Download Using cryo-electron microscopy to determine thermodynamic and

Current Opinion in Colloid & Interface Science 10 (2005) 261 – 268
www.elsevier.com/locate/cocis
Using cryo-electron microscopy to determine thermodynamic
and elastic properties of membranes
Ryan van Zanten, Joseph A. Zasadzinski *
Department of Chemical Engineering, University of California, Santa Barbara, Ca 93106-5080, United States
Available online 10 October 2005
Abstract
Both cryo transmission electron and freeze-fracture microscopy provide the high resolution images necessary to elucidate the structures and
size distributions of spontaneous vesicles formed from cationic and anionic surfactant mixtures, lipid and polymer mixtures, and mixed lipid and
protein mixtures. From the size distributions and structures, estimates of the elastic and thermodynamic properties of the membrane can be
determined to begin to relate molecular properties to bilayer parameters.
D 2005 Elsevier Ltd. All rights reserved.
1. Introduction
An important and still open question is whether unilamellar
vesicles can ever be at thermodynamic equilibrium, or are
simply a metastable organization on the way towards multilamellar liposomes (or more properly, lamellar phase dispersions in excess water) [1&&,2&&,3,4&&,5,6&,7&]. In order for
unilamellar vesicles to be stable relative to a multilamellar
phase, the bilayer interactions must be repulsive to neutral so
that the system does not prefer a multilamellar stacking with a
well-defined layer spacing. Recently, there have been reports of
three distinct ways that unilamellar vesicle phases can be
stabilized with respect to multilamellar phases [1&&,4&&,8,9&],
although questions of ultimate thermodynamic equilibrium will
likely require a significantly longer wait than a typical graduate
student lifetime.
The first way to stabilize vesicles is for the surfactant bilayer
to develop a spontaneous curvature [1&&]. For this to occur,
there must be an asymmetry in the composition of the inner and
outer monolayers (hence, this can only occur in surfactant
mixtures), because the respective curvatures of the monolayers
in a vesicle are opposite in sign but nearly equal in magnitude
whenever the bilayer thickness is small compared to the vesicle
radius [5&,10&&]. Vesicles whose curvatures are similar to the
* Corresponding author. Tel.: +1 805 893 4769; fax: +1 805 893 4731.
E-mail addresses: [email protected] (R. van Zanten),
[email protected] (J.A. Zasadzinski).
1359-0294/$ - see front matter D 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cocis.2005.09.001
spontaneous curvature have a lower energy than the
corresponding flat lamellae. If the bending constant is
sufficiently large, variations from the spontaneous curvature
are sufficiently unfavorable that multilamellar vesicles are
prohibited, especially at low concentrations where entropy also
favors unilamellar vesicles. Hence, a monodisperse population
of unilamellar vesicles at equilibrium implies that stabilization
results from a spontaneous curvature and a large bending
constant (Fig. 1) [1&&,2&&,11&]. At higher concentrations, or in
the presence of attractive bilayer interactions, this mechanism
can lead to stable vesicles with a quantized number of bilayers
as the interaction energy can overcome the increase in
curvature energy for a limited deviation away from the
spontaneous curvature [1&&]. At sufficiently high concentrations, however, packing constraints lead to a coexistence of
vesicles with a multilamellar phase [12&&].
In the second mechanism, vesicles can be stabilized by the
gain in entropy resulting from the large number of finite sized
vesicles as opposed to a few large lamellae or an intermediate
number of multilamellar liposomes. This will always be the
case at sufficiently low surfactant concentration, although in
practice, the concentration may need to be vanishingly small to
stabilize vesicles [5&]. At higher surfactant concentrations, the
bilayer elastic constant, j, must be of order k BT or less to favor
vesicles in the absence of a spontaneous curvature. A low value
for the bending constant is one way to generate sufficient
repulsive steric interactions to overcome van der Waals
attraction between bilayers. The repulsive Helfrich undulation
interaction between bilayers is inversely proportional to the
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R. van Zanten, J.A. Zasadzinski / Current Opinion in Colloid & Interface Science 10 (2005) 261 – 268
Fig. 1. (a) Cryo-TEM image and (b) measured size distribution fit to Eq. (2) of vesicles formed from mixtures of sodium perfluorooctanoate (FC7) and
cetyltrimethylammonium bromide (CTAB) (4 : 1 wt : wt, 2 total wt.%). Spontaneous vesicles of this composition are almost monodisperse and have R 0 = 23 T 0.5 nm
and K = 6 T 2 k BT [1&&]. A narrow size distribution is consistent with stabilization by the energetic costs of deviating from the spontaneous curvature due to the high
value of the bending constant.
