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An Fd3m Lyotropic Cubic Phase in a Binary Glycolipid/ Water System John M. Seddon,*,† Neelofar Zeb,† Richard H. Templer,† Ronald N. McElhaney,‡ and David A. Mannock‡ Department of Chemistry, Imperial College, London SW7 2AY, U.K., and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Received July 8, 1996. In Final Form: September 3, 1996X The lyotropic cubic phase of space group Fd3m has been observed for the first time in a purely binary amphiphile/water system, consisting of fully-hydrated synthetic 1,2-di-O-alkyl-3-O-(R- or β-D-xylopyranosyl)sn-glycerols. These glycolipids are saturated analogues of naturally-occurring membrane lipids, with the polar headgroup chemically modified to render it less hydrophilic. A number of chain lengths of these Rand β-anomers in the range from C12 to C19 have been studied, and we find that the Fd3m cubic phase is suppressed when the chain length is reduced to C14 or shorter. The relative intensities of the X-ray Bragg peaks are similar to those of previously observed Fd3m phases, which have been shown to have an inverse micellar structure, implying that the glycolipid Fd3m cubic phase described here also consists of the same complex packing of inverse micelles.
Introduction Over the past few decades, a great deal of effort has been expended in clarifying the structures of the various translationally-ordered lyotropic liquid-crystalline phases and their locations in phase diagrams.1-3 It is useful to arrange these lyotropic phases according to their average interfacial mean curvatures, as shown in Figure 1. The lamellar LR (fluid bilayer) phase, with zero mean curvature, occupies a central position in this diagram. The phases to the right of LR have a positive mean curvature (the interface curves toward the hydrocarbon chain region) whereas those to the left are inverse, with negative mean curvature (the interface curves toward the water). In general, a given lipid will only exhibit some of these phases, reflecting the hydrophilic/hydrophobic balance of the particular amphiphile. This balance can be altered for a given lipid by changing system variables such as hydration, temperature, or hydrostatic pressure. Complex three-dimensional phases, usually cubic in symmetry, may be found in the four locations labeled a, b, c, and d (see Figure 1). Those in region d are based upon various packings of spherical or slightly anisotropic micelles (see for example ref 4-6). On the other hand, those in locations b and c are bicontinuous cubic phases, with interwoven fluid porous structures, based upon underlying infinite periodic minimal surfaces (those in location b are the inverse version of those in location c). Surprisingly, (although predicted by a theoretical study7 ), it has only quite recently been established that cubic phases in location a, based upon packings of inverse micelles, also exist.8,9 Such a location might be anticipated for amphiphiles having bulky hydrocarbon chains and * To whom correspondence should be addressed: e-mail
[email protected]. † Department of Chemistry, Imperial College, ‡ Department of Biochemistry, University of Alberta. X Abstract published in Advance ACS Abstracts, October 1, 1996. (1) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1. (2) Tate, M. W.; Eikenberry, E. F.; Turner, D. C.; Shyamsunder, E.; Gruner, S. M. Chem. Phys. Lipids 1991, 57, 147. (3) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221. (4) Eriksson, P.-O.; Lindblom, G.; Arvidson, G. J. Phys. Chem. 1985, 89, 1050. (5) Lindblom, G.; Johansson, L. B.-Å.; Wikander, G.; Eriksson, P.O.; Arvidson, G. Biophys. J. 1992, 63, 723. (6) Gulik, A.; Delacroix, H.; Kirschner, G.; Luzzati, V. J. Phys. II 1995, 5, 445. (7) Charvolin, J.; Sadoc, J. F. J. Phys. 1988, 49, 521.
