Langmuir 1996, 12, 2111-2114
2111
Notes The “Egg-Carton” Phase: A New Morphology of Complexes of Polyelectrolytes with Natural Lipid Mixtures
Scheme 1. Structure Formula of the Examined Lipid Complex: Note That Soybean Lecithin Contains a Number of Different Lipid Tails and Head Groups Which Are Not Presented Here
Markus Antonietti,* Antje Wenzel, and Andreas Thu¨nemann Max Planck Institut fu¨ r Kolloid- and Grenzfla¨ chenforschung, Kantstrasse 55, D-14513 Teltow-Seehof, Germany Received July 25, 1995. In Final Form: December 4, 1995
1. Introduction In a previous publication, we have described the formation and phase morphology of the complex between soybean lecithin and a cationic polyelectrolyte.1 This complex with its mixture of head groups and tail lengths exhibits, opposite to all one-component synthetic counterparts, a quite unconventional phase structure where the stack of lamellar bilayers undulates with very high amplitudes. In addition, a rubbery behavior of the lecithin complex film accompanied by the depression of the glass transition of the ionic layers to Tg ) 10 °C was observed. This combination of properties allowed large amplitude deformations and mechanical orientation as well as thermomechanical processing of these complexes; consequently, we call these systems “plastic membranes”. Since these undulations already contain ca. 40% of excess area, it was speculated that a further increase of the spontaneous curvature of the lipid film and the related frustration constant destabilizes the stack of bilayer morphology, and a new phase might appear. For soybean lecithin, this can be performed by increasing the relative amount of the phosphatidylinositol lipids with their steric pretentious head groups and a surfactant ratio well-below unity. As in our previous work, we choose for the complexing polymer poly(diallylldimethylammonium chloride), PDADMAC, since it combines good binding properties with an appropriate charge density (the length of a monomer unit is about 0.54 nm and only weakly influences the original bilayer structure). 2. Experimental Section 2.1. Polymer Synthesis, Complex Purification and Film Casting. PDADMAC is made using a standard technique of a water-in-oil dispersion cyclopolymerization, initiated with AIBN.2 With this technique, an un-cross-linked, linear polymer material is obtained in which the repeat unit mainly consist of the fivemembered ring species. The molecular weight was checked by viscometry in 0.5 N NaCl salt solution and was determined to be on the order of Mv ) 76 000 g/mol. Soybean lecithin which is oil free and enriched in phosphatidylinositole (50%) was purchased from Sigma Chemical Co. (ca. 50% L-R-phosphatidylinositol, 20-30% phosphatidic acids, and 10-15% phosphatidylethanolamine, as declared by the supplier). It must be emphasized that soybean lecithin is a natural product; the composition of which is subject to seasonal and regional fluctuations. For a successful repetition of all experiments, one has to select product charges with similar properties and compositions. For complexation, 2.0 g of the lecithin are dispersed in 50 mL of water to form a vesicular phase, and a 1% solution of PDADMAC is added dropwise with stirring until no further (1) Antonietti, M.; Kaul, A.; Thu¨nemann, A. Langmuir 1995, 11, 2633. (2) Hurlock, J. R.; Ballweber, E. G.; Conelly, L. J.; U.S. Patent 3920599, 1974; Chem. Abstr. 1976, 84, 45246.
0743-7463/96/2412-2111$12.00/0
precipitation is obtained. The spontaneously formed crude complex is separated and redissolved in THF. The complex is purified by repeated precipitation from THF solutions in water. As in case of the other lecithin films, complexation with PDADMAC apparently results in a balance of lipids and polymeric repeat units which is about 1:1 with respect to the charged species, only. This was checked by weight uptake. Scheme 1 gives a graphic representation of the chemical structure of this polyelectrolyte-lipid complex. For film casting, the redissolution of the solid complex in THF is casted on a planar glass plate hydrophobically coated with octadecyltrichlorosilane. The two-dimensional geometry of the film is controlled with glass frames of variable size which are mounted on top of the glass plate. After slow evaporation of the solvent at 25 °C, the transparent films are easily removed with the frames and cut. 2.2. Wide and Small Angle X-ray Scattering (WAXS and SAXS). The details of the SAXS characterization follows the scheme given in our previous publication.1 The measurements were performed in a range of 1.0 × 10-2 nm-1 < s < 9.0 × 10-1 nm-1 (the scattering vector s is defined as s ) 2/λ sin Θ where 2Θ is the angle between incident and scattered light). The data were corrected for parasitic scattering (max 2% of the signal). The beam profile was measured without the detector slit and convoluted with the detector slit profile. The resulting slit length profile and the corrected data were used for desmearing using the method via generalized Laguerre orthogonal functions by Burger and Ruland.3 A newly developed procedure using the interface distribution function and the Kirste-Porod formalism4 was used to determine the Porod length lP and the average curvature of the phases.5 No slit width correction was performed because of the small width of the primary beam (integral width of 1 × 10-2 nm-1) compared to the width of the observed scattering peaks. WAXS measurements were performed with a Nonius PDS120 powder diffractometer in transmission geometry. The unique (3) Burger, C.; Ruland, W. Presented at the IXth International Meeting on Small Angle Scattering, Saclay, 1993. (4) Kirste, R.; Porod, G. Kolloid Z. Z. Polym. 1962, 184, 1. (5) Burger, C.; Antonietti, M.; Micha, M. A. Manuscript in preparation.