bending elastic constant [5&,13&]. The smaller the bending
constant, the larger are the repulsive interactions between
layers; if the bending constant is of order k BT, the net
interaction between bilayers is repulsive, and multilamellar
vesicles are prohibited [1&&]. A polydisperse population of
unilamellar vesicles at equilibrium implies that stabilization
results from entropy and a low bending constant [1&&,11&].
Again, at sufficiently high concentrations, packing constraints
lead to a coexistence of vesicles with a multilamellar phase
[9&].
In the third mechanism, strong, long-range interactions
between the bilayers prevent the formation of a stable multilamellar phase [4&&,14,15&]. Double-layer electrostatic interactions can stabilize vesicles against aggregation at sufficiently
low salt concentrations [4&&14,15&]. The best example of this
mechanism involves mixtures of ion-pair amphiphiles (typically a hydroxide and an acid surfactant pair) whose counterions combine to form water [4&&,14,15&]. As a result, the
background electrolyte concentration remains small (¨10 7
Fig. 2. Icosahedral vesicle formed from a mixture of cetyltrimethylammonium
hydroxide and myristic acid. These vesicles formed from ion-pair amphiphiles
(typically a straight-chain hydroxide surfactant with a straight chain fatty acid)
whose counterions combine to form water make extremely rigid bilayers with
strong electrostatic interactions. The curvature is confined to defect sites such
as the edges and pores (center) where bending is possible [4&&].
molar) and electrostatic interactions between the bilayer are
large. Large, polyhedral vesicles have been imaged with pores
at the vertices of the rigid sheets from mixtures of fatty acids
and hyroxide surfactants (Fig. 2) [4&&]. These vesicles are only
stable at relatively low surfactant concentrations in the absence
of any added salt. Sufficient repulsive interactions between
bilayers can also be generated by adding polymer-lipids such as
polyethyleneglycol dipalmitoylphosphatidylethanolamine
(PEG-DPPE); PEG-DPPE can stabilize vesicles by steric
interactions and lead to unilamellar vesicles at low concentrations [8]. The sizes and size distributions of such electrostatically or sterically stabilized vesicles cannot be simply related
to the bending elasticity or other bilayer parameters [15&].
For vesicles stabilized by the first two mechanisms,
cryogenic transmission electron microscopy (cryo-TEM) is an
especially useful way to determine both the thermodynamic
and elastic properties of the vesicle bilayers. Direct imaging at
Fig. 3. Cylinders, discs and spherical vesicles coexisting in mixtures of FC7
and CTAB (as in Fig. 1). This is one of the few systems where a Gaussian
curvature modulus could be measured so that the two individual elastic
constants could be determined [2&&]. For the cylinders and spheres to coexist,
12j ; 8kj̄. From Fig. 1, K =j + j̄ / 2 = 6 k BT for FC7 : CTAB vesicles.
Combining the two results gives j = 5 T 1 k BT and j̄ = 2 T 1 k BT. Cylinders
and discs have been confirmed by small angle X-ray diffraction measurements;
the discs are a left-over intermediate of vesicle growth from micelles [33&&].
R. van Zanten, J.A. Zasadzinski / Current Opinion in Colloid & Interface Science 10 (2005) 261 – 268
Fig. 4. Cryo-TEM micrographs of cubosomes of glycerol monooleate containing small fractions of a ethylene oxide copolymer. The bulk phases were
prepared in the two-phase region between a bicontinuous cubic phase and
excess water. The cubosomes can be used as drug delivery systems. Bar
represents 100 nm [41&&].
low temperature by cryo-TEM and the related imaging of
freeze-fracture replicas of rapidly frozen samples (FF-TEM),
can be used to directly view the different microstructures of
most self-assembled surfactant systems to resolutions of about
2 nm. There have been several recent reviews showing the
ability of cryo-TEM to image different amphiphilic systems
[16 –18]. However, the additional step of quantifying the image
to determine the complete vesicle size distribution and mean
sizes and shapes can provide information on the bending
constants of the bilayer, as well as suggesting whether or not
the vesicle size distribution is consistent with that predicted at
equilibrium [1&&,2&&,11&].