S0743-7463(96)00664-6 CCC: $12.00
Figure 1. The natural sequence of lyotropic liquid-crystalline phases, arranged according to their average interfacial mean curvature. Cubic phases may be located in regions a, b, c or d (from ref 29). Above is shown the schematic structure of the inverse micellar cubic phase of space group Fd3m. The unit cell contains 24 quasi-spherical inverse micelles, 8 larger ones (polar cores shaded light) and 16 smaller ones (polar cores shaded dark). The remaining volume is filled with the fluid hydrocarbon chains of the lipid. The connecting lines and rods are drawn merely to guide the eye. Adapted from refs 8 and 34.
small, weakly polar headgroups, ideally also with attractive lateral headgroup interactions (such as hydrogenbonding). In fact, the only example of an inverse micellar phase discovered so far, of crystallographic spacegroup Fd3m (No. 227), seemed only to form in hydrated mixtures of a strongly polar lipid such as phosphatidylcholine with (8) Seddon, J. M.; Bartle, E. A.; Mingins, J. J. Phys.: Condens. Matter 1990, 2, 285. (9) Luzzati, V.; Vargas, R.; Gulik, A.; Mariani, P.; Seddon, J. M.; Rivas, E. Biochemistry 1992, 31, 279.
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a very weakly polar amphiphile such as diacylglycerol.10,11 Very recently, the same Fd3m cubic phase has been discovered in a ternary system consisting of an amphiphilic diblock copolymer, p-xylene, and water.12 The structure of the Fd3m cubic phase (Figure 1) has been established by X-ray diffraction7 and by freezefracture electron microscopy.13 It consists of a complex packing of two types of quasi-spherical inverse micelles of different diameters. There are 8 of the larger and 16 of the smaller micelles per unit cell. NMR self-diffusion measurements support a structure based upon a packing of discrete inverse micelles.14,15 The lattice parameter is typically in the range 120-170 Å. A somewhat surprising feature of this structure is that it contains two types of nonequivalent aggregates of different diameters, and hence different interfacial mean curvatures. We had previously suggested16 that the presence of the two lipid components with differing amphiphilicities might facilitate the formation of the Fd3m phase, by the less polar amphiphile being preferentially partitioned into the smaller, more highly curved inverse micelles. However, the results presented in this Letter demonstrate that this possibility for amphiphile partitioning is not a necessary condition for the formation of the lyotropic Fd3m cubic phase and that it can be formed in a purely binary glycolipid/water system, by chemical modification of the sugar headgroup, to lower its hydrophilicity and steric bulk. The effect of this modification is to push the preferred interfacial mean curvature of the lipid layer to a strongly negative value, into the region where an inverse micellar cubic phase can form. Glycolipids are of great interest both biologically, because of their key roles in recognition processes in biomembranes, and in potential pharmaceutical applications, due to their powerful immunomodulating effects.17 Furthermore, for systematic physicochemical studies of self-assembly and lyotropic liquid crystal phase formation, glycolipids offer the possibility of fine tuning the headgroup hydrophilicitysand hence phase behaviorsby making minor modifications to the headgroup chemical structure. Examples of such modifications include varying the number and/or configurations of the hydroxyl groups in the glycopyranosyl ring, the type of anomeric linkage (Ror β-) between the sugar ring and the glycerol, and the chirality at carbon atom C2 on the glycerol (producing two chemically and physically nonequivalent diastereomeric forms). Materials and Methods The 1,2-di-O-alkyl-3-O-(D-xylopyranosyl)-sn-glycerols were synthesized essentially according to previously published procedures.18,19 The lipid samples were chromatographically homogeneous by thin layer chromatography in chloroform/methanol (9:1 (v/v)). The 300 MHz 2D-1H-nuclear magnetic resonance spectroscopy measurements of protonated and deuteriumexchanged samples in DMSO-d6 were used to assign the chemical (10) Seddon, J. M. Biochemistry 1990, 29, 7997. (11) Takahashi, H.; Hatta, I; Quinn, P. J. Biophys. J. 1996, 70, 1407. (12) Alexandridis, P.; Olsson, U.; Lindman, B. Langmuir 1996, 12, 1419. (13) Delacroix, H.; Gulik-Krzywicki, T.; Seddon, J. M. J. Mol. Biol. 1996, 258, 88. (14) Hendrikz, Y.; Sotta, P.; Seddon, J. M.; Dutheillet, Y.; Bartle, E. A. Liq. Cryst. 1994, 16, 893. (15) Ora¨dd, G.; Lindblom, G.; Fontell, K.; Ljusberg-Wahren, H. Biophys. J. 1995, 68, 1856. (16) Seddon, J. M.; Bartle, E. A. In The Structure and Conformation of Amphiphilic Membranes; Lipowsky, R., Richter, D., Kremer, K., Eds.; Springer-Verlag: Berlin, 1992; p 257. (17) Lockhoff, O. Angew. Chem., Int. Ed. Engl. 1991, 30, 1611. (18) Ogawa, T.; Beppu, K. Agric. Biol. Biochem. 1982, 46, 255. (19) van Boeckel, C. A. A.; Visser, G. M.; van Boom, J. H. Tetrahedron 1985, 41, 4557.
