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Pressure-jump X-ray studies of liquid crystal


transitions in lipids
John M Seddon, Adam M Squires, Charlotte E Conn, Oscar Ces, Andrew J
Heron, Xavier Mulet, Gemma C Shearman and Richard H Templer
Phil. Trans. R. Soc. A 2006 364, 2635-2655
doi: 10.1098/rsta.2006.1844

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Phil. Trans. R. Soc. A (2006) 364, 2635–2655


doi:10.1098/rsta.2006.1844
Published online 21 August 2006

Pressure-jump X-ray studies of liquid crystal


transitions in lipids
B Y J OHN M. S EDDON *, A DAM M. S QUIRES † , C HARLOTTE E. C ONN ,
O SCAR C ES , A NDREW J. H ERON , X AVIER M ULET , G EMMA C. S HEARMAN
AND R ICHARD H. T EMPLER

Department of Chemistry, Imperial College London, London SW7 2AZ, UK

In this paper, we give an overview of our studies by static and time-resolved X-ray
diffraction of inverse cubic phases and phase transitions in lipids. In §1, we briefly discuss
the lyotropic phase behaviour of lipids, focusing attention on non-lamellar structures, and
their geometric/topological relationship to fusion processes in lipid membranes. Possible
pathways for transitions between different cubic phases are also outlined. In §2, we discuss
the effects of hydrostatic pressure on lipid membranes and lipid phase transitions, and
describe how the parameters required to predict the pressure dependence of lipid phase
transition temperatures can be conveniently measured. We review some earlier results of
inverse bicontinuous cubic phases from our laboratory, showing effects such as pressure-
induced formation and swelling. In §3, we describe the technique of pressure-jump
synchrotron X-ray diffraction. We present results that have been obtained from the lipid
system 1 : 2 dilauroylphosphatidylcholine/lauric acid for cubic–inverse hexagonal,
cubic–cubic and lamellar–cubic transitions. The rate of transition was found to increase
with the amplitude of the pressure-jump and with increasing temperature. Evidence for
intermediate structures occurring transiently during the transitions was also obtained. In
§4, we describe an IDL-based ‘AXCESS’ software package being developed in our
laboratory to permit batch processing and analysis of the large X-ray datasets produced
by pressure-jump synchrotron experiments. In §5, we present some recent results on the
fluid lamellar–Pn3m cubic phase transition of the single-chain lipid 1-monoelaidin, which
we have studied both by pressure-jump and temperature-jump X-ray diffraction. Finally,
in §6, we give a few indicators of future directions of this research. We anticipate that the
most useful technical advance will be the development of pressure-jump apparatus on
the microsecond time-scale, which will involve the use of a stack of piezoelectric pressure
actuators. The pressure-jump technique is not restricted to lipid phase transitions, but can
be used to study a wide range of soft matter transitions, ranging from protein unfolding
and DNA unwinding and transitions, to phase transitions in thermotropic liquid crystals,
surfactants and block copolymers.
Keywords: lyotropic liquid crystals; phase transitions; hydrostatic pressure;
X-ray diffraction

* Author for correspondence (j.seddon@imperial.ac.uk).



Present address: Cavendish Laboratory, University of Cambridge, Cambridge CB2 1TN, UK
One contribution of 18 to a Discussion Meeting Issue ‘New directions in liquid crystals’.

2635 q 2006 The Royal Society


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2636 J. M. Seddon and others

1. Lyotropic phase behaviour

Lyotropic liquid crystals of one-, two- or three-dimensional periodicity


spontaneously assemble when biological amphiphiles are mixed with a solvent
under various conditions of temperature, pressure and hydration (Seddon &
Templer, 1993, 1995). The mesophases formed by double-chain lipids include the
fluid lamellar (La), inverse hexagonal (HII) and inverse bicontinuous cubic phases
(QII). Biologically, the fluid lamellar phase (figure 1a) is ubiquitous, being the
structure upon which cell membranes are based. However, the inverse
bicontinuous cubic phases have become increasingly accepted as not only being
present in certain cell membranes, but as relevant to cell processes involving
membrane fusion (Hyde et al. 1997). To understand how bicontinuous cubic
phases may arise, it is helpful to consider the curvature frustration that may exist
in a fluid lamellar La phase (Charvolin 1990). This arises because a build-up of
conformational disorder in the chain region, for example upon heating, tries to
bend each lipid monolayer away from the bilayer midplane, avoiding additional
water–chain contact (figure 1b). However, this would create voids and is resisted
by the hydrophobic effect.
One of the ways by which the system can relax is to undergo a transition to an
inverse hexagonal HII phase, where the monolayers wrap around into inverse
cylinders, which then pack onto a two-dimensional hexagonal lattice. This
transition from a lamellar to an HII phase is very commonly observed in fully
hydrated lipids upon heating (figure 2a). However, there is still a packing frustration
in this phase (figure 2b), because circular cylinders fill space only to a packing
fraction of 0.907, and this would leave voids comprising over 9% of the volume of the
phase in the centre of the hydrocarbon region, which cannot be tolerated.
Thus, either the interface must develop a non-uniform mean curvature (by
becoming faceted) or some of the chains must deviate away from their preferred
conformational state, in order to fill the hydrophobic volume. For a given lipid,
the effect of reducing the chain length is to make this packing frustration
relatively more severe, and below a critical chain length, the system finds other
ways of developing inverse interfacial mean curvature, without creating costly
voids. This can be achieved by the bilayer deforming to a saddle surface, with
negative Gaussian curvature everywhere (apart from special flat points, where
KZ0). For a symmetrical bilayer, the midplane should correspond to a minimal
surface, having zero mean curvature at all points. Such surfaces can be extended
to form three-dimensionally ordered arrays, known as infinite periodic minimal
surfaces (IPMS). Three of the simplest IPMS are the cubic P, D and G surfaces.
These are related to each other by a Bonnet transformation, which is one-to-one
isometric mapping between identical surface patches. Draping a lipid bilayer
onto these three IPMS leads to the three most common lipid inverse cubic phases
(figure 3), of crystallographic space groups Im3m, Pn3m and Ia3d, respectively
(Luzzati et al. 1993). The lattice parameters of these cubic phases are typically in
the range of 100–300 Å.
When the underlying minimal surfaces are Bonnet-related, the lattice
parameters of these three cubic phases should be in the ratios: aP : aD : aGZ
1.279 : 1.000 : 1.576 (Hyde et al. 1984). Experimental observation of these ratios
implies that the three cubic phases have the same free energy (to second order in
the principal curvatures), when the free energy is dominated by the curvature