In addition to providing information on the entire size
distribution, direct imaging techniques allow the observation of
coexisting or complex microstructures (Figs. 2 – 4) that may
confuse indirect methods such as scattering [2&&]. For evaluating both size distributions and complex structures, direct
imaging avoids the need for model-dependent interpretation
of scattering data [9&]. For vesicle phases, direct imaging
methods agree rather well with information obtained by
scattering techniques, which diminishes the concern over
sample preparation and imaging artifacts [11&]. However, for
best results, multiple microscopy techniques should be
combined with light, X-ray and neutron scattering to provide
the most reliable evaluation of structural data.
2. Measuring bilayer bending constants
The starting point for the description of bilayer organization in solution is the harmonic approximation to the bending
free-energy:
" #
1
1
1
2 2
1
FB ¼ X dA j
þ
þ j̄
ð1Þ
2
R1
R2
R0
R1 R2
Eq. (1) was derived from the limiting case for thermotropic
smectic phases [19] by Helfrich over 30 years ago and is
263
generally accepted to describe monolayer and bilayer deformations [5&,20&&]. R 1 and R 2 are the principle radii of curvature
(for spherical vesicles, R 1 = R 2 = R, the vesicle
¯ radius), R 0 is the
spontaneous radius of curvature, j and j are the mean and
Gaussian bending constants, respectively, and A is the area of
the bilayer. The harmonic approximation is appropriate when
the bilayer thickness (¨3– 4 nm) and the Debye length [21] for
ionic surfactants, are small compared to R 1 and R 2 (> 30 nm).
The two elastic constants, j and j̄, play very different roles
in
¯
determining bilayer organization. The magnitude of j determines the energy needed to bend the bilayer away from its
spontaneous radius of curvature, R 0. For single component or
otherwise symmetric bilayers, 1 / R 0 = 0 by symmetry
[5&,10&&,13&,19]; a non-zero bilayer curvature is only possible
when non-ideal surfactant mixing causes the interior and
exterior monolayers of the bilayer to have different compositions or environments [5&,10&&].
On the other hand, j̄ only influences the topology (and
hence the number) of the structures formed [2&&]. The Gauss –
Bonnet theorem states that the integral of the Gaussian
curvature over a given surface only depends¯ on the genus of
the structure [22]. Hence, the magnitude of j has little effect at
equilibrium as long as structural fluctuations take place at
constant topology or vesicle number. However, transformations
between discs and closed spheres or between spheres and
cylinders are influenced by j̄ [2&&].
Although Eq. (1) has become the accepted description of the
energetics of bilayer organization, there are relatively few
measurements of j and almost no measurements of j̄ for
surfactant or lipid bilayer structures. Moreover, there are no
generally accepted methods for determining these elastic
constants. j̄ is especially difficult to measure as it influences
topological transformations between structures such as discs,
spheres, and toroids [2&&], the distribution of material between
vesicles [1&&,5&,10&&], or the transition between lamellar and L3
phases. However, j̄ does not influence the more readily
measured fluctuations of an equilibrium structure.
A key requirement to the understanding and possible control
of surfactant structural organization is developing both
experimental and theoretical tools to relate R 0, j, and j̄ to
surfactant molecular structure and solution conditions.
For macroscopic vesicles (>10 microns) j has been
measured by both micropipette aspiration [23] and video
imaging of membrane fluctuations [24] , but there is not good
agreement between the two sets of values. These techniques
limit the investigation to structures on the order of microns due
to experimental limitations. Forming large vesicles requires
special non-reversible methods, and there are additional
questions as to whether the inside and outside monolayers
reach their preferred area per molecule over the course of the
experiments [23]. Shape fluctuations can also be observed for
sub-micron vesicles using quasi-elastic light scattering, but it
has been difficult to extract reasonable values of j from the
data [25&].
Spontaneous vesicles from mixtures of anionic and cationic
surfactants are much better suited for cryo-TEM examination
of the vesicle size distribution [1&&,2&&,9&]. In the vesicle
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R. van Zanten, J.A. Zasadzinski / Current Opinion in Colloid & Interface Science 10 (2005) 261 – 268
regions of the phase diagram, the size distributions are
independent of aging and method of formation, suggesting
that the vesicles are at least metastable for long times (the
most stable systems have maintained the same size distribution for 5 years in our hands) if not at true equilibrium [9&].