structure using the conditions described earlier.20,21 All other analytical data were consistent with the structures depicted in Figure 2a and will be reported in full elsewhere (D. A. Mannock, unpublished experiments). The purity of the samples after the X-ray measurements was assessed by thin layer chromatography using a solvent system of chloroform/methanol (4/1 (v/v)), and no significant chemical degradation was observed. The water used to prepare the samples was HPLC grade. Hydrated lipid samples were examined for optical birefringence using a Nikon Labophot polarizing microscope equipped with a Linkam heating stage. The chain-melting transition between the weakly birefringent Lβ gel phase and the birefringent HII phase was detected by an expansion in volume and an increase in deformability under slight pressure from a spatula. Upon formation of the cubic phase, the samples became uniformly optically isotropic and viscous. All samples were scanned at 2.5 °C/min in excess water (3-10 mg of lipid in 30 µL of H2O) by calorimetry using a Perkin-Elmer DSC-2C differential scanning calorimeter interfaced to a computer. The analysis program determined the transition temperature (estimated accuracy (1 °C) from the intersection of the leading edge of the peak with the baseline and the enthalpy (estimated accuracy (10%) from the area under the peak. The calorimeter was calibrated using indium. X-ray diffraction samples were prepared by transferring 5-10 mg of dry lipid lyophilized from cylohexane into thin-walled glass capillaries (diameter 1.5 mm) and adding sufficient water to ensure that the water concentration was greater than 75 wt %. The capillaries were sealed by using a heat shrink polymer and were then thermally cycled three times between 2 and 95 °C to ensure full and homogeneous hydration (this was assessed by the sharpness and uniformity along their length of the X-ray lines). X-ray diffraction was carried out using a Guinier camera (Robert Huber) operated in vacuum to reduce air scattering. This camera is fitted with a bent quartz crystal monochromator to isolate the Cu KR1 radiation with a wavelength of λ ) 1.5405 Å. The X-rays were produced by a Philips PW 2213/20 generator operated at 40 kV and 30 mA. Temperature regulation of the sample ((0.5 °C) was by electrical heating, employing an electronic controller. In order to record the diffraction pattern as a continuous function of temperature, the film holder was scanned down at different programmed rates (typically 0.2 °C/min). Between the film and the sample, an X-ray beam shield with a narrow horizontal aperture of 3 mm restricts the region of the film exposed to the diffracted beam. The Kodak Scientific Imaging film used to record the diffraction patterns (a stack of one to three films) was developed using standard procedures. The optical densities of the patterns were scanned as a function of the reciprocal spacing s ()2 sin θ/λ, where 2θ is the diffraction angle) using an Agfa Arcus II scanner, then analyzed using the IDL software package (Research Systems, Inc). The measured X-ray spacings, d ) s-1, have an accuracy of (0.5 Å.
Results and Discussion The glycolipid system which we chose to study, the 1,2O-dialkyl-3-O-(R- or β-D-xylopyranosyl)-sn-glycerols (Figure 2a), are structurally very similar to the dialkyl glucosides, which are well-known to adopt inverse bicontinuous cubic and HII phases in excess aqueous solution.22-24 The xylose ring differs from glucose only in that the hydroxymethylene (CH2-OH) group on carbon atom C5 of glucose is replaced by a proton, thereby reducing the headgroup volume and also rendering it less hydrophilic. A previous study had looked at the effect of a different modification to the headgroup of a dialkyl (20) Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Chem. Phys. Lipids 1987, 43, 113. (21) Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Chem. Phys. Lipids 1990, 55, 309. (22) Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N.; Akiyama, M.; Yamada, H.; Turner, D. C.; Gruner, S. M. Biophys. J. 1992, 63, 1355. (23) Sen, A.; Hui, S.-W., Mannock, D. A. Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1990, 29, 7799. (24) Turner, D. C.; Wang, Z. G.; Gruner, S. M.; Mannock, D. A.; McElhaney, R. N. J. Phys. II 1992, 2, 2039.
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Figure 2. (a) Chemical structures of the 1,2-di-O-alkyl-3-O(D-xylopyranosyl)-sn-glycerol R- and β-anomers. (b) X-ray diffraction pattern of fully hydrated di-16:0-R-D-xylopyranosylglycerol at 120 °C, shown as a plot of intensity I (scanned optical density) versus reciprocal spacing s ()2 sin θ/λ). The region from 0.018 to 0.024 Å-1 was scanned from the second film, since it was over exposed on the first film (in order to detect the weaker Bragg peaks). (c) Indexing of the low-angle Bragg peaks of the diffraction pattern of part b. A linear fit, intersecting the origin, is obtained from a plot of the s values of the observed peaks versus x(h2 + k2 + l2) for space group Fd3m. The lattice parameter, from the reciprocal slope of the plot, is a ) 148 ( 1 Å.
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glucosyl lipid, substitution of an O-methyl group for the hydroxyl on carbon C3 of the glucose ring.25 This latter modification, which reduces the hydrophilicity but slightly increases the headgroup volume, was found to promote nonlamellar phase formation, but did not induce any inverse micellar cubic phase to form. We surmised that the structural modification involving replacement of the CH2OH group by a proton might be more favorable to inverse micellar cubic phase formation. This is indeed confirmed by our results reported here for the xylopyranosyl glycolipids. For example, Figure 2b shows the X-ray diffraction pattern from the dihexadecyl (C16:0) R-xylolipid in excess water at 120 °C (the water does not boil although the temperature exceeds 100 °C, because the sample is sealed). The wide-angle region shows a single diffuse band located at a spacing of 4.6 Å, characteristic of fluid hydrocarbon chains. In the lowangle region, 11 Bragg peaks are observed, whose reciprocal spacings are found to be in the ratios x3, x8, x11, x12, x16, x19, x24, x27, x32, x40, and x44. As shown in Figure 2c, a plot of the data fits perfectly the indexing expected for the cubic spacegroup Fd3m.26 All permitted reflections below 444 (x44) are observed, apart from 531, 442, and 533 (it should be emphasized that the intensities of the Bragg peaks from liquid crystal phases invariably fall very steeply with increasing hkl, due to the local liquidlike order). The lattice parameter, given by the reciprocal slope of the linear plot, is a ) 148 Å. This value, and also the pattern of observed intensities of the various Bragg peaks from this binary xylolipid/water system, is very similar to that of the Fd3m cubic phase previously observed in ternary lipid/amphiphile/water systems.9 There is thus almost no doubt that the glycolipid Fd3m cubic phase has the same structure as that previously found in lipid mixtures, as shown in Figure 1. To gain more insight into the lyotropic phase behavior of these xylolipids, we have studied homologous series of fully-hydrated dialkyl R-D-xylopyranosyl glycerols and β-Dxylopyranosyl glycerols, with chain lengths ranging from C12:0 (didodecyl) to C19:0 (dinonadecyl), by polarizing microscopy, calorimetry, and X-ray diffraction (the C17:0 and C18:0 R-anomers were not available for study). A very similar pattern of phase behavior was found for the R- and β-anomers, although shifts in the phase transition temperatures were observed between the two anomers for a given chain length. The results for the β-anomer (Table 1) show that at low temperature each compound adopts the untilted lamellar Lβ gel phase. This is indicated by the appearance in the wide-angle region of a single, quite sharp, and symmetrical X-ray peak at a spacing of 4.1-4.2 Å for each lipid. The Lβ gel phase was observed down to 25 °C in each case. Upon heating to the chainmelting temperature, all of the lipids transform directly to an inverse nonlamellar phase, without forming a fluid lamellar LR phase. The C12-C14 compounds adopt an inverse hexagonal HII phase, which is stable up to at least 100 °C. For the C16 to C18 lipids, however, the HII phase then transforms at higher temperatures to the inverse micellar Fd3m cubic phase. For the longest chain length lipid studied (C19:0), the Lβ gel phase transforms directly to the Fd3m phase upon chain-melting. It should be noted that the presence of cis-unsaturated bonds in the hydrocarbon chains of these glycolipids would be expected to greatly lower the temperatures of both the chain-melting and the nonlamellar transitions. For example, saturated C18:0 (distearoyl) glucopyranosyl glycerol R- and β-ano(25) Trouard, T. P.; Mannock, D. A.; Lindblom, G.; Rilfors, L.; Akiyama, M.; McElhaney, R. N. Biophys. J. 1994, 67, 1090. (26) Hahn, T., Ed. International Tables for Crystallography, Vol. A; Reidel: Dordrecht, Holland, 1983.
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Table 1. Phase Sequences on Heating of Fully Hydrated Dialkyl r- and β-D-Xylopyranosylglycerols as a Function of Chain Lengtha R-anomers chain length C13 C14 C16 C19
transition temperature (°C)/ enthalpy (kJ mol-1) Lβs43.2/29.2sHII Lβs53.1/35.7sHII Lβs59.5/44.1sHIIs90.7/sFd3m Lβs71.3/48.3sFd3m β-anomers
chain length C12 C13 C14 C16 C17 C18 C19
transition temperature (°C)/ enthalpy (kJ mol-1) Lβs36.2/28.8sHII Lβs44.4/30.4sHII Lβs50.9/32.4sHII Lβs61.7/43.2sHIIs74.3/sFd3m Lβs66.3/45.7sHIIs75.0/sFd3m Lβs71.7/50.3sHIIs80.4/sFd3m Lβs78.0/52.4sFd3m
a The transition temperatures and the gel-fluid transition enthalpies were measured by DSC (the HII-Fd3m transition enthalpies were 0.2 kJ mol-1 or less).
mers have gel-fluid transitions at 68.4 and 71.7 °C respectively, and fluid lamellar LR-HII transitions at 74.5 and 73.8 °C, respectively27,28). On the other hand, a cisunsaturated glucopyranosyl glycerol (extracted from Acholeplasma laidlawii) with the same C18 chain length (dioleoyl) was found to have a gel-fluid transition at -15 °C, and an HII phase appeared by 0 °C (or slightly lower).29 This pattern of phase behavior is very similar for the R-anomers (Table 1), with the C13 to C16 xylolipids transforming from the Lβ gel to the HII phase. The C16 compound then transforms at a higher temperature to the Fd3m cubic phase, and the C19 compound melts directly from the Lβ gel to the Fd3m cubic phase. These results demonstrate very clearly that increasing chain length favors formation of the Fd3m cubic phase. There are two basic reasons for this. Firstly, the desire for negative (inverse) mean curvature is normally expected to increase with chain length, due to the increased tendency for splay of the fluid hydrocarbon chains. Secondly, packing constraints become less severe as the chain length increases. Inverse micellar cubic phases have a quite severe packing frustration, arising from the competition between the need to fill uniformly the hydrophobic volume with the fluid hydrocarbon chains and the desire of the interfaces to maintain a uniform mean curvature. A simple measure of the packing frustration of a phase is given by its “packing fraction”, i.e., the fraction of the total volume occupied by the structure elements, taking the structure elements to be close-packed circular cylinders (HII phase) or spheres (inverse micellar cubic phase). For a hexagonal phase the packing fraction is 0.91, whereas for the Fd3m cubic packing it is 0.71. We thus see that packing constraints are much more severe for the Fd3m cubic phase than for the HII phase, and this is another factor which causes the stability of the Fd3m cubic phase to increase with chain length (see Table 1). It is interesting to note that purely binary phospholipid/ water systems appear unable to form inverse micellar (27) Mannock, D. A.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1990, 29, 7790. (28) Mannock, D. A.; Lewis, R. N. A. H.; Sen, A.; McElhaney, R. N. Biochemistry 1988, 27, 6852. (29) Lindblom, G.; Brentel, I.; Sjo¨lund, M.; Wikander, G.; Wieslander, Å. Biochemistry 1986, 25, 7502.