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Pressure-induced transitions in lipids 2637

(a) (b)

Figure 1. (a) The fluid lamellar La phase. (b) Curvature frustration in a fluid bilayer upon heating.

(a) (b)

Lmax
Lmin

rw

Figure 2. (a) The inverse hexagonal HII phase. (b) Packing frustration in the inverse hexagonal
phase where a is the lattice parameter; rw, the water core radius; and Lmax and Lmin, the maximum
and minimum chain extension, respectively.

elastic energy and the two halves of the bilayer form parallel interfaces to the
underlying minimal surface (Templer et al. 1998a). This degeneracy is broken if
the two halves of the bilayer tend to form constant curvature interfaces instead.
Since the three underlying minimal surfaces pack space differently (degree of
compactness increasing from P–D–G), the degeneracy may also be broken by
transverse interactions between the lipid layers across the water regions. The
phase sequence with decreasing water content is thus expected to follow the
order Im3m–Pn3m–Ia3d (Templer et al. 1998a; Schwarz & Gompper 2000), and
this is observed experimentally (Templer et al. 1998b). The Ia3d (G) cubic phase
is very common both in type I and type II versions in surfactants, lipids and
block copolymers (Bates & Fredrickson 1999), whereas Pn3m (D) and Im3m (P)
seem only to occur as type II (inverse) versions in lipid systems so far. We (and

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2638 J. M. Seddon and others

Figure 3. The underlying minimal surfaces and skeletal graphs (centres of water channels) for the
inverse bicontinuous cubic phases. (a) Pn3m (D), (b) Ia3d (G) and (c) Im3m (P).

others) have found that for various lipid systems which exhibit La–HII transitions
upon heating, reducing the chain length, typically to approximately C12, usually
induces the formation of inverse bicontinuous cubic phases between the La and
the HII phases, or may suppress the HII phase completely.
Many cellular processes including endocytosis, exocytosis and membrane
budding involve changes in membrane topology. The inverse bicontinuous cubic
phases may be relevant to, or may even act as intermediates in some of these
processes, since it is likely that the mechanism of formation of the cubic phase
from a corresponding fluid lamellar phase has much in common with the
mechanism of cell membrane fusion and fission (Siegel & Banschbach 1990).
Much is now known about amphiphile–water mixtures in equilibrium but, as
yet, rather little is known about their non-equilibrium behaviour. This is
surprising given that in all of the examples we have mentioned, the structural
dynamics of phase transitions in the lyotropic mesophases play a profound role.
Previous studies of lyotropic phase transitions have concentrated on
transformations between different lamellar structures and from lamellar to
inverse hexagonal phases, with remarkably little work being done on transitions
involving cubic phases (Erbes et al. 1994; Qiu & Caffrey 2000; Zana 2005).
However, a complete understanding of the physical processes governing such
transitions, including the nature of any intermediates formed and the
mechanistic routes taken, is essential if we are to further our knowledge of
their possible roles in cellular processes involving membranes. Rather little is
known yet about the mechanisms and pathways of transitions involving cubic
phases. It was first thought that they might follow the same pathway as the
Bonnet transformation, a view which was quickly realized to be unphysical,
since it would necessarily involve tearing and self-intersection of the bilayer
(Hyde et al. 1984). Later, it was suggested that a continuous P–D–G transition
could occur by a stretching mechanism, without any tearing (Sadoc &
Charvolin 1989; Benedicto & O’Brien 1997). Thus, a sixfold junction of water
channels in the P (Im3m) phase can be converted into two fourfold junctions of

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Pressure-induced transitions in lipids 2639

Figure 4. Stretching mechanism for converting (a) D to G and (b) P to D.


