Article
pubs.acs.org/JPCB

Phase Diagrams and Ordering in Charged Membranes: Binary
Mixtures of Charged and Neutral Lipids
Naofumi Shimokawa,*,† Hiroki Himeno,‡ Tsutomu Hamada,† Masahiro Takagi,† Shigeyuki Komura,§
and David Andelman∥†

School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan
Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Kagawa 761-0395, Japan§
Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, Japan∥
School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv 69978,
Tel Aviv, Israel‡

ABSTRACT: We propose a model describing the phase behavior of two-component membranes consisting of binary mixturesof electrically charged and neutral lipids. We take into account the structural phase transition (main-transition) of thehydrocarbon chains, and investigate the interplay between this phase transition and the lateral phase separation. The presence of
charged lipids signiﬁcantly aﬀects the phase behavior of the multicomponent membrane. Due to the conservation of lipid
molecular volume, the main-transition temperature of charged lipids is lower than that of neutral ones. Furthermore, as comparedwith binary mixtures of neutral lipids, the membrane phase separation in binary mixtures of charged lipids is suppressed, in
accord with recent experiments. We distinguish between two types of charged membranes: mixtures of charged saturated lipid/
neutral unsaturated lipid and a second case of mixtures of neutral saturated lipid/charged unsaturated lipid. The corresponding
phase behavior is calculated and shown to be very diﬀerent. Finally, we discuss the eﬀect of added salt on the phase separationand the temperature dependence of the lipid molecular area.

1. INTRODUCTION
Biomembranes are composed of various components such as
phospholipids, sterols, and proteins, and are believed to havecompositional heterogeneities. In multicomponent membranes,
regions or domains enriched in saturated lipid and cholesterol
are called “rafts”, and are thought to play an important role invarious biocellular functions, such as signal transduction andmembrane traﬃcking.1,2 Artiﬁcial bilayer membranes composedof several phospholipids and cholesterol have been used as
typical model systems for biomembranes. Phase separation insuch model membranes has been widely investigated in orderto understand the mechanism of raft formation.3 Considerable
eﬀort has been given to ternary systems composed of saturated
lipid, unsaturated lipid, and cholesterol, where it is known thatdomains enriched in saturated lipid and cholesterol form aliquid-ordered phase in a matrix of an otherwise liquiddisordered phase.4−10
Two-component lipid bilayers consisting of saturated andunsaturated lipids also show a phase separation between solid© 2016 American Chemical Society

like (Lβ) and liquid-like (Lα) phases. This phase separation canbe directly visualized by ﬂuorescence microscopy, which
demonstrates that the solid-like Lβ domains exhibit various
anisotropic shapes.11−14 The resulting phase diagrams ofdiﬀerent binary mixtures have been experimentally exploredin great detail,14−18 and have been quantitatively reproduced inseveral theoretical works.19−23 In particular, in models
proposed by some of the present authors,19,20,22 a coupling
between the membrane composition and its internal structure
was suggested in order to consider the interplay between thephase separation and the chain phase transition (maintransition). In these experimental and theoretical studies,
however, the lipids were taken to be electrically neutral.
Special Issue: William M. Gelbart Festschrift
Received: March 25, 2016
Revised: April 29, 2016
Published: May 3, 2016
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molecular volume of the lipids, which leads to a coupling
between the electrostatic interactions and the lipid lateral phaseseparation. We calculate the diﬀerent phase diagrams ofcharged membranes by changing the interaction between the
two lipids and/or by changing the amount of added salt. We
also explain the diﬀerent combination of hydrocarbon
saturation and headgroup charge (either CS/NU or NS/CU
as were deﬁned above). On the basis of the calculated phase
diagrams, we discuss the main-transition temperature, and theeﬀect of added salt in a binary mixture of charged/neutral
lipids.
In the next section, we explain the model by Goldstein andLeibler, the phase separation, and the PB theory. In section 3,
we show the calculated phase diagrams and investigate theeﬀects of the interaction between the two lipids, salt
concentration, and combination of the lipids (either CS/NU
or NS/CU) on the phase separation. Moreover, we explore
how the molecular area changes and its dependence on theadded salt. Finally, we oﬀer a qualitative discussion in section 4
and some concluding remarks in section 5.

Figure 1. Schematic illustration of two-component membranes. (a) A
neutral membrane consisting of a binary mixture of neutral saturated(NS) and neutral unsaturated (NU) lipids. (b) A charged membraneconsisting of a binary mixture of charged saturated (CS) and neutral
unsaturated (NU) lipids. (c) A charged membrane consisting of abinary mixture of neutral saturated (NS) and charged unsaturated(CU) lipids.

