Langmuir 1993,9,233-241
233
Osmotic Stress Measurements of Dihexadecyldimethylammonium Acetate Bilayers as a Function of Temperature and Added Salt Yi-hua Tsao and D. Fennel1 Evans' Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455
R. P. Rand Biological Sciences, Brock University, St. Catherines, Ontario L2S 3A1, Canada
V. A. Parsegian National Institutes of Health, Bethesda, Maryland 20892 Received April 6,1992.In Final Form: September 8,1992 Interaction forces between dihexadecyldmethylammonium acetate (DHDAA) bilayers were measured using oemotic streas (OS) and surfaceforces apparatus @FA)techniques as a function of temperatureand sodium acetate concentration (5-600 mM). Below T,,the hydrocarbon chain melting temperature, the two techniques gave identical force.curves. Above T,,the OS-derivedrepulsiveforces were slightly higher and more slowly decaying than the SFA-derivedcurves. The differenceis consistent with a contribution from thermal-mechanicalundulationsallowed to occur in the multilayere used in the OS preparation but suppressed in the SFA by immobilization of the bilayers onto mica surfaces. The OS measurements at surfaceseparationsof 0-8 A showed strong hydration forcesidentical above and below T,. This observation would suggest that neither thermal-mechanical undulations nor molecular protrueiona contribute significantlyto DHDAA hydration forces. Above T,and below a critical osmotic stress, P,,DHDAA bilayera unbind from multilayers to form myelin structures. Visualization by video-enhancedmicroscopy (VEM)ahom that the myelin structures coexist with a graduallydisappearingand progressively dehydrated multilayer phase. Comparison of P, for different salt concentrations with the direct electrostaticP,and mechanical undulatqry Pf components suggests that unbinding occurs at a constant value of P,which is independent of salt concentration and bilayer separation.
Introduction Because intermolecular forces play an important role in surfactant systems (micelles, vesicles, bilayers, and emulsions), and because amphiphilic aggregates are especially sensitive to change in temperature and ionic etrength, we have been measuring forces between surfactant bilayera to learn to controlthese systemsunder varying temperature and concentration of salt. M a t force information in bilayer systems comes from oemotic stress (OS) measuremente1-5 on spontaneously forming multilayers and from surface forces apparatus (SFA) measurementas-8 in thoee cases where bilayers are immobilizedonto oppositelycurved mica surfaces. A pipet aspiration (PA) method*" hae also provided critically important informationon bilayer adhesion and mechanics in excellent agreement with OS measuremente. (1) Pamegian, V. A; Rand, R. P.; Fuller, N. L.; Rau, D.C . Methods Enzymol. 1986,127,400. (2) Pamegian, V. A,; Fuller, N. L.; Rand, R. P. h o c . Natl. Acad. Sci. U.S.A. 1979, 76,2'lM). (3) LeNeveu, D. M.;Rand, R. P.; Pareegian, V. A. Biophys. J. 1977, 18.209. ~. ,~ - (4) Lis, L.J.; McAliabr, M.; Fuller, N.; Biophys. J. 1982,37,667.
Rand, R. P.; Paresgian, V. A.
(5) Rau, D. C.; Lee,B.; Pareegian, V. A. R o c . Natl. Acad. Sci. U.S.A.
1984.81. 2621. -__(6) Tabor,D.; Winterton, R. H.S . h o c . R. S O ~London . 1969, A312, 436. (7) Israelachvili, J. N.; Adame, G. E. J. Chem. SOC.,Faraday Tram. 1 1978, 74,976. I --I
(8) P ~ ~ , R . ~ ; M c G ~ g a n , P . M . ; N i n h aW.;Brady, m , B . J.;Evane, D. F. J. Phys. Chem. 1986,90,1637. (9) E v e , E.; Kwok,R. Biochemistry 1982,21,4874. (10) &m,E. A.; Needham, D. Macromolecules 1988,21,1822. (11) E v m , E. Langmuir 1991, 7,1900.
We continue studies on the synthetic dihexadecyldimethylammoniumacetate (DHDAA) surfactant in sodium acetate (NaOAc) solutions. We use both the OS and SFA methods applied to bilayers at temperatures above (T= 40 "C) and below (T = 23 "C) the bilayer melting temperature (T, 34 "C). In earlier forces were directly measured by OS on frozen-chain bilayers and found to be consistent with SFA results8 at [NaOAcl = 10 mM. An important difference, however, was the identification in the OS measurements of a significant deviation from expecteddouble-layerrepulsion as bilayers approach contact. This extra repulsion, interpreted by resemblance to forces seen by OS applied to neutral lipids, was taken to be a hydration force whose appearance had been suppressed by the curvature of mica and the deformation of mica in an antecedent SFA study.l3 We now extend the OS/SFA comparison with measurements at several NaOAc concentrations. In addition, with video-enhanced microscopy (VEM),'Cle we have watched the formation of vesicles that peel off from melted-chain multilayer8 subject to low osmotic stress. Coupled with force measurements, these observations give a view of bilayer unbinding different from the conventional picture of general multilayer expansion.
-
(12) Parsegian,V. A.; Rand, R. P.; Fuller, N. L. J. Phys. Chem. 1991, 95,4711. (13) Rand, R. P.; Pareegian, V. A. Biochim. Biophye. Acta 1989,988, 351. (14) Kachar, B.; Evane, D. F.; Ninham, B. W. J. Colloid Interface Sci. 1984,100,281. (15) Evans, D. F.; Brady, J.; Kachar, B.; Ninham, B. W. J. Solution Chem. 1986,14, 141. (16) Miller, D. D.; Bellars, J. R.; Evans, D. F.; Talmon, Y.;Ninham, B. W. J. Phys. Chem. 1987,91,674.
Q 1993 American
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234 Langmuir, Vol. 9, No.1, 1993
The transition from ordered lamellar to a disordered state has been studied in two recent papersl'J8 on a near relative of DHDAA, didodecyldimethylammonium bromide (DDAB) in KBr solutione. Here too, electrostatic double-layer forces predominate at separations greater than -10 A and multilayers disperse under low stress. Materials and Methods Dihexadecyldimethylammonium acetate (DHDAA) was prepared by pawing dihexadecyldimethylammonium bromide (DHDABr, Sogo Pharmaceutical Co., Ltd.) dissolved in methanol through an anion-exchange column (Fisher Rexyn 201) in the acetate form. The acetate counterion was selected for this study because unlike the bromide or chloride counterions, the solidliquid transition occurred in an easily accessible temperature range. DHDAA was purified by recrystallization from ether.19 Analytical grade sodium acetate (BDH AnalaR) was used without further purification. Double-distilled water was used in all osmoticstress measurements. For surface forces measurementa, Millipore water was processed using a Water Prodigy (from Labconco) before injecting into the SFA. Osmotio StressMeasurement#. Osmotic strew was applied to the bilayers by setting the water activity eqqilibrated with the bilayerslP2 using either polymer solutions (polyethylene glycol (PEG) or dextran) or vapors of saturated salt solutions. Solutions of polyethylene glycol (PEG, from Sigma Chemicals, MW 16000-2oooO)and Dextran T2000 (from Pharmacia; MW 260000-2000000) used to adjust the osmotic stress were prepared by dissolving the solute in a solution of known ionic strength. The solutions were placed on one side of a semipermeable membrane with DHDAA on the other side. The system reached equilibrium in an incubator after 2 days. During this period, the polymer solution was changed frequently in order to prevent concentration gradients from building up in the system. The osmotic stress was calculated using known formulas relating measured osmotic pressures to weight percentage of the polymer in so1ution.l Application of extremely high pressure (greater than 100 atmospheres) to the lamellar phase was achieved by the vapor pressure method. The vapor pressure was calculated from1p2J6
where is the chemical potential of water vapor, p is the vapor pressure of saturated electrolyte solution, po is the saturated vapor pressure of water, RH is the relative humidity, P is the osmotic preesure, and uwis the molar volume of water. Samples equilibrated by this vapor pressure method contained only the counterion in the aqueous compartment. The relation between repeat spacing, bilayer thickness, and bilayer separation was determined from X-ray diffraction measurementa using stoichiometric mixtures of DHDAA in 3 X 10-4 M NaOAc-2 mM TES aqueous solution with pH = 7.3. The absence of TES buffer did not show detectable differences in the diffraction patterns.12 The measurements were performed from low to high temperature, namely 10, 23, and 40 OC, without removing the sample from the diffractometer. In order to measure forces at 40 OC, samples were thermostated in an incubator for a t least 48 h and then placed in a thermostated sample holder in the X-ray machine. Fluctuations in temperature were less than 0.1 OC. Surface Forces Measurements. The surface forces measurements were carried out with a SFA similar to that designed ~
~~
(17) Duboii, M.; Z",Th. Langmuir 1991, 7, 1362. (18) Zemb, Th.;Belloni, L.; Dubois, M.; Marblja, 5.h o g . Colloid Polym. Sei., in press. (19) Brady, J. E.;Evans,D. F.; Ninham, B. W. J. Am. Chem. SOC.1984, 106,4279.
by TaborP and Israelachvili,21but with a number of modificatiomzZ Two molecularly smooth mica sheets of identical thickness were mounted in a croas-cylindriccll configuration in the SFA. The surface separation was measured with an interferometric technique by observing fringes of equal chromatic order (FECO). A constant temperature air bath was installed to control the temperature of the SFA. Warm water was injected into the SFA to reduce heating time at elevated temperature. The bare mica contact distance was set to be D = 0 after thermal equilibrium was achieved. An aliquot of a DHDAA solution which would give a thermal concentration of 2 X lo-'M was injected into the SFA and left for a t least 12 h in order to allow the bilayers to form on the mica surfaces. The forcecurveswere determined as a function of added NaOAc by subsequently injecting aliquots of electrolyte into the SFA. Video-Enhanced Microscopy. The video-enhanced mimecopy (VEM)1616 with differential interference contrast (DIC, Nomarsky provides an immediate real-time characterization of colloid or surfactant aggregatesby direct visualization on a television screen. Dynamic processes of self-assembliesand their changes in form can be directly viewed, recorded, and analyzed in real time, freeze-frame, slow motion, or time lapse. To increase cohtrast, a computer proceseor ( H a " a t s u systems, Inc., Waltham, MA) was used to store an image out of focus and subtract continuously from succeedingframes. All video images . were recorded on 3 l ~ i n tapes. A small amount of DHDAA was mixed with a 10-mL PEG solution of known concentration for 2 days. A drop of polymer solution containing the surfactant aggregates was placed in a thin glass chamber between a microscope elide and a coverslip. UV-cured glue was used to seal the glass chamber. The glass chamber was clamped to a heating stage c o n n d to a precision temperature controller and placed in the sample holder in the light microscope. The temperature of the sample was monitored by a thin-plate thermocoup1e.attached to the glass chamber.
Results
Gravimetric Mixtures. Dimensions of the lamdar structure were determined by X-ray diffraction. Preliminary expehenta showed that in limited amounta of aqueous solution that restrict bilayer eeparation the ionic strengthof the solutiondoee nota€€&%herelation between the repeat lamellarspacing, bilayer thiclmese, and bilayer separation.12 The chain transition temperature of DHDAA is about 34 OC. Below this chain transition temperature, a sharp wide-angle diffraction at (4.12 A)-1 wae obeerved for all samples and the chain projection area wm calculated to
be 20.2
A?
The repeat spacings from the small angle region were calculatedfrom the Braggequation. T h e DHDAAlamellar system is well-behaved and the repeat spacinge are highly reproducible ( i O . 1 A). The relation between lamellar repeat spacing and water content as a function of surfactant concentration and temperature is shown in Figure 1. Mwurementa at 10 and 23 "Cgive almost identical resultsand agree very well with literaturewithin experimental error.I2 At 40 O C where the hydrocarbon chains are in the melted state the repeat spacings increase by 6-14 A depending on the eoncentration of DHDAA. (20) Tabor, D.; Winterton, R. H. 6.Roc. R.SOC.London 1988, A312, 435. (21) Israelachvili, J. N.; Adams, G. E. J. Chem. Soc., Faraday TrOm. 1 1978, 74, 976. (22) T ~ oY.-H.; , Y w ,S.X.;EVU, D. F.LclngmUir 1098,8,1188. (23) Spence, M. Fundomentab of Light Micrwcopy; Cambridge University Prese: Cambridge, England, 1982.
Osmotic Stress Measurements
Lmgmuir, Vol. 9, No.1, 1993 236
250
1
" I
80
4OOC
a 0
0.2
0.0
0.6
0.4
0.8
1.0
0.0
Weight Fraction HzO
0.2
0.6
0.4
0.8
1.0
Weight Fraction HzO
Figure 1. Repeat lamellarspacingDSva weight fraction of water C as determined from X-ray for DHDAA at 10, 23, and 40 ' diffraction.
Figum 3. Head group area A vs weight fraction of water at 10, 23, and 40 "C. at 23°C- frozen chains 9.5
0
-
O
O
0 00
24
0
at 40°C-melted chains
I
1 0.0
0.2
0.4
0.6
0.8
Weight Fraction H20
Figure 2. Bilayer thicknessDIvs weight fraction of water at 10, 23, and 40 O C .
Bilayer thickness D1 and the bilayer separation D, were calculated from the X-ray repeatingspacingDs as follows12 f$ 3
cv,/(cu, + 1- c)
D,
Ds- D1
where 9 is the volume fraction of the surfactant, c is the weight percentageof the surfactant in gravimetricsample, and 01 is the specific volume of the surfactant. The specific volumes are 1.05 cm3/g for frozen hydrocarbon chains at both room temperature and at 10 OC, 1.17 cm3/g for melted chains at 40 O C , 8 1.47 cm3/g for dimethylamines,0.963 cm3/gfor acetic acid, and 1.0 cm3/g for aqueous eolutions.~ Bilayer thickness,D1, is plotted in Figure 2 as a function of concentration and temperature. For the frozen chains at 10 O C and room temperature the bilayer thickness, D1, is 23.6-26 A depending on the weight percent of H2O. Thie value isconsiderabl smallerthanestimatesof normal bilayer thickness of 40 by assuming two fully extended DHDAA chains. Analysis of these data led to the conclusion that DHDAA bilayers are formed from interdigitated monolayers in the frozen statal2 Thisconclusionisstrengthened by the observationthat the bilayer thickneeseafor DHDAA increase to 28.3-31.7 Generally,the thickness of normal bilayers decreases upon melting because the gauche rotations of
K
uwnmeltT
(24) Hondbook of Chemktry and Phyaicr; CRC Prew Boca Raton,
FL,1967; Vol. 41, p 41.
5
1.0
10
15
20
D, (A) Fig- 4. OS measurementsof DHDAA bilayers at high osmotic pressures (using vapor pressure method) at 23 (from ref 12) and 40 "C.
the chains increase. Figure 2 shows that the bilayer thickness of DHDAA changes little compared to the total repeat spacing, particularly for the frozen lamellar phase. The head group area A was calculated as follows A ~?~,V,/NAD, (3) where ml is the molecular weight of the molecule and N A is Avogadro's number. This area vs water content is plotted in Figure 3 for three different temperatures. The 10 O C measurementa give almost identical resulta as those at room temperature with head group areas ranging from 76 to 80 A2 per four chains depending on concentration of DHDAA. Taken together, the wide-angle and small-angle X-ray scattering patterns indicate that hydrocarbon chains are fully interdigitated and perpendicular to the lamellar arrays as described in ref 12. At T = 40 O C the head group areas are 68-76 A2 per two chains. The average difference between the room temperature and 40 O C measurementa is about 2-8 A2. Osmotic Stress Measurements. OS measurementa at high osmotic pressures and corresponding small values of D, are shown in Figure 4. Within experimental error, the data pinta for 23 and 40 O C fall on the same line with a decay length of 2.6 A. These observationsestablish that the short-range hydration force is identical for DHDAA bilayers in the frozen and melted state. For D, S 8 A the force is given by
-
Phyd 108*32 exp(-Dd2.6) dyn/cm2 (4) OSmeasurementa over a more extended range of pressurea at 23 and 40 O C and four ionic strengths (5,10, 100, and
Tea0 et al.
236 Langmuir, Vot. 9, No. 1, 1993
1
9,
6
pr
5
4O0C
o
0
67 \
B
(dynel:;:?)
23OC
23'C
log P ( dynes/cm2) O:;k6
c O*
*O 0
--------*
- - ? 4
** > 4
50
150
100
23OC o
60
40
80
100
Dw (A) Figure 8. OS measurements of DHDAA bilayers in 600 mM NaOAc, 2 mM TES a t 23 and 40 "C. T = 23'C
'.
7i
i
20
0
2b0
Dw (A) Figure 6. OS measurements of DHDAA bilayers in 5 mM NaOAc, 2 mM TES at 23 (from ref 12) and 40 "C. The star symbol gives the X-ray spacing determined as the DHDAA bilayers undergo the order-disorder transition at 40 O C ; it is not an equilibrium value. The bar labeled P,gives an estimate of the value of the osmotic pressure at which bilayer unbinding occurs.
7 i
%eo t o
.
- - - - - - - - - - - $
'6
log P (dyneslcm2)
4OoC
[NaOAc] = 10 mM
I
40°C
OO
..
0
50
i oo (A)
200
Dw
1 44
50
0
150
100
200
Dw (A) Figure 6. OS measurements of DHDAA bilayers in 10 mM NaOAc, 2 mM TES at 23 (from ref 12) and 40 O C .
Figure 9. Force data from OS (from ref 12) and SFA (from ref 8) measurements of DHDAA in [NaOAcl = 10 mM at 23 O C . The SFA data points are fitted to the DLVO theory using a surface potential of 185 mV with a Debye length of 30 A together with the hydration force as measured in the OS experiments. The upper dashed line is obtained by differentiating the SFA fitting curve (solid line). This differentiated curve coincides well with the OS data points (open squares).
g 1 appears to decrease, but over a period of time the lamellar 1
23°C
0
., 0
.
I
20
.
1
40
.
8
60
.
4OoC
I
80
.
,
100
Dw (A) Figure 7. OS measurements of DHDAA bilayers in 100 mM NaOAc, 2 mM TES at 23 and 40 "C.
600 mM NaOAc) are shown in Figures 6-8. In general,
data points at the two temperatures coincide over most of the pressure range, there is a slight discrepancy at the highest ionic strength. At low osmotic pressures there is a major difference in the data at the two temperatures. At 23 OC where the bilayers are in the frozen state, D, increases as the osmotic stress is decreased in accordance with expectations. However, at 40 OC, there is a pronounced break in the curveswhich occurs below some osmotic pressure, P,.The values of P, are indicated by the bars on the vertical axis of Figures 6-8. If the applied osmotic pressure is lowered below this value, the initial separation between the bilayers
arrays become less ordered. When the osmotic pressure is lowered sufficiently,for example, log P < 4.6 for the 10 m MNaOAc sample,no diffraction pattern can be detected. This loss of order appears to be irreversible. We attribute thisbehavior to the spontaneoustransformationof bilayers to myelin structures as described in more detail in the sections below. Surface Forces Measurements. At 23 OC,the bilayer thickness measured using the SFA is 27.6 A,which is very close to the value obtained in OS studies. AFM images of bilayers self-assembled onto mica indicated the formation of interdigitated bilayers.22 At 40 O C , the increases in repulsive force due to an increase in surface potential prevent bilayer thickness from being accuratelymeaswed using the $FA. The bilayer thickness aa determined from the OS measurement, 67 A, was used to calculate Dw. SFA measurementa (log (FI2rR)vs D,) for [NaOAc] = 10 and 100 mM at 23 and 40 "C are shown in the bottom half of Figures 9-12. The data were analyzed using an equation of the form
F12rR
E(London Dbp) + &EL Repubion)
+ E(Hydntion) where F/2rR = E b according ~ to the Derjaguin appro.imation26 of energy of interaction between two parallel plates, and R is the radius of curvature of the mica surfaces. E(Londo,, D L ~ was ) evaluated using a Hamaker mutant of 2.2 X J,E(EL bpulrion) was evaluated using a numerical (25) Derjaguin, B. V. Kolloid-Z. l9S4,69, 155.
Osmotic Stress Measurements
Langmuir, Vol. 9, No. 1,1993 237 T = 23OC [NaOAc] = 1 0 0 mM
log P or
P (dynedcm2) 5
1
F/27cR (mdynedcm)
1 , 0
V
I
V
40
20
80
60
D w (A)
Figure 10. Force data from OS and SFA measurements of DHDAA in [NaOAc] = 100 mM at 23 "C. The SFA data points are fitted to the DLVO theory using a surface potential of 95 mV with a Debye length of 9.6 A together with the hydration force. The dashed line is obtained by differentiating the SFA fitting curve (solid line). "
.%
T = 4OoC [NaOAc] = 10 mM
h 7:
log P or log (FI2xR)
%R
=tk
65-
-e-
0
50
06
0-
P (dynedcmz)
-*-
100
--
- I
150
200
D w
Figure 11. Force data from OS and SFA measurements of DHDAA in [NaOAc] = 10 mM at 40 "C. The SFA data points are fitted to the DLVO theory using a surface potential of 210 mV with a Debye length of 35 A together with the hydration force as measured in the OS experiments. The dashed line which is equivalent to the pressure curve as measured in the OS measurementsis obtained by differentiatingthe SFA fitting curve (solid line). T = 4OoC [NaOAc] = 1 0 0 mM
0
20
40
Dw
60
80
(A)
Figure 12. Force data from OS and SFA measurements of DHDAA in [NaOAc] = 100 mM at 40 "C. The SFA data points are fitted to the DLVO theory using a surface potential of 110 mV with a Debye length of 9.5 A together with the hydration force. The dashed line is obtained by differentiating the SFA fitting curve (solid line). The OS data points show higher repulsions at surface separation from 30 to 60 A than the SFA data.
solution to the Poisson-Boltzmann equation26 and employed the values given in eq 4. The lower, solid lines were obtained by fitting the SFA data (solid
E(Hy&etion)
(26) Chan, D.Y.C.; Paahley, R.M.;White, L.R.J. CoZZoidInterface Sci. 1980, 77, 283.
Figure 13. (A, top) VEM image of DHDAA in 45% PEG (in water) at 23 "C. The clear phase in the middle represents the PEG solution. The surfactant aggregates show features of lamellar phase. (B, bottom) VEM image of DHDAA in 45% PEG (in water) at 40 "C. The surfactant aggregates change slightly in shape upon melting. Bar length shown is 10 pm and applies to all photographs.
squares) to eq 5; surface potentials and Debye lengths are given in the figure captions. Since the SFA data give energies of interaction per unit area while the OS data give force per area, one can compare the two sets of measurements by taking derivatives of curves fitted to the SFA data. The derivatives of these solid lines are the dashed lines which give an excellent description of the OS points (open squares). One may conclude that the agreementbetween the two techniques is satisfactory both above (Figures 11and 12) and below (Figures 9 and 10) the chain melting temperature. Video-Enhanced Microscopy. In order to gain insight into the discontinuities in the OS force curves (Figures 5-8) at 40 "C,we used video-enhanced microscopy to visualize how the structure of the DHDAA bilayers changed as a function of OS and temperature. In the VEM experiments we simply added surfactant to the polymer solutions, unlike the OS experiments where the two were separated by a membrane. Figures 13-15 show VEM images of DHDAA in 45,15, and 1%PEG solutions, respectively. The top panel in each of the figures (labeled A) shows the structures expected for a lamellar phase. The second panel (labeled B) shows the corresponding images obtained when the samples are heated to 40 OC. In 45% PEG the aggregate changes shape slightly upon melting, but no other transformations in either the DHDAA or PEG phases can be discerned. In the 5%
238 Langmuir, VoZ.9, No.1,1993
Tsao et aZ.
Figure 14. (A, top left) VEM image of DHDAA in 5 % PEG (in water) a t 23 "C. The clear phase in the lower-left corner represents the PEG solution. Surfactant aggregates show features of lamellar phase. (B, bottom left) VEM image of DHDAA in 5 % PEG (in water) at 40 "C. Some myelin figures form a t the surfactant-PEG interface upon melting, while in the middle of the aggregates (upper-right and -left corners) melted lamellar features remain. (C, top right) VEM image of DHDAA in 5 % PEG (in water) at 40 "C under polarized light shows that these myelin figures are highly birefringent. (D, bottom right) VEM image of DHDAA in 5 % PEG (in water) after cooling back from 40 to 23 "C. The upper-right corner indicates the presence of frozen lamellar phase. The polygonal figures are frozen myelins.
PEG solution myelin figure^^'-^ formed at the surfactantPEG interface. Under polarized light, these myelin structures are highly birefringent as shown in Figure 14C. When the temperature is decreased to 25 "C,the myelin figures persist. In 1 % PEG solutions the aggregatechanges its shape dramatically and the lamellar phase is hardly discernible. Myelin figures form at the surfactant-PEG interface, but if the temperature is held constant, they stop growing after 1 or 2 min. We believe that as the myelin structures form, they concentrate the adjacent PEG solution thereby decreasing the local osmotic pressure, and myelin growth stops. When the-temperatureof the 1% PEG solution was increased to 50 "C,the aggregates spontaneouslydisrupted and vesiclesformed and dispersed into the solution as shown in Figure 15C. Discussion Pertinent properties of the DHDAA bilayer system can be summarized by three main points: (1)hydration forces for DHDAA bilayers are identical in the frozen and melted states; (2)SFAand OS measurements give almost identical force curves in both the melted and frozen states,although there might be small differences at high ionic strength in (27) Sakurai, I.; Kawamura, Y.Biochim. Biophys. Acta 1984,777,347. (28) Sakurai, I. Biochim. Biophys. Acta 1985,815, 149. (29) Mishima, K.;Yoshiyama, K. Biochim. Biophys. Acta 1987,904, 149.
the melted state; (3)in OS measurementsabove the chain melting temperature, the force curves show a pronounced break at a low osmotic pressure that is weakly dependent on, or even independent of, ionic strength. VEM images suggest a transformation from bilayers to vesicles. We develop the implications of these observations below. Hydration Forces. At separations less than 10A there is an upward break in the OS vs separation curves.12 Because this part of the force-distance curve could not be described in terms of electrostatic double layer interactions, and because of its -2.6 A exponential decay rate, it was previously suggested that this was a hydration force in which electrostatic contributions are relatively weak (see Figure 4,ref 12). Such a repulsion, it was argued, had been missed in earlier SFA measurements8 because the opposite curvature of the mica surfaceseffectsa systematic obfuscation of shorter range hydration forces, especially those measured against a background of longer range electrostatic double layer forces. It is remarkablethat to within experimental scatter the hydration forces are virtually indistinguishable in frozen (solid points) and melted (open points) bilayer states. Similarity of forces argues against a thermal/mechanical origin for the observed repulsion. Otherwise one would have to show that this important thermal/mechanical property of the bilayer was negligibly affected by chain melting to a normal bilayer from an interdigitated frozen
Osmotic Stress Measurements
Langmuir, Vol. 9, No.1, 1993 239 play an important role in controllingthe interaction forces between bilayers has stimulated considerable interest including development of numerous theoretical models.10*31932 A major experimental challenge has been to devise experiments in which the role of thermal-mechanical undulations can be unambiguously determined. Comparison of force curves generated by SFA and OS measurements provides one such approach. In SFA measurements, bilayers are immobilized on mica, effectively suppressingundulations, while in OS measurements bilayers are free to undergo thermally induced fluctuations. A t 23 "C,the SFA and OS results are largely identical, as shown in Figures 9 and 10. This confirms that at temperatures below the chain transition temperature, T,, OS and SFA give identical results. At temperatures above T,,there is a small difference between the OS data and the log P curve calculated from the SFA data, particularly at separations of 30-60 A in the [NaOAc] = 100 mM solution (Figure 12). This raises the question of how large a contribution thermal-mechanical undulations make to the interaction forces in DHDAA bilayer systems. In pursuing this question, we follow to first approximation, the formulation of Evans and P a r ~ e g i a nand ~~ distinguish between the direct or elastic pressure and a fluctuational repulsion In these equations k is the Boltzmann constant, T the temperature in Kelvin, X the Debye length, D, the surface separation, B the bending modulus, and Pose= 64n&Tyo2 (no = electrolyte concentration, ro= [exp(ze'cp/2kT) l]/[exp(ze'cp/BkT) + 11, 'cp = surface potential).33 Comparison of eqs 6 and 7 shows that we can immediately express Pf as a function of Pe
Pf' CP,1I2
(8)
where C = ( ~ k T ) / ( 3 2 B ~ / ~ X ~Thus, / 2 ) . we can write the total pressure P, as
Figure 15. (A, top) VEM image of DHDAA in 1% PEG (in water) a t 23 O C . The lower-right side is the surfactant aggregate showing lamellar phase features. The upper-left side is the PEG solution. (B, middle) VEM image of DHDAA in 1%PEG (in water) at 40 "C. The surfactant aggregateschange dramatically. No lamellar phase can be found on the surfactant aggregates. (C, bottom) VEM image of DHDAA in 1% PEG (in water) at 50 "C. Upon increasing temperature, these myelin figures are disrupted and form vesicles which disperse into the PEG solution.
bilayer. Our observations suggest the importance of molecular area since the small differences in area in the melted and frozen DHDAA bilayers are not sufficient to change the hydration force. Comparison of Bilayer Interaction Forces As Determined by SFA and OS Measurements. The suggestion by Helfrich30that thennal-mechanical undulations (30) Helfrich, W. 2.Naturforsch. 1978,330,305.
log P = log (Pe+ Pf)= log (Pe)+ log [1+(Pf/PJl (9) With eq 9 we can evaluate the relative contributions of Peand Pftothe force curves in the DHDAA bilayer system at 100 mM NaOAc. Using the bending modulus B measured for phospholipid bilayers of 10-l2erg34 and X = 9.5 A,we obtain a value of C = 145 erg112 We focus our attention on the pressure range, log P = 5.5 to 6.0, since it is in this region that the SFA and OS force curves show the greatest deviation. Here, Pf/Pe equals 0.29 and 0.16 for log P = 5.5 and 6.0, respectively. On a log plot one expects a shift of log (1.16) = 0.06 to log (1.29) = 0.11 from the values predicted without the contribution of fluctuations. These shifts are big enough to suggest the presence of mechanical undulations, but not big enough to demonstrate their contributions conclusively. The calculated results are plotted in Figure 16. Discontinuities in the Osmotic Stress Curves at Low Osmotic Pressures. A major focus in amphiphilic systems involves understanding the interaction forces which lead to microstructuraltransformations such as the spontaneous conversion of bilayers to vesicles and/or (31) Podgomik, R; ParSegian, V. A. Langmuir 1992,8,557. (32) Evans, E.A.; Paraegian, V. A. h o c . Natl. Acad. Sci. U.S.A.1986, 83, 7132. (33) Hiemenz,P. C. Principles of Colloid and Surface Chemistry, 2nd ed.; 1986;p 706. (34) Bo, L.;Waugh, R E.Biophys. J. 1989,55,509.
240 Langmuir, Vol. 9, No.1, 1993 10
-I
8
-
2 -
Tea0 et 41. [NaOAc] = 100 mM [NaOAc] = 500 m~
[NaOAc] = 500 mM
I
[NaOAc] = 100 mM
micelles. With ionic surfactants there appear to be three ways of driving such transformations. The fmt is illustrated by the behavior of ditetradecyldimethylammoniumbromide (2C14N2ClBr).3~Below Tc(31"C), crystalsof 2Cla2ClBr do not dissolvein water. At temperaturesslightlyhigher than T,,2C1a2C1Br forma turbid dispersions even at concentrations less than 0.05 wt 7%. With the more ionic 2C1&12C10Ac1the acetate surfactant forms clear, isotropic solutions up to 20 wt % at room temperature. Fluorescence quenchingand smallangle neutron scattering measurements establish that the microstructurein 2C&J2C1OAcis predominately micelles with aggregation number of The second method of driving bilayer transformations involves modulating head groupcounterion interactions by using either mixtures of counterions like acetate and bromide or complexing agents like the cryptates. For example,additionof the C222 cryptate to sodium 8-phenyln-hexadecyl-p-sulfonatebilayer systems transforms them into vesiclesand eventually to micellesas the ratio of C222: Na+ in~reases.3~ The thiid method involves the control of osmotic preseure on a bilayer systempoised to undergospontaneous dissociationlike that described in this paper. Of the three methods, the OS method is potentially the most valuable because it provides information on the force and distances at which bilayer dissociation occurs. The OS curves at 40 OC (Figures 5-8) show that as P decreases, the bilayer spacing increases as expected until a critical pressure, P,,is reached. Below this pressure, the X-ray scattering data indicates asmaller value of D, from a transient lattice that itself disappears with time. The measured data points below P,are enclosed with brackets to emphasize that they are NOT equilibrium values. Before analyzing the OS data in more detail, we first consider the VEM studies on DHDAA solutions because they provide direct visual information on bilayer transformations below P,. Figures 13-15 illustrate the changes in DHDAA structure as a function of osmotic pressure (polymer concentration) and temperature. Below DHDAA's chain transition temperature, lamellar arrays are observed at all polymer concentrations. In sharp contrast, at tempera(36)Miller, D. D.; Bellare, J. R;Kaneko, T.;Evans, D. F. Langmuir
1988,4,1363.
(36)Miller, D. D.; Magid, L. J.; Evans, D. F. J. Phys. Chem. 1990,94,
rnn.
OJLL.
(37)Miller, D. D.; Evans, D. F.; Warr, G.G.;B e h e , J. R.J. Colloid
Interface Sei. 1987, 116, 698.
tures above T,and at applied osmotic stress lese than P,, myelin figures begin to form. Analysis of the change in refractive index of the PEG solutione used in the osmotic stress measurements below P,shows that it dehydratee aa myelin structures form. As a consequence,the real PEG osmoticstressbecomes higher than the original, nominal value. The remaining lamellar array, presumably subjected to an osmotic stress higher than what is represented by the bracketed points, shows D, spacings that are lower than what would be seen from extrapolatingthe exponentiallydecayingforce curve. Thia transition is reminiscent of the transition from the lamellar phase seen by Dubois and Zemb.17J8 Under osmotic control and the conditions of this experiment, however, the transition appears to occur at lower water contents and bilayer separations. This simultaneous loss of water from the stressing polymer solution and from the multilayer phase indicates that water is being sucked into the myelins; indeed, the VEM images show that myelin formation slowsdown after a short time. When the sample is incubated long enough, the X-ray diffractionpattern completelydisappears. This is consistentwith the VEM observation that at sufficiently low osmotic streas, DHDAA bilayers transform to vesicles on a readily observable time scale. The VEM and X-ray diffraction data together might suggest that the procese of vesiculation need not be a general unbinding, Le., where fluctuations cause multilayers to expand uniformly. Rather, bilayers seem to be sloughed off from the multilayer. It is possible that the peeling off of one layer at a time is dictated by the kinetics of getting water into the multilayer, a limitation which would preclude a general and simultaneous swelling of the lattice. Inspection of the osmotic stress curves suggests that there is a critical P, and corresponding distance D, associated with each concentration of added salt. At thew bilayer separation distances, hydration forcesand van der Waals forcesare negligibly small; assuming the validity of eqs 7 through 9 we can write P, = P, + Pf (10) We take critical pressure for bilayers in each of the solutions of different salt concentrations to be between the last point seen on the exponential part of the data and the f i s t clearly deviant point. From eq 8 the form
Pf = CP,1/2
(11)
p y Ix
(12)
with gives us
P, = P, + Pf = x 2 + cx for a simple quadratic equation x 2 + cx -P,= 0 From this relation one can solve IC=
(13) ( 14)
-c + (C2 + 4PC)'/2 2
with
= %1 2
+ 4PC/C2)"2 - 1)
(15)
for
Pf = C2
-p(l+ 4PC/C2)1'2 - 1)
with values given displayed in Table I.
(16)
Osmotic Stress Measurements
Langmuir, Val. 9, No. 1, 1993 241
Table I. Summary of Surface Potentiah, Y, Debye Lengths,h, and Elwtrortatic, Pw and Fluctuational, Pi, Repulrionr at Critical Prerrurer, P., ar a Function of [NaOAc]. 5 10 100 600 a
210 210 110
43 36 9.6 4.25
50
4.86-5.10 6.07-5.29 4.W6.24 4.78-5.24
4.83-5.07 5.065.28 4.56-5.10 4.03-4.77
3.6c3.73 3.83-3.95 4.44-4.71 4.70-5.07
P,,P., Pi in dyn/cm*.
How do we interpret these numbers? P,appears to be relatively insensitive to changes in salt concentration, consistent with what was reported by Zemb et al. with DDAB.l* However, values of the critical separation distance, D,, change several-fold between 0.006 and 0.5 M. The electrostatic pressure Pe seems to vary even less with salt concentration. Within the precision of the data, Pr does not appear to havejust one single interwting value. It appears though that DHDAA multilayers decompose when their electrostatic interaction pressure is some 1.5 X l@ dyn/cm2 regardless of bilayer separation, fluctuation repulsion, or salt concentration.
Conclusions At T = 23 OC, below the temperature T, = 34 OC at which hydrocarbonchains freeze, the electrostatic double
layer repulsion between DHDAA bilayers is identical measured either by OS and X-ray diffraction on multilayers or by SFA where bilayers are immobilized onto oppositely curved mica surfaces. At T = 40 OC > T,, electrostatic double layer repulsion is slightly but noticeably stronger between free bilayers in multilayer assembly, a difference that can be due to the additional contribution of mechanical-undulatoryforces in multilayers. Very strong hydration forces seen by OS at 0 to 8 A separations are identical above and below the chainmelting temperature, suggesting the unlikelihood of a mechanical-undulatory origin of this force. Initially subjected to an applied osmotic stress less than a critical value, P,,melted-chain bilayers detach from the multilayer to form vesiclea whose own osmotic pressure exceeds P,. The remnant multilayers gradually shrink under this higher pressure, but finally all convert to vesicles.
Acknowledgment. Support by NIH Grant 2RO1-GM34341-06is gratefullyacknowledged. R.P.R. acknowledgea the support of the National Science and Engineering Research Council of Canada. We have relied a lot on the advice and experience of Mrs. Nola Fuller.
