Some electrical properties of thin lipid films formed from cholesterol and cetyltrimethylammonium bromide

Some Electrical Properties of Thin Lipid Films Formed from Cholesterol and Cetyltrimethylammonium Bromide 1 GEORGE D. SWEENEY ~ AND M A R T I N BLANK Department of Physiology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 Received May 9, 1972; accepted July 20, 1972 Bimolecular lipid membranes were formed from decane solutions of cholesterol in contact with an electrolyte of i m M NaC1 and 20-100~M cetyltrimethylammonium bromide (CTAB). At the extremes of CTA + concentration in the range studied, films have stable high resistances exceeding 106 f~ cm ~. At concentrations of CTA + slightly above the minimum concentration required for film formation, the resistance falls rapidly with time and stabilizes at levels around 104 f~ cm 2. The composition an d properties of these films may be altered by the passage of currents of the order of l0 ~ A cm-2. The electrical properties of this simple system are similar to those reported for more complex membranes of biologically derived material, and suggest possible interpretations of these effects. INTRODUCTION

Following the predictions of Gorter and Grendel (1) regarding the organization of lipids in cell membranes, attempts were made to reconstitute extracted membrane lipids. In 1961 Mueller et al. (2) reported the formation of artificial membranes from a mixture of natural lipids, and there is now considerable evidence that such lipid bilayers are a suitable model for the lipid component of the plasma membrane (3). Thin lipid films separating two aqueous phases also represent an interfacial phenomenon with novel properties that require interpretation in terms of the known behavior of phase boundaries. Because of this twofold interest there have been extensive studies of lipid bilayers in recent years. Tien and Diana (4) have described the formation of bilayers from cholesterol dissolved in hydrocarbon in contact with an aqueous phase containing the cationic surfactant, 1 This work was supported in part by research grant GB-6847 from the National Science Foundation. 2 Present address: Faculty of Medicine, McMaster University, Hamilton, Ontario, Canada.

cetyltrimethylarmononium bromide (CTAB', We saw several advantages in the furthe study of this system, since it appeared tha independent control of the bilayer surfac charge might be achieved by varying the con centration of CTAB in the aqueous phase. Th interracial tension would likewise depend upol the concentration of surfactant in the aqueou phase, making it possible to investigate th formation of thin films as a function of varyinl interracial tension. We were also interested t, know how cholesterol-CTAB films would be have when conducting an electric current, be cause changes in transport number at phas boundaries affect the ionic concentrations an~ interracial tensions at such boundaries (5) Therefore, in this system it was possible t, investigate the role of CTA + ions as curren carriers across the bilayer as well as the rela tion between CTA + concentration ant conductance. METHODS

A beaker (o.d. 19 ram) was made from solid rod of Teflon, and an area of the 2-ran 410

Journal of Colloid and Interface Science, Vol. 42, No. 2, February 1973

Copyright ~ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved

BILAYERS OF CHOLESTEROL AND CTAB

thick wall about 1 cm 2 was milled inside and outside to a thickness of 0.0175 ram. A circular hole, 1.5 mm in diameter, was punched through the thinned area. The beaker was mounted rigidly in a vessel made of optically flat glass measuring 3.9 cm ;< 4.1 cm ;< 4.0 cm. The "inside" and "outside" compartments were distinguished with reference to the beaker and two sets of electrodes were provided, platinum electrodes for alternating current measurements and calomel fiber-junction electrodes for measurements using direct current. The temperature of the electrolyte was monitored by means of a thermistor probe in the outer chamber, and kept at 25.0 4- 0.5°C. Reagents were of analytical grade; cholesterol was recrystallized from ethanol before use. Cetyltrimethylalmnonium bromide (CTAB) was obtained from Lachat Chemicals Inc. with the purity given as 99.5%. Decane (99O-/o) was from Aldrich Chemical Co. The electrical arrangements were as shown in Fig. l; low-loss rotary switches (S1A and $1~) allowed either calomel or platinum electrodes to be connected to the appropriate circuits. Capacitance was measured by connecting the platinum eiectrodes to a General Radio 1 kHz impedance bridge (Model 165 OA, General Radio Co., West Concord, Mass.) using a cathode-ray oscilloscope as an external detector. This bridge permits separate balancing of the complex and real portions of the impedance over a wide range, which is essential in measurements in dilute electrolyte (1 m M NaC1) with the consequent low conductance of the solutions. Results with this technique for determining capacitance were verified by determining the charging-time constant when a constant current from an external source was applied across the membrane. Direct current measurements were made using a Keithley 610 electrometer in various configurations; in addition to voltage ranges from 10 mV to 30 V at an input resistance of 1014 ohms, this instrument provides a variety of shnnt resistors from 101 to 1011 ohms which can be placed across the input providing current ranges. The calomel electrode in the outer