2. MODEL
In order to calculate the phase diagrams of binary mixtures ofcharged lipid membranes, we consider a model that takes into
account both the lipid main-transition and the lateral lipid
phase separation. For simplicity, we consider a perfectly ﬂatmembrane ignoring any possible out-of-plane membrane
undulations. To avoid further complexity, we do not consider
any speciﬁc bilayer attributes such as asymmetric lipid
composition between the two leaﬂets, or ionic strength
diﬀerence between the electrolytes in contact with the twosides of the membrane. Therefore, our model describes thephase behavior of an isolated symmetric bilayer.
2.1. Two-Component Neutral Membranes. On the basisof the Goldstein−Leibler model,33,34 we ﬁrst review the freeenergy of a single-component neutral lipid membrane, whichleads to the main-transition. The main-transition involving thechain ordering and stiﬀening is described via a scalar orderparameter

Since a certain amount of charged lipid is always present inorganelles such as mitochondria and lysosomes,24 electrostaticinteractions are signiﬁcant in biomembranes. Recently, several
experiments have investigated the phase separation of multicomponent lipid mixtures containing at least one type of
charged phospholipid.25−30 In binary mixtures of neutral
saturated lipid and negatively charged unsaturated lipid, thephase-separated region was suppressed as compared with thecase when both lipids are neutral. This phase-separated region
reappears when salt (e.g., NaCl), which screens the electrostaticinteractions, was added. This is a strong indication that theelectrostatic interaction aﬀects the phase behavior. Althoughsuch behavior has been qualitatively explained by phenomenological models using the Poisson−Boltzmann (PB)
theory,26,31,32 the structural phase transition (the maintransition) of the membrane was not considered.
In membranes composed of binary mixtures of neutral andcharged lipids, either the saturated or unsaturated lipids can be
charged. Hence, it is convenient to deﬁne two types of charged
membranes: (i) mixtures of charged saturated (CS) and neutral
unsaturated (NU) lipids (denoted as CS/NU, see Figure 1b)and (ii) mixtures of neutral saturated (NS) and charged
unsaturated (CU) lipids (denoted as NS/CU, see Figure 1c). In
recent experiments by Himeno et al.,29 it was reported that thephase separation was suppressed in the NS/CU lipid mixture ascompared with the neutral membrane (NS/NU as in Figure1a), while it was enhanced in the CS/NU case. This result
indicates that the phase behavior of charged membranes is very
diﬀerent between these two types of charged mixtures.
Being motivated by this experimental work, we consider inthis paper charged membranes, and propose a phenomenological model that distinguishes between the above-mentioned
two cases. In particular, we discuss the interplay between the
main-transition and the lateral phase separation between the
two lipid species. The main-transition of lipid chains is taken
into account by extending the model of Goldstein andLeibler33,34 to include electrostatic interactions using the PB
theory.35,36 Furthermore, we employ the constraint of constantm=δ − δ0δ0(1)where δ is the actual membrane thickness and δ0 a reference
membrane thickness in the Lα phase. Notice that m represents
the changes that may occur in several degrees of freedom,including the conformation of the hydrocarbon chains, theirinterchain correlations, molecular tilt, and positional ordering.
For an isolated lipid bilayer, a Landau expansion of thestretching free energy per lipid molecule in powers of the orderparameter m is given by
fst (m) =aaa 2′(T − T *)m2 + 3 m3 + 4 m 4
2
3
4

(2)

where a′2 > 0, a3 < 0, and a4 > 0 are phenomenologicalcoeﬃcients. Here a3 is taken to be negative, since the maintransition is a ﬁrst-order transition,16 while a4 is always positive
to ensure thermodynamical stability. In the above, T is thetemperature and T* is a reference temperature. The maintransition temperature is diﬀerent from T* and is denoted by
T**. It can be calculated from the conditions fst = 0 anddfst/dm = 0, yielding
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T ** = T * +2(a3)29a 2′a4(3)Note that the main-transition temperature described by eq 3 isappropriate only for neutral lipids.
Next, we describe the phase separation in membranes
containing a binary mixture of neutral saturated (NS) andneutral unsaturated (NU) lipids, as shown in Figure 1a. We
model the two-component membrane as an incompressible
NS/NU lipid mixture containing ϕ molar fraction of NS lipid
and 1 − ϕ molar fraction of NU lipid. (Throughout this paper,
ϕ denotes the molar fraction of saturated lipid.) For simplicity,
we assume the same area per molecule for the two species inthe Lα phase and ignore any lipid exchange with thesurrounding solvent. The mixing free energy (per lipid
molecule) consists of the mixing entropy and enthalpy. It canbe expressed within mean-ﬁeld theory as

Figure 2. Schematic representation of the electrostatic potential of a
two-component charged membrane consisting of charged saturated(CS) and neutral unsaturated (NU) lipids. The lipid head groups arelocated at the plane z = 0. The dimensionless electrostatic potential insolution is ψ(z) = eΨ/kBT, and ψs = ψ(z = 0) is the surface potential at
the membrane. The dielectric constant of water solution is εw.

