Interaction of surfactants with vesicle membrane of dipalmitoylphosphatidylcholine. Effect on gel-to-liquid-crystalline phase transition of lipid bilayer

Chemistry and Physics of Lipids, 42 (1986) 261-270

261

Elsevier Scientific Publishers Ireland ktd.

INTERACTION OF SURFACTANTS WITH VESICLE MEMBRANE OF DIPALMITOYLPHOSPHATIDYLCHOLINE. EFFECT ON GEL-TO-LIQUIDCRYSTALLINE PHASE TRANSITION OF LIPID BILAYER

TOHRU INOUE a, KENJI MIYAKAWA b and RYOSUKE SHIMOZAWA a

aDepartment oj Chemisto' and bDepartment of Applied Physics. Faculty of Science. Fukuoka Unirersity, Nanakuma. Fukuoka 814.01 (Japan) Received September 8th, 1986 accepted October 29th, 1986

revision received October 29th, 1986

Tile gel-to-liquid-crystalline phase transition of dipalmiloylphosphatidylcholine (I)PP(') vesicle membrane was observed in the pre~nce of various types of surfactants; sodium alkylsulfates, alkyhrimethylammonium bromides, alkam)yl-N-mcthylglucaluides, and hcxaethylencglycol mono n-dodecyl ether. The phase transition was monitored by a change in scattered light intensity of the lipid suspension. I:or all the surfactants examined, the phase transition temperature was depressed linearly with the surfactant concentration in the measured concentration range, from which the partition coefficient, K, of the surfactant between bulk solution and lipid membrane was estimated. Except alkyltrimethylammonium bromides, log K and log ('M(" showed a linear relationship, which indicates that the driving t'olce to transfer the surfactant from bulk solution to lipid membrane is a hydrophobic interaction. The addition of surfactants increased the transition width. The extent of widening the transition width was in the order of sodium alkylsulfate > alkyhrimethylammoniulu bromides hexaethyleneglycol mono n-dodecyl ether; in the case of alkanoyI-N-mcthylglucamides, lhe transition width was not affected by the addition. These effects on the transition width ~as interpreted qualitatively in terms of the cooperativity of the transition.

Keywords: surfactant-membrane interaction; phospholipid membrane; phase transition: partition coefficient.

Introduction The i n t e r a c t i o n o f s u r f a c t a n t s w i t h p h o s p h o l i p i d m e m b r a n e has been studied f r o m i n t e r e s t in the s o l u b i l i z a t i o n or fusion o f biological m e m b r a n e s [1,2]. In a d d i t i o n , m o r e r e c e n t l y , s u r f a c t a n t s have b e e n used to prepare the large unilamella~ vesicle o f various lipids (i.e., d e t e r g e n t r e m o v a l m e t h o d ) , a n d the p h y s i c o c h e m i c a l p r o p e r t y o f l i p i d - s u r f a c t a n t s y s t e m has b e e n progressively investigated [3 5]. C o m p a r e d w i t h the above practical i n t e r e s t , it seems t h a t the f u n d a m e n t a l aspects o f the i n t e r a c t i o n o f s u r f a c t a n t s w i t h lipid have received less a t t e n t i o n . It is well k n o w n t h a t small ligand m o l e c u l e s i n t e r a c t w i t h lipid m e m b r a n e a n d a f f e c t the m e m b r a n e p r o p e r t y s u c h as fluidity a n d gel-to-liquid-crystalline 0009-3084/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

262 phase transition. The investigation of the interaction between a small molecule and biological membrane is important for understanding the action mechanism of drugs such as anesthetics, the transport phenomena through the membrane, etc. Surfactants are useful agents to study the interaction between a foreign molecule and a lipid membrane, because the surfactants are readily available with a wide variety of structures and properties according to the combination of various types of head group and hydrocarbon tail, which allows the systematic study of the interaction. In the present work, we studied the interaction of surfactants with the vesicle membrane of DPPC by observing the effects on the phase transition behavior, using various types of surfactants with different head groups (anionic, cationic, and nonionic) and different hydrocarbon chain length.

Experimental Materials

Synthetic DPPC was obtained from Sigma. The following 11 surfactants were used; sodium octyl-, decyl- and dodecylsulfate (SOS, SDeS, SDS, respectively), octyl-, decyl-, dodecyl- and tetradecyltrimethylammonium bromide (OTABr, DeTABr, DTABr, TTABr, respectively), octanoyl-, nonanoyl- and decanoyl-Nmethylglucamide (MEGA-8, MEGA-9, MEGA-10, respectively), and hexaethyleneglycol mono n-dodecyl ether (C12E6). SOS (Eastman), SDeS (Kao Co.), and SDS (Nakarai Chemicals) were purified twice by recrystallization from methanol. OTABr, DeTABr, DTABr and TTABr (Tokyo Kasei Co.) were purified by recrystallizing from ethanol for two or three times. MEGA-8, MEGA-9 and MEGA10 (Dojin Chemicals) were purified by recrystallization from ethanol/ether. C~2E6 with homogeneous head group was obtained from Nikko Chemical Co. and used without further purification. Water was deionized and doubly distilled, once from alkaline potassium permanganate solution. The stock suspension of DPPC in water was prepared by sonication in the cuphorn of a Branson Sonifier Model 185 at above the gel-to-liquid-crystalline phase transition temperature for about 30 min; the size of vesicles thus prepared was measured by quasielastic light-scattering technique and was found to be about 160 nm diameter with good reproducibility. After sonication, the vesicle suspension was stored at 4°C. The sample suspension was prepared by mixing the stock DPPC suspension and surfactant solution to give a desired concentration of the surfactant, and by sonicating again above the phase transition temperature for about 5 min. The DPPC concentration was kept at about 0.5 × 10 -3 moldm -3 throughout the experiments. Methods

The gel-to-liquid-crystalline phase transition of DPPC vesicle membrane was

263

monitored optically; it is well known that the phase transition is accompanied by a drastic change in turbidity or scattered light intensity of vesicle suspension [6,7]. The light intensity scattered from the lipid suspension was measured with a JASCO FP-550A fluorescence spectrophotometer in the 90 ° light-scattering mode at 400 nm, to which a temperature programmable sample holder was equipped. The sample was heated from 30°C to 45°C at a rate of 0.5°C/min. The temperature of the sample was monitored using a platinum resistance thermometer, the tip of which was inserted into the sample. The lipid suspension was continuously stirred by a Teflon-coated magnet during the measurement. The photometer output was recorded together with the temperature signal on an X - Y recorder. Typical traces of the scattered light intensity change with an increase of temperature are shown in Fig. 1. The transition temperature, Tin, was taken as the temperature corresponding to the half-height between two intersections of a line drawn through the steep portion of the curve and two lines drawn through the linear portions above and below the transition. The transition width, W, was also taken as the temperature interval between the two intersections described above.

i11 C

~v

l

I

30

I

,

,

,

I

,

40 Temperature (°C)

,

,

,

1

50

Fig. 1. T y p i c a l t r a c e s o f c h a n g e in s c a t t e r e d light i n t e n s i t y at 4 0 0 n m ~ i t h the t e m p e r a t u r e rise. D P P C c o n c e n t r a t i o n is 0.5 × 10 -3 m o l d m -3. A d d e d s u r f a c t a n t s are (a) n o n e , (b) 0 . 3 9 9 × 10 -3 m o l d m -3 SDS, (c) 1 3 . 0 × 10 -3 m o l d m - 3 0 T A B r , (d) 2 . 4 2 × 10 -3 m o l d m -3 M I ( G A - 9 a n d (e) 3 . 5 9 × 10 -s m o l d m -3 GI2E 6.

264

Results and Discussion The phase transition temperature, Tin, was depressed by the addition of surfactants, as is seen in Fig. 1 where typical examples of phase transition pattern with and without added surfactants are shown. The depression of the transition temperature, AT = T m -- Tm,o where Tm,o represents Tm without additives, is plotted against the surfactant concentration in Figs. 2 - 4 for anionic, cationic, and nonionic surfactants, respectively. The transition temperature for pure DPPC, Tin,o, was in the range 40.9-41.1°C which is in agreement with literature [8,9]. As shown in these figures, --AT increases linearly with increasing surfactant concentration within the measured concentration range, and the slope increases with increasing hydrocarbon chain length in a given series of surfactants. It should be noted here that at higher concentration of surfactants, a different behavior of transition was observed; the main phase transition disappeared and the scattered light intensity decreased drastically in lower temperature range (near about 30°C), which was not recovered by cooling process. Further increase of surfactant concentration leads to a clear solution of lipid rather than suspension; this may due to the formation of mixed micelle. Thus, we focused our attention only on the low concentration range of surfactants where normal behavior of the phase transition was observed. The depression of phase transition temperature of lipid membrane induced by small ligand molecules has been frequently analyzed in terms of freezing point

3.0

~ o

/ []

o

2.0

I-I

/

1.0

0

1

0

i

/

|

I

I

I

I

I

I

5.0 Surfactant concentration

I

I

1

I

I

10.0

(10"3moldm-3)

I.ig. 2. Plot of depression of the phase transition temperature, -AT, against the concentration of added surfactants. Surfactants are SOS (e), SDeS (s), and SDS (o).

265

3.0

! v

2.or

I//

/

/7

,,/

o / D

,/.

9

•

0

J

10

20

30

Surfactant concentration (10-3mol dm -3 ) l'ig. 3. Plot of - A T against the concentration of added surfactants. Surfactants arc OTABI jR), DeTABr (o), DTABr (*) and TTABr (o).

3.0

,/ o/•

2.0 I--

/

o/

J°

I

[] v

0

i

0

o

5

|

I

I

5

10

15

Surfactant concentration (10-3 mo[ dm-3) Fig. 4. Plot of --AT agains the concentration of added surfactants. Surfactants are ME(;A-8 (~), MEGA-9 (o), MEGA-10 (o) and C~2E6 (inserted figure).

266 depression [10-12]. We analyze the present experimental results along the line of this treatment. Basic assumptions are that (i) surfactants are incorporated into the lipid membrane and a partition equilibrium of surfactant between bulk solution and membrane phases is established and (ii) the surfactants in a lipid membrane in the liquidcrystalline state are in a fashion of random mixing, while those in the gel state membrane are in a fashion of phase separation. The validity of the second assumption may be seen from the observation of spectroscopic behavior of a surfactant-like derivative of azobenzene in lipid membrane [13]. Then, for the ideal case where the solute concentration in the lipid membrane is sufficiently low as in the present case, the depression of phase transition temperature is related to the solute concentration in the membrane by a well known relation, RT2mo , " xA ,SJ/

--AT=

(1)

where AH is the enthalpy change associated with the phase transition, R the gas constant, and x~ the mole fraction of surfactant in the membrane. Let the partition coefficient of surfactant between the bulk solution and the lipid membrane be K, i.e.,

(2)

K = x'A/XA

where x A represents the mole fraction of surfactant in the bulk solution. The mole fractions are expressed in terms of the number of moles as t

XA =

t

?

(3)

n A / ( n L + nA )

x A = (n~4 -- n'A ) / ( n w + n~4 -- n'A)

(4)

where n L, nw, n'A, and n,~l represent the number of moles of lipid, water, surfactant in membrane phase, and the total number of surfactant, respectively. Combining Eqns. (2) - (4) under the condition of n L ~" nJA and nlv >>n~4, one can obtain t

(s)

XA = n~4K/(nw + nL K )

Thus,

-~XT =

R T2m,o AH

x

K

n)

(6)

nl,¢ + nLK

Converting the number of moles to molar concentration for dilute solution with

267 respect to b o t h lipid and surfactant, Eqn. (6) leads to

--AT=

RTZmo ' ,SJ-/

X

K 55.5 +

CLK

~A

(7)

where CL and CA denote the lipid c o n c e n t r a t i o n and the total concentration of surfactant, respectively. Equation (7) predicts that --2~T increases linearly with the surfaclanI concentration, CA. F r o m the slopes o f the straight lines shown in Figs. 2 4, the values of partition coefficient, K, were d e t e r m i n e d according to Eqn. (7"1, using the following values of parameters: Tin.o = 314 K and / 5 / / = 36.4 kJ tool -1 [8]. The values of K thus obtained for various surfaclants are listed in Table 1 together with the values of critical micelle c o n c e n t r a t i o n (CMC) o f the surfactants. It is of interest to compare the values of K and CMC; the former reflects the interaction between surfactanl and lipid molecules, while the latter reflecls tire interaction between surfactant molecules themselves. In Fig. 5, log K is plotted against log CMC. With anionic and nonionic surfactanI series, straight lines with the slope o f 1 are obtained, whereas w i t h cationic surfactant series, alkyltrim e t h y l a m m o n i u m bromides, no linear relationship is obtained. For h o m o l o g o u s series of surfactants, the d e p e n d e n c e o f CMC on the carbon n u m b e r o f hydrocarbon chain, No, is expressed by Shinoda et al. [18] In CMC = A - - BN c

(8)

where A and B are constants. The partition coefficient, K, for anionic and nonionic surfactant series showed a similar dependence on the carbon n u m b e r o f TABLE I PARTITION COEFFICIENT (K) OF SURFACTANTS BETWEEN WATER AND VESI('LI MEMBRANE OE DPPC AND CMC OF THE SURFACTANTS Errors are standard errors estimated from linear leasl-squares analysis. Surfactant

K

CMC(moldm ~)

Refs.

SOS SDeS SDS OTABr DeTABr DTABr TTABr MEGA-8 MEGA-9 MEGA-10 CItE 6

(4.71 _+0.25) X 102 (2.00 _+0.04) X 103 (5.42 _+0.15) X 103 (1.86 _+0.05)X 102 (7.02 _+0.21)X 102 (2.20 _+0.09) X 103 (3.06 _+0.09) × 103 (5.59 _+0.07) X 102 (1.33 _+0.02) x 103 (5.74_+0.10) X 103 (2.91 _+0.25) X 10 s

1.36 3.41 8.88 1.5 7.0 1.65 3.51 6.60 1.81 5.31 7.2

14 14 14 15 15 15 16 Unpublished Unpublished Unpublished 17

× 10-' (40°C) X 10 ~ (40°( ") × 10 -3 (40°C) X 10 i (40Oc) X 10 2 (40°(,) X 10 -2 (40°( ") × 10 -3 (30°C) X 10 -~ (30°C) × 10 `2 (40°( ") X 10 -3 (40°C) × 10 -s (350( `)

268

\

,,¢-

4 1210

o

%0 \

0

I

-4

I

I

I

I

-3

-2

-1

0

1

log CMC

Fig. 5. Plot of log K against log ('MC. Surfactants are sodimn alkylsulfates (~, left scale), alkyltrimethylammonium bromides (o, right scale) and MEGA and ('~:E, (n and i, left scale). hydrocarbon chain, i.e., in K = A' + B'N c

(9)

where A' and B' are constants depending on the type of surfactant. On considering Eqns. (8) and (9), the linear relationship between log K and log CMC with the slope of - 1 indicates that B = B'. The physical meaning of constant B is the standard free energy change per methylene unit associated with micelle formation divided by R T [ 1 8 ] . On the other hand, R T B ' corresponds to the standard free energy change per methylene unit to transfer surfactant molecule from bulk solution to lipid membrane. Thus, it may be concluded from the above results that in the case of sodium alkylsulfates and nonionic surfactants, the driving force for the transfer of surfactant molecule from the bulk solution to the lipid membrane is just the same as that for micelle formation of the surfactant. In other words, hydrophobic effect is a main factor for the interaction of these surfactants with the vesicle membrane of DPPC. Contrary to anionic and nonionic surfactant series, departure from the linearity was seen in log K vs. log CMC plot for alkyltrimethylammonium bromides. The importance of hydrophobic effect was reported recently for the adsorption of alkyltrimethylammonium ions on lipid membrane of dipalmitoylphosphatidylethanolamine [19]. The present results for alkyltrimethylammonium bromides suggests that other factor(s) than hydrophobic interaction, probably arising from head group, may also contribute to the interaction with lipid membrane of DPPC.

269

It can be seen in Fig. 1 that the addition of surfactants other than MEGA-9 increases the transition width accompanied with the depression of the transition temperature, whereas MEGA-9 depresses the transition temperature without affecting the transition width. The transition width, W, is plotted against --AT in Fig. 6. Although the plots are somewhat scattered, an almost linear correlation is observed between W and --AT in each series of surfactant. The extent of the widening effect depends on the type of head group, but is independent of the hydrocarbon chain length in a given series of surfactant. The transition width is a measure of cooperativity in the phase transition. The cooperativity depends on the range of correlation between lipid molecules in the membranes, i.e., size of cooperative unit. The increased transition width means that the cooperativity is weakened by the addition of surtactants. This may be interpreted as that the surfactant molecules incorporated in the membrane tend to disrupt the correlation between lipid molecules responsible for the cooperativity of the transition, and reduce the size of cooperative unit, or in other words, the transition becomes somewhat localized within the nrembrane due to this 'blocking' effect of the surfactant molecules present in the membrane. Then, it may be expected that W increases with the fraction of surfactant molecule present in the lipid membrane, and this is in accord with the observation shown in Fig. 6, where --AT is proportional to the mole fraction of surfactant molecules in the membrane. The increasing rate of W with the mole fraction of surfactant in thc membrane is independent of the hydrocarbon chain length in a homologous series of surfactant, but depends on the type of head group of the surfactant. This suggests

5

(c)

(b)

(a)

4

[]

3

~'f~/"~ []

"7"

o [] eo% o []/

2

/ I

/= /==/=<=

~/o

0 J 0

/

.

I

I

I

I

I

I

I

I

I

I

I

1

2

3

0

1

2

3

0

1

2

3

-zxT(*C)

- zxT

(*C)

-zxT(*C)

Fig. 6. Plot of transition width, W, against --AT. (a) Anionic surfactants: SOS (o), SDeS {L,) and SDS (o). (b) Cationic surfactants; OTABr try), DeTABr (e), D I A B r ( t ) a n d TTABr (::,). (c) N o n i o n i c s u r f a c t a n t s ; M E G A - 8 ( o ) , M E G A - 9 { ) . M E G A - 1 0 t , ~ ) a n d C , , E , ( = ) .

270 that the interaction between head groups of lipid molecules is more dominant for the cooperativity of the phase transition rather than that between hydrocarbon chains, at least for the lipid membrane of phosphatidylcholine. As seen in Fig. 6, the effect of widening the transition width increases in the order of nonionic < cationic < anionic surfactants. It can be speculated from this fact that the electrical charge on head group of surfactant participates in disrupting the interaction associated with the transition cooperativity. MEGA series are unique surfactants in terms of their behavior not affecting the transition width. It is sure that these surfactants are incorporated in the lipid membrane, because they depress the transition temperature. Hence, it is considered that MEGA series surfactants do not perturb the interaction between lipid molecules associated with the cooperative transition, even when they are incorporated in the membrane. This suggests that the nature of a MEGA-lipid interaction in the membrane is essentially the same as that of a lipid-lipid interaction. The difference in the influence on the transition cooperativity from other surfactants may be attributed to the unique structure of head group of MEGA, although the details are not clear at the present time.

Acknowledgments We are grateful to Dr. Gohsuke Sugihara of Fukuoka University for permission to use the CMC data of MEGA prior to publication. This work was supported in part by funds from the Central Research Institute of Fukuoka University.

References 1 A. Alonso, R. S~iez,A. Villena and F.M. Gofii, J. Membrane Biol., 67 (1982) 55-62. 2 A. Alonso, R. Sfiez and F.M. Gofii, FEBS Lett., 137 (1982) 141-145. 3 J. Brunner, P. Skrabal and H. Hauser, Biochim. Biophys. Acta, 455 (1976) 322-331. 4 0 . Zumbuehl and H.G. Weder, Biochim. Biophys. Acta, 640 (1981) 252 262. 5 M. Ueno, C. Tanford and J.A. Reynolds, Biochemistry, 23 (1984) 3070-3076. 6 H. Tr/iuble, in: F. Kreuzer and J.F.G. Sleyers (Ed.), Biomembranes, Vol. 3, Plenum Press, New York, 1972, pp. 197 227. 7 M.B. Abramson, Biochim. Biophys. Acta, 225 (1971) 167-170. 8 S. Mabrey and J.M. Sturtevant, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 3862-3866. 9 T.W. Tsong and M.I. Kanehisa, Biochemistry, 16 (1977) 2674-2680. 10 M.W. Hill, Biochim. Biophys. Acta, 356 (1974) 117 124. 11 H. Kamaya, S. Kaneshina and I. Ueda, Biochim. Biophys. Acta, 646 (1981) 135-142. 12 S. Kaneshina, H. Kamaya and I. Ueda, J. Colloid Interface Sci., 93 (1983) 215-224. 13 M. Shimomura and T. Kunitake, Chem. Lett. (1981) 1001-1004. 14 E.D. Goddard and G.C. Benson, Can. J. Chem., 35 (1957) 986-991. 15 A.B. Scott and H.V. Tartar, J. Am. Chem. Soc., 65 (1943) 692-698. 16 R.L. Venable and R.V. Nauman, J. Phys. Chem., 68 (1964) 3498-3503. 17 J.M. Corkill, J.F. Goodman and S.P. Harrold, Trans. Faraday Soc., 60 (1964) 202-207. 18 K. Shinoda, T. Nakagawa, B. Tamamushi and T. Isemura, Colloidal Surfactants, Academic Press, New York, 1963, pp. 1-96. 19 H. Matsumura, M. lwamoto and K. Furusawa, Bull. Chem. Soc. Jpn., 59 (1986) 15331537.