatant was removed. The centrifugation and resuspension procedure was repeated three times to remove any remaining vesicles from the bulk. The idea of the second protocol was to directly form lipid layers on the polyelectrolyte support by means of adsorbing lipid molecules from a saturated methanol solution. Capsules were suspended into a 1 mg/mL solution of lipids in methanol. The methanol was slowly evaporated at 60 °C until the volume was reduced to approximately 1/20 of its initial value of 2 mL. Then 100 µL water was added dropwise in aliquots of 20 µL. The sample was kept for 20 min in the water bath. During this procedure lipid adsorption onto the polyelectrolyte surface is supposed to occur in parallel with vesicle formation. Again, centrifugation and washing cycles followed to remove nonadsorbed lipids. Confocal Laser Scanning Microscopy. Confocal images were taken with a confocal laser scanning microscope “Aristoplan” from Leica (Germany), equipped with a 100× oil immersion objective. For confocal microscopy L-R-phosphatidylcholine (NBD-βaminohexanoyl)-γ-palmitoyl at a concentration of 5% w/w was used as a fluorescent label for the lipid layer visualization. Scanning Force Microscopy. SFM images have been recorded in air at room temperature using a Nanoscope III Multimode SFM (Digital Instrument Inc., Santa Barbara, CA) in contact mode. Microlithographed tips on silicon nitride (Si3N4) cantilevers with a force constant of 0.58 N/m (Digital Instrument) have been used. Scanning Force Microscopy (SFM) images were processed by using the Nanoscope software. Samples have been prepared by applying a drop of the capsule solution onto a freshly cleaved mica substrate. After allowing the capsules to settle the substrate was extensively rinsed in Millipore water and then dried under a gentle stream of nitrogen. Electrorotation. The electrorotation of PE capsules referred to as control below was measured in a microchamber with an electrode-electrode distance of 100 µm.22 For the lipid-coated capsules a “macrochamber” with a distance between the platinum needle electrodes of 1 mm was used.23 A computer-controlled generator (FOKUS Giesenhorst, Germany) provides four 90° phase shifted, symmetrical square-wave signals.23 The applied field strength was kept constant during the experiments and ranged from 5 to 40 kV/m. The electrorotational behavior of PE and lipid-PE composite capsules was investigated as a function of the bulk conductivity. Electrorotation spectra were recorded over a frequency range from 500 to 16 MHz. Separately, frequencies up to 200 MHz were applied for the determination of the characteristic frequency and the maximal rotation speed of the control at high solution conductivities (500 MHz generator, HP 8131A). The capsule rotation was recorded by means of a video microscopy system. Electrorotation spectra were modeled applying the algorithm of Pastushenko et al.24 The torque acting upon a spherical particle consisting of a homogeneous core covered with n layers of different dielectric properties in an external rotating electric field was calculated numerically. Although the capsules have an ellipsoidal shape the error introduced by modeling them as spheres is small since their excentricity is not large.25

Results and Discussion Figure 1 gives an overview about the electrorotational behavior of PE and lipid-PE composite capsules in salt free solution. The PE capsules (curve 1) show a pronounced rotation in the frequency range from 105 to above 107 Hz. They rotate in the same direction as the externally applied electric field. At lower frequencies rotation is not observed. This behavior is typical for a conducting particle suspended in an aqueous solution of smaller conductivity.22 (22) Gimsa, J.; Mu¨ller, T.; Schnelle, Th.; Fuhr, G. Biophys. J. 1996, 71, 495. (23) Neu, B.; Georgieva, R.; Ba¨umler, H.; Shilov, V. N.; Knippel, E.; Donath, E. Colloids Surf. A 1998, 140, 325. (24) Pastushenko, V. F.; Kuzmin, P. I.; Chizmadjev, J. A. Biologicheskije Membranij 1988, 5, 65.

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Figure 1. Typical electrorotation spectra of (PAH/PSS)5 (1), DPPA-(PSS/PAH)5 (2), DPPC/DPPA(95%/5%)-(PAH/PSS)5 (3), and DPPC-(PAH/PSS)5 (4) capsules. The conductivity of bulk solution was 1 mS/m in the curves 1, 3, and 4 and 0.5 mS/m in curve 2.