4~11

Fro. 1. A diagram of the apparatus used to study bilayer properties.

chamber was connected to the shielded lead of the electrometer in series with two sources of variable dc potential and of low internal resistance. One source was used to buck out any asymmetry between the liquid junctions of the two calomel electrodes, the other was used to provide a dc potential across the membrane in series with the Keithley electrometer input shunt. Current through, and potential across the thin lipid film were thus known and the dc resistance of the film defined by Ohm's law. A micrometer syringe containing electrolyte was used to inject electrolyte into the outer compartment of the chamber allowing small pressure differentials to be either corrected for, or created, between inside and outside. Light was focused on the orifice in the Teflon beaker so the film could be viewed through a lowpower, long focal-length microscope; planarity of the membrane was checked by observing even reflection from the surface. Films were formed by injecting 10 ~,i of cholesterol-decane solution directly into the orifice in the Teflon beaker, through a micrometer syringe fitted with a curved spout, tipped with Teflon tubing and shaped to mate well with the orifice, To obtain reproducible results in studying film resistance it was important to prevent excessive contamination of the electrolyte with cholesterol or decane. The procedure arbitrarily adopted was to clean the

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SWEENEY AND BLANK

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A. Thinning Films When a drop of lipid solution was placed in the orifice of the Teflon beaker, the shape of the hydrophobic material gave an indication of the interracial tensions. In the absence of CTAB the lipid assumed a spherical shape and excess material drained slowly or not at all into the surrounding medium. If black film appeared, its area would enlarge rapidly, and rupture of the black film inevitably occurred. As the concentration of CTAB was increased the interfacial tension decreased, since the lipid in the orifice formed a torus around the Teflon support with a relatively thick planar film occupying the orifice itself. Further thinning of this central planar area occurred at a velocity dependent upon the CTAB concentration. The minimum concentration of CTAB required for stable black film formation was 20 /zM in 1 m M NaC1 and somewhat less (in the range 10 to 15 #M CTAB) in 2.5 m M NaC1. As the CTAB concentration was increased, the rate of film formation increased. In 1 m M sodium chloride, 20 vM CTAB permitted stable black film formation in 1 to 2 rain, whereas doubling the CTAB concentration reduced the time required for black film formarion by a factor of about 5.

The appearance of interference fringes al: altered with increasing CTAB concentratio At the mininmm surfactant concentration r quired for stable film formation drainage w~ slow, and the interference fringes were a ranged horizontally, indicating that the drai] ing film tapered towards its base as the light, solution drained upwards. As the CTAB col centration was increased, the regular horizol tal fringes became disorganized by swirlir patterns of color. At the highest concentratioi of CTAB used (100 ~M in 1 m M NaC1 thinning to stable black films was almost ii stantaneous and interference films were usuall not observed.

B. Film Capacitance Capacitance measurements were highly r~ producible and the limiting factor in repro ducibility appeared to be the stability of th measuring apparatus. The capacitance of film depends on the area, the lipid dielectric cot. stant and the thickness of the film. While th area of films can be accurately known, electrica measurements do not make the dielectric con stant and thickness independently accessible The capacitance of films formed in a meditm of given ionic strength is constant within 5~, from film to tim and also during the life of in dividual flms. There is, however, a systemati variation of capacitance with ionic strength and in sodium chloride the capacitance in creases with an increase in the ionic strength as shown in Fig. 2. This is probably the resul of an increased "adsorption" of CTA + ions a; found in the case of quaternary amines at th, mercury/water interface (6).

C. Resistance The resistance of these films is subject t( wide variations. Examining films formed in ar aqueous electrolyte of 1 m M NaC1, three situa. tions could be distinguished. At the minimux~ concentration of CTAB required for filrr stability, films had a constant high resistance in the region 1.5 X 106 to 1.5 X l0 T ohm cm 2 Films formed with a high concentration ot

Journal of Colloid and Interface Science, Vol. 42, No. 2, F e b r u a r y 1973

BILAYERS OF CHOLESTEROL AND'CTAB

413

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Fie. 3. The resistance of a bilayer, in ohms, as a function of time, ill minutes, when the aqueous phases contain 1 mM NaC1 and the following CTAB concentrations: * 20 vM, • 30 #M, and X 80 #M.

FIG. 4. The resistance of a bilayer, in ohms, as a function of time, in minutes, when the aqueous phases contain 2.5 mM NaC1 and the following CTAB concentrations: * 20 #3//, • 30 #M, and X 60 ~M.

CTAB in the aqueous phase (80 ~M) had resistance which stabilized at the relatively high value of 7.5 X 105 ohm cm ~. However, films formed with CTAB concentration slightly greater than the minimum required for stability (30/~M CTAB) showed progressive fall in resistance from 1.5 X 107 down to 1.5 X 104 ohm cm 2. Not all films formed in a high CTAB concentration exhibited a stable high resistance, but the higher the detergent concentration (approaching 100 /zM CTAB), the greater the chance of a stable high-resistance film forming. The time-dependent resistance changes in these three types of films are shown in Fig. 3. Figure 4 plots similar time-dependent resistance changes at various CTAB concentrations in 2.5 m M NaCL. Stable high-resistance films at the minimal concentration of CTAB required for film formation were not obtained, a failure which m a y be due to the short life of such films. The foregoing results, which are summarized in Fig. 5, suggest that there are two types of stable films, stable high-resistance films formed in the presence of relatively high concentrations of surfactant and relatively low resistance films formed at a critical but low concentration of surfactant. We have observed some interesting phenomena in the low resistance films. Films formed in an electrolyte containing 2.5 m M

NaC1 and 30 ~M CTAB develop stable lowresistance after some 10 to 12 rain. Slight variation of the hydrostatic pressure on either side of the film leads to bulging and an increase in the surface area of the film (verified by capacitance measurements). I t would be expected that the conductance of the film would increase together with the surface area, but surprisingly the application of a pressure up to 30 dynes cm-2 led to as muchas a 400-/0 increase in resistance, which was reversed by removing the pressure gradient across the film. R

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Journal of Colloid and Interface Science, VoI. 4 2 , N o . 2, F e b r u a r y 1 9 7 3

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SWEENEY AND BLANK

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cases we also observed oscillations of membram potential during the passage of current.

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A second phenomenon in the stable lowresistance films concerned the effects of applying small dc potentials across the films. Potentials as low as 30 mV were found to rupture the films although such potentials are considerably lower than those reported to cause dielectric breakdown in other studies. Rupture was preceded by complex voltage-dependent resistance changes and also in some films by visible local changes in the appearance of the film. A typical record is shown in Fig. 6. Behavior of the film was approximately ohmic until the potential across it reached 30 inV. The current was allowed to flow for 20 sec and then switched off. A small reverse current was then seen to be flowing, suggesting that the film was acting as a current source with a polarity opposing the current originally applied. This effect was repeated on application of 40 mV, with polarization again appearing when the 40 mV was switched off. On again switching on the 40 mV source, the film resistance rose steeply for several seconds at which time the film ruptured. In each instance, during the first application of 30 and 40 mV potentials circular opacities appeared in the bilayer which slowly disappeared when the applied potential was switched off. In some

A. The Effects of CTA + Ions on Bilayer Properties From the observations it is clear that cer tain properties of the bilayer depend criticall) upon the concentrations of surfactant and sol1 in the aqueous solutions. The aqueous sur. factant concentration appears to control th~ bilayer composition, prestnnably by equilibra. tion with the bulk phase, and increases in th~ aqueous CTA concentration would be expectei to increase its "adsorption" or presence in th~ bilayer. The results of the study are compatible with this assumption, and also with th~ idea that the behavior of the CTA in the bilaye~ system parallels the behavior of long chai~ quaternary ions in other interracial systems The permeability of the bilayer to ions appears to be affected bythe same factors as in th( case of monolayer systems (7) where there is relation between the resistance of the monolayer to the diffusion of ions, R, and the surface concentration of long chain ions, ~ (i~ charges/area). The relation can be given approximately as : R = R0 exp ( -- a~),

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where R and ~ refer to univalent ions havin~ the same sign, R0 is the resistance when ~ = ( and the factor a is a constant that depends o1~ the ionic strength. Although the experiments with bilayers are far more complicated and involve the simultaneous transport of cations and anions, the resistance varies with the adsorption of CTA + ions, and the biphasic change shown in Fig. 5, can be explained if the bilayer formed in very low CTA solutions is initially negatively charged. CTA adsorptio~ first neutralizes the negative charge and at higher concentrations introduces a positive charge, with the minimum resistance, R0 corresponding to the point of zero net charge. It is hard to see what part of the cholesterol-decane system contributes the negative charge, but

JournaI of Colloid and Interface Science, Vol. 42, No. 2, February 1973

B I L A Y E R S OF C H O L E S T E R O L A N D CTA]3

cholesterol monolayers show about the same surface potential as fatty acid (8), and the effective negative charge at the bilayer surface may be due to the water surface structure. In any case the presence of a relatively large negative charge on the bilayer surface would also explain why it is unstable at very low CTA concentrations. These assumptions are consistent with our other observations on this system, such as the qualitative estimates of drainage rates. Since drainage proceeds most rapidly when the surfaces are uncharged the data show that the lowest responding films are those with the lowest CTA + present, while the rate increases as the CTA + concentration increases. At high CTA +, drainage is usually complete long before the resistance stabilizes. The data given in Fig. 3 and 4 present kinetics that are due to adsorption as well as drainage, and they are compatible with the notion that adsorption of CTA + ions into a negatively charged film proceeds more rapidly than into a positively charged one. The resistance changes on bulging lowresistance bilayers and the kinetics of the resistance variation at high CTA are also consistent with the above picture. It was found that increasing the area of the bilayer film caused an increase in the value of R under certain conditions, rather than the expected decrease. In general R = pq/A),

E2]

where p is the resistivity, 1 = thickness and A = area. Therefore, if 1 is fixed by the thickness of two monolayers, & l n R = 2~lnp -- 2~inA.

[-3]

R should decrease as A increases, but the change in p depends on the CTA concentration in the bilayer. The amount of CTA in the bilayer is fixed initially, the area increase being due to cholesterol and decane, and p should slowly return to its former value as CTA diffuses into its "equilibrium" concentration in the bilayer. The direction of change of p on bulging should depend upon the CTA con-

415

tration relative to the CTA concentration at the minimum o, Co. If C > Co, then ~ in p < 0, while for C < Co, zXIn p > 0. Therefore, when C > Co, zX In R is always negative. However, when C < Co both A In A and zXIn p are positive, and the change in R depends on the relative magnitudes of the two terms. It is therefore possible to observe an increase in R when the bilayer area is increased in the region where C ~ Co. The kinetics of the resistance changes shown in Figs. 3 and 4 usually indicate a decrease from the initial high resistance to the steady state value. However, at the high CTA concentrations there are cases where the first readings are for lower resistances than the final plateau values. These results indicate that the resistance changes in a single bilayer with time follow the pattern predicted on the basis of Fig. 5. As the CTA diffuses into the bilayer the resistance first decreases and then increases. This is not always seen because the kinetics are complicated by the fact that the film drainage kinetics are not reproducible and they obscure the kinetics of the resistance change due to CTA penetration. The ability of high salt concentration to shift the R (CTA) curve can also be explained in terms of earlier results obtained with interfacial films (6). It has already been mentioned in connection with the capacitance measurements that the degree of adsorption of a quaternary ammonium ion at an interface increases as the salt concentration increases. If the cholesterol-decane bilayer behaves as an interface, the adsorption of CTA + ions should be greater in higher salt concentrations. This is observed for the CTA + concentrations studied. The behavior of the drained films can also be understood in terms of the variations in surface charge, since the concentration of surfactant will influence the interfaciai tension. When black film first forms the membrane lies fiat in the orifice; gradually the structure becomes less rigid and the film has a tendency to expand beyond the confines of the orifice. This is probably due to the development of strong

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SWEENEY AND BLANK

electrostatic repulsion between "adsorbed" ions since the increases in area that follow initial formation of the film are more marked at higher concentrations of CTA +.

A C/C is 10-3, and there is a relatively small

change in the concentration under these conditions. Assuming that the transport numbers of the Na + are 0.4 in solution and 0 in the bilayers, we would calculate the same low value B. The Effects of Current Flow of A C / C for NaC1 at the surfaces of the bilayer. These concentration changes are too The results that have been discussed thus small to account for the observed changes in far are consistent with the idea that the CTA + resistance, even when the changes in [-CTA~ ions present in the aqueous phases become inand ENaC13 act simultaneously. corporated into the bilayer, depending on the However, the assumption that CTA carries CTA + and salt concentrations. Therefore, if no current across bilayer m a y not be correct there is a change in the ionic concentration for our system. If CTA + ions are major curadjacent to the bilayer, one should be able to rent carriers across the bilayer we would predetect changes in the bilayer resistance that dict large changes in the bilayer concentration reflect this. Such changes can occur when curof CTA and in the resistance as a result of the rent is flowing in the system, and in earlier flow of current. (In the nitrobenzene/water work (5) on an interfacial system containing system the relative mobility of CTA + ions is CTAB, it was shown that the passage of a greater in the nonaqueous phase (5,11), and steady current, i, gives rise to changes in the it m a y be so in the bilayer as well.) For a interfacial concentration of ions. In cases CTA + concentration of 0.01 ions/h 2 a current where there was an increase in the ionic conof 6 X 1012 ions/cm2/sec would cause a ~ 6 % centration, the balance between the constant depletion of CTA concentration per second or rate of supply of ions by the current, and the --~60°-/o in ten seconds. This is bound to cause diffusion away from the interface gave rise to a a large change in the measured R value. Therecharacteristic time dependence for small values fore, the large resistance changes observed in of time. The change in concentration at the this system can be explained in terms of CTA + interface (5), ions carrying current across the bilayer. Furthermore, the existence of oscillatory beAC = (iAn/A)(t/TrD)i E4] havior under some circumstances can also be where An = the difference in the cation trans- explained in terms of the assumption that port number, D - - d i f f u s i o n coefficient and CTA + ions carry appreciable current across t = time. the bilayer. If C > C 0 t h e decrease in CTA + Let us assume that the transport nmnber of concentration in the bilayer due to transference CTA + in the bilayer is negligible, since Tien's will lead to a fall in R. This should lead to a recent measurement suggest that this is true larger current and a more rapid fall in CTA confor phospholipid films (9). The transport num- centrations. However, since R goes through a ber in aqueous solution is about 0.4 (10). A minimum there is a point where R should begin current of 10-8 amps through 10-2 cm 2, as in to increase as the CTA + concentration in the Fig. 6, is passing 6 X 1012 ions/cm 2 sec, and bilayer decreases. This will lead to a gradual in an aqueous solution containing --~20 times decrease in i, allowing the CTA + ion conas much Na + as CTA +, the CTA + carries --~0.02 centration to build up slowly and the resistance of the current. If the CTA + does not carry any to decrease toward the minimum. If the concurrent across the bilayer, CTA + ions are con- centrations and voltage are in a certain range centrated at the anode side of the bilayer and it is conceivable that this process could be diluted at the cathode side. Assuming D ~ 10-5 made to repeat and the system would oscillate cm2/sec and t = 10 sec, AC = 6 >( 1018 ions/ around the minimum in the R (CTA) curve. cm 3. In'~a"~CTA solution that is 100 /~M, If one starts with C < Co the decrease in Journal of Colloid and Interface Science, Vol. 42, No. 2, February 1973

BILAYERS OF CHOLESTEROL AND CTAB

CTA + concentration should lead to an increased R and eventually to rupture of the film unless the system reaches a steady state on the straight line portion of the R (CTA) curve. (It should be noted that these speculations are based on the assumption that the resistance of the bilayer is due to its average CTA + ion concentration. Undoubtedly, the concentration differences between the two sides of the bilayer would arise by either of the two possibilities considered above, but the effect of the asymmetry is not known. Asymmetry in another bilayer system (12) has been shown to lead to instability.) CONCLUDING REMARKS

417

in terms of simple physical properties of the components. It should be noted that the phenomena encountered in this simple system, have previously been described in much more complex and biologically derived systems. We feel that further elucidation of mechanisms in this simple system will contribute to our understanding of more complex biological systems. ACKNOWLEDGMENTS The authors wish to express their thanks to H. T. Tien for his help at the outset of this work and to John S. Britten for many helpful discussions during the course of the work. REFERENCES

It is not possible to make any definite statement concerning the nature of the resistance changes that follow film formation in the presence of electrolyte and CTA +, because the nature of conduction across these films is not known. However, on the basis of our results we are led to suggest that: 1. CTA + ions are incorporated into the cholesterol-decane bilayer depending on the concentration in the aqueous phases. 2. The resistance of the bilayer varies with the concentration of CTA + ions in the bilayer. At low concentrations the bilayer is negatively charged, while at high concentrations the bilayer is positive. 3. When current flows across the bilayer it is possible to account for changes in resistance (and oscillations) if the CTA + ions are significant current carriers. With these assumptions it is possible to explain many of the properties of these bilayers

1. GORTER, E., AND GREND~L, F., J. Exp. Med. 41, 439 (1925). 2. MUELLER, P., RUDIN, D. O., TIEN, H. T., AND WESCOTT, W. C., Circulation 26, 1167 (1962). 3. TIEN, H. T., AND DIANA, A. L., Chera. J:'/~ys. Lipids 2, 55 (1968). 4. TIEN, H. T., AND DIANA, A. L., J. Colloid Interface Sd. 24, 287 (1967). 5. BLANK, M., in "Physics and Physical Chemistry of Surface Active Substances" (Overbeek, ed.), ~Vol. II, p. 233]. Gordon and Breach, University Press, Belfast, 1967. 6. BLANK, M., AND MILLER, I, R., J. Colloid Interface Sci., 26, 26 (1968). 7. MILLER,I. R., ANDBLANK,M., J. Colloid Interface Sci., 26, 34 (1968). 8. GAINES, G. L., "Insoluble Monolayers at LiquidGas Interfaces," Interscience, New York, 1966. 9. TIEN, H. T., in "Surface Chemistry of Biological Systems" (M. Blank, ed.), p. 135. Plenum, New York, 1970. i0. HARTLEY, G. S., COLLIE, B., AND SAMIS, C. S., Trans. Faraday Soc. 32, 795 (1956). 11. BLANK, M., J. Colloid I~.terface Sci. 22, 51 (1965). I2. OHKI, S,, AND PAPAItADJOPOULOS,D., in "Surface Chemistry of Biological Systems, (M. Blank, ed.), p. 155. Plenum, New York, 1970.

Journa~ of Colloid and Interface Science. VoL 42, No. 2, February 1973