© 1996 American Chemical Society
2112 Langmuir, Vol. 12, No. 8, 1996
Figure 1. WAXS diffractogram of PDADMAC-Ino50; the scattering vector s is defined as s ) 2/λ sin Θ where 2Θ is the angle between incident and scattered light. features of the diffractometer are a FR590 generator as the source of Cu KR radiation and monochromatization of the primary beam with a curved Ge crystal of the scattered radiation with a CPS120 position sensitive detector. The resolution of this detector is below 0.018°. For our purpose, the measured scattering intensity as a function of the scattering vector was sufficient without further data correction.
Notes
Figure 2. Smeared SAXS diffractogram of PDADMAC-Ino50 in a semilogarithmic presentation corrected for background scattering; s is defined as in Figure 1. Inset: linear presentation of the desmeared data set which is used to demonstrate the sharpness of the peak.
3. Results and Discussion The resulting complex of the inositol-enriched lecithin, PDADMAC-Ino50, is a very brittle material, in sharp contrast to the rubbery appearance of the complex of the natural soybean lecithin mixture, PDADMAC-Lec. With DSC, a broad endothermic transition at -38 °C related to the side chain melting of the lipid tails is observed. This transition is also typical for the parental lecithin; due to the mixture of hydrophobic tails in the lipids, the liquid lamellar LR phase is preserved over the complete temperature range of our measurements. In addition, a glass transition centered at 45.4 °C of the polymeric ionic interlayers was detected. It was already stated that the PDADMAC-Lec complex exhibits a glass transition at Tg ) 10 °C with similar width. We relate this increase with the increased amount of lipid molecules coupled to the polyelectrolyte backbones. The amorphous nature of the alkyl tails of the lipids at room temperature is also supported by the WAXS diffractogram which is shown in Figure 1. The WAXS profile is typical for a liquid packing of tails; the maximum is translated to an area requirement of each lipid tail of Atail ) 0.228 nm2. A similar behavior is well known from polyelectrolyte-surfactant complexes.6 Contrary to WAXS, the SAXS measurements show a very marked and rich scattering behavior which is presented in Figure 2. We observe no less than five peaks which can be indexed according to a lamellar superstructure (stack of bilayers). In addition, a strong but broader peak between the first and second lamellar peak is observed which contradicts a simple lamellar indexing. Such peaks are also observed at solid polyelectrolytesurfactant complexes6-9 and are usually related to frustration wiggles onto the lamellae which are less ordered than the carrying lamellae. The PDADMAC-LEC complex showed only a broad scattering peak and no higher order peaks. Using a stack model according to J. J. Hermans,10 we are able to calculate the average thickness of the alkyl (6) Antonietti, M.; Conrad, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1869. (7) Antonietti, M.; Conrad, J.; Thu¨nemann, A. Macromolecules 1994, 27, 6007. (8) Antonietti, M.; Burger, C.; Effing, J. Adv. Mater. 1995, 7, 751.
Figure 3. Radial density autocorrelation function γ(r) as calculated from the raw scattering data. The beginning of the curve is used in a virial expansion to determine the inner specific surface as well as a measure for the averaged curvature.
phase, d1 ) 3.13 nm, the average thickness of the ionic layer, d2 ) 2.17 nm, and the length of the repeat unit, d ) d1 + d2 ) 5.31 nm. Both values significantly exceed the corresponding thicknesses of the original lecithin complex, which were determined to be 1.45 and 2.35 nm, respectively. This increase is attributed to a better packing of the alkyl tails perpendicular to the lamellae normal and a related increase of the number of head groups per unit area. From the integral width of the first reflection which is in the order of the primary beam width, we can only estimate the correlation length ξ to be significantly larger than 75 nm, which is unusually high for a liquid lamellar mesophase. To our knowledge, it is unusual to observe a fifth-order scattering peak or a ξ being so large for liquid structures. We can deduce from all these data that the superstructure consists of stiff and nonbending lamellae (high bending modulus) which explains the high degree of order and the brittle mechanical behavior. The normalized autocorrelation function of the radial density γ(r) of PDADMAC-Ino50 which is calculated from the scattering data by inverse Fourier transformation via generalized Laguerre polynoms5 is shown in Figure 3. This characteristic function can be expanded according to (9) Antonietti, M.; Burger, C.; Micha, M. A. Submitted for publication to Macromol. Chem. Phys. (10) Hermans, J. J. Recl. Trav. Chim. Pays-Bas 1944, 63, 211.