This allows the use of free-energy minimizing mass-action
models in conjunction with Eq. (1) to relate the size
distribution to R 0, j and j̄ [1&&,2&&,9&,11&]:
CN ¼
8pK r0 2
1
CM exp
kB T
R
R22
r
0
ð2Þ
C N (= X N / M) is the molar or number fraction of vesicles of
size N and C M is the mole fraction of vesicles of radius r 0.
For spherical vesicles of radius, R, the size distribution is
determined by the sum of the bending constants, K, and the
spontaneous curvature:
2K ¼ 2j þ j̄
r0 ¼
2j þ j̄
R0
2j
ð3Þ
The individual constants, j and j̄, cannot be determined
independently from the size distribution. A consequence of
Eqs. (2) and (3) is that vesicles stabilized by thermal
fluctuations (K ¨ k BT) have a much broader size distribution
than vesicles stabilized by the spontaneous curvature
(K H k BT). This is the opposite of vesicle size distribution
models that do not include a spontaneous curvature; larger
bending constants predict more polydisperse vesicles of larger
size [11&,26]. If the size distribution is narrow, Eq. (2)
resembles a Gaussian distribution and K can be simply related
to R 0 (which is given by the mean, <R>, of the size
distribution) and the standard deviation, r [26,27&&]:
2
kB T
R0
K¼
ð4Þ
16p
r
Eq. (4) can give a rough estimate of the parameters, but a
more reliable measure is to fit the entire size distribution
measured by cryo-TEM to Eq. (2) to determine the best
values of K and R 0 [1&&,11&] (Fig. 1). However, it is possible
to estimate <R> and r from an analysis of freeze-fracture
images of vesicles and use Eq. (4) to determine R 0 and K
[1&&,26].
3. Experimental results
Relating the size distribution of vesicles to their elastic
constants is experimentally complex and requires extensive
cryo-TEM imaging, followed by the rather tedious analysis of
the size distribution. Hence, the number of available bending
constants of different systems is still rather small. The first
attempts at using cryo-microscopy to estimate bending elastic
constants was by Denkov et al. [27&&] who examined a rather
complex vesicle system incorporating several lipids and
proteins. While the vesicles involved were probably not at
equilibrium, the distribution was stable for weeks. The
population was quite monodisperse, resulting in a value of
K ¨ 5 k BT.
Systems of spontaneous vesicles made from mixtures of
anionic and cationic surfactants (also known as catanionic
vesicles) are likely the best hope for a systematic use of cryoTEM analysis of size distributions to measure bilayer elastic
properties. Such vesicles form spontaneously [9&,12&&], the final
size distribution is independent of the method of vesicle
formation [1&&,2&&,11&,26] and changes only slightly with time
after an initial period of equilibration [7 &,9&]. Hence, the
assumptions involved in deriving Eqs. (1) –(4) are reasonably
well met, although the ultimate question of equilibrium is still
open.
The first system examined was a 3 : 7 (wt : wt) mixture of
cetyltrimethylammonium bromide (CTAB, cation) with sodium
octyl sulfonate (SOS, anion) at 2 wt.% total surfactant in water.
The measured size distribution was well fit by Eq. (2) for
values of R 0 = 37 T 3 nm and K = 0.7 T 0.2 k BT. For the 7 : 3
CTAB : SOS, R 0 = 30 T 2 nm and K = 0.2 T 0.1 k BT. Changing
the surfactants to a 3 : 7 mixture of cetyltrimethylammonium
tosylate (CTAT) and sodium dodecylbenzene sulfonate (SDBS)
resulted in an equally good fit of Eq. (2) to the size distribution
for R 0 = 36 T 1 nm and K = 0.54 T 0.05 k B T. For 7 : 3
CTAT: SDBS, R 0 = 55 T 10 nm and K = 0.15 T 0.03 k BT. There
are two conclusions to draw from this limited data: it appears
that surfactant mixing leads to low bending constants and
bilayers with an excess of cationic surfactant have lower
bending constants than bilayers with excess anionic surfactant.
A number of molecular parameters have been theoretically
predicted to influence the bilayer elasticity. For example, the
tosylate counterion of CTAT is hydrophobic, and remains
strongly associated with the surfactant aggregate in solution.
