The formation of bilayer membranes and liquid-crystalline properties of amphiphiles

45

Bioelectrochemistry and Bioenergetics, 22 (1989) 45-56 A section of J . Electroanal. Chem., and constituting Vol . 276 (1989)

Elsevier Sequoia S .A., Lausanne - Printed in The Netherlands

The formation of bilayer membranes and liquid-crystalline properties of amphiphiles Yurii A . Shchipunov, Irina G. Maslennikova, Alexander F . Kolpakov and Elena V. Schumilina Institute of Chemistry, Far East Department, Academy of Sciences of the US.S.R., 690022 Vladioostok (U S . S. R.)

(Received 12 January 1989 ; in revised form 17 April 1989)

ABSTRACT Dialkylammonium salts and alkylethylenediamine derivatives have been shown to be capable of forming planar bilayer membranes and myelin figures in aqueous solutions when the amphiphiles contain charged functional groups . This is a major distinction between the species studied and neutral phospholipids, whose liquid crystals swell in water due mainly to repulsive hydration forces . As has been shown, the charge on the molecule is not conducive to an effective stabilization of bilayer membranes . It has been suggested that stable bimolecular films can be generated from amphiphiles carrying functional groups capable of adding a significant number of water molecules to the hydration shell .

INTRODUCTION

The most common liquid-crystalline structure in living systems is the bimolecular lipid layer constituting the framework for biological membranes [1-3] . A single bilayer, in the strict sense of the term, cannot be referred to as a liquid crystal ; nevertheless, it represents a basic structural unit of lamellar mesophases, one of three types into which the lyotropic liquid-crystalline phases are classified [4] . Lamellar mesophases are capable of forming, under definite conditions, many of the known amphiphilic, or surface-active, substances, i .e . those containing functional groups and one or more hydrocarbon radicals [4] . Nonetheless, in contrast to phospholipids, none of these is capable of forming stable planar bilayer membranes . This unique feature is found in lipids only ; properly speaking, owing to this

* Presented at the 4th International A .N . Frumkin Symposium "Bioelectrochemistry Today and Tomorrow", Moscow-Suzdal, U .S .S .R., 24-28 October 1988 . 0302-4598/89/$03 .50

© 1989 Elsevier Sequoia S.A .

46

property biomembranes and living systems exist as they are encountered on Earth . It should be emphasized that to date, a full understanding of the mechanisms for formation and stability of planar bimolecular films and a criterion for the choice of amphiphiles suitable for fabrication of such films are actually non-existent . Extensive studies in this domain of science were conducted in the 60s, stimulated by the pioneering work of Mueller and co-workers [5], who developed a technique for the preparation of planar bilayer lipid membranes ; since then, no alternative has been proposed for phospholipids (see, for example, refs . 6-8) . Besides, the searches were conducted on a purely empirical basis . Starting from the middle 70s, a resurge of interest in this problem has been observed . A research group headed by Kunitake has synthesized a very impressive number of amphiphiles and studied their membrane-forming properties [9,10]. It should be noted, though, that the formation of vesicles and liposomes rather than of planar bilayer membranes has been considered . The major distinction between these two types of systems is that the former are mostly spherical particles whose internal aqueous medium is confined within an envelope composed of one or more than one bimolecular layer . The problem whether synthetic amphiphiles, constitutive of liposomes and vesicles, are capable or not of forming stable planar bilayer membranes has remained an unsettled issue . This problem has never been the subject of special investigation, nor has the relationship between the stabilities of a single bilayer and lamellar mesophases been duly considered. The present work is concerned with a study of the swelling of synthetic amphiphiles and the formation of bilayer membranes in aqueous solution ; its major objectives are to specify the nature of surfactant molecules responsible for the formation of lamellar mesophases, liposomes, and planar bilayers . The experiments have been carried out using neutral phospholipid and a number of derivatives of dialkylammonium salts and ethylenediamine . A diagnostic sign for the formation of lyotropic lamellar mesophases was the observation of myelin figures . Myelin figures, observable as tubular structures in which a multitude of bimolecular layers are arranged coaxially around a central axis [3,11,12], appear when dry liquid crystals come in contact with an excess of water . Their occurrence is indicative of a definite liquid-crystalline state of surfactants . The following specific features should be pointed out : (i) Myelin figures are observed only in smectics A [12-14] which are made up of a large number of planar bimolecular layers (lamellae) arranged in parallel . (ii) Myelin figures are observable at or above the temperature of the gel-to-liquid crystal phase transition [12,15] . (iii) Myelin figures can exist only in the concentration gradient of substances in lamellar mesophases [16,17] . As the concentration gradient decreases with time, the myelin figures seem to have a tendency to disintegrate into liposomes [14,16,18] . Hence we may conclude that the observation of myelin figures in a swollen material in aqueous solution is indicative of the liquid-crystalline state of the system in question, its multilamellar structure and the temperature of its gel-to-liquid crystal phase transition . These features may be presumed to suffice for the char-