‘stretch’

‘untwist’

Figure 5. Visualization of a possible continuous pathway between the QIIG and QIID cubic phases.

the D (Pn3m) phase; each of these can then, in turn, be converted into two
threefold junctions of the G (Ia3d) phase (figure 4).
Recently, it has been suggested that such continuous cubic transitions could
involve non-cubic (tetragonal, rhombohedral) distortions of the underlying
minimal surfaces, yet with the surfaces remaining minimal during the processes
(Fogden & Hyde 1999; Schroeder et al. 2004). We can try to visualize this
continuous process for the G–D phase transition that involves (figure 5), first, a
tetragonal stretch, changing c/aZ1 to c/aZ2, followed by an ‘untwisting’ of the
water channels with a merging of each pair of three-way junctions to form single
four-way junctions.

2. Effects of hydrostatic pressure

In order to predict the pressure dependence of lipid phase transition


temperatures Tt from the Clapeyron equation
dTt DVm T DV
Z Z t m; ð2:1Þ
dp DSm DHm

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2640 J. M. Seddon and others

(a)
0

−0.002

Cp (cal.°C−1)
−0.004

−0.006

−0.008

(b)
0

−2000

−4000
Q (µcal)

−6000

−8000

−10000

20 30 40 50 60
T (°C)

Figure 6. Calorimetric scans of the gel–fluid transition of DPPC/water. (a) DSC scan; (b) PPC scan.
The tilted gel–rippled gel transition at 358C is detected by DSC but not by PPC.

it is necessary to determine the molar entropy DSm and molar volume DVm
changes at the transitions. These can be conveniently measured using a
combination of differential scanning calorimetry (DSC) for the enthalpy DHm
(figure 6a) and the rather new technique of pressure perturbation calorimetry
(PPC) for the volume change DVm (figure 6b). DSC measures heat flow to/from
the sample upon a change of temperature, whereas PPC measures the heat flow
induced by a small change in pressure, which is directly proportional to the
coefficient of thermal expansion aZ(1/V )(vV/vT)p (Lin et al. 2002). The DSC
values from the scan shown in figure 6a for the transition temperature Tt
and enthalpy change DHm at the gel–fluid (chain-melting) transition of the
lipid dipalmitoylphosphatidylcholine (DPPC) in excess water are 41.58C and
33.8 kJ molK1, respectively, yielding a transition entropy DSm of 108 J KK1 molK1.
The PPC value from the scan shown in figure 6b for the volume change DVm is
23.5 cm3 molK1. In combination, our data predict a pressure dependence of the
gel–fluid transition of DPPC/water of 228C kbarK1, in perfect agreement with
the measured pressure dependence of the transition temperature for this lipid
(Kusube et al. 2005). The Clapeyron equation predicts a linear relationship

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Pressure-induced transitions in lipids 2641

between pressure and transition temperature when DSm and DVm are
independent of pressure, or have the same pressure dependence; in practice,
the linearity is often maintained until at least 2 kbar.
There is an interesting consequence of the fact that the Clapeyron equation
contains the ratio of the volume change and the entropy change; a phase
transition with a smaller enthalpy (entropy) change, such as a fluid–fluid
transition, also tends to have a correspondingly smaller volume change, and thus
the pressure dependence of the transition temperature is little changed. In
practice, for fluid lamellar–bicontinuous cubic, lamellar–hexagonal and hexago-
nal–inverse micellar cubic phases, the value of dTt/dp lies within the narrow
range of 20–308C kbarK1 (Duesing et al. 1997).
The possibility that pressure might induce topological transitions in
membranes was suggested by Gruner and co-workers, although for the lipid
system studied, they concluded that pressure increased the rate of formation of
the cubic phase, rather than inducing its formation (So et al. 1993). For the
phospholipid ditetradecylphosphatidylethanolamine/H2O, we found that QIID and
QIIP inverse bicontinuous cubic phases (space groups Pn3m and Im3m) were
induced to form between the fluid lamellar La and HII phases when the pressure
was increased above 700 bar (Duesing et al. 1997). On the other hand, for 1 : 2
phosphatidylcholine/fatty acid mixtures in water, having a range of symmetric
chain lengths, pressure-induced formation of cubic phases was not observed
(Winter et al. 1999).
The application of hydrostatic pressure to lyotropic phases in excess water
tends to increase the lattice parameters of the phases. This effect is caused by the
increase in conformational order of the hydrocarbon chains by pressure, and is
the opposite effect to that of increasing temperature. The effect is generally small
for lamellar phases (less than 2 Å kbarK1), slightly larger for the HII phase, but
can be as much as 80 Å kbarK1 for inverse bicontinuous cubic phases, where the
reduction in chain splay tends to reduce the magnitude of the (negative)
interfacial curvature, thereby swelling the phase if it is in contact with an excess
water phase (Duesing et al. 1997; Winter et al. 1999). This can provide a method
for isothermally tuning the diameter of the water channels of lipid bicontinuous
cubic phases, which could be useful for certain biotechnological applications.
To second order in the principal curvatures, the curvature elastic energy per
unit area of a thin amphiphile film is given (Helfrich 1973) by
gc Z 2kðH KH0 Þ2 C kG K; ð2:2Þ
where H and K are the surface-averaged mean and Gaussian curvatures, H0 is the
spontaneous mean curvature, and k and kG are the mean curvature (bending)
and Gaussian curvature (saddle-splay) moduli, respectively. k is a measure of the
energy required to bend the film where kG is a measure of the energy cost of
changing its Gaussian curvature; from the Gauss–Bonnet theorem, this second
term integrates to a constant value unless a change occurs in topology. A positive
value of kG favours saddle deformations of the film, since these have negative
Gaussian curvature K. For systems which tend to form inverse phases, for the
lipid monolayer, the spontaneous curvature H0 is negative and k is positive;
typical values for monoolein are K0.025 ÅK1 and 1.2!10K20 J (approx. 3 kT),
respectively (Vacklin et al. 2000). On the other hand, kG for a lipid monolayer in
an inverse cubic phase is negative, with typical values of K0.75k (Templer et al.