Inserting the tosylate into the membrane effectively increases
the area per surfactant headgroup, which is predicted to reduce
membrane rigidity [28,29]. However, the bromide counterion
of CTAB is much less hydrophobic, so this does not explain the
lower bending constants observed in the CTAB excess bilayers.
SDBS is a branched chain aromatic surfactant; such
branching can influence hydrocarbon packing in the bilayer
interior, which may in turn affect the bending rigidity. It is
theoretically predicted that branched hydrocarbons pack more
loosely in the bilayer, and thus are more accommodating to the
gauche hydrocarbon conformations associated with membrane
deformations [30,31]. Molecular relaxation (chain rearrangement and flip-flop) in the bilayer could be enhanced by the
presence of branched hydrocarbons. However, there is little
difference between the K measured for the branched SDBS and
the straight chain SOS. Hence, mixing surfactants by itself is
apparently sufficient to lower the bending rigidity to ¨k BT
[30,31]. As a result, most spontaneous vesicles formed from
mixtures of anionic and cationic hydrocarbon surfactants are
stabilized against formation of multilamellar phases by
Helfrich steric interactions [1&&], the second mechanism
discussed in the Introduction.
However, before making too many conclusions regarding
the influence of molecular structure on bending elasticity, it
R. van Zanten, J.A. Zasadzinski / Current Opinion in Colloid & Interface Science 10 (2005) 261 – 268
must be noted that the vesicle size distributions measure
K = j + j̄/ 2. There are very few predictions
on how j̄ changes
¯
with molecular architecture and j can be either positive or
negative, while j must always be positive [20&&]. K can remain
small as observed for the cationic –anionic vesicles if both j
and j̄ increase in magnitude, but with opposite signs. What
really is necessary is another method of measuring either j or
j¯ independently so that the variation of the individual
parameters with molecular structure can be ascertained. Brocca
et al. [25 &] have measured the shape fluctuations of ¨100 nm
diameter phospholipid vesicles by light scattering, but have
not been able to relate the observed fluctuations directly to a
value of j. Another route may be analysis of the small angle
X-ray scattering from the chemically similar multilamellar
phases of the cation –anion surfactant pair. The X-ray line
shape depends on the value of j [26,32]; however, it is not
known how the bending elasticity depends on overall
surfactant concentration.
One clear example of the effect of surfactant chemistry on
bending elasticity occurs for mixtures of sodium perfluorooctanoate (FC7) and CTAB (4 : 1 wt : wt, 2 total wt.%)
Spontaneous vesicles of this composition (Fig. 1) are almost
monodisperse and have R 0 = 23 T 0.5 nm and K = 6 T 2 k BT
[1&&]. However, reducing the length of the fluorinated
surfactant to 6 carbons (sodium perfluorohexanoate, FC7)
with CTAB at the same 4 : 1 weight ratio reduces K to 0.5 k BT,
the same order of magnitude as for the hydrogenated
surfactant mixtures. If they are long enough, the rigid fluorine
tails increase the bending elasticity of the membrane by an
order of magnitude compared to similar mixed hydrogenated
surfactants [1&&]. The higher rigidity of the bilayer leads to
higher energy penalties for deviations from the spontaneous
curvature, and an essentially monodisperse vesicle population.
The CTAB/FC7 vesicles are stabilized against multilamellar
liposome formation by the high energy costs of deviating from
the spontaneous curvature [1&&], the first mechanism discussed
in the Introduction.
To independently test the validity of Eq. (1) and a nonzero spontaneous curvature, salt was added to the rigid, FC7/
CTAB vesicle system. Simple calculations based on the same
Helfrich elastic energy and mass action models suggest that
attractive forces between the bilayers can overcome slight
deviations from the spontaneous curvature, leading to a
distribution of vesicles with two bilayers [1&&]. These vesicles
with a discrete number of bilayers confirm that the
spontaneous curvature is non-zero; no other explanation can
lead to a stable distribution of vesicles with a discrete number
of layers. This confirms that the vesicles are stabilized by the
combination of a spontaneous curvature and a large elastic
constant [1&&]. Adding salt to the vesicles with the lower
bending constants did not alter the distribution of vesicles,
confirming that the repulsive Helfrich interaction [13 &] was
sufficient to stabilize the vesicles against forming multilamellar phases.