The lipid coating of the capsules, however, led to qualitative changes of the electrorotation spectrum. DPPC-PE composite capsules rotate in antifield direction over the frequency range from 4 kHz to 1 MHz (curve 4). At higher frequencies no rotation occurs. This behavior is typical for a particle with conductivity lower than the conductivity of the bulk solution.22 DPPA-PE (curve 2) and DPPC/DPPA (95%/5%)-PE (curve 3) composite capsules show antifield rotation, in the frequency range from 4 to 10 kHz and from 0.5 to 100 kHz, respectively. At higher frequencies, however, cofield rotation reappeared. Such electrorotation spectra are characteristic for conducting particles surrounded by a thin isolating layer, in case, that the bulk conductivity is lower than the conductivity of the particle interior. It is the typical rotation behavior one would observe for biological cells.13-19 Hence, it was concluded that the isolating properties of the lipid-PE composite capsule walls indicate the presence of a continuous lipid layer on the PE capsule. The existence of a lipid coat adjacent to the PE matrix was further confirmed by means of confocal microscopy. When fluorescent lipids are used the thin layer of lipids on top of the PE matrix can be visualized. Indeed, Figure 2 shows an image of a DPPA (5% w/w labeled DPPC)coated capsule. A continuous fluorescence with almost constant intensity can be seen over the whole capsule surface. For the DPPC/DPPA mixture an analogous fluorescence distribution was found pointing to a homogeneous lipid coverage. Let us now discuss the electrorotation spectra in more quantitative terms. PE Capsules. It is well-known that the speed and direction of electrorotation of particles depends strongly on the difference between the particle conductivity and the conductivity of the bulk solution.13,15,16,22 At bulk conductivities equal to the conductivity of the particle the rotation disappears. Cofield rotation is expected to occur at solution conductivities lower than the conductivity of the particle and antifield rotation should occur if the solution conductivity exceeds the particle conductivity. Therefore, it is possible to directly assess the particle conductivity by means of varying the bulk electrolyte conductivity. The particular bulk electrolyte conductivity at which the rotation in the MHz range becomes zero should be a measure of the particle conductivity. Therefore, the electrorotation behavior of capsules consisting of 10 layers PAH/PSS was investigated as a function of in-

Figure 2. Confocal laser scanning image of an erythrocyte templated polyelectrolyte capsule covered with DPPA (5% labeled DPPC). The width of the image is 12 µm.

Figure 3. Maximal rotation of (PAH/PSS)5 capsules as a function of conductivity of the bulk solution.

creasing bulk conductivity in the range between 1 mS/m to 1.53 S/m. A considerable decrease of the cofield rotation speed was obtained with increasing bulk solution conductivity. At all bulk electrolyte conductivities below or equal to 0.82 S/m cofield rotation was measurable. At conductivities equal to or larger than 1.53 S/m antifield rotation occurred. The peak of cofield rotation is represented for the conductivity range from 60 mS/m to 1.53 S/m in Figure 3. It was found by interpolation to zero rotation that the polyelectrolyte layer conductivity should be about 1 S/m. This conductivity corresponds roughly to 0.1 M NaCl solution. This is a surprisingly large conductivity. If one calculates the concentration of fixed charges in the film from the mean coverage of 1 mg PE/m2 per layer distributed in a layer with a thickness of 1-2 nm3, this fixed charge should have a concentration of 1-3 M. The observed conductivity of 1 S/m can then be understood assuming that roughly 10% of the PE charges of the layer are not used in the layer build-up. Their mobile counterions would be responsible for the conductance, even if their mobility is smaller than in the bulk electrolyte. It can be that these mobile ions migrate through nanometer-sized pores created by the release of the cell interior during the dissolution process. The conductance of the diffuse part

