Langmuir 1997, 13, 2655-2664
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Pressure Effects on Lamellar and Inverse Curved Phases of Fully Hydrated Dialkyl Phosphatidylethanolamines and β-D-Xylopyranosyl-sn-glycerols P. M. Duesing,† J. M. Seddon,*,† R. H. Templer,*,† and D. 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 January 16, 1997X We report on the effects of hydrostatic pressure and temperature on the phase and structural behavior of the complete range of the known, excess water, inverse lyotropic mesophases. This range of interfacial topologies is exhibited by homologous series of saturated C12, C14, and C16 dialkyl phosphatidylethanolamines (PE) and saturated C12 and C16 dialkyl xylopyranosylglycerols (xylolipids) in the p-T region 0-2.5 kbar and 30-130 °C. The PEs cover the mesophases with gentle interfacial curvature while the xylolipids cover the more curved mesophases. We demonstrate that temperature and pressure have noncongruent effects on the structural and the phase behavior. Quantitatively, mesophase lattice parameters and phase boundaries show small but observable differences in their functional dependence on pressure and temperature. Qualitatively, increasing pressure stabilizes inverse bicontinuous cubic phases in C14 PE. At atmospheric pressure only the C12 PE contains such phases. Conversely we find that increasing pressure destabilizes the inverse micellar cubic phase observed in the C16 xylolipid. In general a trend is seen for a stronger pressure effect in eliminating phases with larger packing costs, which we infer is due to differences in the effects of pressure and temperature on the monolayer’s spontaneous mean curvature and its bending rigidity.
Introduction Many biological amphiphiles have a rich lyotropic liquid crystalline phase behavior, exhibiting both lamellar and nonlamellar liquid-crystalline phases.1,2 It is well-known that a substantial proportion of cell membrane amphiphiles form nonlamellar liquid-crystalline phases. For the typical double-chain lipids found in the membrane, the polar/apolar interface curves toward the water (such phases are called inverse or type II). Structures which form with such interfaces are by their very nature porous, and there has therefore been much interest in the possible role of such lipids in the membrane. Particular interest has been shown in the idea that it is the monolayers’ desire for interfacial curvature while still in the lamellar phase which may control the function of bilayer spanning proteins.3 Compelling evidence supporting this idea has been collected in a number of systems.4 It has also been pointed out that events such as cell fusion and division involve intermediate structural forms which are identical to the morphology of some of the inverse lyotropic liquid crystalline phases.1,2,5,6 Most recently a reanalysis of a large number of published electron micrographs of cell membranes has shown that inverse bicontinuous cubic structures abound in cells.7 Given these observations, it is clear that understanding lyotropic phase behavior is a critical element in understanding the behavior of the biological membrane. * To whom correspondence should be addressed. † Imperial College. ‡ University of Alberta. X Abstract published in Advance ACS Abstracts, April 15, 1997. (1) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1 and references therein. (2) Seddon, J. M.; Templer, R. H. Philos. Trans. R. Soc. London, Ser. A 1993, 344, 377. (3) Gruner, S. M. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3665. (4) For example: Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221. Jamil, H.; Hatch, G. M.; Vance, D. E. Biochem. J. 1993, 291, 419. (5) Gruner, S. M. In Liposomes; from Biophysics to Therapeutics; Ostro, M. J., Ed.; Marcel Dekker, New York, 1987; p 1. (6) Bouligand, Y. Colloq. Phys. 1990, C7, 35. (7) Landh, T. FEBS Lett. 1995, 369, 13.
S0743-7463(97)00050-4 CCC: $14.00
Not only are a large variety of phases witnessed in different systems but also phase sequences also vary. In some systems, for instance, increasing temperature gives rise to a phase transition from the fluid lamellar to the inverse hexagonal phase, while in others one observes an inverse bicontinuous cubic phase interposed between these two phases. Nevertheless, there remains a concept that lyotropic phase behavior can be explained in terms of a small set of parameters, irrespective of the precise chemical nature of the molecules. Helfrich8 described the surface curvature energy, gc, associated with amphiphile films, in terms of three curvature elastic parameters: the spontaneous mean curvature, H0, the mean curvature modulus, κ, and κG, the Gaussian curvature modulus
gc ) 2κ(H - H0)2 + κGK
(1)
H is the mean interfacial curvature, equal to half the sum of the principal curvatures at the interface, and K is the Gaussian curvature at the interface, given by the product of the principal curvatures. In a binary system, where all the hydrophobic volume has to be filled by the amphiphile chains, there will be another energetic contribution, quantifying the resultant packing frustration,9,10 which might add further independent parameters. To probe the concept that any energetic description and any resultant set of such phenomenological parameters is sufficient to provide a general explanation of lyotropic phase behavior and can thus predict a universal phase diagram, one needs to scan this parameter space experimentally. Most of the experimental work so far has relied on only temperature and sample composition as the tools to attack this task. In order to tune the phenomenological parameters independently, it would be desirable to have another thermodynamic intensive experimental variable at our disposal. (8) Helfrich, W. Z. Naturforsch. 1973, 28c, 693. (9) Kirk, G. L., Gruner, S. M.; Stein, D. E. Biochemistry 1984, 23, 1093. (10) Duesing, P. M.; Templer, R. H.; Seddon, J. M. Langmuir 1997, 13, 351.