The rigid fluorinated/hydrogenated vesicles also establish an
equilibrium with disks and cylindrical objects [2&&] (Fig. 3).
This is one of the few systems where a Gaussian curvature
265
modulus could be measured so that the two individual elastic
constants could be determined [2&&]. For the cylinders and
spheres to coexist, 12j ; 8kj̄. The analysis of the size
distribution of the FC7 : CTAB vesicles showed that K =
j + j̄/ 2 = 6 k BT. Combining the two results gives j = 5 T 1
k BT and j̄ = 2 T 1 k BT [2]. Cylinders and discs have been
confirmed by small angle X-ray diffraction measurements; the
discs are a left-over intermediate of vesicle growth from
micelles [33&&].
A large value of bending elasticity also appears to be
important to the structure and stability of giant polyhedral
vesicles formed from mixtures of ion-pair amphiphiles
(typically a straight-chain hydroxide surfactant with a straight
chain fatty acid) whose counterions combine to form water
[4 &,14,15 &] (Fig. 2). In the high temperature, fluid state, the ionpaired surfactants exhibit in-bilayer miscibility, as is the case
for the catanionic vesicles described earlier. However, as the
temperature is lowered, the mixed surfactants crystallize into
polygonal frozen bilayers at fixed mole ratios of anion to
cation. Any excess of anion or cation is expelled from the
growing crystal and accumulates at the polygonal edges and
vertices of the polygonal vesicles; these edges and vertices then
act as defect sites where the bilayer curvature can be
concentrated [4 &&,14,15 &]. For these crystallized bilayers, j is
estimated to be of order 200 k BT from the in plane coherence
length of the faceted bilayers [15 &]. These giant polyhedral
vesicles are stabilized against aggregation by the low background electrolyte concentration, which leads to large, repulsive electrostatic interactions between the bilayers. The vesicles
are unstable at higher concentrations, or in the presence of
added electrolytes.
4. Phase behavior of spontaneous vesicles
Cryo-TEM is also necessary to validate vesicle microstructure; it is more than a complement to scattering
techniques. The vesicle lobe in several phase diagrams of
catanionic mixtures has been mapped by using EM to image
structure; cryo-TEM and freeze-fracture can readily distinguish between unilamellar vesicles and multilamellar liposomes [1&&,2&&,4&&,9&,11&,15&,34]. It is difficult, if not impossible, to characterize unilamellar vesicles without direct
imaging; scattering methods are usually not sufficient,
especially for polydisperse systems less than a micron in
diameter [9 &].
The micelle to vesicle transition has also been explored
using cryo-TEM by a number of investigators, as has the
equilibrium between micelles, vesicles and discs. In several
studies, direct imaging shows the coexistence of micelles and
vesicles, bringing up the concept of a critical aggregation
concentration versus just a critical micelle concentration [35].
One of the intermediate structures in the micelle to vesicle
transition are bilayer discs; discs have been shown to coexist
with vesicles in a number of these catanionic systems
[2&&,14,36]. Small angle X-ray scattering studies have shown
the importance of discs in the transition from micelles to
bilayer structures [33&&]. The lamellar to vesicle unbinding
266
R. van Zanten, J.A. Zasadzinski / Current Opinion in Colloid & Interface Science 10 (2005) 261 – 268
transition of charged membranes has been explored using
FF-TEM, elucidating the thermodynamics of this phase
change [37].
The phase behavior of other surfactants has been
investigated with cryo-TEM, revealing that bilayer-forming
phases result from several different amphiphiles and mixtures. The addition of certain amphiphiles suppresses the
lamellar phase, causing multilamellar vesicles to form [38].
Vesicles form spontaneously from dimeric arginine-based
cationic surfactant solutions and bolaamphiphile/anionic
surfactants [39,40]. Bicontinuous phases can also be explored by cryo-TEM allowing imaging of cubic phases
which are also of interest for drug delivery applications (Fig. 4)
[41&&].
The effect of PEG-lipids on the stability and structure of
liposomes has been investigated with cryo-TEM, showing a
change from a dispersed lamellar phase to a micellar
phase, including discoidal micelles, depending on the
molecular weight and the concentration of the PEG-lipid
[42]. With the addition of PEG-lipid, flat bilayer discs can
be the preferred structure due to the decoration of the
bilayer rim with the PEG-lipid [43]. Triblock copolymers
work well in maintaining the stability liposomal dispersions
against aggregation [44]. The effect of PEG-lipids on
equilibrium vesicles has also been explored and is shown
to influence both the bilayer spontaneous curvature and
elasticity [45].