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of the electrical double layer adjacent to the PE capsule wall may also contribute to the total conductivity. Conductivity measurements on macroscopically supported PE layers yielded very different results. Seven orders of magnitude smaller than reported above conductivity values were found for the conductivity of a PE film consisting of 8 layers PAH/PSS on a cysteaminecovered gold electrode.26 This very high resistance was explained with the absence of free ions in the PE film. Others, however, found such a small resistance of the PE layer that it could not be measured by impedance spectroscopy.27 From the large difference between the capsule wall conductivity measured here and the PE supported multilayer conductivity provided in ref 26 it can be concluded that these structures have different permeability properties although both of them have been originally assembled by means of consecutive adsorption. This difference is currently attributed to the existence of pores and to a modified chemical composition of the capsule walls compared with PE multilayers as a result of the NaOCl treatment.28 The electrorotation behavior of PE capsules was then theoretically modeled. Theoretical calculations showed that the torque and the characteristic frequency are strongly influenced by the solution conductivity, the capsule wall conductivity and the layer thickness. The radius of the particle has almost no influence on the characteristic frequency but determines the torque. The dielectric constant of the capsule wall has no effect on the spectrum at bulk conductivities up to 0.82 S/m but it is important when the bulk electrolyte conductivity is close to the shell conductivity. For example, at a conductivity of 1.53 S/m the theory predicts cofield rotation for pe ) 50 and antifield rotation for pe ) 60. The conductivity of the capsule interior, Gi, cannot be smaller than the conductivity of the bulk electrolyte because the theory would predict antifield rotation in such a case, which is contradictory to the experimental results. The capsules were assumed as electrolyte spheres surrounded by a thin conducting layer with a conductivity of 1 S/m representing the PE film. A particle radius of 4 µm was used. The conductivity of the particle interior was supposed to be equal to the bulk electrolyte conductivity since the polyelectrolyte layer conductance of 1 S/m indicates a large permeability for ions. The only free parameters left are polyelectrolyte layer thickness and the dielectric constant of the PE layer. In Figure 4 the theoretically calculated spectra are compared with the experimental data for three different bulk conductivities. The modeling leads to the conclusion that the experimental spectra could be only described assuming a thickness of the PE layer of 60 nm and a dielectric constant of 60. Other parameter combinations did not provide a reasonably good fit. A dielectric constant of 60 can be explained by the large amount of water present in PE multilayers.29 The polyelectrolyte layer was originally expected to be considerably thinner than 60 nm. About 15 nm for 11 layers were measured by single particle light scattering (SPLS);3 however, these measurements were made on PE-coated colloidal particles and not on capsules obtained by coating red blood cells followed by an oxidative removal of the (25) Gimsa, J.; Wachner, D. Biophys. J. 1999, 77, 1316. (26) Cassier, T.; Sinner, A.; Offenha¨user, A.; Mo¨hwald, H. Colloids Surf. B 1999, 15, 215. (27) Lindholm-Sethson, B. Langmuir 1996, 12, 3305. (28) Moya, S.; Da¨hne, L.; Donath, E.; Mo¨hwald, H. Unpublished results. (29) Ruths, J.; Essler, F.; Decher, G.; Riegler, H. Langmuir, in press.

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Figure 4. Electrorotation spectra of (PAH/PSS)5 capsules at solution conductivities of 1 mS/m (squares), 9 mS/m (circles), and 15 mS/m (triangles). Parameters used for the theoretical spectra (dashed curves): radius of the capsule, R, 4 µm; thickness of the PE layer, hpe, 60 nm; relative dielectric constant of the PE layer, pe, 60 and of the solution, e, 80; conductivity of the PE layer, Gpe, 1 S/m. The conductivity of the particle interior, Gi, was equal to the bulk conductivity, Ge. The experimental and the theoretical spectra were plotted in units relative to the maximal measured rotation and to the maximal calculated torque, respectively.