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2642 J. M. Seddon and others

1998c) or K0.83k (Siegel & Kozlov 2004). For a symmetric lipid bilayer, H0
is zero (by symmetry), kb is twice the value for a single monolayer, but a more
complex form is found for the Gaussian modulus,
kbG Z 2ðkG K4kH0 [Þ; ð2:3Þ
where all the terms on the right-hand side, including the thickness [, refer to the
lipid monolayer. Since the second term on the right-hand side is overall positive
for a lipid that tends to form inverse phases (negative monolayer H0), kbG can be
positive even when kG for a monolayer is negative. This is one of the main driving
forces behind the formation of inverse bicontinuous cubic phases in lipid systems
(Seddon & Templer 1993; Templer et al. 1998a).
Our results (Duesing et al. 1997; Winter et al. 1999) indicate that increasing
pressure tends to reduce the magnitude of the spontaneous curvature H0,
increase the bending rigidity k and increase the rigidity of chain extension. We
surmise from the pressure-induced formation of inverse bicontinuous cubic
phases (Duesing et al. 1997) that pressure also tends to increase kbG , and thereby
stabilizing inverse bicontinuous cubic phases; although this is difficult to predict
from equation (2.3), as the effect of pressure on kG is not known, and the effect of
pressure on k and H0 will tend to cancel out. In contrast, Mariani and co-workers
(Mariani et al. 1996; Pisani et al. 2001, 2003) report similar effects on H0, but
deduce that k decreases with pressure, and that the ratio kG/k is positive and
increases strongly with pressure (from 0.14 to 1 by 1.8 kbar).

3. Pressure-jump X-ray diffraction

We have used the X-ray pressure-jump technique (Mencke et al. 1993; Österberg
et al. 1994; Erbes et al. 1996; Winter et al. 1999; Winter 2002; Winter & Koehling
2004) to investigate the rate and mechanism of lyotropic phase transitions in
monoglyceride/water and fatty acid/phospholipid/water systems, by monitoring
the time evolution of these structural conversions using time-resolved X-ray
diffraction. The use of pressure as a trigger mechanism has several advantages:
(i) the solvent properties are not significantly altered, (ii) pressure propagates
rapidly, indicating that equilibrium is achieved rapidly, and (iii) pressure-jumps
can be both in the pressurization and depressurization directions. The Dortmund
millisecond pressure-jump X-ray cell was used for our experiments (Woenckhaus
et al. 2000). This cell has flat diamond windows that can be used at pressures up
to 8 kbar, and the pressure-jumps occur in 5–7 ms. In order to reduce the
attenuation of the X-ray beam by the diamond windows, it is best to carry out
the experiments at short wavelengths, such as 0.75 Å (17 keV), which gives
typically a transmission of 65%. Beamline ID02 at the ESRF is ideally suited to
these experiments, as will be beamline I22 at the Diamond synchrotron, once this
is operational.
We have used time-resolved X-ray diffraction to monitor transitions, induced
by pressure-jumps, between various inverse lyotropic phases of a 1 : 2
dilauroylphosphatidylcholine/lauric acid (DLPC/LA) system in 50 wt% water
(Squires et al. 2000, 2002, 2005; Seddon et al. 2003). The pressure–temperature
(p–T ) phase diagram (figure 7) of this sample was first assessed by static X-ray
diffraction measurements.

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Pressure-induced transitions in lipids 2643

65 H2O
+ HO C O
O
HII O
T (°C)

D O O
G -P
+ O O
O
O
D O
HO C
60 N

0 0.5 1.0
p (kbar)

Figure 7. The pressure–temperature ( p–T ) phase diagram of 1 : 2 DLPC/LA in 50 wt% water. The
exact position of the G–D phase boundary was found to be somewhat dependent on sample history.