2enb
d2Ψ
eΨ
=
sinh
2
εw
kBT
dz

fmix (ϕ) = kBT[ϕ ln ϕ + (1 − ϕ) ln(1 − ϕ)]+Jϕ(1 − ϕ)2

where e is the elementary charge, nb the salt concentration in
the bulk, and εw the dielectric constant of water. Using thedimensionless electric potential ψ = eΨ/kBT, we obtain thefollowing dimensionless PB equation(4)

where kB is the Boltzmann constant and J > 0 is an attractive
interaction parameter between the lipids, which enhances the
NS/NU demixing.
In general, the two lipids have diﬀerent main-transition
temperatures originating from diﬀerent molecular parameterssuch as their chain length, degree of saturation, and hydrophilic
headgroup. The dependence of the main-transition temperatureon the composition ϕ cannot be calculated from such aphenomenological approach. Therefore, we proceed by further
assuming the simplest linear interpolation of T* as a function ofϕ
T *(ϕ) = ϕTS* + (1 − ϕ)TU*d2ψ
= κ 2 sinh ψ
dz 2

(8)

where κ −1 = εw kBT /2e 2nb is the Debye screening length. Far
from the membrane, the bulk electric potential should vanish,i.e., ψ(z → ∞) = 0. With the use of the Gauss law, theboundary condition at the membrane (z = 0) is
εw

(5)

dψ
eσ
=−
dz z = 0kBT(9)Under these boundary conditions, the dimensionless electric
potential ψ(z) can be obtained35−37 by integrating twice eq 8

where TS* and TU* are the reference temperatures of pure NS
and NU lipids, respectively. In general, saturated lipids havehigher transition temperatures than the unsaturated ones,
TS** > TU**, for the cases when the numbers of hydrocarbons in
the lipid tails are the same. Therefore, we generally use
T*S > T*U .
By combining eqs 2 and 4, we obtain the total free energy ofa neutral NS/NU membranefNS/NU (m , ϕ) = fst (m , ϕ) + fmix (ϕ)(7)ψ (z) = 2 ln

1 + Γ exp( −κz)
1 − Γ exp( −κz)

(10)

where
Γ=

(6)

exp(ψs/2) − 1
exp(ψs/2) + 1(11)Here ψs = ψ(z = 0) is the dimensionless surface potential given
byThis total free energy will be minimized with respect to both mand ϕ to obtain the phase behavior of a neutral membrane.
2.2. Electrostatic Free Energy of a Charged Membrane. In what follows, we extend the above model for the caseof membranes consisting of a binary mixture of charged andneutral lipids. In order to estimate the electrostatic interactions
in membranes at a ﬁxed surface charge density (chosen to be
negative throughout this paper), σ < 0, we obtain the freeenergy of a charged membrane on the basis of the PB
theory.35,36
As shown in Figure 2, we consider a ﬂat charged lipid bilayerthat lies at z = 0. The bilayer is in contact with a monovalentsalt solution located at z > 0. We assume that the two leaﬂets
constituting the bilayer do not interact with each other. Withina mean-ﬁeld treatment, the electric potential Ψ(z) satisﬁes thePB equation⎛ 2π S σ ⎞⎛1⎞ψs = 2 sinh−1⎜ B ⎟ = −2 sinh−1⎜ ⎟⎝ bκ ⎠⎝ eκ ⎠(12)

where S B = e2/4πεwkBT is the Bjerrum length and b = e/2πS B|σ|is the Gouy−Chapman length.
The electrostatic free energy per lipid molecule can becalculated from the charging method37,38
fel (σ ) =

kBT Σ
e

σ

∫0 dσ′ψs(σ′)(13)

where Σ is the cross-sectional area per lipid. By substituting thesurface potential, eq 12, into eq 13, we obtain the expression
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kBT Σ ⎢bκ −π SBb ⎢⎣fel (σ ) =

+ ln

1+

1 + (bκ )2⎤
1 + (bκ )2 ⎥
⎥⎦
bκ

(14)

2.3. Two-Component Charged Membranes. In the caseof membranes consisting of a binary mixture of charged
saturated (CS) and neutral unsaturated (NU) lipids, the surface
charge density, σ < 0, is related to the CS molar fraction, ϕ, by
σ=−

eϕ
Σ

Figure 3. Schematic illustration of molecular volume conservation.
The molecular area is indicated by a gray square, and the molecularvolume is enclosed by the dashed lines. We assume that the molecularvolume v is conserved, v = Σβδβ = Σαδα, for all the temperatures. The
larger the membrane thickness δ, the smaller the molecular area Σ.

(15)

As illustrated in Figure 3, we further assume that the lipid
molecular volume, v = Σδ, is conserved and stays constant at
any temperature. From eq 1, the cross-sectional area Σ isrelated to the order parameter, m, by
Σ0
Σ=
1+m

μ=

where Σ0 is the cross-sectional area of a neutral lipid in the Lαphase. By substituting back eqs 15 and 16 into eq 14, fel can be
written as⎤
1 + [pϕ(1 + m)]2 ⎥
⎥
⎦

)

(17)

where p = 2πS B/Σ0κ is a dimensionless parameter.
The total free energy of the CS/NU membrane is ﬁnallygiven byfCS/NU (m , ϕ) = fst (m , ϕ) + fmix (ϕ) + fel (m , ϕ)