cytoplasm. When, however, these smaller values of 1530 nm were used for the modeling of the spectra the peak frequencies could not be matched regardless of the assumed dielectric constants. Instead, much lower characteristic frequencies than measured experimentally were calculated at bulk conductivities up to 15 mS/m. So, the conclusion was drawn that the PE wall of the capsules should be thicker than the PE layer on the fixed erythrocyte prior to the decomposition of the template. Some remains of the destroyed template may have caused the apparent thickness increase. AFM measurements were therefore conducted to verify the layer thickness estimates from electrorotation. As it can be seen in Figure 5 the capsules have minimum vertical heights of about 60 nm in the dry state. This corresponds to a single layer thickness of 2.7 nm in the dry state, approximately twice as much as found in capsules prepared on melamine formaldehyde templates.4 When dispersed in water the wall thickness can be even larger because swelling may occur. It has to be kept in mind that the electrorotation measurements refer to the PE capsule wall together with the diffuse part of the double layer. Hence, at very low bulk electrolyte concentrations the film thickness estimated by electrorotation could be larger than the one measured with SPLS because the effective thickness derived from electrorotation is equal to the sum of twice the Debye length and the layer thickness. This apparent change of the layer thickness with the electrolyte concentration may be responsibel for asymmetry of the measures spectra. It has also be kept in mind that polarization of the double layer is an additional factor influencing the shape of the spectrum. Finally, it cannot be excluded either that intrinsic mechanisms of dielectric dispersions exist. Lipid-Polyelectrolyte Composite Capsules. Electrorotational Data. Next, the electrorotation behavior of lipid-PE composite capsules was investigated by varying the conductivity of the bulk electrolyte. The electrorotation spectra of DPPA-PE composite capsules are presented in Figure 6. Now antifield and cofield rotation was observed at all investigated solution conductivities. It can be seen that the capsules rotate in antifield direction at frequencies

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Figure 7. Electrorotation spectra of DPPC/DPPA (95%/5%)(PAH/PSS)5 composite capsules measured as a function of Ge: 1 mS/m (squares); 7 mS/m (circles), and 11 mS/m (triangles). The dashed lines represent theoretical curves. The following parameter were used: R ) 4 µm, hpe ) 60 nm, pe ) 60, Gpe ) 0.05 S/m, l ) 5, hl ) 8 nm, Gl ) 0.002 mS/m at Ge ) 1 mS/m, Gl ) 0.015mS/m at Ge ) 7 mS/m and Gl ) 0.025 mS/m at Ge ) 11 mS/m. Gi was equal to Ge.

Figure 5. (Top) SFM contact mode image of a (PAH/PSS)5 capsule derived from a fixed erythrocyte template. (Bottom) Section, as indicated by arrows in the top image showing the height profile.

Figure 6. Electrorotation spectra of DPPA-(PAH/PSS)5 capsules measured as a function of Ge: 0.5 mS/m (squares); 7 mS/m (circles), and 11 mS/m (triangles). The dashed lines representing theoretical curves were obtained on the basis of a two layer model. The following parameters were used: R ) 4 µm, hpe ) 60 nm, pe ) 60, Gpe ) 0.05 S/m, l ) 15, hl ) 5 nm, Gl ) 0.01 mS/m at Ge ) 0.5 mS/m, Gl ) 0.05mS/m at Ge ) 7 mS/m and Gl ) 0.1 mS/m at Ge ) 12 mS/m. Gi was equal to Ge. Here the subscript l indicates parameters of the lipid layer.

between 4 and 10 kHz in water and from 10 kHz to 1 MHz in the salt solutions. The first characteristic frequency (minimum) shifts to higher values with increasing bulk conductivity. The speed of the antifield rotation of the DPPA-PE capsules was larger in the salt solutions than in water. Cofield rotation was observed in the frequency