47

acterization of the definite liquid-crystalline properties that are required for amphiphiles potentially capable of forming planar bilayer membranes and lamellar mesophases . EXPERIMENTAL

Chromatographically pure egg yolk phosphatidylcholine was a gift of Prof . E.Ya. Kostetsky . Didodecyldimethylammonium bromide (2C 12 2C,NBr) was prepared by alkylation of dimethylamine with dodecyl bromide in an autoclave at 90-100 ° C for 9-10 h. The synthesized salt was purified by triple recrystallization from ethyl acetate. The betaine derivative of didodecylmethylammonium (2C 12 1C 1 NC 1 000HC1) was obtained by heating a mixture of didodecylmethylamine and chloroacetic acid at 80'C for 120 h . The product was purified by triple recrystallization from acetone . N-Hexadecylethylenediamine (C 16 NHC 2 NH 2 ) was prepared by alkylation of ethylenediamine, taken in a 10-fold excess, with hexadecyl bromide by stirring the mixture at room temperature for 2 h . The reaction product was extracted and then purified by triple recrystallization from hexane . N,N-Dihexadecylethylenediamine (2C 16 NC 2 NH 2 ) was synthesized and purified as described in ref . 19 . N,N,N',N'-Tetrahexadecylethylenediamine (2C 16 NC2 N2C 16 ) was synthesized and purified as recommended in ref . 20 . To obtain N-hexadecyl-1,2-ethylenediamine-N'-acetic acid (1C 16 NHC 2 NHC 1 COOH), N-hexadecylethylenediamine and sodium chloroacetate were dissolved in an ethanol + water mixture and kept at 70 ° C for 30 h . On addition of sodium carbonate, a precipitate was deposited . After subsequent evaporation, the crystalline reaction product was purified by multiple recrystallizations from acetone and hexane . Distilled or, if necessary, doubly distilled water was used to prepare aqueous solutions . HCI (chemically pure) and NaOH (chemically pure) were used for acidification and alkalization of the solutions . Non-aqueous films were formed from n-heptane or n-decane (both chemically pure) . Prior to starting the experiments, the organic solvent and doubly distilled water made to saturate mutually as described in ref . 21 . The swelling of amphiphiles in aqueous solutions (prepared using distilled water) and the growth of optically anisotropic myelin figures was followed through a MBI-15 microscope in transmitted light with crossed polaroids . In taking photomicrographs of the myelin figures, it was necessary to lower the growth velocity of the tubelike structures by adding some glycerol (pure grade) to the water. The ability of amphiphiles to form bimolecular films was assessed by observing the spontaneous appearance of black spots in the middle of a biconcave drop of non-aqueous solution submerged in water in the orifice of a Teflon cell . The experimental technique was described in detail a previous report [21] .



48 RESULTS

Myelin figures In the literature, evidence for the formation and mechanism of growth of myelin figures is available for phospholipids only [14,18] . In our experiments with swollen amphiphiles we have observed that tubelike structures, once formed, pass through roughly the same developmental stages as in the case of neutral lipids. Now, this process will be illustrated and exemplified with didodecyldimethylammonium bromide. The salt 2C 12 2C I NBr undergoes a gel-to-liquid crystal phase transition at 16'C [9] which enables the swelling experiments to be carried out at room temperature . Photomicrographs showing the morphological features of lamellar mesophases at three stages of myelin figure development are presented in Fig . 1 . The initial stage is characterized by an extensive growth of simple rod-like forms (Fig . 1A) . As the rods grow they become bent and form, in the second stage, folded tubelike structures (Fig. 1B) . The process is terminated at the third stage as the myelin figures break down into liposomes and lyotropic mesophases with mosaic texture (Fig . 1C) . Some of the amphiphiles studied swell in aqueous solution in a similar manner . The evidence of their ability to generate myelin figures is presented in Table 1 . The indicated ion-molecular forms are produced as the aqueous medium pH is shifted to either the acidic or the alkaline region . The following reactions may be envisioned : C 16 NH 2C2 NH 3

'

C 16 NH 2C2 NHC I 000 - ;

C 76 NH 2 C2 NH 2 + H + C 16 NH 2 C 2 NC 1 000 + H + E

2C 12 1C 1 NC 1 000H acidic region pH 2-3

C16 NHC 2 NH 2 + H + C 16 NHC 2 NC I 000 - + H +

2C12 1C 1 NC 1 COO - + H + neutral region

alkaline region

pH 5-7

pH 9-11

Bilayer membranes These are formed spontaneously from non-aqueous films as they reach a critical thickness of several tens of nm . The abrupt thinning can have two results : rupture of the films or the formation of bilayers when the surfactants adsorbed on opposite surfaces of the oil/water interface come into contact . The latter case is manifested by the appearance of single spots with areas up to about 0 .01 mm2 . These spots have been named black spots, since bilayers with thicknesses up to several nm look stained black in reflected white light . The lowest amphiphile concentration in non-aqueous solution at which single bilayer areas start forming into a non-aqueous film has been named the critical concentration (C bl ) for black spot formation . In colloid chemistry, this concentration serves to characterize the emulsifying efficiency of surfactants [8] . The formation of black spots is a random process and may be characterized in

49

Fig. 1 . Formation and disintegration of myelin figures on swelling of didodecyldimethylammonium bromide in a water+glycerol mixture (75% glycerol, by volume). (A) Initial stage ; (B) formation of folded tubelike structures ; (C) spherulites (liposomes), the final stage of myelin figure disintegration and salt swelling in water . Photomicrographs taken in transmitted light with crossed polaroids . Temperature 20'C.



0 11 R -COCH

Phosphatidylcholine

+ -

2C 16 NHC2 NH 3 2C 16 NHC2 NH 2 2C 16 NC 2 NH

-

2C 121C 1 NC,000 -

2C 16 NC2 NH 2

+

2C121C 1 NC 1 000H

2C121C 1 NC 1 000HCI

+

2C 12 2C 1 N +

20-100 20-100

77

20-60 b

44

20

-

4.3 x 10 -5

10 -4 a

-

4 .8 x 10 -5

1 .5 X 10 -4

1 .2x10 -4

10 -5

20

c bl /mol dm -3

+ [3,11,14,18]

c bm /mol dm -3

Bilayer membranes

Formation

Temp ./ oC

Myelin figures

2C 12 2C 1 NBr

O 11 R„-COCH + I + H 2C-O-P02 -OCH 2CH 2 N(CH 3 ) 3

Ion-molecular

Amphiphile

Formation of myelin figures and bilayer membranes

TABLE 1

20-60 20-60

45

20-60

20-60 b

16 [10]

-5

Temp ./ oC



62 90 90 84

+ + + +

1C 16 NHC2 NH 2

1C 16 NH 2C 2 NH 2 C 1 000 -

1C 16NH 2C 2 NHC1 000 1C 16NHC2 NHC I 000 -

47

+

1C 16 NH 2C 2 NH 2

20-100

10 -3 (P = 0 .7)

2 x 10 -2 (P = 0 .4)

-

-

20-100 20-100

-

-

2C 16 NHC 2 N2C 16 2C 16 NC2 N2C 16

-

98

1C 16 NH 2 C 2 NH 3

+

2C 16 NHC 2 NH2C 76

20

20-60 30

20-60

20-60

20-60

-

20-60 20-60

20-60

-

-

a In the presence of 0 .1 mol dm -3 KBr in water . b Temperature interval in which swelling of amphiphiles and formation of bilayers were investigated . ` Concentration at which black spots in non-aqueous films were observed . Probabilities for black spot formation are shown in parentheses .

1C 16 NHC2NHC 1000H

1C16 NHC2 NH 2

2C 16 NC 2 N2C16



52

terms of a probability P [22] . A quantitative estimation of the probability P is the ratio of the number of experiments in which the black spot formation has been noted to the overall number of experiments performed . Commonly, the value of P is seen to increase markedly as the concentration C b , is attained, which should be attributed to a denser filling of the interfacial boundaries with adsorbed molecules and an increased stability of the bimolecular structures [8,21] . As the black spot lifetime becomes longer, the number of black spots and, consequently, the bilayer area also become larger . At a certain concentration c bm , the lifetime of the bilayer structures becomes long enough to enable the film to transform into a bimolecular one over the whole film area . The relationships between the probability of black spot formation in non-aqueous films and the concentration of phosphatidylcholine, 2C 72 2C 1 NBr, and 2C 16 NC 2 NH 2 are presented in Fig. 2. The numerical experimental results illustrative of the bilayer membrane formation are summarized in toto in Table 1 . It should be noted that the most stable bilayer membranes are those produced from phosphatidylcholine. Their lifetime ranges over several tens of min . Among the other amphiphiles studied, 2C 12 2C 1 NBr and 2C 16 NC2 NH 2 should be mentioned : they form bimolecular films remaining stable for several minutes . In all instances, the onset of membrane formation is observed at temperatures corresponding to the gel-to-liquid crystal phase transition . These temperatures are shown in Table 1 .

P I

1 .0

P' .

0

0 .8

0 .6

0 .4

0 .2

0 .0 0 .0

I I I 0 .5

1 .0

1 .5

2 .0

10`C Imol dm 3 Fig. 2 . Probability of black spot formation vs . concentration of N,N-dihexadecylethylenediamine (1), phosphatidylcholine (2), and didodecyldimethylammonium bromide (3) in alkane . Aqueous medium pH is 2 .86 (1) and 5-6 (2,3) ; 0 .1 mol/dm3 potassium bromide is present in water (2) . Temperature 45 (1), 20 (2), and 30 ° C (3) . The concentrations C bi are marked by arrows.

53 DISCUSSION

The data presented in Table 1 provide evidence that the synthetic amphiphiles that form lamellar mesophases are most commonly also capable of generating bilayer membranes . It should be admitted, though, that a single planar bilayer exhibits a low stability . For example, the lifetime of a 2C 12 2C 1 NBr membrane is only a few minutes, while vesicles possessing a single bimolecular layer are stable for weeks [23] . Nonetheless, as shown by detailed analysis, the processes conducive to the formation of myelin figures and bimolecular films do not appear to be related directly . In order to get a deeper insight into the origin of different conditions for the existence of these systems, let us consider the forces operative in the formation and stability of a single planar bilayer and in the swelling of liquid crystals . The myelin figures, consisting of a manifold of coaxially stacked hydrated bimolecular layers, are expelled from the crystal by the action of a disjoining pressure which arises due to the penetration of water molecules into the space between the lamellae and to mutual repulsion of the latter . In the initial stages, when the distance between the neighbouring bilayers in the dry smectic A is minimal, the repulsion is defined chiefly by hydration forces (by the structural component of the disjoining pressure) [24] . It seems probable that these repulsion forces reach their maximum among the surfactants studied in phosphatidylcholine . The phosphatidylcholine molecule is capable of taking up 30 to 40 water molecules [3], with a resultant increase in molar volume from 1 .622 to 2 .266 nm3 [25] . The synthetic amphiphiles that have been considered in this study, take up much less water . For example, the hydration shell of didodecyldimethylammonium bromide contains 4-5 water molecules [26], i .e . 7-8 times less than phosphatidylcholine . For this reason, the hydration forces are distinctly more pronounced in the lamellar phospholipid mesophase (see, for example, refs . 24, 27, 28) than in the case of swelling structures of synthetic amphiphiles . As shown by the data in Table 1, the myelin figures are observed in charged synthetic surfactants . It may therefore be presumed that a major contribution to the disjoining pressure in the swelling process is due to electrostatic forces . This presumption is supported by the fact that myelin figures fail to develop if the electrostatic component is made smaller . This is exemplified by didodecyldimethylammonium bromide : the replacement of one methyl group in the molecule by a negatively charged acetic acid residue, resulting in the neutralization of the positive charge at the quaternary nitrogen, makes it impossible for the myelin figures to occur (see Table 1) . On the other hand, in C 16NHC2 NHC 1 000H in aqueous solution, the ability to generate myelin figures is retained within a wide pH range ; in this amphiphile, although one of its forms is a zwitterion, the polar moiety is larger in size and, consequently, more hydratable as compared to 2C 12 1C 1 N000HCI, which is conducive to stronger repulsive hydration forces in the lamellar mesophase . In this connection it should be emphasized that the occurrence of a non-compensated charge on the phosphatidylcholine molecule leads to a superposition of electrostatic and hydration forces, and their resultant effect produces a strong

54

repulsion between the bimolecular layers and facilitates the disintegration of the lamellar mesophase [29,30] . Thus, myelin figure formation in the synthetic amphiphile-water systems should be explained as due to electrostatic repulsion between neighbouring lamellae in the smectic A . The hydration forces in these systems are much weaker than in neutral phospholipids ; in the latter, according to evidence in the available literature, the hydration forces are the major contributors to the disjoining pressure in the lamellar mesophase . The formation of planar bilayer membranes from non-aqueous films proceeds in a manner basically distinct from the swelling of amphiphiles in water . Indeed, as the initial double concave drop gradually becomes thinner, the non-aqueous solution is expelled into the Plateau-Gibbs border by a combination of static and dynamic processes [6-8] . In films with a critical thickness of several tens of nm, an abrupt increase in film thinning rate is observed . If adsorbed surfactants are present at the interface, the process terminates in contact of the monomolecular films from apposition of the hydrocarbon radicals. The polar groups are oriented in water solution . The formation of bilayer membranes is thus rendered possible because of a mutual repulsion between the two adsorbed monolayers . As has been recognized [8,24], this repulsion is due mainly to electrostatic interactions between the electric double layers at opposite interfacial boundaries . Stabilization of the bilayer membranes is achieved owing to the positive electrostatic component of the disjoining pressure . Thus, if the swelling of smectic A is caused by the moving apart of neighbouring bilayers interacting through a water interlayer, the formation of bilayer membranes is effected by approaching monolayers interacting through a non-aqueous film. The specific features of repulsive interactions provide a clue to an understanding of the similarities and differences in the formation of myelin figures and planar bilayers (see Table 1) . The similarity between the two processes consists in the fact that on conversion of the synthetic amphiphiles into an uncharged form, they fail to form myelin figures and bilayer membranes . Therefore, electrostatic forces are equally important in the stabilization of both bimolecular films and lamellar mesophases . However, the formation of bilayer membranes is more sensitive to the magnitude of the charge. For example, planar bilayer membranes of didodecyldimethylammonium bromide form by screening of the charged functional groups on the film surface ; otherwise, the interaction between the charged groups in the lateral plane leads to disruption of the bilayers [31] . From the standpoint of the surplus charge concept, the destabilizing effect due to the second charged amino group in the studied alkylethylenediamines is also amenable to explanation . In these amphiphiles, as is seen in Table 1, bilayer membranes are formed only if the molecule bears a single charge . Asymmetric ethylenediamine derivatives with two hydrocarbon radicals (2C 16 NC2 NH 2 ) are representative of a special group of amphiphiles . They form bimolecular films only if two charges are present in the molecule .

55

To explain the cause of the high stability of planar bilayer lipid membranes, it is essential to gain a deeper understanding of the factors affecting the formation of liquid crystalline structures of synthetic amphiphiles and phosphatidylcholine . It should be noted that the lipid molecule is electrically neutral, since its positively and negatively charged groups compensate each other . A stabilization of bilayer lipid membranes owing to electrostatic repulsions appears to be highly improbable . Moreover, even a small number of uncompensated charges developed on the bilayer surface are conducive to bilayer rupture [32] . Therefore, the stability of bilayer lipid membranes should be attributed, in all likelihood, to the steric component of the disjoining pressure. Dialkyldimethylammonium salts are in many respects similar to phospholipids . In particular, the properties of the liposomes and vesicles they form are close to those of phospholipids [23,26] . Dialkyldimethylammonium salts are also capable of forming bimolecular films, but their lifetimes are shorter by far than those of bilayer lipid membranes [31] . Taking into account that the charge on the 2C 12 2C 1 N+ molecule leads to destabilization of the bilayer [31], we have replaced one methyl group in this molecule by a carboxyl . We anticipated that the synthetic amphiphile 2C 12 C 1 NC 1 000 - , due to its membrane-forming properties, would exhibit a greater resemblance to phospholipids . But contrary to expectation, the negative charge on the molecule produces an opposite effect : in no manner could bimolecular films be formed from 2C 121C 1 NC 1 000 - . Now, a question arises as to the cause of such a striking difference between the properties of 2C 121C 1 NC 1 000HCI and phosphatidylcholine. We presume that it is due to distinct differences in the hydration properties of these two compounds, for example the fact that 2C 121C 1 NC 1 000 - on swelling in water produces no myelin figures (see Table 1) . It follows, therefore, that the repulsive hydration forces in this salt are less pronounced as compared to those in the lipid . It is easily seen now that these forces exert no effect on the film stabilization, since hydration forces are operative in the water interlayers between the lamellae only [24,27,28], but the stability of the bilayer lipid membranes may presumably be enhanced owing to the significant increase in geometrical size of the molecule - roughly 1 .5-fold [25] produced by the addition of 30-40 H 2O molecules . This effect must be reflected in the steric component of the disjoining pressure whose occurrence may, in all likelihood, explain the stabilizing effect of phospholipid hydration . In the case of 2C 121C 1 NC 1 000H, no similar steric effect is observable because of the small number of water molecules in the hydration shell of this compound . Thus, to summarize briefly, in order to improve the membrane-forming properties of synthetic amphiphiles, the polar moiety in their molecules should be made more hydratable, for example, with the aid of an increase in the number of functional groups, to allow the addition of a larger number of water molecules to the hydration shell .

56 REFERENCES 1 V.G . Ivkov and G .N. Berestovsky, Lipid Bilayers of Biological Membranes, Nauka, Moscow, 1982 . 2 G.H . Brown and J .J. Wolken, Liquid Crystals and Biological Structures, Mir, Moscow, 1982 (Russian transl .). 3 D.M . Small, The Physical Chemistry of Lipids : From Alkanes to Phospholipids, Plenum, New York and London, 1986 . 4 P . Ekwall, Adv. Liq. Cryst ., 1 (1975) 1 . 5 P . Mueller, D .O . Rudin, H.T . Tien and W .C. Wescott, Nature (London), 194 (1962) 979 . 6 H .T. Tien and A.L . Diana, Chem . Phys . Lipids, 2 (1968) 55 ; H .T . Tien, Bilayer Lipid Membranes (BLM). Theory and Practice, Dekker, New York, 1974 ; Prog . Surf. Sci ., 19 (1985) 169 . 7 M .K . Jain, The Bimolecular Lipid Membrane ; A System, Van Nostrand Reinhold, New York, 1972 . 8 P .M . Kruglayakov and Yu .G. Rovin, Physico-Chemistry of Black Hydrocarbon Films, Nauka, Moscow, 1978 . 9 Y . Okahata, S . Tanamachi, M . Nagai and T . Kunitake, J. Colloid Interface Sci ., 82 (1981) 401 . 10 T. Kunitake, A . Ando and Y . Ishikawa, Mem . Fac . Eng. Kyashi Univ ., 46 (1986) 221, 245 . 11 D .G. Dervichian, Prog . Biophys ., 14 (1964) 265 . 12 D . Chapman and D .J . Fluck, J . Cell Biol., 30 (1966) 1 . 13 M . Kleman, C .E . Williams, M.J . Costello and T. Gulik-Krzywicki, Philos . Mag., 35 (1977) 33 . 14 I. Sakurai and Y . Kawamura, Biochim . Biophys. Acta, 777 (1984) 347 . 15 H . Sandermann, Jr ., H. Fulk and G . Schumacher, Anal . Biochem., 82 (1977) 587 . 16 A. Saupe, J. Colloid Interface Sci ., 58 (1977) 549. 17 I. Sakurai, Biochim . Biophys . Acta, 815 (1985) 149 . 18 I. Sakurai, Y . Kawamura, T. Sakurai, A . Ikegami and P . Seto, Mol. Cryst. Liq . Cryst ., 130 (1985) 203 . 19 J .-H . Fuhrop, V. Koestig and G . Schonberger, Liebigs Ann . Chem., 10 (1984) 1634. 20 V.A . Semenov, T .N . Krischko, L.I . Sokol'skaya and N .G. Zukov, Zh. Prikl . Khim . (Leningrad), 57 (1984) 2315 . 21 Yu .A . Shchipunov and A.F. Kolpakov, Kolloidn . Zh ., 44 (1987) 530 . 22 D .R. Exerova, Commun . Dep . Chem . Bulg . Acad. Sci ., 11 (1978) 739 . 23 J .H . Fendler, Ace. Chem . Res., 13 (1980) 7. 24 B .V. Derjaguin, N .V. Churaev and V .M. Muller, Surface Forces, Nauka, Moscow, 1985 . 25 S .H. White and G.L. King, Proc . Natl . Acad . Sci. USA, 82 (1985) 6532 . 26 T . Kajima, A . Kumano, M . Takangi, Y . Okahata and T. Kunitake, Contemp . Top . Polym. Sci ., 4 (1984) 829 . 27 R.P. Rand, Annu. Rev. Biophys . Bioenerg., 10 (1981) 237 . 28 J . Marra and J. Israelachvili, Biochemistry, 24 (1985) 4608 . 29 Y . Talmon, D .F. Evans and B .W. Ninham, Science, 224 (1983) 1047 . 30 H . Hauser, 4th Proc . Eur. Found. Workshop : Reverse Micelles, Plenum, New York, 1984, p . 37 . 31 Yu .A . Shchipunov, I .G. Maslennikova, A .F . Kolpakov and E.V . Schumilina, Izv . Akad. Nauk Latv . SSR, Ser . Khim ., N2 (1988) 196 . 32 S . Ohki and O. Aono, J . Colloid Interface Sci ., 32 (1970) 270.