3. RESULTS
Having described our model in the previous section, we present
the resulting phase diagrams and the temperature dependenceof the molecular area. First, we show the phase diagrams for theneutral membrane case. This calculation essentially reproduces
the theoretical results of refs 19 and 20. Then, the phase
diagrams for charged membranes will be presented.
3.1. Choice of System Parameters. As examples fortypical neutral saturated (NS) lipids and neutral unsaturated(NU) lipids, we consider, respectively, dipalmitoylphosphocholine (DPPC) and dioleoylphosphocholine (DOPC), whose
main-transition temperature is T*S * = 41 °C and T*U * = −20 °C,
respectively. From eq 3, the reference temperature of NS and(18)

On the other hand, when the membrane consists of neutral
saturated (NS) and charged unsaturated (CU) lipids, the total
free energy of an NS/CU membrane becomes
fNS/CU (m , ϕ) = fst (m , ϕ) + fmix (ϕ) + fel (m , 1 − ϕ)(19)Note that the only change between the two cases, eqs 18 and
19, is that ϕ ↔ 1 − ϕ in the electrostatic free energy. The
phase behavior of two-component charged membranes in thesetwo cases is analyzed using either eq 18 or 19.
2.4. Equilibrium Phase Behavior. Since the electrostatic
free energy described by eq 17 depends on the order parameterm, the main-transition temperature of a pure charged lipid(either CS or CU) is diﬀerent from eq 3. Instead, it is given by
the following conditions
fst (mβ , 1) + fel (mβ , 1) = fst (mα , 1) + fel (mα , 1)∂(fst + fel )∂m=
m = mβ∂(fst + fel )∂mTable 1. Parameter Values of the Modelparameter
a2′
a3
a4
T*S
T*U
Σ0
εw

(20)

=0
m = mα(23)

The two-phase coexistence region in the (T, ϕ) plane iscalculated by using the common tangent construction toaccount for the constraint value of the molar fraction ϕ. In
other words, the two-phase coexistence has to satisfy theconditions g(ϕ1) = g(ϕ2) and μ(ϕ1) = μ(ϕ2), where the slopeof the common tangent is equal to the chemical potential. Fromthe coexisting two points in the (T, ϕ) plane, we can also
construct the phase diagram in the (T, μ) plane. The Lβ phaseis characterized by positive m values, while its value in the Lαphase becomes negative.

pϕ(1 + m)

(

(22)

m=m*

g (ϕ) = ftot (m = m*, ϕ) − μϕ

1 + [pϕ(1 + m)]2

+ ln pϕ(1 + m) +∂ϕ

where f tot is taken either from eq 6 for neutral membranes or
eqs 18 and 19 for the two cases of charged membranes. The
optimal order parameter, m*, is obtained by minimizing thetotal free energy with respect to m, ∂f tot/∂m|m=m* = 0.
As the lipid molar fraction ϕ is a conserved quantity, it isconvenient to introduce another thermodynamic potential
given by

(16)

⎡
1−
fel (m , ϕ) = 2kBTϕ⎢
⎢
⎣

∂ftot

(21)

For lipid mixtures, the chemical potential μ is given by
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value
−21

2.4 × 10−1.1 × 10−18
2.2 × 10−18
−10
−71
65
7.08 × 10−10

unit
J/K (Ref 34)
J (Ref 34)
J (Ref 34)
°C
°C
Å2
F/m
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Figure 4. Phase diagrams of a neutral NS/NU membrane as a function of the molar fraction, ϕ, of the NS lipids, and temperature, T, reproducing thetheoretical results of refs 19 and 20. The attractive interaction parameter J is chosen to be 1.2 × 10−20 J in part a and 1.8 × 10−20 J in part b. The
other parameters are listed in Table 1 and are mentioned in the text. The critical point located at (ϕc, Tc) = (0.5, 53 °C) in part b is indicated by aﬁlled circle. The horizontal dashed lines at T = −19.2 °C in part a and T = −19.7 °C and T = 36.4 °C in part b indicate a three-phase coexistence(triple point).

Figure 5. Phase diagrams of a neutral NS/NU membrane as a function of the chemical potential, μ, of the NS lipids and temperature T. The
attractive interaction parameter J is chosen to be 1.2 × 10−20 J in part a and 1.8 × 10−20 J in part b. The other parameters are listed in Table 1 andmentioned in the text. The critical point located at (μc, Tc) = (0 J, 53 °C) in part b is indicated by a ﬁlled circle. The ﬁlled squares located at (μ, T) =(−1.44 × 10−20 J, −19.2 °C) in part a and (−1.45 × 10−20 J, −19.7 °C) and (0 J, 36.4 °C) in part b indicate the triple points.

NU lipids is set as T*S = −10 °C and T*U = −71 °C, respectively.
The molecular area of the neutral lipid in the Lα phase is ﬁxed
to Σ0 = 65 Å2. The interaction parameter is J = 1.2 × 10−20 or
1.8 × 10−20 J as considered before,19,20,22 and the bulk salt
concentration is chosen either as nb = 1 M or 10 mM. All theparameters and physical constants used in our calculations are
summarized in Table 1.
3.2. Two-Component Neutral Membranes. For the NS/
NU membrane, the total free energy is given in eq 6. As
mentioned before, the NS lipid (whose molar fraction is ϕ) has
a higher main-transition temperature than that of the NU lipid.
Following the previous theoretical studies,19,20 we reproduce inFigures 4 and 5 the phase diagrams of two-component NS/NU
neutral membranes in the (T, ϕ) and (T, μ) planes,
respectively. Figures 4a and 5a are calculated for J =1.2 × 10−20 J, while Figures 4b and 5b are obtained for J =1.8 × 10−20 J. The main-transition temperature of NS (ϕ = 1)
and NU (ϕ = 0) lipids is T*S * = 41 °C and T*U * = −20 °C,
respectively. As shown in Figure 4a, we obtain three two-phase(2)(1)
coexistence regions denoted by Lα + L(1)β , Lα + Lβ , and Lβ +L(2)β , and in Figure 4b, there are ﬁve two-phase coexistence
(2)
(1)
(1)
(1)
(2)
(2)
regions indicated by L(1)α + Lα , Lα + Lβ , Lα + Lβ , Lα +
19,20
(2)
(1)
(2)
the phaseLβ , and Lβ + Lβ . As discussed before,
separated region is more extended when the interaction
parameter J is larger.
3.3. Two-Component Charged Membranes. Next, we
calculate the phase diagrams for two-component charged
membranes. We ﬁrst consider the charged saturated/neutral
unsaturated (CS/NU) (Figure 1b) lipid mixtures whose total
free energy is given by eq 18. In Figures 6a and 7a, we show thecalculated phase diagrams for J = 1.2 × 10−20 J and nb = 1 M,
recalling that ϕ is the molar fraction of the CS lipids. The maintransition temperature of the CS lipid is calculated numericallyfrom eqs 20 and 21, yielding TS** ≃ 29 °C. On the other hand,
the main-transition temperature of the NU lipid is TU** =−20 °C, as before. In this phase diagram, there is only one
coexistence region between the Lα and Lβ phases.
In Figures 6b and 7b, we obtain the phase diagrams for J =1.8 × 10−20 J, while the other parameters are the same as thosein Figures 6a and 7a. The main-transition temperatures of the6362

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Figure 6. Phase diagrams of two-component charged membranes as a function of the molar fraction, ϕ, of saturated lipid (either CS or NS) andtemperature T. (a) CS/NU membrane with J = 1.2 × 10−20 J and nb = 1 M; (b) CS/NU membrane with J = 1.8 × 10−20 J and nb = 1 M; (c) CS/NU
membrane with J = 1.2 × 10−20 J and nb = 10 mM; and (d) NS/CU membrane with J = 1.2 × 10−20 J and nb = 1 M. The other parameters are listed
in Table 1 and are mentioned in the text. The horizontal dashed lines located at T = −15.3 °C in part b and T = −28.0 °C in part d indicate thethree-phase coexistence (triple point).

neutral. Due to the lipid volume conservation, the maintransition temperature of the charged lipid (either CS or CU) is
reduced as compared with that of the neutral counterpart.
These ﬁndings will be further discussed in section 4.
3.4. Molecular Area in Charged Membranes. In Figure
8, we show the temperature dependence of the molecular area,
Σ, which is calculated from the optimal order parameter, m*,and eq 16. For the CS/NU mixture, the molar fraction ϕ of the
CS lipid is varied, and the salt concentration is either nb = 1 M(high salt) or 10 mM (low salt), as shown in Figure 8a and b,
respectively. In Figure 8c, we show the molecular area of the
NS/CU mixture for nb = 1 M. Since Σ is independent of theinteraction parameter J, the calculation in Figure 8a has beenused in generating Figure 6a and b. Similarly, the calculated Σvalues shown in Figure 8b and c were used to obtain Figure 6cand d, respectively. In order to see the change of the moleculararea clearly, we ignore here the multiphase coexistence and lookat only the minimum of the total free energy, f tot.
In Figure 8a and b, for a pure NU membrane (ϕ = 0), Σ
gradually increases when the temperature is increased
approaching the main-transition temperature. As mentioned
in section 2, the molecular area discontinuously jumps to alarger value as appropriate for the Lα phase at TU** = −20 °C
because the main-transition is ﬁrst-order. In the Lα phase of thetwo lipids are unchanged, because the conditions in eqs 20 and
21 are independent of the interaction parameter J. In this case,there are three two-phase coexistence regions denoted by Lα +
(2)
(1)
(2)
L(1)
β , Lα + Lβ , and Lβ + Lβ .
In order to further explore the eﬀect of electrostaticinteraction in CS/NU mixtures, we looked at the case oflower salinity. Figures 6c and 7c are the phase diagrams
corresponding to the low salt case of nb = 10 mM. The otherparameters are the same as those in Figures 6a and 7a. The
main-transition temperature of the CS lipid is now T*S * ≃23 °C, while that of the NU lipid is unchanged (T*U * =−20 °C). In this case, there is only one two-phase coexistence
region between the Lα and Lβ phases.
Finally, we show in Figures 6d and 7d the phase diagrams forthe neutral saturated/charged unsaturated (NS/CU) lipid
mixtures whose total free energy is given by eq 19. Noticethat the molar fraction ϕ refers here to that of the NS lipids.
The other parameters, J and nb, are the same as those in Figures
6a and 7a. The main-transition temperature of the NS lipid is
TS** = 41 °C, and that of the CU lipid is decreased to TU** ≃−30 °C. There are three two-phase coexistence regions
(2)
(1)
(2)
denoted by Lα + L(1)β , Lα + Lβ , and Lβ + Lβ .
The phase behavior of two-component membranes depends
on various quantities such as the interaction parameter J, thesalt concentration nb, and whether the lipid is charged or
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Figure 7. Phase diagrams of two-component charged membranes as a function of the chemical potential, μ, of saturated lipid (either CS or NS) andtemperature T. (a) CS/NU membrane with J = 1.2 × 10−20 J and nb = 1 M; (b) CS/NU membrane with J = 1.8 × 10−20 J and nb = 1 M; (c) CS/NU
membrane with J = 1.2 × 10−20 J and nb = 10 mM; and (d) NS/CU membrane with J = 1.2 × 10−20 J and nb = 1 M. The other parameters are listed
in Table 1 and are mentioned in the text. The ﬁlled squares located at (μ, T) = (−0.50 × 10−20 J, −15.3 °C) in part b and (−2.48 × 10−20 J,−28.0 °C) in part d indicate the triple points.

Figure 8. Plots of molecular area Σ as a function of the temperature T. (a) CS/NU lipid membrane with nb = 1 M, (b) CS/NU lipid membrane with
nb = 10 mM, and (c) NS/CU lipid membrane with nb = 1 M. Black, red, blue, green, and orange lines correspond, respectively, to ϕ = 0, 0.25, 0.5,
0.75, and 1.

NU membrane, Σ takes a constant value, Σ0 = 65 Å2, because
the order parameter m* always vanishes there.
In the presence of the CS lipid (ϕ > 0), Σ also increases andexhibits a discontinuous jump at the main-transition temperature. At the transition temperature, as compared with theneutral membranes, Σ for the charged membranes is slightly
smaller for the Lβ phase but larger for the Lα phase. Thistendency can be clearly seen for higher molar fractions of CS
lipid (ϕ ≈ 1) or for lower salt concentrations (Figure 8b). As
the temperature is further increased from the main-transition
temperature, T**, Σ in the Lα phase asymptotically approaches
the constant value, Σ0 = 65 Å2.
The temperature dependence of Σ for the NS/CU mixture isshown in Figure 8c. The molecular area ﬁrst increases in the Lβ
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not taken into account. In the present work, we have included
this phase transition in order to fully describe the phasebehavior of two-component charged membranes. The advantage of our model is that it describes the diﬀerent phasebehavior, distinguishing CS/NU and NS/CU lipid mixtures.
As mentioned in section 1, there are two types of twocomponent charged membranes: CS/NU and NS/CU lipid
mixtures. These two cases are presented in Figure 6a and d,which manifest that the phase-separated region in the formercase is narrower than that in the latter one. This is because thediﬀerence in the main-transition temperature between thesaturated and unsaturated lipids is larger in the NS/CU case
(Figure 6d). We note again that the reduction of the transition
temperature for charged lipids is induced by the molecularvolume conservation.
In general, the larger the diﬀerence in the transition
temperatures, the stronger the segregation, and the phaseseparation is enhanced. Himeno et al.29 reported that the phasebehaviors of CS/NU and NS/CU lipid mixtures are quite
diﬀerent. In particular, they obtained the liquidus lines andcompared the phase separations in CS/NU and NS/CU
mixtures with that in an NS/NU mixture. According to theirresults, the phase separation is enhanced for a CS/NU mixture,
whereas it is suppressed for an NS/CU mixture. Although it isdiﬃcult to compare directly this experiment with our theory,
our model clearly distinguishes between the two diﬀerent typesof charged membranes, and the trends agree with experiments.
In the previous experimental work,29 it was considered that
there is a counterion mediated attraction between charged
saturated lipids, which overcomes the electrostatic repulsion
between the charged head groups. For simplicity, this eﬀect was
not considered in the present study. However, it will be ofinterest to consider such added interactions in future theoreticalstudies in order to make a direct comparison with theexperimental results.
We further comment now on the discontinuous jump of themolecular area at the main-transition temperature. For a pureNS lipid membrane (ϕ = 1 in Figure 8c), the molecular area
jumps from Σ = 48.7 to 65 Å2 at T*S * = 41 °C. For a pure CS
lipid membrane (ϕ = 1 in Figure 8a or b), on the other hand, itvaries from Σ = 47.4 to 67.6 Å2 at TS** = 29 °C in Figure 8a andfrom Σ = 46.9 to 68.8 Å2 at T*S * = 23 °C in Figure 8b. Thismeans that the main-transition of a CS lipid occurs when thetemperature is lower and the molecular area is smaller, as
compared with an NS lipid. This behavior can be explained by
the electrostatic repulsion between the charged head groups(see Figure 3). In general, the main-transition is induced when
the attraction between the lipids becomes weaker. For CS
lipids, the electrostatic repulsion between the lipids acts inaddition to the attraction. Hence, the main-transition occurs at
a lower temperature and smaller molecular area. Moreover, themolecular area of a CS lipid in the Lα phase is larger than thatof an NS lipid (Σ = 65 Å2) due to the electrostatic repulsion.
The increase in the molecular area at the main-transition ismore enhanced when the molar fraction of charged lipid ishigher.
For higher temperatures, the molecular expansion in twocomponent charged membranes is suppressed. In the presenceof charged lipid, Σ in the Lα phase asymptotically approaches
Σ0 as the temperature is further increased. We recall here thatthe total free energy of CS/NU or NS/CU membranes is given
by eq 18 or 19. Taking into account the temperaturedependence of p = 2π SB/Σ0κ = e/Σ0 8nbεw kBT , we note

phase and then jumps discontinuously at the main-transition
temperature. This molecular area expansion is larger for higher
molar fraction of the CU lipid (ϕ ≈ 0). In the next section, weshall discuss in more detail the eﬀect of the lipid volumeconservation on the molecular area.

4. DISCUSSION
Several points merit further discussion. First, we discuss theeﬀect of the interaction parameter J on the phase behavior. As
shown in Figures 4 and 6 for neutral and charged membranes,
respectively, the phase-separated region becomes larger as J
increases. In particular, we ﬁnd diﬀerent coexistence regions(2)
such as liquid−liquid (L(1)α + Lα ) in Figure 4b and solid−solid(1)(2)(Lβ + Lβ ) in Figure 6b. These coexistence regions do notappear for smaller J value as in Figures 4a and 6a. Such adependence on J was also reported in previous theoreticalworks.19,20
As seen in Figure 6, the main-transition temperature, T**, is
reduced for a single-component charged lipid membrane. The
transition temperature of NS lipid is T*S *= 41 °C, while it isdecreased to T*S * ≃ 29 °C for CS lipid as in Figure 6a and b, or
to TS** ≃ 23 °C as in Figure 6c. The electrostatic repulsion
between the charged lipid molecules tends to expand themolecular area, resulting in a decrease of the membranethickness, while keeping the molecular volume v = Σδ constant(see Figure 3). Hence, the molecular volume conservation
enhances the phase transition toward the Lα phase, and themain-transition temperature is decreased.39 For a singlecomponent unsaturated lipid, the reduction of the transition
temperature from TU** = −20 °C (for NU) to −30 °C (for CU)
can also be explained by the volume conservation constraint(see Figure 6d).
Next, we comment on the eﬀect of the electrostaticinteraction on the extension of the phase-separated regions.
We have presented the phase behavior of a neutral NS/NU
membrane (Figure 4a), a charged CS/NU membrane with ahigh salt concentration (Figure 6a), and a charged CS/NU
membrane with a low salt concentration (Figure 6c). The phaseseparation takes place below T = 41 °C in Figure 4a, between
T = 29 °C and −20 °C in Figure 6a, and between T = 23 °C
and −20 °C in Figure 6c. As the electrostatic interaction
strength is increased in the order of Figures 4a, 6a, and 6c, thephase-separated region becomes narrower. For the NS/CU
membrane in Figure 6d, the phase coexistence occurs below
T = 41 °C. Although this temperature range coincides with thatof neutral NS/NU membranes as in Figure 4a, the phasecoexistence region in charged membranes (Figure 6d) isnarrower than that in neutral membranes (Figure 4a). Hence,the phase separation is generally suppressed in charged
membranes as compared with neutral ones.
Some experiments 25−29 reported that the two-phasecoexistence region for charged membranes (containing mainly
CU lipid) is narrower than that for neutral membranes, and thephase separation in these membranes can be enhanced again byadding a salt. Compared with two-component neutral
membranes (Figure 4a), the phase-separated region of an
NS/CU membrane (Figure 6d) becomes narrower due to theelectrostatic interactions. This is indeed consistent withexperiments. Previous theoretical models for charged membranes26,31,32 have considered only the contributions from thephase separation and the electrostatic interactions. Althoughthese works also reported the suppression of the phaseseparation in charged membranes, the main-transition was
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The Journal of Physical Chemistry B

In particular, we have considered a coupling eﬀect between theorder parameter and the lipid composition through themolecular volume conservation. We showed that the phaseseparation in charged membranes is suppressed as comparedwith the neutral case. Furthermore, as the salt concentration isincreased because of screening, the phase separation in charged
membranes is enhanced again, signaling the importance ofelectrostatic interaction.
We have focused on the combination of the lipidhydrocarbon saturation and the charge on the headgroup(either CS/NU or NS/CU). The phase behavior of these two
types of binary mixtures of charged/neutral lipids is very
diﬀerent, and is in agreement with recent experiments.29 We
also ﬁnd that the molecular area of charged/neutral mixtures issmaller in the Lβ phase and larger in the Lα, as compared withneutral mixtures. In other words, the discontinuous jump of themolecular area for charged/neutral mixtures is larger than thatfor the neutral ones. This tendency is clearly seen when the
charged lipid fraction is large and/or the salt concentration islow, and can be understood by the electrostatic repulsion
between the charged head groups. In general, the phasebehavior is signiﬁcantly inﬂuenced by the presence of thecharged lipid.
Our model describes the phase behavior of binary mixturesof charged/neutral lipids. In most of the recent experimental
studies,25−29 however, the phase separation has been observedin ternary charged lipid mixtures containing cholesterol. Since
these studies are motivated by the formation of raft domains
enriched in cholesterol, lipid mixtures containing cholesterol
are also important from the biological point of view. Putzel and
Schick proposed a model42 for the phase separation in neutral
membranes consisting of saturated lipids, unsaturated lipids,
and cholesterol. In the future, it will be of interest to consider amodel for ternary charged lipid membranes, which will take
into account explicitly the speciﬁc role of cholesterol, in
addition to the eﬀect of charges in lipid mixtures.

that the relative contribution of the electrostatic free energy
vanishes in the high temperature limit. Namely,fel/( fst + f mix) → 0 as T → ∞. This means that the electrostaticinteraction becomes irrelevant with respect to the nonelectrostatic free energies in the high temperature limit, andΣ → Σ0.
In neutral membranes, the optimal order parameter m*vanishes in the Lα phase. On the other hand, in charged
membranes, m* in the Lα phase becomes negative due tomolecular volume conservation. Since the order parameter
represents the membrane thickness, negative m means an
expansion of the molecular area, as shown in Figure 3. In orderto understand why m* becomes negative, we expand theelectrostatic free energy fel of eq 17 in powers of mfel (m , ϕ)
kBT

⎡
1−
≈ 2ϕ⎢
⎢
⎣

(

+ ln pϕ +

(

1−

−

1 + (pϕ)2
pϕ2

1 + (pϕ)

1 + (pϕ)22

p 1 + (pϕ)

⎤
2 1−
⎥−
⎥
⎦

(

)

1 + (pϕ)2
p

)m

2

)m

2

(24)

The order parameter m is determined by minimizing the total
free energy, eq 18, which includes eq 24. The latter equation isminimized for nonzero m due to the linear term in m because
1 + (pϕ)2 > 1. Since the stretching free energy fst in eq 2
does not include any linear term in m, the total free energy is
minimized for a negative m* < 0. As mentioned above, theelectrostatic interaction becomes negligible at higher temperatures. Therefore, m* asymptotically vanishes when thetemperature is increased.
In this study, we have used the Poisson−Boltzmann theory
to obtain the electrostatic potential for symmetric monovalentsalt. Such a mean-ﬁeld treatment would break down when ion−
ion correlations become signiﬁcant. For example, when
multivalent ions are present or a crowding state of charged
lipids occurs, it would be important to include ion−ion
correlations beyond the mean-ﬁeld theory. This point will be
addressed in a future study. Furthermore, modulated phases inneutral lipid membranes were discussed theoretically by taking
into account both the lipid main transition and the spatial
dependence of the lipid composition.22 In the case of a charged
lipid membrane, the formation of modulated phases waspredicted within the Debye−Hückel approximation without
considering the lipid main transition.40 However, theoccurrence of such a microphase separation has not yet beenreported experimentally for charged bilayer membranes using
optical microscopes.25−30 This is because the characteristic
wavelength of the microphase separation is considered to be of
the order of nanometers.41 Further theoretical and experimental studies are needed in order to investigate the possibilityof such microphase separations in charged lipid membranes.

■

AUTHOR INFORMATION

Corresponding Author*E-mail: nshimo@jaist.ac.jp. Phone: +81-(0)761-51-1657.
Fax: +81-(0)761-51-1525.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
We thank R. Okamoto for useful discussions. N.S. acknowledges support from the Grant-in-Aid for Young Scientist (B)(Grant No. 26800222) from the Japan Society for the
Promotion of Science (JSPS) and the Grant-in-Aid for
Scientiﬁc Research on Innovative Areas “Molecular Robotics”(Grant No. 15H00806) from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (MEXT).
T.H. acknowledges support from the MEXT KAKENHI Grant
No. 15H00807 and 26103516, JSPS KAKENHI Grant No.
15K12538, and AMED-CREST, AMED. M.T. acknowledges
support from the Grant-in-Aid for Scientiﬁc Research on
Innovative Areas “Thermal Biology” (Grant No. 15H05928)
from MEXT and the Grant-in-Aid for Scientiﬁc Research (B)(Grant No. 26289311) from JSPS. S.K. acknowledges support
from the Grant-in-Aid for Scientiﬁc Research on Innovative
Areas “Fluctuation and Structure” (Grant No. 25103010) from
MEXT, the Grant-in-Aid for Scientiﬁc Research (C) (Grant

5. CONCLUSION
In this work, we have considered phase transitions andseparations in binary mixtures of charged membranes. Taking
into account the chain main-transition, we have calculated thephase diagrams of two-component charged and neutral lipids.
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No. 15K05250) from JSPS, and the JSPS Core-to-Core
Program “International Research Network for Non-equilibrium
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