range between 30 kHz and 16 MHz in water and between 1 and 16 MHz in salt solutions. The second characteristic frequency (maximum) increases with increasing conductivity of the bulk electrolyte, while the rotation speed decreases. In the low-frequency range from 0.25 to 2 kHz a slow cofield rotation at small bulk conductivities was observed. It was higher at a bulk solution conductivity of 7 mS/m than in water and was not measurable at a bulk conductivity of 12 mS/m. This is typical for the electroosmotically induced low-frequency electrorotation (LFER).30-34 This is primarily caused by the surface conductance and the surface charge of the particle. As expected, LFER was not found for the DPPC-PE composite capsules, since these lipids are not charged. A similar electrorotational behavior as for DPPA-coated capsules with both anti- and cofield rotation present was found for DPPC/DPPA (95%/5%)-PE composite capsules (Figure 7). In salt solutions antifield rotation occurs at frequencies in approximately the same range as for DPPAPE capsules. In water antifield rotation was observed over a frequency range from 4 to 90 kHz. The antifield rotation speed was highest in water and decreased in salt solutions with increasing conductivities. The first characteristic frequency shifted to higher frequencies with increasing bulk conductivity. Cofield rotation was found at frequencies between 100 kHz and 16 MHz in water and between 1 and 16 MHz in salt solutions. The second characteristic frequency increased with increasing conductivity of the bulk electrolyte, while the rotation speed decreased. The antifield rotation of DPPC/DPPA (95%/5%)-PE composite capsules was larger than that of DPPA coated composite capsules, while the cofield rotation was more pronounced for the DPPA-PE capsules than for the mixture. The dependence of the cofield rotation peak on the bulk electrolyte conductivity was identical to that of the DPPA-PE capsules. For both samples approximately the same characteristic frequencies were measured. (30) Grosse, C.; Shilov, V. N. J. Phys. Chem. 1996, 100, 1771. (31) Burt, J. P. H.; Chan, K. L.; Dawson, D.; Patron, A.; Pethig, R. Ann. Biol. Clin. 1996, 54, 253. (32) Maiar, H. Biophys. J. 1997, 73, 1617. (33) Zhou, X. F.; Mark, G. H.; Pethig, R.; Eastwood, I. M. Biochim. Biophys. Acta 1995, 1245, 83. (34) Georgieva, R.; Neu, B.; Shilov, V. M.; Knippel, E.; Budde, A.; Latza, R.; Donath, E.; Kiesewetter, H.; Ba¨umler, H. Biophys. J. 1998, 74, 2114.

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Figure 8. Electrorotation spectra of DPPC-(PAH/PSS)5 composite capsules measured as a function of the bulk conductivity: 1 mS/m (squares), 7 mS/m (circles), and 11 mS/m (triangles). The dashed lines represent theoretical curves. The following parameter were used: R ) 4 µm, hpe ) 60 nm, pe ) 60, Gpe ) 0.01 S/m, l ) 5, hl ) 8 nm, Gl ) 0.0001 mS/m at Ge ) 1 mS/m, Gl ) 0.0007 mS/m at Ge ) 7 mS/m and Gl ) 0.0011 mS/m at Ge ) 11 mS/m. Gi was equal to Ge.

Compared to the uncoated PE capsules both samples show a considerably smaller cofield rotation speed, and the second characteristic frequency of lipid coated capsules was by about 1 order of magnitude shifted to lower frequencies. The experimental spectra of DPPC-PE composite capsules are given in Figure 8. Here, the rotation is only in antifield direction from the kilohertz range up to 10 MHz. Cofield rotation could not be observed. The antifield rotation peak decreased only very weakly with increasing bulk conductivity. The first characteristic frequency increased from 10 kHz for measurements in water to above 100 kHz in the salt solutions. Modeling. Assumptions. It was found by means of differential scanning calorimetry that lipids assembled onto polyelectrolyte films on colloids observe a gel to liquid crystalline phase transition. The presence of this phase transition ensures that lipids are forming a well-defined phase on the capsules and are not just assembled on a single molecular basis.12 Therefore, a theoretical model consisting of two sandwiched layers was used to describe the rotation of the composite capsules. A composite capsule was assumed as an electrolyte sphere surrounded by one conducting PE and one isolating lipid layer. For of the PE layer as shown above a value of 60 for the dielectric constant and a thickness of 60 nm were assumed. With regard to the parameters of the lipid layer the following assumptions were introduced: For DPPA bilayers thicknesses of 3.6 and 6 nm have been reported corresponding to different states of the bilayer in water and in 150 mM buffer solution.35 DPPC bilayers have a thickness of 6.3 nm in water and of about 9 nm in buffer solution.36 Single-particle light-scattering measurements showed an increase of the capsule wall thickness of 3.5-5 nm after adsorption of DPPA.12 When DPPC was assembled the thickness increase was 15 nm.12 If PAH was deposited on the top of DPPC the total thickness decreased by 5 nm.12 This decrease was understood as a result of the removal of external lipid layers or vesicles adsorbed on the surface of the lipid layer by the next incoming polyelectrolyte layer.12 (35) De Meijere, K.; Brezesinski, G.; Mo¨hwald, H. Macromolecules 1997, 30, 2337. (36) Estrela-Lopis, I.; Brezesinski, G. Small-angle X-ray Scattering (SAXS), personal communication.

