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Langmuir 1994,10, 1040-1043
Isotopic Effect in Phase Separation of Dioctanoylphosphatidylcholine/Water Micellar Solutions Pierandrea Lo Nostro,+JNadia StubicarJ and Sow-Hsin Chen Department of Nuclear Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received April 23,1993. In Final Form: December 8,1993@ Dioctanoylphosphatidylcholine (diC8-PC) forms micellar dispersions in water. At a certain lowconcentration range the solution phase separates into two micellar phases below a certain transition temperature To,and the coexistence curve shows an upper consolute temperature Tcat a lecithin molar fraction x,. The phase separation phenomenon can be understood in terms of an adaptation of a thermodynamic model due to Blankschtein, Thurston, and Benedek (BTB). In this paper we report the results of a study of diC8-PC micellar solutions in a solvent consisting of D2O/H2O mixtures. The dicg PC/D20/H20 system shows phase separationwith significantvariations of Tcand of the criticallipid molar fraction xc, depending on the relative amount of D20. This indicates that an increasing fraction of D2O in the solvent primarily affects the system by modifying lipid-water interactions in such a way that the effective monomer-monomer interaction increases.
Introduction Phospholipids, as well as glycolipids and cholesterol, are very active biomolecules. They are typically used by living cells as signal molecules and as biomembrane components. Phospholipids are made up of a glycerol molecule linked to two carboxylicacid moieties in positions 1and 2, and to a phosphate group in position 3. In turn, the phosphate group can also be linked to another residue such as serine, ethanolamine, glycerol, inositol, choline, and so f0rth.l A “phosphatidylcholine” is thus composed of two acyl chains, one glycerol molecule, a phosphate group, and a choline residue, as illustrated in Figure 1.We shall use a short abbreviation, diC,-PC, meaning that the lipid molecule contains two acyl chains with n carbon atoms each. Structurally speaking, the two aliphatic chains are almost parallel but not equivalent, both perpendicular to the phosphocholine polar head group, and the molecule is a zwitterion, with a negative charge attached on the phosphate group, and a positive charge on the nitrogen atom of choline. Several studies on aqueous dispersions of diC,-PC molecules have revealed that the shortest members, that is, for 6 In 5 8, form micelles, whereas diCg-PC forms smallunilamellar vesicles upon appropriate sonication and ultracentrifugation.2 In particular dice-PC’s form a globular micelle, the size of which stays constant as the lipid concentration increase^,^ while diC7-PC and diC8PC organize into rod-shaped structure^^*^ that grow preferentially in the longitudinal direction with increasing concentration of the lipid, as shown by a series of smallangle neutron scattering experiments.4 + Present address: Department of Chemistry, University of Florence, 50121 Firenze, Italy. 1 Present address: Laboratory of Physical Chemistry, Faculty of Sciences, University of Zagreb, 41001 Zagreb, Croatia. Abstract published in Advance ACS Abstracts, February 15, 1994. (1) Stryer, L.Biochemistry, 3rd ed.; W .H. Freeman & Co.: New York, 1988. (2) Tausk,R. J. M.; Oudshoorn,C.; Overbeek,J. Th. G. Biophys. Chem. 1974,2,53. Tausk, R. J. M.; Karmiggelt, J.; Oudshoom, C.; Overbeek, J. Th. G. Biophys. Chem. 1974,1,175. (3) Lin,T. L.;Chen, S. H.; Gabriel, N. E.;Roberts, M. F. J. Am. Chem. SOC.1986,108, 3499. (4) Lin, T. L.;Chen, S. H.; Gabriel, N. E.; Roberta, M. F. J. Phys. Chem. 1987,91,406. @
0743-7463/94/2410-1040$04.50/0
polar head groups region
Figure 1. Structure of a diCB-PC/HgO micelle.
An interesting application of the aggregation properties of such phospholipids in water is the study of the mechanism that is involved in the enzymatic hydrolysis of the two fatty acid ester bonds. It has been shown that the activity of the porcine pancreatic phospholipase A strongly depends on the state of aggregation of the lipid, and on the hydrocarbon chain length. In fact previous reports show that micellar formation dramatically enhances the enzymatic a ~ t i v i t y . ~ , ~ Another important issue to be addressed is the phase separation phenomenon occurring slightly above room (5) Burns,R. A.; Donovan, J. M.; Roberts, M. F. Biochemistry 1983, 22,964. Lin, T. L.;Liu, C. C.; Roberts, M. F.; Chen, S. H. J. Phys. Chem. 1991,95,6020.