Notes
Langmuir, Vol. 12, No. 8, 1996 2113
Kirste and Porod4 for a two-phase system, resulting in a series having the relevant topological parameters as the leading coefficients:
γ(r) ) 1 -
1 b r + r3 + O(r4) lP lP
(1)
lP is the so-called Porod length. The Kirste-Porod parameter b is defined via eq 2 as the difference between the mean Gaussian curvature, 〈K〉, and the mean square of the averaged curvature, 〈H2〉.
1 1 b ) 〈H2〉 - 〈K〉 8 24
(2)
The expansion of γ(r) of PDADMAC-Ino50 with these equations using data up to 3 nm results in values of lP ) 2.14 ( 0.01 nm and b ) 0.025 ( 0.005 nm-2. The KirstePorod parameter b might be used to calculate the amplitudes of undulations onto the lamellae. However, this calculation sensitively depends on the assumed shape of the wiggles. On the other hand, it is easy to calculate the roughness or wavyness A/A0 of the bilayers which is defined as
〈
〉
2d1d2 1 A ) ) A0 dlP cos R s0
(3)
Ao/A is the real interface area of the layered structure projected to a plane unit area parallel to the bilayer director. This ratio can also be expressed by an averaged angle R between the elements of the real interface and the bilayer director. For PDADMAC-Ino50, we obtain A/Ao ) 1.20 which is rather high for lamellar structures, but clearly below the value of the plastic membranes.1 Vesicles with standard bending energies of the bilayer show a thermal population of undulations which are characterized by A/Ao ) 1.051.20.11 Undoubtedly, it is not possible to extract the morphology of the wiggles from the broad additional peak in the SAXS diffractogram, only. However, measurements on oriented samples were not possible since the brittleness of the material even above glass transition prohibits orientation by simple mechanical treatment; therefore, different methods have to be found. On the other hand, a closer examination of theoretical predictions is helpful to allow for some speculations on how this undulation structure might look. In a recent paper, Goetz and Helfrich described a phenomenon for a bilayer membrane where an increase of the spontaneous curvature results first in the formation of wiggles which secondly get periodically packed in a quadratic arrangement.12 They named this proposed phase the “egg-carton” phase. Although both works where stimulated independently by different aspects of research, it seems that the Helfrich prediction is closely related to the present data set, since we are able to interpret all our experimental findings within this theoretical approach. In this picture, our extra scattering can be attributed to a (011) packing of wiggles onto the lamellae. A schematic drawing of such a modulated egg-carton phase is given in Figure 4 Assuming a sinusoidal shape of the ozillations, we can even estimate the amplitudes of the wiggles to be in the order of 0.8 nm, which results in the correct values of lP as well as A/A0. We want to mention again that due to the width of the undulation peak and the related low order (11) Evans, E.; Needham, D. J. Phys. Chem. 1987, 91, 4219. (12) Goetz, R.; Helfrich, W. J. Phys. France II, accepted for publication.
Figure 4. Computer model of the phase architecture of PDADMAC-Ino50. The picture shows the lamellae consisting of the polyelectrolyte chains and the lipid head groups which exhibit tetragonal thickness undulations. The presentation of the alkyl tails between the ionic layers is omitted for simplicity. The squarelike arrangement in each layer is stabilized by the A-B-A type packing of undulations between the layers. The distance between the undulations equals the long period of the layers.
of the structure of undulations the proposed structure model is not unique. A more definite assignment requires more ordered samples or high-resolution electron microscopy, both being out of our current possibilities. It is also plain to see that such a phase structure also explains the observed high layer stiffness, since we know from daily life experience on macroscopic objects that a rippled sheet is much harder to bend than a planar structure. 4. Conclusion and Outlook We have described a new phase structure of a polyelectrolyte-lipid complex which is characterized by an extremely well-defined stack-of-bilayer type order carrying a second, less-defined type of order which is attributed to a periodic packing of undulations onto the lamellae. The high degree of order is also seen in the mechanical behavior; although it is noncrystalline and even above its glass transition, this complex is very brittle. This is in strong contrast to all other liquid polyelectrolyte-lipid complexes which are rubbery or at least flexible and ductile. Again assuming that our special three-dimensional model can be transferred to the more general and important case of a membrane structure itself (which is not trivial), it is nice to speculate about some deductions related to the natural origin of these materials. (1) Stiffening a membrane by localized, periodic undulations is the nanomechanical analog to corrugated iron or corrugated cardboard. Mother nature knows how to play “molecular origami”. (2) The transition between the very bendable, wavy plastic membrane phase and the stiff eggcarton phase by exceeding a critical value of the frustration energy or certain lipid components is rather pronouced
2114 Langmuir, Vol. 12, No. 8, 1996
and might be also locally induced by the enrichment of the lipids with inositole head groups. The described example shows again that natural lipid mixtures with their (at least) five components are far from being random mixtures but show pronounced synergistic effects. Although a general phase diagram for the variation of all five components is rather difficult, it is obviously attractive to learn about some of the influences
Notes
of these components on structural and dynamic phase properties by examination of artifically blended lecithin fractions. Acknowledgment. We thank C. Burger for helping with the data evaluation/computer graphics. Financial support by the Max Planck Society is gratefully acknowledged. LA950620R