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Therefore, a thickness of 5 nm for the DPPA layer and of 8 nm for the DPPC/DPPA (95%/5%) layer was assumed for the theoretical modeling of the electrorotation spectra. Antifield rotation in the megahertz frequency range should occur at least at conductivities of the bulk solution higher than 5 mS/m, if the bulk conductivity would be larger than the conductivity of the capsule interior. The interior conductivity of the capsule was assumed equal to the conductivity of the bulk since such behavior was never observed. Hence, as free parameters for modeling the spectra remained the conductivities of the lipid and PE layers as well as the dielectric constant of the lipid layer. The DPPC-PE composite capsule was modeled as a multilayer system consisting of one conducting PE layer and one, two or more isolating lipid layers. The presence of a conducting layer, such as the PE layer, should generally lead to the appearance of cofield rotation and the presence of an isolating layer to antifield rotation. When the conductivity of the conducting layer, however, is not very different from the conductivity of the bulk electrolyte and the isolating layer has a low dielectric permittivity the anti- and cofield rotation peak should come very close and the resulting torque of the capsule could be in antifield direction over the whole frequency range. Modeling Results and Discussions. From the modeling, we found that the antifield rotation peak of DPPA-coated capsules could be described by assuming a dielectric constant of 15 for the DPPA layer corresponding to a capacitance of 2.7 µF/cm2. This is about 5 times higher than the well-known value of 0.5 µF/cm2 for a lipid bilayer.37 In the work of Lindholm-Sethson 27 the capacitance of a DPPA monolayer was found to be 1.2 µF/cm2 when the lipids are assembled on PE multilayers with a negative PE layer on the top. For a DPPA layer on gold a significantly higher but not very reproducible capacity was found.27 This was interpreted as a result of the strong interaction of DPPA with the crystalline gold surface. The high capacity value of 2.7 µF/cm2 found in this work may be attributed an interdigitated state of the DPPA with a thickness of the hydrophobic part of 2 nm. The interdigitated state of DPPA could be the result of the coupling with the oppositely charged polylectrolyte matrix producing larger head to head separations.35 Another factor worth to consider is that DPPA has a small headgroup. The spacing of the DPPA headgroups as a result of the interdigitation could thus result in the presence of water inbetween the headgroups. One to two water molecules per lipid molecule would be sufficient to explain the relatively large dielectric constant of the DPPA layer. For the mixed lipid (DPPC/DPPA) layer, however, dielectric constant of 5 and accordingly a capacitance of 0.55 µF/cm2 were obtained. Concerning the conductivity of the lipid layer, it was calculated that its conductivity was much smaller than that of the bulk solution. However, the lipid layer conductivity increased with increasing bulk conductivity. For example, the conductivity of the DPPA layer increased from 0.01 mS/m in water to 0.1 mS/m in a salt solution with a conductivity of 12 mS/m, otherwise the rotation behavior could not be described. The conductivity of the mixed lipid layer increased from 0.002 mS/m in water to 0.025 mS/m at a bulk conductivity of 11 mS/m. It was generally not possible to obtain a proper theoretical description for the experimental curves assuming a constant conductivity of the lipid layer. (37) Hanai, T.; Haydon, D. A.; Taylor, J. J. Theor. Biol. 1965, 9, 422.