We initially studied transitions between the G and D inverse bicontinuous cubic


phases, and between D and the inverse hexagonal HII phase. Typical small-angle
X-ray diffraction patterns from the G and D cubic phases are shown in figure 8. The
effect of a pressure-jump from 600 to 240 bar at TZ59.58C is shown in figure 9.
The transition from the G to the D cubic phase is clearly seen from the changes
in the diffraction patterns. The lattice parameters of the coexisting G and D
phases when the D phase initially forms are in the ratio 1.58–1.59G0.015, very
close to the Bonnet ratio of aG/aDZ1.576. Plots of the peak intensities with time
(not shown) are fitted quite well by single exponentials, indicating that the
transition appears to follow first-order kinetics. For experiments with an end
pressure of 290 bar, increasing the temperature from 57.5 to 62.58C decreased the
transition half-time from 4 to 0.3 s. Apparent activation energy can be calculated
from the temperature dependence, but the interpretation of the resulting value is
open to some questions. The transition kinetics are also sensitive to the
magnitude of the pressure-jump. The half-time at TZ59.58C with a starting
pressure of 600 bar increases from approximately 3 to 180 s on changing the final
pressure from 240 to 360 bar. Thus, the rate becomes slower when we jump less
deeply into the D phase. This is consistent with the thermodynamic driving
force becoming larger; further, the final pressure takes the sample away from the
phase boundary (Erbes et al. 2000; Squires et al. 2005).
Another interesting feature of the diffraction patterns is that they show
evidence for transient, weak additional peaks, labelled (a) and (b) in figure 9,
which are not associated with either the G or the D cubic phases. The first such
peak (a), close to a spacing of 54 Å, appears 1–2 s after the pressure-jump and only
survives for approximately 0.5 s. A long-lived intermediate peak (b) is observed at
a spacing of 51 Å. We have now established that the latter is the first-order peak of
a transient HII phase, but we have not yet been able to identify the initial, short-
lived peak. There are two possible roles of these intermediate structures: (i) they
could be true intermediates in some well-defined pathway between the initial and
final cubic phases, or (ii) they could transiently form as coexisting phases, possibly
in order to act as sources/sinks of water. This could facilitate the cubic–cubic
transition by allowing it to occur initially at constant curvature (as previously
mentioned, the G, D and P cubic phases fill space with differing degrees of
compactness, and thus have different water contents at equivalent curvatures).

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2644 J. M. Seddon and others

800 500
√2
G (Ia3d) √6 D (Pn3m)

600 400

√3
300
400

200
√8 √22
200 √24
√20 √26 √6 √9
√16 100
√4 √8
√14
0
0

Figure 8. Small-angle X-ray diffraction patterns of the G (Ia3d ) and D (Pn3m) cubic phases. Both
exposure times were 0.1 s. The squared ratios of the reciprocal spacings of the observed peaks are
indicated on the integrated one-dimensional patterns (insets). The lattice parameters are 176.2 and
108.5 Å.

On carrying out pressure jumps from D to a region of coexisting (DCHIICexcess


water; see figure 7), we found evidence for the transient formation (surviving for
some tens of seconds) of a P (Im3m) cubic phase, with a lattice parameter of 138.5 Å
(Squires et al. 2000). The ratio of the P and D cubic phase lattice parameters was
1.276G0.015, which is very close to the Bonnet ratio aP/aDZ1.279.
We have also studied the kinetics of the lamellar La–G (Ia3d ) cubic phase
transition of the same 1 : 2 DLPC/LA/50 wt% water system, induced by
pressure-jumps (Squires et al. 2002). The time-scale of the transition was found
to vary from 4 to 193 s for the small range of temperatures and pressure-jumps
studied so far. During this transition, transient intermediate X-ray peaks were
also observed, which we tentatively ascribed to an intermediate D cubic phase,
but this interpretation is still somewhat speculative at this point in time.

4. Batch processing/analysis of time-resolved X-ray data

The IDL-based AXCESS software package, developed in our laboratory, allows for
batch processing and analysis of the large X-ray datasets produced by pressure-
jump synchrotron experiments. It has a widget-based front end for user
accessibility, and can process thousands of images sequentially, allowing for a
large reduction in analysis time.
Figure 10a shows a small-angle diffraction pattern from a silver behenate test
sample in a highly ordered lamellar mesophase. The program automatically finds
the centre of the image and integrates over a butterfly-shaped area to produce a

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Pressure-induced transitions in lipids 2645

(b)
10000
÷2
1000 ÷3
÷4 ÷6 ÷8 ÷9
100

10

28
t (s–1)

59.1
41.4
29.1
20.4
14.4
10.1
7.10
5.00
3.40 ÷22
2.20 ÷6 ÷8 ÷16
1.40 ÷20 ÷24
÷14 ÷26
0.70 (a)
0.20
–0.20
0 0.01 0.02 0.03 0.04 0.05
s (Å–1)

Figure 9. Time-resolved X-ray diffraction plots of the G–D transition induced at TZ59.58C by a
pressure-jump from 600 to 240 bar (Squires et al. 2005). The intensities are plotted logarithmically
to emphasize the weak peaks. The time increment between successive 0.1 s frames increases
geometrically from bottom to top.

one-dimensional plot of intensity versus pixel number, as shown in figure 10b.


Each peak in this one-dimensional plot may be fitted individually within pre-
specified constraints to a specified functional form, for example a modified
Gaussian. Indexing of these peaks allows us to identify the symmetry of the lipid
mesophase, provided the system has been calibrated using a known standard,
such as silver behenate (dZ58.38 Å), to assign a d-spacing to the phase.
Following a pressure-jump, a sequence of diffraction images are generated. Such
a sequence may be integrated sequentially by the program producing a ‘stacked’
plot of one-dimensional images with time. Figure 10c shows such a plot for a typical
cubic–cubic transition, the QIID –QIIG transition in 1-monoolein (1-MO), following a
pressure-jump from 470 to 1500 bar at 60.58C. Such stacked plots can be viewed
from a variety of orientations and are a useful visual aid when analysing changes in
intensity and spacing occurring during the transition. Figure 10d shows the fitting
of the O2, O3, O4 and O6 peaks characteristic of the QIID cubic phase. Initial peak
constraints must be manually set; however, if the lattice parameter changes with
time, then the system may be set up so that the constraints follow the moving peaks.
The d-spacing of each image is then automatically generated and an output file is
created, showing the lattice parameter as a function of image number, along with
the indexing of the phase and the fit of each peak analysed. This offers a very
considerable reduction in the analysis time (figure 10e).

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2646 J. M. Seddon and others

Figure 10. AXCESS software package for batch processing/analysis of X-ray patterns.

5. Lamellar La to Pn3m cubic phase transition

We have studied the fluid lamellar to Pn3m (QIID ) inverse bicontinuous cubic
phase transition, effected by means of jumps in temperature and pressure, for
the single chain amphiphile monoelaidin in excess water (Conn et al. 2006).
On increasing temperature or decreasing pressure, the system displays the
phase sequence Lb–La–QIIP –QIID –HII (Czeslik et al. 1995). In particular, the fluid
lamellar to Im3m cubic (QIIP ) transition is interesting, as it is not well defined,
proceeding via a kinetically hindered intermediate, termed X, of unknown
structure and characterized by a broad featureless ring of scatter at low angle in
the diffraction pattern (Caffrey 1987).

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Pressure-induced transitions in lipids 2647

(a) (b)
1717
806 QIIP(2) QIIP
÷4 647 ÷4 Q D (2)
÷2 ÷2 Q D (2) ÷2
261 ÷4 ÷3 II
÷2 ÷3 II
220 ÷6
÷4
85 ÷6
QIIP(1) 76
t (s–1)

÷2 D
QII(1)
28 ÷3 ÷2 QD(1)
26 ÷3
II

9 9
X X
2 3
Lα Lα
0.005 0.010 0.015 0.020 0.025 0.005 0.010 0.015 0.020 0.025
s (Å–1) s (Å–1)

Figure 11. Pressure-jumps from (a) 1110 to 240 bar and (b) 1110 to 260 bar at TZ46.78C are
shown. Each original two-dimensional X-ray image has been integrated to produce a one-
dimensional plot of intensity versus scattering vector, s, which is then stacked vertically as a
(nonlinear) function of time.

Figure 11 shows the structural changes following pressure-jumps from (a) 1110
to 240 bar and (b) 1110 to 260 bar, at TZ46.78C. The two jumps, carried out on
the same sample, are remarkably similar and are observed to follow a common
mechanistic route. The disappearance of the fluid lamellar phase is accompanied
by the appearance of a broad, featureless ring of scatter (marked X in figure 11).
After a period of a few seconds, the diffuse scatter resolves itself into a set of
peaks from intermediate inverse bicontinuous cubic phases, QIID and QIIP , which
replace it. These intermediate cubic phases are initially significantly more
hydrated than expected from equilibrium results and dehydrate over time. In
both jumps, a final QIID phase forms at fairly constant lattice parameter.
A major and long-standing problem in the field of lipid phase transition kinetics
is the lack of reproducibility in results. The requirement for intense synchrotron
X-ray sources for adequate time resolution indicates that a transition may be
observed only a few times, yet a number of studies have noted an increase in the
speed of transition with successive jumps. We have overcome the variability in
such dynamical measurements and, for the first time, this has allowed us to record
the dynamical process reproducibly in temperature- and pressure-jump experi-
ments. Figure 12 shows the lattice parameters of all phases present during the
transformation, superimposed for both jumps (a) (filled symbols) and (b) (hollow
symbols). With the exception of the QIIP (2) phase, which forms during jump (a) but
not jump (b), the two transformations are virtually identical. We observe the
transition to be highly reproducible not only in terms of the sequence of phases and
their lattice parameters, but also in the kinetics of the process, i.e. the time at
which each successive phase appears. We believe that the effect is owing to the
homogenization of domain size on repeated thermal cycling and strongly linked to
the transfer of water throughout the sample. The relative amounts of each phase
present throughout the transformation may be approximated by the area under
one of the characteristic diffraction peaks.

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2648 J. M. Seddon and others

(a)
180
(b)
160
55.5
55.0
140 54.5
54.0 Lα
a (Å)

53.5
120
53.0
52.5
100 52.0
51.5
–1.0 –0.5 0 0.5 1.0 1.5 2.0
80 t (s–1)

60 Lα

0 200 400 600 800 1000 1200 1400


t (s–1)

Figure 12. (a) The change in lattice parameter with time following the pressure-jumps shown in
figure 11 superimposed for both jumps (a, 1110 to 240 bar) (filled symbols) and (b, 1110 to 260 bar)
(hollow symbols). (b) The inset is a close-up view of the behaviour of the lamellar phase in the first
2 s following the jump.

Immediately following the pressure-jump, the intensity of the lamellar phase


begins to drop sharply (figure 13), the complete disappearance of this phase
occurring within 2 s, by which point its layer spacing has dropped by nearly 4 Å.
The QIID (1) phase appears 1.4 s later and grows steadily with time until the
appearance of the QIIP (1) phase at the point in which its intensity begins to drop
sharply. This is strongly indicative of a direct transformation between these two
cubic phases. However, the structural aspects of the transformation, specifically
the fact that the QIIP (1) phase is initially swollen and decreases towards an
equilibrium value, are more usually associated with the formation of a cubic
phase directly from the fluid lamellar phase. In contrast, during a cubic–cubic
transition, the growing cubic phase maintains a fairly constant lattice parameter.
This is displayed here during the growth of the final two cubic phases, QIIP (2) and
QIID (2), which form directly from the QIIP (1) phase, both of which maintain a
constant lattice parameter throughout the transformation. It is interesting to
note that, even in such non-equilibrium conditions, both sets of cubic phases
maintain a ratio of lattice parameters close to that predicted by the Bonnet ratio
(1.279). The QIIP (1) : QIIP (1) ratio (estimated error G0.015) is initially 1.317, but
drops sharply to 1.287 as the transition proceeds, while the QIIP (2) : QIID (2) ratio
is maintained between 1.295 and 1.305.
We have achieved the same level of reproducibility with our temperature-
jump experiments as that seen in the pressure-jump results previously. Figure 14
shows the changes following a temperature jump from 30 to 608C, at a nominal
jump rate of 508C minK1.
In this case, the transition to the first (QIIP ) cubic phase, via the X phase,
occurs much more slowly, and the appearance of the QIID cubic phase does not
occur until more than 1000 s have passed (the time for the temperature-jump to

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Pressure-induced transitions in lipids 2649

Q
Q
Q
Q
I (Q) (arbitrary units)

t (s–1)

Figure 13. The change in relative intensity (area under one of the characteristic diffraction peaks)
of each phase with time following the pressure-jump shown in figure 11a.

equilibrate at the new temperature is estimated to be 40 s). Thus, the


temperature-jump seems to follow the equilibrium phase diagram but very
slowly, whereas the pressure-jump induces a more complex pathway between the
La phase and the final QIID phase, but the various steps occur much more quickly.
The lattice parameters of all phases present during the transformations are shown
in figure 15. Again, the disappearance of the lamellar phase is accompanied by the
appearance of the intermediate phase X. However, some coexistence is observed
between the fluid lamellar and phase X during a pressure-jump, whereas for
temperature-jumps, the process is entirely discontinuous. Peaks characterizing a
swollen QIIP phase grow directly from the diffuse scatter, bypassing the intermediate
QIID phase observed during pressure-jumps. We are actively investigating the
dependence of the transition pathway on the thermodynamic parameters.

6. Future prospects

Time-resolved studies of lyotropic liquid crystal transitions are still at an early


stage, but clearly will be invaluable in helping to clarify complex transition
pathways and mechanisms. It would be particularly useful—although experimen-
tally challenging—to carry them out on aligned samples, which would allow any
epitaxial relationships between the phases to be determined at the same time.
The pressure-jump technique is not restricted to lipid phase transitions, but can

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2650 J. M. Seddon and others

1680 ÷2
QIID
÷3
1440

1200

÷2 P
960 QII
t (s–1)

÷4
÷6
720

480

240 X

0.001 0.002 0.003
s (Å–1)

Figure 14. Temperature jump from 30 to 608C, using a laboratory-based X-ray beamline. Each
two-dimensional X-ray image was integrated to produce a one-dimensional plot of intensity versus
scattering vector, s, which was then stacked vertically as a function of time.

180

160

140 QIIP

120
QIID
a (Å)

100

80

60 Lα

40
0 200 400 600 800 1000 1200 1400 1600 1800
t (s–1)

Figure 15. The change in lattice parameter following the temperature-jump shown in figure 14.
Intensity data are not shown owing to the poor resolution of the in-house X-ray equipment used.

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Pressure-induced transitions in lipids 2651

be used to study a wide range of soft matter transitions, ranging from protein
unfolding and DNA unwinding, to phase transitions in liquid crystals,
surfactants and block copolymers.
We anticipate that the most useful technical advance will be the development
of X-ray pressure-jump apparatus on the microsecond time-scale, involving the
use of piezoelectric stacks of pressure actuators. Such a device, for pressure-
jumps of up to G200 bar in 80 ms, has recently been developed for optical studies
of protein solutions (Pearson et al. 2002). Beamline I22 at the new Diamond
synchrotron, with its planned fast detectors with enhanced count-rate and
energy-range capabilities, will be an ideal beamline at which to establish a
UK-based microsecond X-ray pressure-jump facility.
This work has been supported by Platform grant (GR/S77721) and Ph.D. studentships from the
EPSRC. We thank Aurelien Huisman (Socrates student, Imperial College London) for measuring
the DSC and PPC scans shown in figure 6, and Nick Brooks, Chandrashekhar Kulkarni, Sarra
Sebai and Christina Turner for their help with carrying out the synchrotron experiments. We
also thank Prof. R. Winter and Ms J. Kraineva (Dortmund University) and Drs S. Finet and
T. Narayanan (ESRF) for their invaluable contributions.