Many fluorinated surfactants show a vesicle region,
especially in mixtures with hydrogenated surfactants [1&&,2&&].
The different hydrophobic properties of fluorinated alkyl
chains make these interesting systems to investigate. Vesicles
have been seen in aqueous solutions of anionic fluorocarbon/
hydrocarbon surfactants [46]. Unique single-chain sugar-based
fluorinated surfactants have also been seen to spontaneously
produce a vesicle phase [47]. Both of these systems are
interesting because vesicles form with only one surfactant
present, which suggests that vesicle stabilization must not be
due to a spontaneous curvature, which requires at least two
components [5&]. An alternative explanation is that these
nominally pure, sugar-based systems actually consist of a
mixture of different isomers, which allows for a spontaneous
curvature.
5. Stability of spontaneous vesicles
The stability of spontaneous equilibrium vesicles has also
been examined by cryo-TEM. In some systems, light
scattering suggests that the vesicles continue to evolve
slowly with time [7 &]. The diameter of vesicles has been
shown to increase suggesting that they are not at Ftrue_
thermodynamic equilibrium. In other work, the size distributions measured between different samples prepared over
months to years have provided similar size distributions
[11&,26]. As shown in Ref. [11&], rather small changes in the
number of large vesicles can skew the mean size measured
by light scattering. Ref. [7 &] shows that the microstructure is
altered after 2 months of aging, suggesting possible chemical
changes to the surfactants. The actual phase boundaries
between the single unilamellar phase and the coexisting
multilamellar/unilamellar phase are difficult to determine with
any certainty, and the least stable vesicle phases are often
those at the highest total concentrations [7 &], where multilamellar phases are likely. Hence, there have been systems in
which the size distribution is quite stable, and others that
evolve slowly over time.
In addition to being invariant with time, equilibrium
requires that the phases and microstructure observed at a given
concentration and temperature be independent of the method of
sample preparation. One interesting technique is to form
vesicles by a chemical reaction, without any input of energy
either by shear forces or temperature changes [48&&]. Hao et al.
[48&&] have shown that vesicles form spontaneously via
chemical reactions that create the necessary anionic and
cationic surfactants from various precursor. In the dilute
regime, vesicles form spontaneously and remain stable for
long periods of time. At higher concentrations, vesicles do not
form spontaneously because the system is too concentrated for
the vesicles to exist without a coexisting multilamellar phase
[9 &].
Another interesting method is to use zinc as the counterion for the anionic surfactant and add hydrogen sulfide to
the system [49]. The hydrogen sulfide protonates the
surfactant, and the zinc ions precipitate by complexing with
sulfur. This leaves a true ternary system in which spontaneous vesicles are created without the introduction of any
outside energy, similar to the chemical synthesis scheme
mentioned above.
6. Summary
Cryo-TEM and freeze-fracture microscopy methods are
invaluable to assign microstructure to complex surfactant
mixtures, as well as to determine important bilayer parameters. As a complement to light, X-ray and neutron
scattering, direct imaging provides unambiguous structural
information and is perhaps the only method to identify
unilamellar vesicles, cylindrical vesicles, discs, worm-like
micelles, etc. coexisting in a single solution. With careful
sample preparation, conservative image interpretation, and a
systematic analysis of the images, it should be possible to
begin to understand the relationship between molecular
structure and bilayer elasticity, and from this understanding,
develop new microstructures for applications in drug delivery,
microreactors, catalysis, etc.
Acknowledgements
The authors wish to thank Hee-Tae Jung and Bret Coldren at
UCSB, Eric Kaler and his group at the University of Delaware
for ongoing collaborations, and M. Almgren and T. Zemb for
generous use of their results on catanionic vesicles. This work
was supported by National Science Foundation Grant #CTS0436124 and the Petroleum Research Foundation Grant
#41016-AC7.
R. van Zanten, J.A. Zasadzinski / Current Opinion in Colloid & Interface Science 10 (2005) 261 – 268
References and recommended readings
[1] Jung HT, Coldren B, Zasadzinski JA, Iampietro DJ, Kaler EW. The
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