cubic phases, even when the lipid headgroup is weakly polar and/or the water content is very low. Why then can binary glycolipid/water systems adopt such phases? One possibility is that the monolayer bending modulus of glycolipids might be significantly smaller than that of phospholipids, making the chain packing constraints less severe. However, too little data are yet available to assess this point properly. A further possibility is that the xylopyranosyl sugar ring may be able to adopt different conformations in the two nonequivalent inverse micelles of the Fd3m cubic phase, leading to two different effective headgroup hydrophilicities, and hence behaving more like a ternary system. We have recently discovered further examples of other purely binary amphiphile/water systems which adopt the Fd3m cubic phase, both glycolipid-based (M. C. Ward, J. M. Seddon, R. H. Templer, and D. A. Mannock, unpublished results) and nonionic surfactant based (S. A. Roberts, R. H. Templer, J. M. Seddon, G. J. T. Tiddy, and D. Parrott, unpublished results), which means that the results reported here are not peculiar to the xylolipid system, but are a more general phenomenon. Inverse micellar cubic phases have the potential for a wide range of important applications. Apart from biomedical aspects such as providing a periodic matrix for enzyme immobilization, or a vehicle for very slow drug release, there might also be chemical uses such as the fabrication of semiconductor “quantum dot” superlattice arrays.30 Furthermore, biological membranes typically contain significant amounts of phospholipids1,2 or glycolipids3,22,31 which do not on their own spontaneously form fluid bilayers in aqueous solution. Instead, these lipids tend to adopt inverse nonlamellar phases such as the hexagonal HII phase and/or bicontinuous cubic phases.1,2,32-34 There has been intense speculation as to the possible biological roles of such “nonlamellar lipids”, either in the static structure of membranes or in dynamic processes such as fusion.35-37 Convincing evidence for the existence of nonlamellar structures in biomembranes is accumulating.38-41 Acknowledgment. N.Z. was supported by a Scholarship from the High Commission for Pakistan; the results presented in this Letter form part of her Ph.D. thesis (University of London, 1996). This work was supported in part by Grant GR/K20309 to J.M.S. from the EPSRC (U.K.). D.A.M. was supported by an operating grant to R.N.M. from the MRC (Canada). LA960664F (30) Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Science 1995, 270, 1335. (31) Hinz, H.-J.; Kuttenreich, H.; Meyer, R.; Renner, M.; Fru¨nd, R.; Koynova, R.; Boyanov, A. I.; Tenchov, B. G. Biochemistry 1991, 30, 5125. (32) Mariani, P.; Luzzati, V.; Delacroix, H. J. Mol. Biol. 1988, 204, 165. (33) Luzzati, V.; Vargas, R.; Mariani, P.; Gulik, A.; Delacroix, H. J. Mol. Biol. 1993, 229, 540. (34) Seddon, J. M.; Templer, R. H. Philos. Trans. R. Soc. London, A 1993, 344, 377. (35) Ellens, H.; Siegel, D. P.; Alford, D.; Yeagle, P. L.; Boni, L.; Lis, L. J.; Quinn, P. J.; Bentz, J. Biochemistry 1989, 28, 3692. (36) Nieva, J. L.; Alonso, A.; Basanez, G.; Goni, F. M.; Gulik, A.; Vargas, R.; Luzzati, V. FEBS Lett. 1995, 368, 143. (37) Epand, R. M., Ed. Structural and Biological Roles of Lipids forming Non-lamellar Structures. In Advances in Lipid Research; Academic Press, in press. (38) Gulik, A.; Luzzati, V.; DeRosa, M.; Gambacorta, A. J. Mol. Biol. 1988, 201, 429. (39) Meyer, H. W.; Richter, W.; Gumpert, J. Biochim. Biophys. Acta 1990, 1026, 171. (40) Brandenburg, K.; Koch, M. H. J.; Seydel, U. J. Struct. Biol. 1992, 108, 93. (41) Landh, T. FEBS Lett. 1995, 369, 13.