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Discussion
H. F. GLEESON (School of Physics and Astronomy, University of Manchester,
UK ). Do you see flow as part of the p-jump or T-jump transition process? Work
of Julia Yeoman (Oxford) simulating flow in cubic (blue phase) structures show
structural changes in the unit cell—could this kind of process be contributing to
the intermediate signals in the transitions from cubic to cubic?
J. M. SEDDON. In our experiments, we have been careful to create sample
environments, in which no shearing of the hydrated lipid samples can occur,
nor is extraneous water able to flow in or out of the samples. However, in
transforming from one phase to another there may indeed be local flows of

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2654 J. M. Seddon and others

water through the sample, related to the mechanism of the transition. As an


example of this, we typically see swollen cubic phases that appear as
intermediates as we make jumps from a lamellar to an inverse bicontinuous
cubic phase. This evidence is consistent with the hypothesis that these out-
of-equilibrium bicontinuous structures appear as a low-energy means of
transporting water from one part of the sample to another, while the
additional bicontinuous channels are being created in order to form the
equilibrium structure.
V. PERCEC (Department of Chemistry, University of Pennsylvania, USA). Your
pressure giving X-ray experiments were carried out on lyotropic liquid crystalline
phases. What is the lowest lipid concentration on which these experiments can be
carried out? Can single molecules in solution be analysed by p-jump X-ray
experiments?
J. M. SEDDON. The amphiphiles we study have very low critical micelle
concentrations, and hence aggregate even at very low concentrations in aqueous
solution. It is becoming feasible to carry out single molecule X-ray experiments,
but, of course, the kind of information obtained will bear little or no relation to
the structural data we are obtaining from collective, cooperative transitions
between different self-assembled interfacial phase structures.
S. T. LAGERWALL (The Royal Swedish Academy of Sciences, Sweden). When you
heat the lamellar phase the layers curve in a certain fashion, giving the flexible
carbon chains more space. Is this sign of bending a general feature for all
molecules with a polar head?
J. M. SEDDON. Yes, so long as they have a single polar headgroup, attached to one
or more flexible chains.
P. PALFFY-MUHORAY (Liquid Crystal Institute, Kent State University, USA).
What is the status of theory? Are there estimates for the time-scales on which
pressure-jump-induced phase transitions occur?
J. M. SEDDON. Theories are being developed to describe certain steps which may
be involved in the transition process, for example the dynamics of hemifusion in
lipid bilayers (Hed & Safran 2003). However, we are not aware of any theory that
can successfully predict the time-scales of pressure-jump-induced lyotropic cubic
phase transitions. We hope that our experimental results may stimulate such
development.
C. R. SAFINYA (Department of Materials, University of California at Santa
Barbara, USA). The Gaussian curvature modulus kG should be positive for the
minimal surfaces to be preferred over spherical phases. You appeared to say that
you had measured a negative kG. How is that possible?
J. M. SEDDON. Although for a monolayer kG may be negative, however, one can
show that for a bilayer kG can be positive, as is required for the thermodynamic
stability of the inverse bicontinuous cubic phases. For the bending modulus, the
bilayer value kb should be simply twice that of the monolayer bending modulus k,
and both should have positive values. However, as discussed in detail in a
forthcoming review article (Shearman et al. 2006), a more complex situation
exists when considering the link between the monolayer and bilayer Gaussian

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Pressure-induced transitions in lipids 2655

moduli, kG and kbG . The monolayer Gaussian modulus kG has been found to be
negative for systems forming inverse curved phases, and with a magnitude less
than that of the bending modulus k. The bilayer Gaussian curvature modulus kbG
is directly related to the monolayer Gaussian modulus, but also includes a term
that contains the bending modulus k. The explicit expression (Helfrich &
Rennschuh 1990) is

kbG Z 2ðkG K4kH0 [Þ;

where the bilayer is taken to be symmetric, H0 is the spontaneous mean curvature of


the monolayer, and [, the monolayer thickness. Since for inverse-phase forming
amphiphiles, H0 is negative, the second term tends to make kbG positive.
We have, however, found it more instructive to consider the energetics of inverse
bicontinuous cubic phases from the point of view of the monolayer curvature
elasticity. Here we have found that although saddle-shaped interfacial curvature is
energetically more costly than cylindrical or spherical curvature, the cost of packing
hydrocarbon chains into the saddle-shaped geometry of the inverse bicontinuous
cubics is significantly less costly than in packings of cylindrical or spherical
monolayers. When chain lengths are short this difference becomes great enough
that the inverse bicontinuous cubic phases are the energetically preferred structure.

Additional references
Hed, G. & Safran, S. A. 2003 Initiation and dynamics of hemifusion in lipid bilayers. Biophys. J. 85,
381–389.
Helfrich, W. & Rennschuh, H. 1990 Landau theory of the lamellar-to-cubic phase-transition.
J. Phys. 51, 189–195.
Shearman, G. C., Ces, O., Templer, R. H. & Seddon, J. M. 2006 Inverse lyotropic phases of lipids
and membrane curvature. J. Phys.: Condens. Matter. 18, S1105–S1124. (doi:10.1088/0953-
8984/18/28/S01)

Phil. Trans. R. Soc. A (2006)

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