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Pressure is an obvious choice. However, the effects of temperature and pressure on an amphiphilic system are qualitatively very similar. Increasing temperature introduces disorder in hydrocarbon chains, leading to larger chain splay and greater desire for interfacial curvature toward the hydrophilic region. Increasing pressure will encourage denser chain packing, which is generally equivalent to straightening the chains. This then decreases the desire for interfacial curvature toward the hydrophilic region, which means that increasing pressure will cause an increase in the lattice spacing of curved inverse (or indeed lamellar) systems. Thus, it is crudely true to say that increasing pressure cancels out the effects of an increase in temperature (at a rate of typically about 20-30 K/kbar, depending on the chain length and its saturation). However, there are more subtle effects at play that allow interesting new information to be gained from highpressure experiments. A system at higher temperature and correspondingly higher pressure will not be fully identical to the initial system. While for some chosen values it might retain the same preferred amphiphile shape and thus the same value of H0, the bending rigidity must have increased and chain extension must necessarily be more difficult. In particular, bicontinuous cubic phases, where packing constraints are comparatively insignificant,11 should be favored under higher pressures. Similarly inverse micellar phases with their comparatively bad packing properties10 should be penalized. Consequently we might expect an exploration of the complete p-T phase diagram to yield an increase in accessed phases, which would otherwise only be possible through altering the lipid chain length, headgroup, or other chemical properties of the amphiphile molecules. Thus it is these more subtle deviations from direct p-T equivalence which make pressure an interesting experimental variable for work on lyotropic systems. Varying both pressure and temperature in the right way must then provide a capacity to alter two of the proposed phenomenological phase-determining parameters independently or three or more noncongruently. So far, no generally recognized full theoretical description of lyotropic phase behavior exists. Nevertheless, it is a frequent assumption that the phase behavior is dominated by the interfacial curvature energetics. Any description of the interfacial curvature must consist of two competing surface curvature terms, as surface curvature is a two-dimensional problem. In binary phases, a stretching term must be added to quantify the packing frustration. Each of these terms can then be described, to first order, by a Hookean law for small deviations, giving rise to three optimum parameters and three elastic constants. One of each of these can be eliminated to scale, as we are only interested in comparative values, and one surface curvature optimum will vanish for rotationally isotropic amphiphiles.8,12-14 We are thus left with three free parameters, two of which might be interdependent, and thus either a two- or three-dimensional phase space as a direct consequence of current assumptions about curvature and packing frustration dominating the phasedetermining energetics. Using both pressure and temperature will allow us to scan a surface through this space, while only using temperature or pressure would be (11) Anderson, D. M.; Gruner, S. M.; Leibler, S. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5364. (12) Fischer, T. M. J. Phys. II 1992, 61, 327. (13) Templer, R. H.; Seddon, J. M.; Warrender, N. A.; Syrykh, A.; Huang, Z.; Duesing, P. M.; Winter, R.; Erbes, J. Submitted for publication in J. Phys. Chem. (14) Duesing, P. M.; Templer, R. H.; Seddon, J. M. In preparation.
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equivalent to only scanning a line. In either case we will not be able to scan the whole of phase space with a single sample, as the chemical composition will invoke absolute limits on the range. For no value of temperature and pressure can we exceed a certain chain length, nor can we transgress certain values of preferred curvature. Much quantitative modeling remains to be done, before the task of systematically probing this postulated phase space experimentally can be fulfilled. The aim of this work is to show qualitatively that pressure data do contain new information and most importantly that the application of pressure will yield a phase behavior in one system that is equivalent to the phase behavior of a chemically distinct system at another pressure, thereby adding credence to the entire concept of a universal lyotropic phase diagram. Remarkably little pressure work on curved lipid phases has been done to date. This is partly due to the experimental difficulties inherent to work involving pressures of several kilobars. Yet lamellar phases have been studied under pressure and the main transition with its direct bearing on the two-dimensional fluidity of the bilayer has been probed extensively,15-20 shedding light on some pressure effects of inherent interest: Deep sea fish display markedly different membrane compositions to related species subjected to smaller pressures.15 Pressure has been found to reverse the effects of anaesthesia.21 In this work we wanted to commence the systematic study of the effects of hydrostatic pressure on all the known inverse lyotropic mesophases. In particular we wanted to examine the possibility that one might induce the appearance of the inverse bicontinuous cubic phases by applying hydrostatic pressure and that the reverse effect would be observed in the case of the discontinuous inverse micellar cubic phases. As far as we are aware no data have been collected previously on the effects of pressure on the latter phases. The only published account of the possible unfolding of an inverse bicontinuous cubic phase in dioleoylphosphatidylethanolamine22 concluded that its appearance was in all probability a kinetic effect. In order to address these issues, we concentrated on two systems, the ether-linked, saturated double-chain phosphatidylethanolamines (henceforth PE) and the 1,2dialkyl-3-O-(β-D-xylopyranosyl)-sn-glycerols in excess water. Both have been explored in some detail at atmospheric pressure in our research group23-25 and together cover all the stable, excess water lyotropic mesophases observed to date. We selected a homologous sequence of the PE system such that the short chain PE formed inverse bicontinuous cubic phases at atmospheric pressure, but the longer chain lengths did not. Similarly with the xylolipid we selected a sequence of chain lengths which at the longer chain length stabilized the discontinuous inverse micellar phase, (15) MacDonald, A. G. Philos. Trans. R. Soc. London, Ser. B 1984, 304, 47. (16) Braganza, L. F.; Worcester, D. L. Biochemistry 1986, 25, 2591. (17) Wong, P. T. T. Physica B 1986, 140, 847. (18) Prasad, S. K.; Shashidhar, R.; Gaber, B. P.; Chandrasekhar, S. C. Chem. Phys. Lipids 1987, 143, 227. (19) Jones, J.; Winter, R; Grandinetti, P. J.; Driscoll, D. J. Magn. Res. 1990, 87, 536. (20) Bo¨ttner, M.; Ceh, D.; Jacobs, U.; Winter, R. Z. Phys. Chem. 1994, 184, 205. (21) Winter, R.; Christmann, M. H.; Bo¨ttner M. In The Structure and Conformation of Amphiphilic Molecules; Lipowsky, R., Richter, D., Kremer, K., Eds.; Springer Proceedings in Physics 66; Springer-Verlag: Berlin, 1992. (22) So, P. T. C.; Gruner, S. M.; Shyamsunder E. Phys. Rev. Lett. 1993, 70, 3455. (23) Seddon, J. M.; Cevc, G.; Kaye, R. D.; Marsh D. Biochemisty 1984, 23, 2634. (24) Seddon, J. M.; Hogan, J. L.; Warrender, N. A.; Pebay-Peroula, E. Prog. Colloid Polym. Sci. 1990, 81, 189. (25) Seddon, J. M.; Zeb, N.; Templer, R. H.; McElhaney, R. N.; Mannock, D. A. Langmuir 1996, 12, 5250.
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but at the shorter chain length did not. This destabilization of the inverse bicontinuous cubic phases with increasing chain length and stabilization with increasing chain length of the discontinuous inverse micellar cubics appear to be a universal feature of lyotropic mesomorphism. The phase diagrams of three different chain length, saturated phosphatidylethanolamines in excess water were investigated, in the expectation that we would be able to witness the unfolding of a cubic phase in samples with C14 chains. These systems form a major constituent of real cell membranes. The 1,2-dialkyl-3-O-(β-D-xylopyranosyl)-sn-glycerols studied here contained two alkyl chains containing either 12 or 16 carbon atoms, henceforth denoted C12 and C16 xylolipids, respectively. The headgroup of these lipids is significantly smaller and less hydrophilic than the corresponding β-D-galacto- or glycosyl-sn-glycerols, allowing access to more curved phases. Xylolipids have only recently received significant attention, and the compound with C16 chains has been found to feature the first example of an inverse micellar cubic phase in a purely binary system25 and is thus the most appropriate candidate to explore the effect of pressure on the stability of the inverse micellar cubic phase.
for the identification of bicontinuous cubics disallow the determination of many interesting results. The procedure for deciding on the reliability of any data point would ideally involve purity checks after each exposure. Unfortunately such a demand is incompatible with the number of exposures required to probe a two-dimensional phase diagram, as well as the cost of the lipids involved. Instead, doubtful results which were not reproducible with the first exposure on a fresh sample were immediately discarded and the remaining data points carefully scrutinized for an unexpected tendency to prematurely exhibit curved phases, while extrapolating from the data obtained at lower, “safe” pressures. This tendency can be considered to give a very precise indication, as some samples had to be discarded in this way, despite not showing any degeneration through purity checks by thin layer chromatography. The tendency to degrade is a surprising feature, as the samples are quite stable to rather high temperatures at atmospheric pressure. It was thus at first assumed that some external contamination had taken place. After thorough elimination of all possible causes and the occurrence of the same effect even in carefully flame-sealed capillaries which successfully survived pressurization, we must presume that the saturated PE degradation is due to the effects of high pressure and temperature. Fortunately the saturated xylolipids displayed no tendency to decay over the time scale required for p-T scans and a greatly reduced number of samples was thus required.
Materials and Methods
Results
Excess water samples for X-ray diffraction were prepared in thin-walled glass capillary tubes of 1.5 mm diameter. We recorded the mass of the pure lipid addedsabout 6-9 mgsto ensure complete hydration. The sample was centrifuged down to the bottom of the tube, triply distilled, deionized water was added to a minimum mass fraction of 70%, and the sample was centrifuged again and left to equilibrate. Care was then taken to eliminate air bubbles as indicated by lipid floating on the water. The final result was a well-confined sample at the bottom of the tube, white in appearance, with a layer of clear water above it. In order to accommodate the sample into its pressure cell, the capillary tube had to be shortened. Other than for a few reference samples in flame-sealed capillaries, samples were not isolated from the triply distilled, deionized water used as pressurizing medium. The pressure system was predominantly constructed of stainless steel. The X-ray windows were manufactured from beryllium, but this was isolated from water contact by a thin Mylar sheet. The only other material in contact with the pressurising water was the lead O-ring used in the Bridgmann seal to the beryllium windows. The exposed area of lead was extremely small, but we did test to see if this material had any effect on our results, by comparing data taken from the sealed capillary tubes and the normal unsealed type. We found no detectable difference. The samples were then analyzed using small angle X-ray line diffraction in a pressure- and temperature-controlled sample cell, which is described in detail elsewhere.26 Details of the synthesis of the three PE samples have been reported in the literature.23 A disturbing general result in all three PE systems is the degeneration of the samples after long exposures to both high temperature and pressure. The extent of decay is restricted, giving rise to no change in optical appearance or any loss in lyotropic behavior. Instead we simply observe an increased tendency for the formation of curved phases. This is what would occur if we were to add alkane to the system, so we surmize that in this case we are observing the gradual fracturing of hydrocarbon chains. Eventually both the fluid lamellar (LR) and inverse bicontinuous cubic phases (QbII) are lost and replaced by the inverse hexagonal phase (HII). While the reaction rate is slow, and thus useful data can be observed even in p-T regimes where the sample does decay with time, it nevertheless severely restricts the exploration of significant parts of the phase diagram. In particular, the longer exposures needed
(i) PE Phases. (a) Didodecyl PE. The didodecyl PE (DDPE) system has a strong tendency to form bicontinuous cubic phases.23,24 These will in general need longer exposure times, due not only to a larger number of peaks required for strict identification of the symmetry but also to a form factor typically leading to lower peak intensities. A further complication arises through the complex topology of cubic phases. In the first instance they are often reluctant to appear and require longer equilibration times than other mesophases. On appearance, they tend to form large monodomains, at odds with identification on a line source without time-consuming quenching techniques. All these complications contribute to the time required at a certain point in the p-T diagram, which here soon exceeds the time at which sample deterioration becomes evident. The pressure results for the curved phases in the DDPE system are thus restricted. The fluid lamellar to lamellar gel (LR/Lβ) transition as well as the exploration of these phases in all the systems poses no such problems. A p-T plot of this phase boundary, which is typical for all the transitions presented in this paper, is shown in Figure 1a. The phase boundary is linear to within the precision of our measurements with a slope of 25.0 ( 0.4 K kbar-1. This is compared to the other transitions described in this paper in Table 1. Also typically for the main transitions of all the PE systems covered, there is a constant jump in the lamellar lattice parameter, ∆a, associated with the transition which is independent of pressure. In this case the lattice parameter jumps by 4 ( 0.3 Å from 51 Å in the LR phase to 47 Å in the Lβ phase. The lattice parameter of the Lβ phase, in all systems, is rather insensitive to isobaric and isothermal changes, while the effects of pressure and temperature on the lattice parameter of the LR phase are at least twice as large. In DDPE over the pressure and temperature range indicated in Figure 1a the lattice parameter of the Lβ phase lies in the range of 50.5-51.5 Å and varies linearly with isobaric and isothermal changes. The lattice parameter of the LR phase is smaller but varies over a wider range, Figure 2. The pressure and temperature dependence of the lattice parameters in these and all other phases are recorded in Table 2 for all the systems studied.
(26) Duesing, P. M.; Templer, R. H.; Seddon, J. M. Rev. Sci. Instrum., in press.
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Figure 1. p-T phase diagrams in excess water. (a) In all cases the phase transition temperature is determined from the X-ray data and in the case of DDPE the error bars on the Lβ/LR transition are shown to indicate the reliabilty of this phase boundary. The phase transition to the inverse bicontinuous cubic phases, QbII, is shown by a heavy gray line to indicate that the phase boundary is extremely tentative. At higher temperatures we observe a transition to the inverse hexagonal phase. (b) For DTPE in excess water the boundaries on the phase diagram are somewhat tentative, especially those bounding the bicontinuous cubic phases and the precise location of the triple point. (c) The p-T phase diagram for DHPE in excess water. (d) The p-T phase diagram for C12 xylolipid in excess water. (e) The p-T phase diagram for C16 xylolipid in excess water.
At higher temperatures, first a sequence of two QbII phases are found before eventually being destabilized by the HII phase. For the reasons explained above, the cubic transitions are difficult to resolve. In particular, the transition from LR to the Im3m cubic phase proves entirely too subtle to probe under the time constraints imposed by the degeneration of the samples. At atmospheric pressure this phase occurs at lower temperatures than the Pn3m QbII phase but requires longer equilibration times and displays significant coexistence with the LR phase, all of which contributes to the difficulties experienced in making X-ray measurements on this phase. Indeed the original published data on the system23 missed this phase completely. Even further work24 was unclear about the precise
phase boundaries. Tentatively, we claim that pressure gradually destabilises the Im3m inverse bicontinuous cubic phase. However we cannot exclude the possibility that at increased pressures there is simply a requirement for longer equilibration times, which are incompatible with our experimental needs. No reliable data can be obtained on the QbII/HII transition, which occurs in a very unfavorable pressure/ temperature regime, while the LR transition to the Pn3m inverse bicontinuous cubic phase is found to have a slope very similar to the Lβ/LR transition at the more reliable lower pressures (up to 600 bar). Furthermore, the QbII lattice parameter displays the very strong pressure and temperature dependence one would expect, which for the
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Table 1. Phase Boundary Dataa
phase boundary
transition temp at 1 bar (°C)
Lβ/LR LR/QbII DTPE Lβ/LR LR/QbII LR/HII DHPE Lβ/LR LR/HII C12 xylolipid LC/HII C16 xylolipid LC/HII HII/Fd3m
36 91 (Im3m) 55 not applicable 92 67 80 36 59 90.3
system DDPE
change in phase lamellar boundary lattice gradient parameter (K kbar-1) (Å) 25 26 23 23 24 22 25.5 21 21 27.5
4 6
8
a The p-T phase boundary data for the five systems investigated is summarized in this table. The lamellar lattice parameter increases by an amount which is independent of pressure, during the gel to fluid transition.
Figure 3. Effect of (a) isobaric and (b) isothermal changes on the lattice parameter of the Pn3m cubic phase for DDPE in excess water: (a) data taken at 200 bar are shown as open circles and at 400 bar as filled circles; (b) the data are taken at 105 °C. Table 2. Lattice Parameter Dependence on Pressure and Temperature in the Various Phases system DDPE
DTPE DHPE
phase
(∂a/∂T)p/Å K-1
Lβ LR QbII (Im3m) Lβ LR HII Lβ LR HII LC HII LC HII Fd3m
-0.03 -0.1 -0.75 -0.05 -0.1 -0.27 -0.07 -0.15 -0.4 -0.02 -0.15 -0.025 -0.15 -0.35
(∂a/∂P)T/Å kbar-1
(∂T/∂P)a/K kbar-1