Lipid-Polyelectrolyte Capsules

Langmuir, Vol. 16, No. 17, 2000 7081

The conductivity of the lipid layer is certainly related to ions migrating through it. Defects of lipid layers may significantly contribute to ion penetration in the isolating hydrophobic region of the lipid layer. Thus, it may be expected that the ion content in the lipid phase is proportional to the bulk electrolyte concentration. Another explanation for the large lipid layer conductivity could be that the strong interaction of the charged DPPA molecules with the polyelectrolyte support induced separations between the lipid molecules. In the case of a bilayer coupled only at one side with the polyelectrolyte support this effect may result in an increased number of defects. The coupling of the polyelectrolyte support with the zwitterionic DPPC has probably only a small effect on the separation distance between lipid molecules and, hence, on the probability of inducing additional defects.36 This can be understood because of the weaker Coulombic interaction and the larger headgroup. For mixtures of DPPA with DPPC the effect may be an intermediate one. A significantly lower conductivity of the PE layer of only 50 mS/m was calculated for both samples of lipid-PE composite capsules as compared with the value of 1 S/m obtained for the naked PE capsules. This smaller PE conductivity may be explained by a decrease of the mobile ion concentration within and adjacent to the PE layer as a result of the interaction between the free charges in the PE layer and the headgroups of the lipid molecules. It may be also conceivable that the lipid coating blocked some nanometer-sized pores within the PE layer. In the case of the DPPC coated capsules the modeling yielded a lipid layer conductivity of 0.0001 mS/m in water. It increased nearly proportional with increasing conductivity of the bulk solution. A capacitance of 0.45 µF/cm2 was found. The conductivity of the PE layer supporting the DPPC layer was about 10 mS/m. The lipid layer conductivities obtained above are several orders of magnitude larger than the conductivities measured for black lipid bilayers. For black lipid membranes conductivities between 10-9 and 10-13 mS/m were measured.37-41 Lipid bilayers supported on indium-tin oxide electrodes consisting of dimyristoyl phosphatidyl choline (40%), cholesterol (40%), and 10% positively charged lipids were investigated by Gritsch et al.42 A conductivity value of 5 × 10-7 mS/m was measured in this

case. Lindholm-Sethson27 obtained for DPPA layers supported on bare and polyelectrolyte covered gold electrodes conductivities between 2 × 10-4 and 5 × 10-4 mS/m. Conductivities of about 4 × 10-4 mS/m are common for biological membranes.41,43 From these considerations it was concluded that the lipid coverage of the capsules has defects contributing to the conductivity. Assuming these defects as “pores” filled with bulk solution one could calculate their effective area as follows:

(38) La¨uger, P.; Lesslauer, W.; Marti, E.; Richter, J. Biochim. Biophys. Acta 1967, 135, 20. (39) Benz, R.; Janko, K. Biochim. Biophys. Acta 1976, 455, 721. (40) Baba, T.; Toshima, Y.; Minamikawa, H.; Hato, M.; Suzuki, K.; Kamo, N. Biochim. Biophys. Acta 1999, 1421, 91. (41) Ti Tien, H. In Biomembrane Electrochemistry; Blank, M., Vodyanoy, I., Eds; American Chemical Society: Washington, DC, 1994; p 513. (42) Gritsch, S.; Nollert, P.; Ja¨hnig, F.; Sackmann, E. Langmuir 1998, 14, 3118.

Acknowledgment. This work was supported by a grant from BMBF 03C0293A1. R. Georgieva thanks the DAAD for financial support. We are grateful to Professor Fuhr (Institute of Biology, Humboldt University, Berlin) for support with electrorotation and confocal microscopy.

A1G1 + A2G2 ) (A1 + A2)Gexp

(1)

where A1 is the area of the pores; A2, area of the lipid layer; G1 ) 1 mS/m, conductivity of the bulk solution; G2 ) 10-9 mS/m, conductivity of the BLM; and Gexp ) 10-4 mS/m, the conductivity of the DPPC lipid layer obtained from electrorotation. For DPPC the apparent area of “pores” constitutes approximately 0.01% of the capsule surface. It has to be once more underscored that the presented data about the conductivity of the PE matrix refer to a multilayer after an oxidizing treatment with NaOCl. It is known that this system is different from the initial multilayer assembly. Work is under way to study the electrical properties of melamine templated capsules. Conclusions Electrorotation has been proved to be a valuable method to characterize the dielectric properties of polyelectrolyte capsules and lipid polyelectrolyte composite capsules. The PE wall of capsules templated on erythrocytes represents a conducting layer with a conductivity of about 1 S/m and a dielectric constant of 60. A lipid coating on the top of the PE support yields a considerably lower conductivity of the composite PE layer. Lipid layers assembled on polyelectrolyte capsules had a higher conductivity than BLM. This difference in conductivities has been attributed to the presence of defects. Charged lipid layer showed higher conductivities than layers assembled from zwitterionic lipids. This might be a consequence of the stronger interaction between polyelectrolytes and charged lipids, which can induce defects or possibly interdigitation of lipid molecules due to the mismatch of PE charge and lipid headgroup spacing.

LA0000421 (43) Zimmermann, U. Electromanipulation of Cells 1996, CRC Press: