185

Biochimica et Biophysica Acta, 470 (1977) 185--201

© Elsevier/North-Holland Biomedical Press

BBA 77810

THEORY OF SELF-ASSEMBLY OF LIPID BILAYERS AND VESICLES

JACOB N. ISRAELACHVILI, D. JOHN MITCHELL a and BARRY W. NINHAM

Department of Applied Mathematics, Research School of Physical Sciences, and a Depart-
ment of Neurobiology, Research School of Biological Sciences, Institute of Advanced
Studies, The Australian National University, Canberra, A.C.T. 2600 (Australia)
(Received January 11 th, 1977 )
(Revised manuscript received June 7th, 1977)

Summary

A simple theory is developed that explains the formation of bilayers and

vesicles and accounts quantitatively for many of their physical properties:
Properties including vesicle size distributions and bilayer elasticity emerge from
a unified theory that links thermodynamics, interaction free energy, and
molecular geometry. The theory may be applied to the analysis of more com-
plicated membrane structures and mechanisms.

Introduction

Any fundamental theory of self-assembly of small molecules, such as phos-

pholipids, into large well-defined structures, such as bilayers, must involve (1)
the interaction free energies of the molecules, (2) the geometry of the mole-
cules and (3) a correct thermodynamic treatment of the whole system.
The thermodynamics of self-assembly of lipid molecules into micelles has
long been well understood [1--4], though only recently has the role of molec-
ular geometry been appreciated [3--6]. On the other hand, in the area of
bilayer and vesicle research, while the importance of the geometry (or packing)
of lipids has been recognized [5,6], the role of thermodynamics in self-assem-
bly has been ignored. Theories of micelles use thermodynamics together with
the ideas of "opposing forces" and "hydrophobic forces", with emphasis on
such micellar properties as the critical micelle concentration and micelle
aggregation numbers [3]. By contrast, theories of bilayers and membranes
have been modelled on certain a priori assumptions involving such concepts
as "fluid mosaics" [7], "lipid packing" [5,6] or "bilayer elasticity" [8--10],
with the emphasis on bilayer and membrane structure. Yet the only difference
between bilayers and micelles is that lipids containing two (or more) hydro-
carbon chains form bilayers, whereas single chained lipids form micelles. A
single theory should describe both.
186

We have shown [4] that when thermodynamics, interaction free energies,

and molecular geometry are taken together a unified theory emerges that
accounts for many previously unexplained properties of micelles (both spheri-
cal and cylindrical), and also explains why diacyl chained lipids form into
extended bilayers and vesicles. Here we develop the theory further for diacyl
chained lipids, and consider both one- and two-component bilayer vesicles. For
a more detailed account of the theoretical background, see ref. 4.

Mechanism of self-assembly
The assembly of lipids into well defined structures such as micelles and
bilayer vesicles derives from the hydrophobic interaction between the hydro-
carbon tails, which induces the molecules to congregate, and the hydrophilic
nature of the head groups, which imposes the requirement that the head groups
remain in contact with water. These two effects compete to give rise to the idea
of two ' o p p o s i n g forces" [3]: the one tending to decrease and the other tend-
ing to increase the head group area in contact with water. These interactions
lead to the concept of an "optimal surface area" per head group at which
the total interaction free energy per lipid molecule is a minimum.
Geometric considerations can now be applied to establish the possible
structures. These depend on the optimal surface area and the hydrocarbon
chain volume, and are limited by the maximum length that the hydrocarbon
chains can extend. Such packing considerations illustrate why single-chained
lipids (e.g. lysophosphatidylcholine) can assemble into small micelles while
double-chained lipids (e.g. diacyl phosphatidylcholines) cannot. However, geo-
metry does allow double-chained lipids to form into bilayers and vesicles, as
indeed they do. But so can the single-chained lipids, which do not in practice.
The deciding factor is entropy, which tells us that those structures with large
lipid aggregation numbers, while possible on energetic and geometric grounds,
are improbable entropically.
Thus the formation of bilayers from lysophosphatidylcholine does not occur
because of entropy, whereas the formation of micelles from diacyl-phosphati-
dylcholine does not occur because of energy and geometry. Diacyl lipids form
vesicles of homogeneous size: larger vesicles are disNlowed because of entropy
while smaller vesicles are disallowed because of energy and packing.
The concepts of interaction free energies, molecular geometry and entropy,
when taken together, furnish a framework for a theory of self-assembly.

Thermodynamics, interaction energies and packing constraints o f lipids in

aggregated structures
Thermodynamics of self-assembly. Equilibrium thermodynamics demands
that the chemical potential of all molecules in a system of aggregated struc-
tures (micelles, bilayers, etc.) will be the same. This may be expressed as [1--4]

kT
pO +-~:-ln(XN/N) = const. N = 1, 2, 3, ..., (1)
N

where XN is the mol fraction of molecules incorporated into micelles of

aggregation number N (XN = concentration in mole fraction units, i.e. in Mol/
 187

55.5, for an aqueous solvent), po is the free energy per molecule in the micelle,
h is Boltzmann's constant and T is the temperature. N = 1 corresponds to iso-
lated molecules or monomers in solution. Eqn. 1 can be written more con-
veniently in the form
XN = N(XM/M)N/M eN(U~l -- U~V)/kT (2)
where M is any arbitrary reference state of micelles with aggregation number M.
Eqn. 2 is the basic thermodynamic equation for any self-assembly analysis. It
shows, for example, that in the absence of the free energy contributions small
aggregates are entropically favoured over larger ones; in fact, when p o = p o ,
most of the molecules will be in the m o n o m e r state (N = 1). Little more can be
said without spelling out the form and magnitude of the free energies p~.
I n t e r a c t i o n free energies. We shall assume that the temperature of the hydro-
carbon chains is above the melting temperature. The interaction forces between
lipids in an aggregate will therefore arise mainly from the hydrocarbon-water
interface and polar head group region. Contributions to the interaction free
energy po will be of two kinds: (1) an attractive interaction arising from attrac-
tive hydrophobic or interfacial tension forces, and (2) a repulsive interaction
arising from electrostatic head-group repulsion, steric head-group repulsion and
steric hydrocarbon side-chain repulsion.
(1) The attractive contribution can be represented by an interfacial free
energy per unit area, 7 ~ 50 erg/cm 2, characteristic of a liquid hydrocarbon-
water interface [4]. The hydrophobic free energy contribution to p~ will
therefore be 7a, where a is the molecular area measured at the hydrocarbon-
water interface.
(2) The repulsive contributions are more difficult to formulate explicitly,
and we adopt an expression of the form C/a, where C is a constant. For zwit-
terionic and ionic head-groups an electrostatic repulsive energy of the form
C/a is appropriate. This choice for the repulsive energy is not critical [4]; all
the subsequent analysis can be carried o u t with other repulsive forms, e.g.
C/a 2 .
The repulsive free energy contribution to p o is therefore taken as C/a,
where C is a constant, into which all repulsive interactions are incorporated. C
may be expected to be larger for larger head-groups and, in the case of anionic
head-groups, should increase with increasing pH and decreasing ionic strength
[4,11]. Note that the area a in the repulsive energy term C/a is not necessarily
measured at the hydrocarbon-water interface, but may be slightly above it (if
the head-group repulsion dominates) or below it (if the side-chain repulsion
dominates). This effect is important when curvature corrections are introduced
into the free energy b u t can be ignored in the present case.
The free energy per molecule is thus
g o = 7a + C/a . (3)
The minimum free neergy p°(min) is given when
0u o
-7--C/a 2=0 i . e . a = a o = Cx/-C-~ (4)
0a
188

whence

p ° ( m i n ) = 2~a o. (5)
a0 will be r e f e r r e d t o as the o p t i m a l surface area per m o l e c u l e (defined at the
h y d r o c a r b o n - w a t e r interface), being t h a t area at which the free energy per
m o l e c u l e in a micelle or bilayer is a m i n i m u m . The free e n e r g y per m o l e c u l e
m a y n o w be expressed in the c o n v e n i e n t f o r m

pO = ~/(a + a2/a) = 2ao~ + ~ ( a -- ao) 2 . (6)

a

E q n . 6 shows t h a t p~ has a parabolic (elastic-type) variation a b o u t the m i n i m u m

energy, as e x p e c t e d f o r a n y t y p e o f i n t e r a c t i o n . If p h o s p h o l i p i d s can p a c k into
a variety o f s t r u c t u r e s in which t h e i r surface areas r e m a i n equal or close t o a0,
it follows f r o m Eqn. 2 t h a t e n t r o p y will f a v o u r the s t r u c t u r e with the smallest
aggregation n u m b e r . We m u s t t h e r e f o r e n o w c o n s i d e r h o w the m o l e c u l a r geo-
m e t r y o f lipids d e t e r m i n e the sort o f s t r u c t u r e s t h e y can pack into such t h a t
their surface areas are close t o a0 and at t h e same t i m e the aggregation n u m b e r
N is a m i n i m u m .
P a c k i n g c o n s t r a i n t s . Given t h a t the h y d r o c a r b o n interior o f a p h o s p h o l i p i d
in a bilayer is in a liquid-like state, we m a y derive g e o m e t r i c expressions t h a t
relate the h y d r o c a r b o n - w a t e r interfacial area a, the h y d r o c a r b o n chain v o l u m e
v, the h y d r o c a r b o n thickness l, and t h e radius o f c u r v a t u r e R o f the s t r u c t u r e
(bilayer or micelle) m e a s u r e d at the i n t e r f a c e (Fig. 1).
F o r a spherical micelle o f radius R = l, we have 4 u R 3 / 3 v = 4 n R 2 / a = N , i.e.,
via = I/3. F o r a cylindrical micelle o f radius R = l, we o b t a i n v / a = 1/2.
F o r a spherical bilayer vesicle (Fig. 1) o f o u t e r radius R, the g e o m e t r i c
" p a c k i n g e q u a t i o n " for the o u t e r layer molecules is [4]

v l 1----+ (7)
a R 3

f r o m w h i c h , in t h e limit o f large R ( R > > l), we o b t a i n the obvious result f o r

a planar bilayer: v/a = l, w h e r e I is the bflayer half-thickness. N o t e t h a t in going
f r o m spherical micelle -~ cylindrical micelle -~ spherical vesicle -~ planar bilayer
the aggregation n u m b e r increases.
Our packing c o n s t r a i n t is t h a t the h y d r o c a r b o n region I m u s t n o t e x c e e d
some m a x i m u m length lc [ 3 , 4 ] . T h e desired s t r u c t u r e is t h e n t h a t which has
the smallest aggregation n u m b e r N and l equal t o or less t h a n Ic.

One-component vesicles
Consider a 1 2 - c a r b o n single-chained anionic lipid f o r which t y p i c a l l y [4] a0
= 70 A z, v = 350 £ 3 . T h u s v/ao = 5 A. F o r this lipid t o pack i n t o a spherical
micelle with a = ao t h e micellar radius w o u l d have t o equal l = 3v/a ~ 15 £ .
This w o u l d o n l y be possible if the h y d r o c a r b o n chain can e x t e n d this far,
which for a 1 2 - c a r b o n chain m a y just be possible [ 3 , 4 ] . Such lipids w o u l d
t h e r e f o r e f o r m into spherical micelles o f radii ~ 15 £ . T h e y c o u l d also f o r m
i n t o bilayers, with their areas r e m a i n i n g equal t o ao; b u t since the spherical
micelle has a l o w e r aggregation n u m b e r t h a n a bilayer t h e micelle w o u l d be t h e
 189

IMICELLES
) (~ monomer conc. ~,10.3 M

liquid-like
interior . ~ ~ ~_~.

,x" ~o~,~~ ~ '~.

,2o~,, ~*~g-'~"e
~ )

IVESICLEI
'
interfacial
hydrophobic
monomer_,°
conc.~10 M
attraction
head-group
repulsion
side chain
repulsion

! 5oi !

Fig. I . S c h e m a t i c i l l u s t r a t i o n o f m i c e H e and vesicle f o r m a t i o n in w a t e r . E n t r o p y favours s t r u c t u r e s w i t h

s m a l l a g g r e g a t i o n n u m b e r s . S i n g l e - c h a i n e d llpids c a n f o r m m i c e D e s . D o u b l e - c h a i n e d lipids c a n n o t a l w a y s
d o t h i s d u e to g e o m e t r i c p a c k i n g c o n s t r a i n t s . T h e s e f o r m b i l a y e r s a n d vesicles.

favoured structure entropically. If now the ionic strength of the aqueous

solvent were increased, the electrostatic head-group repulsion would decrease
and the optimal area would fall below 70 A 2 to, say, 60 A 2 . Thus now v / a
5.8 A, and the radius of a spherical micelle would now have to be equal to
l ~ 17.5 A. But the hydrocarbon chains cannot extend more than 15 A; hence
they can no longer pack into spherical micelles. To form cylindrical micelles,
the radius would only have to be l = 2 v / a ..~ 11.6 A, so that we may conclude
that the chains could comfortably pack into such cylindrical micelles and this
they can do at elevated ionic strengths even though the aggregation number is
higher than for a spherical micelle [12,13]. A rigorous analysis of the transition
from spherical to (finite) cylindrical micelles is complicated [4], but the thread
of the argument outlined above will be a recurring theme in all that follows: i.e.
lipid structures are determined by a subtle interplay of entropy which favours
small structures, and packing constraints which energetically resist the packing
of molecules into arbitrarily small structures.
190

If we d o u b l e the n u m b e r o f chains o f o u r lipid, we shall have v = 700 h3, b u t

the o p t i m a l area a0 w o u l d r e m a i n largely u n c h a n g e d a n d still close to 60 h 2. (This
is an i m p o r t a n t s u p p o s i t i o n , and is a c o n s e q u e n c e o f a s s u m i n g t h a t the chains
are in a liquid-like state. T h e o p t i m u m area ao will r e m a i n the s a m e if the
interfacial t e n s i o n 7 and repulsive c o e f f i c i e n t C in Eqns. 3 and 4 d o n o t change.
T h e value o f the interfacial t e n s i o n (7 ~ 50 e r g / c m 2) o f a h y d r o c a r b o n - w a t e r
i n t e r f a c e is clearly n o t d e p e n d e n t on t h e n u m b e r o f chains a n d so m a y safely
be a s s u m e d to r e m a i n c o n s t a n t . If t h e chains are in a liquid-like state, the repul-
sive c o e f f i c i e n t C, arising f r o m h e a d - g r o u p r e p u l s i o n , s h o u l d likewise r e m a i n
c o n s t a n t . T h e o p t i m a l area a0 s h o u l d n o t be m u c h a f f e c t e d b y d o u b l i n g or
e x t e n d i n g t h e h y d r o c a r b o n chains. This is b o r n e o u t b y e x p e r i m e n t s : b o t h
single-chained lipids, w h i c h f o r m i n t o micelles, a n d d o u b l e - c h a i n e d lipids,
which f o r m i n t o bilayers, have very m u c h t h e s a m e surface areas [ 4 ] , n o r m a l l y
in t h e range 5 0 - - 7 0 A 2 . F u r t h e r . the surface areas o f lipids m e a s u r e d a t the oil-
w a t e r i n t e r f a c e have b e e n f o u n d to be insensitive to chain length for areas
a b o v e the t r a n s i t i o n area (ref. 14 a n d Mingins, J., p e r s o n a l c o m m u n i c a t i o n . )
T h u s f o r t h e diacyl c h a i n e d lipid we have v/a ~ 7 0 0 / 6 0 ~ 12 h . Since the fully
e x t e n d e d length o f t h e chain is still the s a m e as b e f o r e (close to 15 A) n e i t h e r
spherical micelles n o r cylindrical micelles m a y be p a c k e d , since t h e i r radii
w o u l d have to be 36 A or 24 A, w h i c h is far b e y o n d the fully e x t e n d e d length
o f 15 A. H o w e v e r , p l a n a r bilayers can easily be p a c k e d since these w o u l d have a
h a l f - t h i c k n e s s via -~ 12 h . But t h e aggregation n u m b e r w o u l d n o w be v e r y
large. A l t e r n a t i v e l y , t h e lipids c o u l d p a c k i n t o spherical vesicles which, f r o m
Eqn. 7, p u t t i n g a = a0 = 60 A, v 70 A 3, l = 15 A, w o u l d have an o u t e r radius o f
R I ~ 62 A. Such a s t r u c t u r e w o u l d have t h e l o w e s t possible aggregation
n u m b e r c o n s i s t e n t w i t h t h e free e n e r g y p e r m o l e c u l e being a m i n i m u m and at
t h e s a m e t i m e satisfying t h e p a c k i n g c o n s t r a i n t s .
T h e o r e t i c a l p r o p e r t i e s o f vesicles and bilayers. Certain lipids c a n n o t p a c k
into small micelles or even small vesicles since this w o u l d require t h e i r h y d r o -
c a r b o n chain region t o be l o n g e r t h a n t h e h y d r o c a r b o n chains can e x t e n d . This
fully e x t e n s i b l e h y d r o c a r b o n region is d e n o t e d as the "critical l e n g t h " le, a n d
we e x p e c t Ic t o be s o m e w h a t less t h a n t h e fully e x t e n d e d m o l e c u l a r length o f
t h e h y d r o c a r b o n chains [3,4]. R e a r r a n g i n g eqn. 7 we o b t a i n f o r t h e o u t e r
radius R o f a spherical vesicle

l[3 + v ~ ( 4 v / a l -- 1)] l
(8)
611 - - v/al] (1 - - v/al)

If t h e surface area a is t o r e m a i n e q u a l t o t h e o p t i m a l area a0, a n d if t h e h y d r o -

c a r b o n t h i c k n e s s l is n o t t o e x c e e d lc, t h e vesicle radius R c a n n o t be less t h a n a
c e r t a i n radius, t h e "critical r a d i u s " R c, given b y Eqn. 8 p u t t i n g l = le, a = a0.
F o r e x a m p l e , if v = 1 , 0 0 0 A 3, a0 = 65 A 2, le = 18 A, we o b t a i n R e = 1 1 8 A (the
a p p r o x i m a t e e q u a t i o n gives R e = 124 A).
F o r vesicles t h a t are larger t h a n t h e critical radius R e t h e r e are n o p a c k i n g
r e s t r i c t i o n s o n a n y o f t h e m o l e c u l e s , w h i c h can a s s u m e t h e i r m i n i m u m e n e r g y
c o n f i g u r a t i o n w i t h a = a0. T h u s w h e n R > R c we have a = a0, and f o r all t h e
molecules
pOT : 2a0~/. (9)
 191

Note that the inner layer molecules are always in a favourable packing state
whatever the value of R; their hydrocarbon chains simply fill up the inner
volume with their inner surface areas remaining at their optimal value a0 [4],
i.e. packing constraints only affect the outer layer molecules. On the other
hand, for R < R e packing constraints have to be taken into account. We there-
fore have, from Eqn. 6,
po = 2ao7 for the inner layer molecules (10)

pO = 2ao7 + ~[a -- a0] 2 for the outer layer molecules (11)

a

Thus the mean pO per molecule in a vesicle of aggregation number N is

po = 2a07 + ~ [ a - - a 0 ] 2 n (12)
a N'
where n = 47rR:/a is the number of molecules in the outer layer. Now using
Eqn. 8 we obtain to a sufficient degree of approximation [4] that, for R < R e ,

47rlc27 ( R) 2
po = 2a07 + ~- 1 --~-~ (13)

To obtain the vesicle size distribution it is necessary to substitute Eqns. 9

and 13 into Eqn. 2. We take our arbitrary reference state as that when R = R e
(M = N c and pO = 2a07) and finally obtain the distributions:

Xn = N for R > R e (14)

and

X R =N e forR<R e, (15)

where N ~ 47r[R ~ + ( R - - t)2]/ao. In these expressions N / N c is the ratio of the

aggregation numbers of vesicles with radii R and R e, and is given by

N_ [R 2 + ( R - t ) 2] (16)
N c [(Re) 2 + (R e -- t)2] '

where t is the bilayer hydrocarbon thickness. This may be shown to be effec-

tively constant and equal to that of the planar bilayer t = 2V/ao.
Eqns. 14 and 15 give the concentration of molecules X R in vesicles of radius
R; the vesicle concentration being given by X R / N .
The results strictly apply only to low lipid concentrations where vesicle-
vesicle interactions may be ignored. At higher concentrations these interactions
become important and modify the free energy p o , and can lead to vesicle
aggregation and the formation of ordered mesomorphic phases [4,15--20].
C o m p a r i s o n w i t h e x p e r i m e n t s . As an example we consider the theoretical
predictions as they apply to egg phosphatidylcholine vesicles (see also ref. 4):
192

p u t t i n g [ 1 6 , 1 7 , 4 ] v = 1063 A 3, a0 = 71.7 A 2, t = 2 v / a o = 29.6 .,\, l~, = 17.5 A

(giving R c = 108 A ), 7 = 50 erg/cm 2, the d i s t r i b u t i o n is p l o t t e d in Fig. 2 (D = 0
curve) for a total lipid c o n c e n t r a t i o n o f XR = 10 -s (~ 0.5 nag p h o s p h o l i p i d / m l
water, or ~ 0 . 5 mM). T h e distribution peaks at a b o u t R , , . ~ k ~ 105 A [21].
T h e main features revealed by the t h e o r y are:
(1) T h e d i s t r i b u t i o n o f vesicles has a near-Gaussiml profile e x p ( R - - R p e a k ) 2 /
202 which peaks at an o u t e r radius Rpeak close to, b u t slighly smaller than, the
critical packing radius R " . T h e s t a n d a r d deviation

= Rpea " 't T (17)

is o f the o r d e r o f a few per cent o f R (for R = 105 A, lc = 17.5 A, 7 = 50 erg/

cm 2, we o b t a i n o ~ 3.5 A). The d i s t r i b u t i o n is t h e r e f o r e f o u n d to be nar-
r o w l y p e a k e d ( h o m o g e n e o u s ) , in a g r e e m e n t with o b s e r v a t i o n [ 4 , 2 1 ] .
(2) T h e d i s t r i b u t i o n shifts to l o w e r radii as the t o t a l p h o s p h o l i p i d c o n c e n -
t r a t i o n is l o w e r e d , b u t the m a g n i t u d e o f this shift is small. A million-fold reduc-
t i o n in c o n c e n t r a t i o n shifts the peak radius b y o n l y a few A n g s t r o m units.
(3) T h e surface areas o f the inner m o l e c u l e s should be the same as in a
planar bilayer a0, while the o u t e r areas s h o u l d be slightly greater. T h e outside-
inside ratio o f m o l e c u l e s is given b y R 2 / ( R - - t ) 2, w h e r e t is the h y d r o c a r b o n
bilayer thickness and n o t t h a t o f the w h o l e bilayer. F o r egg lecithin we o b t a i n
a ratio o f ~ 2 . 0 [4].
(4) T h e factors t h a t are e x p e c t e d to a f f e c t vesicle size are changes in the
h y d r o c a r b o n v o l u m e v, the critical length lc and the o p t i m a l surface area a0.
for anionic lipids the o p t i m a l area a0 m a y be e x p e c t e d to decrease with
d e c r e a s e d pH and increased ionic strength [ 1 1 ] , resulting in larger R ~ values
and c o r r e s p o n d i n g l y larger vesicles [ 2 2 ] . Z w i t t e r i o n i c lipids s h o u l d be less sen-
sitive to such changes, as observed [22].
Increased u n s a t u r a t i o n should change the critical length le b u t s h o u l d n o t
m u c h a f f e c t the h y d r o c a r b o n v o l u m e v (cf. the densities o f liquid h e x a d e c a n e
and h e x a d e c e n e d i f f e r b y o n l y 1%). We estimate f r o m a b o n d - l e n g t h and bond-
angle analysis t h a t a c i s d o u b l e b o n d s h o r t e n s l~ b y a b o u t 0.9 A, while a t r a n s
d o u b l e b o n d leaves le practically the same. F r o m Eqn. 8 we find t h a t t h e addi-
t i o n o f a c i s b o n d (to b o t h chains) s h o u l d increase the vesicle radius, assuming
a0 remains u n c h a n g e d , and l o w e r the outside-inside ratio, r ~ [ R / ( R - - t ) ] 2,
while the a d d i t i o n o f a t r a n s b o n d s h o u l d have little e f f e c t on the size and ratio
as the critical length l~ varies. Expressing this ratio as
r ~ [RC/(R c -- t)] z (18)
where R e is given b y Eqn. 8 and t = 2 V / a o ~ c o n s t a n t , we o b t a i n b y differentia-
t i o n the variation in r as l~ changes:

-- -- 1 r 3/z (19)

We can see t h a t f o r typical values t ~ 30 A, lc ~ 17.5 A, for a 1 6 - c a r b o n or

1 8 - c a r b o n diacyl chain, we have A r / A l c ~ 0.14 r 3j2. De Kruijff et al. [23] have
 193

found that the outside-inside ratio of phosphatidylcholine vesicles, at 30°C,

increased from r = 1.75 for a 18:1 c/s diacyl-chain to r = 2.0 for a 18:1 trans
phosphatidylcholine, i.e. r increased by 0.25 on replacing a cis bond by a trans
bond in each chain. The theoretical estimate using r ~ 1.85 and Ale = +0.9 A, is
Ar ~ +0.41 (1.85) 3j: 0.9 ~ 0.3. The theoretical estimate for Ar of 16:0 and
16:1 cis phosphatidylcholine vesicles also agrees with De Kruijff et al.'s [23]
results (Ar = 0.4) but not with their 18:0 c/s results (Ar = 0.05). These
measurements, however, were made at very different temperatures so that a
comparison with theory is not strictly possible.
On the other hand, increasing the chain length should leave v/lc unchanged
since both v and l¢ are roughly proportional to the number of carbons per
chain [3,4]. Eqn. 8 shows that for v/l~ ~- constant, and a0 ~- constant, R e is
proportional to l~. Thus, increased chain length should lead to an increased
vesicle radius. However, the outside-inside ratio r will not change since the
bilayer thickness t ~- 2V/ao is also proportional to the number of carbon atoms,
leaving r ~ [ R C / ( R ¢ - t)] ~ practically unchanged. This conclusion is not in
accord with observation: De Kruijff et al. [23] found that the outside-inside
ratio of saturated diacyl-chained phosphatidylcholines 18:0, 16:0 and 14:0
were equal to 1.7, 2.2 and 2.65 respectively, though the measurements were
made at different temperatures (60 °, 50 ° and 30°C). For 16:1 cis and 18:1 cis
vesicles, both measured at 30°C, the ratios were about the same (1.8 and 1.75).
Further, they found that for chains shorter than 14:0 no stable vesicles could
be prepared, in agreement w i t h earlier work [23,24]. This, too, is not borne
out by the theory, which allows for the existence of vesicles even for very short
chains. We return to this matter in the next section.
(5) The theory allows us to calculate values for the elastic moduli of bilayers.
For a planar bilayer under compression or tension the free energy of a molecule
in the bilayer is given by Eqn. 6. For small expansions or compressions in area
Aa = (a -- a0) about the equilibrium optimal area a0 the energy change per unit
area of bilayer is

A(2p0N)
= 2")'(Aa/a) 2 = lks(Aa/a)2 . (20)
a

An energy dependence of the form ~ k s ( A a / a ) 2 is the same as that of an elastic

membrane of elastic stretch modulus ks equal to 47, or ks ~ 200 erg/cm 2. This
value agrees well with measured values on lipid bilayers and red cell membranes
[4,25,26]. This elasticity is only for planar bilayers under vertical compression
or tension, or lateral stretch and does not extend to the bending of bilayers. On
the contrary, the analysis has shown that the bending of a bilayer is favoured
down to the critical packing radius R c if the lipids in each layer can freely
move or slide past each other), and that "bending elasticity" sets in only for
radii smaller than R c .
The theory as it stands appears to account fairly well for many but not all
of the observed physical properties of vesicles. The development so far is
model-independent and relies only on thermodynamics, simple packing
considerations and the notion of opposing forces. We shall now extend the
analysis by considering curvature corrections to the free energy #o.
194

Effects o f finite head-group on vesicle properties

So far, the forces between adjacent lipids have all been assumed to occur at
the h y d r o c a r b o n - w a t e r interface, at which the surface area a per molecule has
been defined. This is likely to be true for the interracial tension, but not neces-
sarily for the head group repulsive forces which are more likely to be centred
above this interface. If the head group region is of length D, then the centre of the
repulsion will be located at a distance D/2 above the h y d r o c a r b o n - w a t e r inter-
face.
The repulsive energy C/a of Eqn. 3 must now be redefined such that its area
a is located at a distance D/2 away. This area is equal to a(1 + D / R ) , where R is
the radius o f curvature of the surface. Thus for a spherical surface the repulsive
energy C/a of Eqn. 3 must now be replaced by C/(a(1 + D/R}). This equation
has the same form as that of the electrostatic repulsion between zwitterionic
head-groups o f dipole length D [4]. We may now write for the free energy (cf.
Eqn. 3)

C
g o = a7 ÷ (21)
a[1 + D/R]

The magnitude of D will depend on factors like the head-group length, size
and c o n f o r m a t i o n , and for ionic head-groups on the ionic strength [4]. The
free energy per molecule in a vesicle can now be shown to be [4] :

47:7Dt
pO=2a07 , forR>R c. (22)
N

and

47rlc27 47rD t 7
p o ~ 2a07 + [1 - - R / R C ] 2 - - , for R ~ R c (23)
N N

which reduces to Eqns. 9 and 13 when D = 0. Thus, a finite head-group repul-

sion results in a small additional term to the free energies which favours smaller
vesicles. Its effect may be obtained by minimizing Eqn. 23 with respect to R.
Assuming th at N ~ R 2 , we obtain

Rneak ~ R~[1 - - D t / l 2] (24)

which gives a good estimate for the peak radius R in terms of R c and D.
The ef f ect o f a finite head-group does n o t m o d i f y the qualitative picture of
vesicles already obtained, but certain quantitative aspects are significantly
altered:
(1) The peak radius Rpeak is appreciably shifted below R e even for fairly
small values o f D. For egg phosphatidylcholine, using t = 29.6 A, lc = 17.5 A,
R c = 108 A, D = +4 A, the peak radius shifts from about 105 A down t o a b o u t
70 A. Thus f o r eggg phosphatidylcholine vesicles to peak at R ~ 105 A [21]
with D = +4 A, the value of lc must be less than 17.5 A (and closer to 16.5 A).
When we consider lipids of different chain lengths we find that, since both t
and lc are roughly p r o p o r t i o n a l to the n u m b e r o f carbons per chain, the fact or
 195

[1 - - D t / l ~ ] in Eqn. 24 falls rapidly for progressively shorter chains. As the

chain lengths are shortened we now expect the vesicle radii to fall, and con-
sequently the outside-inside ratio r ~ [ R / ( R -- t)] 2 to increase. This effect,
which relies entirely on the existence of a positive non-zero D, explains w h y
shorter chained lipid vesicles have higher outside-inside ratios [23]. Further,
Eqn. 24 shows that for sufficiently short chains, such that D ~ l~/t ~ lc/2, no
stable vesicles should exist. We conclude that once the hydrocarbon chain
length falls to a b o u t twice the head-group length no stable vesicles should form;
instead, micelles should be formed. For phosphatidylcholine no stable vesicles
have been reported for chains with less than 14 carbons [23,24]. For a 12-car-
bon chain we may expect lc to be in the range 10--14 A, yielding an empirical
value for the "effective length" of the phosphatidylcholine head-group of
5--7 h.
(2) The vesicle distribution still has a near-Gaussian profile. The standard
deviation in the radius being now

Rpeak (1--Dt/21~) ~ kT (25)

The standard deviation of egg phosphatidylcholine vesicles (R ~ 105 A) should

therefore rise from o ~ 3.5 h (for D = 0) to o ~ 5.0 A for D = +4 A, and to
o ~ 8 A for D = +6 A, as shown in Fig. 2. Further, for progressively shorter
chain lengths vesicles should become not only smaller b u t also progressively
less homogeneous.
(3) Both the outer and inner surface areas should be roughly the same and
slightly greater than the optimal area a0 in lamellar bilayers [27,28]. Due to
the different packing boundaries of the inner and outer hydrocarbon chain
regions we should also expect these to have slightly different chain configura-
tions [29].
The agreement of some of these theoretical predictions with the limited

10 -$ m

xR iii
0 i i I ~ i , ' J
0.5 1.0 1.5
R/RpEAK
Fig. 2. T h e o r e t i c a l c o n c e n t r a t i o n d i s t r i b u t i o n X R in m o l f r a c t i o n units of lipid i n v e s i c l e s o f n o r m a l i z e d
o u t e r radius R / R p e a k ( t h e o r e t i c a l p a r a m e t e r s t a k e n f o r e g g p h o s p h a t i d y l c h o l i n e ) . T h e d i s t r i b u t i o n s are
n e a r Gaussian with a s t a n d a r d d e v i a t i o n o t h a t d e p e n d s o n t h e " e f f e c t i v e " h e a d - g r o u p l e n g t h D.
196

experimental data available gives support to the notion that the repulsive forces
are located within the polar head-group region (at least for phosphatidyl-
choline), and are therefore due more to head-group repulsion than to side-chain
repulsion.

Two-component vesicles
In a t w o - c o m p o n e n t system the competing forces of entropy and curvature
effects, and packing constraints, are still operating, but two additional factors
must be taken into account. First, the molecules may distribute asymmetrically
and still satisfy the packing constraints. This can result in a vesicle of lower
aggregation number than in the symmetrically distributed vesicle. Since a lower
aggregation number is favoured both entropically and energetically we
conclude that in general all t w o - c o m p o n e n t vesicles should be asymmetrical
(Fig. 3). Opposing this a s y m m e t r y is an additional entropic force: the entropy
of mixing, and the problem reduces to one of establishing the balance of these
competing effects.
In extending the theory to t w o - c o m p o n e n t vesicles, we shall assume that
there are no specific interactions between the different lipids, so that they mix
ideally. To include such effects as phase separations arising from specific inter-
actions would certainly be desirable b u t would require a much deeper theoreti-
cal understanding of head-group interactions than we have at present. We shall
also assume that the fully extensible "critical length" Ic of the hydrocarbon
region is the same for both lipids. This considerably simplifies the geometric
packing analysis, but is not otherwise justified.
To analyse a t w o - c o m p o n e n t vesicle we first determine h o w two lipids with
different geometric packing constraints assemble to form a vesicle. Such an
analysis is purely geometric. Second, we determine the free energy for a two-
c o m p o n e n t lipid system. Finally, we determine the properties of the "equilib-

SYMMETRIC ASYMMETRIC

lipid A

A,, Ai F A° Ai
= -- > F > - -
Ao+B o Ai+B i Ao÷B o Ai '" B i
F i g . 3. S y m m e t r i c a n d a s y m m e t r i c v e s i c l e s c o m p o s e d o f a m i x t u r e o f l i p i d s A a n d B. T h e p a c k i n g o f t h e
two lipids are the same in both vesicles, and the total tool fractions of lipids A and B are also the same.
Asymmetric vesicles are favoured due to head-group repulsion contributions to the free energy, but are
o p p o s e d b y t h e u n f a v o t t r a b l e e n t r o p y o f d e m i x i n g . T h e e q u i l i b r i u m a s y m m e t r i c a l v e s i c l e is d e t e r m i n e d
by the balance of these two effects.
 197

r i u m " vesicle, and compare the results with experimental data. We cannot
assume a priori that the two lipids distribute asymmetrically on the inner and
outer layers of the vesicle. Any asymmetry, if it exists, must emerge naturally
in the determination of the equilibrium vesicle.
The different lipids will be denoted by A and B. For a vesicle of outer and
inner radii Ro and Ri (measured at the hydrocarbon-water interfaces as before)
we de~ine: Ao and Bo (Ai and Bi) = number of A and B lipids in the outer
(inner) vesicle layer; N = Ao + Bo + Ai + Bi; F = (Ao + Ai)/N; Xo = Ao/(Ao + Bo);
Xi = Ai/(Ai + Bi); f = (Ao + Bo)/N. Note that for no asymmetry, Xo = Xi = F
(Fig. 3).
P a c k i n g e q u a t i o n o f a t w o - c o m p o n e n t vesicle. We denote the hydrocarbon
volumes of lipids A and B as VA and Vs, and their optimal surface areas as aA
and aB. The critical hydrocarbon length, lc, is assumed the same for both lipids.
The equation for the critical outer radius R e is still given by Eqn. 8, i.e.

R~=Ic {3+~3(4vale1)} {1--~/e} /6 (26)

but v and a are now the mean outer layer values:

v = XoVA + (1 -- X o ) v n , a = XoaA + (1 --Xo)aB. (27)

We remark that an alternative way of analysing the packing of a mixed lipid
system, based on a solid-angle approach [6], yields values of vesicle radii that
differ by 1% or less. We adopt the present approach for convenience only.
For any two-component system the value of F, the mole fraction of A lipids
in the vesicle, is fixed. The mol fraction Xo of A lipids in the outer layer will
be adopted as our yardstick of asymmetry. For Xo = F there is no asymmetry.
For any Xo the value of R~ is readily obtained from Eqns. 26 and 27. This in
turn determines Ao and Bo, since, for the outer vesicle layer:

4~(R~o)2 Ao Bo
(28)
[XoaA+(l--Xo)asl A°+B° Xoo (1--Xoi"
Similarly, for the inner vesicle layer:
47rR 2 4rr[(R c -- lc)3 -- R 3]
[XiaA + (1 -- gi)aB] = Ai + Bi 3 [X i VA + (1 -- X i) VB] " (29)

Finally, Ai and Bi are related through F = (Ao + Bo)/(Ao + Bo + Ai + Bi). Thus

for a given total mole fraction F we can solve the above equations to obtain all
the relevant parameters R~, Ao, Bo, Ai, Bi, Xi, f and N, at any value of Xo.
As an example we take the case of an egg yolk phosphatidylcholine (lipid
A)-phosphatidylethanolamine (lipid B) mixture, and use the following rough
values for these lipids based on recent estimates [6,4]: aA ----72 A ~, VA = 1060
A3, aB = 42 A2, vB = 700 A3, lc = 17 A. Berden et al. [30] have studied mixed
phosphatidylcholine/phosphatidylethanolamine vesicles in which phosphatidyl-
ehtanolamine/phosphatidylcholine = 1.08, i.e. F = 0.48. They found that
the vesicles were asymmetric with (outside phosphatidylethanolamine)/(out-
198
side p h o s p h a t i d y l c h o l i n e ) = 0.92, i.e. Xo = 0.52. Using F = 0.48, X,, = 0.52,
and solving Eqns. 26 to 29 we o b t a i n the following results and c o m p a r e with
e x p e r i m e n t a l values (in brackets): Ro = 175 A ( ~ 1 8 0 A, [ 3 1 ] ) , Bi/A i = 1.36
(1.38), Bo/Bi = 1.17 (1.17), Ao/Ai = 1.52 (1.76), (Ao + Bo)/(Ai + Bi) = 1.41
(1.41), N = 11 4 0 0 ( n o t m e a s u r e d ) . T h e t h e o r e t i c a l and e x p e r i m e n t a l results all
agree t o within 2%.
Having established t h a t the a s y m m e t r i c d i s t r i b u t i o n o f lipids in a vesicle is
well described by their packing properties, we have y e t to predict the sign and
m a g n i t u d e o f Xo. T o d o this we require the expression for the m e a n inter-
a c t i o n free e n e r g y per m o l e c u l e in a m i x e d system.
Free energy o f a two-component vesicle. If we ignore any specific interac-
tions b e t w e e n the lipids, it m a y be s h o w n t h a t their surface areas will remain
close to their o p t i m a l areas (as in a o n e - c o m p o n e n t bilayer). T h e n the free energy
per vesicle N p ° is simply the sum o f the separate c o n t r i b u t i o n s o f the lipids,
t o g e t h e r with the e n t r o p y o f mixing, i.e.

+ 23'aB Bi
(1 +
D B ) -- leT In
l(ho 4- no) ! (h i -b Bi)! 1 . (30)
2Ri \ Ao!Bo! Ai! Bi! /
Eqn. 30 is valid in t h e absence o f packing constraints and r e d u c e s to Eqn. 22
for a o n e - c o m p o n e n t vesicle. A f t e r s o m e simple algebra, Eqn. 30 b e c o m e s

pO=27aAF+27aB(l_F)+_~ '( DA aA + DBaB

+ k T [ f ( X o In Xo + (1 - - X o ) ln(1 - - X o ) )

+ (1 -- f ) { X i In X i + (1 -- Xi) ln(1 -- Xi)}] . (31)

Eqn. 31 m a y be simplified b y use o f Eqns. 26 t o 29, assuming t h a t devia-
tions f r o m s y m m e t r y are small, i.e. t h a t Xo is close t o F. We m a y t h e n express
the free e n e r g y pO in t e r m s o f t h e deviation AXo = (Xo -- F ) :

4"tc
pO = c o n s t . - - - -
N
/
FD A 1 + (1 - - F ) (aA aB)1
aA

r>o,/1 a,a'>/>
47r~[ R2o kT( AXo) 2
- - - (Ro(1 + Ro/Ri) (DA - - D B ) } AXo ~ (32)
N R 2 2F(1 -- F)
All t h a t remains is t o calculate the value o f AX0 at which the m e a n molec-
ular free e n e r g y #~ is a m i n i m u m . We d i f f e r e n t i a t e Eqn. 32 and a f t e r s o m e
m o r e simplifications we o b t a i n the c o n d i t i o n which gives a m i n i m u m p o :
 199

7F(1 -- F)
AXo ~ ---RokT -- I[FaA + (1 - - F ) a B I ( D A - - D B )

+___2[FDA + (1--F)DB] t . (33)

\Ro!
Here Ro is the vesicle radius, RA and RB are the radii of the pure c o m p o n e n t
vesicles as given by Eqn. 8, * and t the hydrocarbon thickness of the bilayer
(Ro -- Ri).
Properties of two-eomponen t vesicles. The main qualitative and quantitative
aspects of t w o - c o m p o n e n t vesicles, as predicted by Eqn. 33, will now be sum-
marized:
(1) In general, mixed lipid vesicles should always be asymmetrical. Those
lipids which have a longer head group D and larger a/R will go preferentially
to the outer layer. This is consistent with experimental studies on mixed phos-
phatidyleholine/phosphatidylethanolamine and phosphatidylcholine/cholesterol
vesicles, as well as with the general conclusions of Berden et al. [30] that lipids
with larger head groups tend to distribute preferentially in the outer layers.
(2) Asymmetries should be small at small mol fractions F (F close to 0 or 1).
Maximum asymmetry should occur near F = 0.5 where F(1 -- F) is maximum.
But since the vesicle radius Ro also varies with F, this value could be apprecia-
bly shifted away from F = 0.5. With phosphatidylcholine/cholesterol vesicles,
significant asymmetry has only been observed at mol fractions F above 0.3
cholesterol [ 32,331.
(3) For phosphatidylcholine/phosphatidylserine vesicles (phosphatidylserine
being negatively charged), an increased pH will increase the head group area
as (through an increased electrostatic repulsion) and thereby decrease RB. At
elevated pH we should therefore expect phosphatidylserine to go progressively
more to the outer layer in a mixed phosphatidylcholine/phosphatidylserine
vesicle, as observed [30].
(4) The first term in Eqn. 33 usually dominates; the head group areas aA and
aB and head group lengths DA and DB being the major lipid properties that
determine asymmetry. Variations in side chain composition and unsaturation
should have a secondary effect (insofar as they vary RA, RB and Ro), in agree-
ment with observation [34].
(5) Unless the asymmetry is large, the equilibrium vesicle radius is the same
as if there were no asymmetry, given b y Eqns. 26 and 27, or alternatively, as
in a previous analysis [6]. Finally, large vesicles should have small asymmetries.
Eqn. 33 yields qualitative predictions which agree with the limited experi-
mental data available at present. Quantitatively, it predicts equilibrium values
for AXo that are close to those observed. Thus taking the phosphatidylcholine/
phosphatidylethanolamine system as an example, using (roughly) F = 0.48, ~/=
50 erg/cm 2, aA ~ 72 A 2, as ~ 42 A 2, R A ~ 120 A, RB ~ 860 A, t ~ 30 A, Ro ~
175 A, D A ~ DB ~ 5 A, DA -- DB ~ 3 A, we obtain the measured value AXo

* F o r a o n e - c o m p o n e n t v e s i c l e E q n . 8 g i v e s R ~ / c / [ 1 - - v/alc]. F o r s o m e lipids, vial c ~ 1, a n d R

t a k e s a n e g a t i v e v a l u e . S u c h l i p i d s , (e.g. c h o l e s t e r o l ) c a n n o t f o r m v e s i c l e s or b i l a y e r s , a n d w e h a v e
p r e v i o u s l y [ 6 ] t e r m e d s u c h lipids " f r a y e d " lipids.
200

+0.04 (i.e. phosphatidylcholine preferentially distributed in the outer layer).

Unfortunately, the theoretical value for AXo depends critically on (D A -- DB)
which depends on the relative head group conformations of phosphatidylcho-
line and phosphatidylethanolamine. This is still a matter of controversy. How-
ever, for values of D A and D~ in the range 5--10 A, the present theory would
predict that the phosphatidylcholine head group is longer than the phosphati-
dylethanolamine head group by between 2 A and 3 2k, in accord with an experi-
mental measurement of 3 A [35]. The values o f D A and DB are not necessarily
the same as the head group lengths. Strictly, they represent the length of the
polar region above the hydrocarbon-water interface where the effective repul-
sive forces are operating.

Discussion

We have presented a general theory of lipid self-assembly in which the self-

assembly mechanism involves the interplay of thermodynamics, interaction
forces, and molecular geometry. We have shown how the theory yields simple
analytical expressions for a number of vesicle and bilayer properties in terms
of measurable parameters, and we have demonstrated how these predicted
properties are in quantitative accord with experiments.
The theory also attempts to give a very definite picture of the mechanism of
self-assembly. Vesicles emerge as thermodynamically stable, homogeneous
structures, unaffected by drastic changes in the surrounding lipid concentra-
tion, so long as this is above the critical micelle concentration (CMC) of ~ 10 -1°
M [3]. At high lipid concentrations, above about 10 -3 M, vesicles begin to
interact significantly with each other and form into ordered mesomorphic
phases [15--20]. We stress that our treatment applies only to dilute systems,
where vesicles are sufficiently far apart on average for these interactions to be
ignored. Whereas we conclude that spherical vesicles, if allowed by packing, are
thermodynamically favoured over isolated bilayers, we cannot say without
further analysis whether they are generally more stable than multi-layered
structures (liposomes) [4]. We note, however, that stable vesicles do form spon-
taneously free of multilamellar structures [36--38].
It is well known that the ability of vesicles to compartmentalize and
segregate an inner region from the outside is utilised in many cellular transport
processes. The existence of asymmetrical bilayer vesicles is now well established
and we find theoretically that these too are thermodynamically stable and that
the asymmetry is expected to be homogeneous. For asymmetric bilayer vesicles
composed of charged and uncharged lipids, the different surface charge den-
sities on the inner and outer vesicle surfaces would result in different surface
potentials in the inner and outer surfaces depending on the magnitude of this
asymmetry. The total membrane potential, as measured between the inner and
outer bulk aqueous solutions, would be zero; but two different potentials
would exist at each surface, these being compensated by a potential difference
across the hydrocarbon region of the bilayer. * There is now compelling evi-
* F o r a c a l c u l a t i o n o f t h e s e p o t e n t i a l s f o r p l a n a r b i l a y e r s , e q u a t i o n s 2 t o 4 g i v e n bY M c L a u g h l i n a n d
Harary [41] may be solved assuming fixed surface charge densities a I and a 2 (a 1 ~ a2), and
p u t t i n g V = O.
 201

dence which shows that these electrostatic potentials at membrane surfaces

play a determining role in many transport and conduction processes [39,40].
We have only to take the argument one step further to conclude that areal mem-
brane potential may be established if one of the membrane constituents is a
molecule that selectively conducts cations or anions across the membrane.
Such a membrane potential could form spontaneously without requiring a
pump or metabolic energy. There is no violation of thermodynamics here since
the system is always at thermodynamic equilibrium and the membrane poten-
tial can do no work. Indeed, the possibility that the inner and outer environ-
ments of vesicles may be asymmetric is conceptually no different from the
equilibrium asymmetry in the membrane constituents.

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