0 1994 American Chemical Society
Langmuir, Vol. 10, No. 4, 1994 1041
Phase Separation of DiCB-PCIH20 Micellar Solutions
temperature shown by diC8-PC in water for concentrations above the critical micelle concentration (cmc). Such a phase separation has a consequence that the lipids avoid fast enzymatic hydrolysis at room temperature. The work by Tausk and co-workers made 20 years ago indicated the existence of a miscibility gap for the diCa-PC/water system.2 They also found that addition of NaCl and LiI has a tremendous effect on the coexistence curve of this lipid in water.2 However, the regular theory of liquidliquid phase separation formulated for molecular liquids could not account for the skewed shape of the coexistence curve? More recently, a new thermodynamic theory of phase separation in micellar solutions has been formulated7and successfully applied to the cloud point curve of diCE-PC/ water and diCs-PC/water/ureasystems? This new theory, elaborated by Blankschtein, Thurston, and Benedek, depicts the micellar state as a multicomponent system containing water molecules, lipid monomers, and micelles of various sizes, in a multiple chemical equilibrium.7 The theory takes into account both the dispersion entropy of the polydisperse micellar system and the mean field attractive interactions between different monomers. The theory predicta experimental parameters such as the upper consolute temperature T,, the critical molar fraction of lipid x,, the osmotic compressibility, and the shape of the cloud point curve in terms of three basic phenomenological parameters. The first parameter is Ap, defined as Ap=A-No6
(1)
A p is called the “free energy gain of micellar growth”. In fact in eq 1,A is the free energy gain of forming a minimum size micelle from NOfree monomers in the solvent and 6 is the free energy gain of transferring one monomer from the spherical end cap region of the micelle to the cylindrical portion of the micelle. This is the terminology adapted from the picture of a sphere-to-rod transition model of micelle growth called the “ladder m ~ d e l ” . ~ In this ladder model the cmc of the solution is given by eXp(~i/N&eT). The quantity A/N&gT for diC8-PC aqueous dispersions can be inferred from small-angle neutron scattering (SANS)studies of a series of homologous shortchain lipids in DzO solutions by Lin et al.lo From this paper it can be deduced that A/N&BT = -12.0 at room temperature and is approximately independent of the HzO/ DzO solvents. However, the quantity A p as deduced from the fitting of the coexistence curve does depend on the solvent HzO/DzO. This is because Ap depends also on 6, the value of which is controlled by the difference between the area per monomer available in the end cap region and in the cylindrical portion of the micelle. This quantity is thus dependent on the strength of the hydrophobic interactions in the solvent. In other words if the experiment indicates that Ap changes from solvent HzO to DzO, it does not automatically imply that cmc changes as well, even though it has been documented that hydrophobic interactions are stronger in D20 than in HzO at the same temperature (12). (6) Guggenheim, E. A. Mixtures: The Theory of the Equilibrium Properties of Some Simple Classes of Mixtures, Solutions and Alloys; Clarendon: Oxford, 1952. (7) Blankschtein, D.; Thurston, G. M.; Benedek, G. B. J. Chem. Phys.
1986,85,7208. (8) Carvalho, B. L.; Briganti, G.; Chen, S. H. J. Phys. Chem. 1989,93, 4282. (9) Missel, P. J.; Mazer, N. A.; Benedek, G. B.; Young, C. Y.J. Phys. Chem. 1980,84, 1044. (10) Lin, T. L.; Chen, S. H.; Roberta, M. F. J. Am. Chem. SOC. 1987, 109,2321.
The second parameter is C, related to the effective free energy of monomer-monomer interaction:
c = u/n,
(2)
where U is the mean field potential of all lipids forming aggregates and Q, is the effective molecular volume of a lipid monomer, 780 AS in the case of diC8-PC. The third parameter y is the ratio between the average effective volume of a lipid molecule Q, and of a water molecule Q, (30 AS),namely 7 = QJQ,
(3)
and it has a value of 26. After the diCE-PC/water system has phase separated, the solution will consist of two homogeneous phases with different concentrations of lecithin: we shall use Y and 2 to mean the lipid molar fractions in the two phaseseparated micellar solutions. A quite complicated nonlinear equation relates Ap, C, y, Y ,and 2,and allowsthe precise description of the cloud point c ~ r v e . ~Therefore, 3~ the experimental determination of the cloud point temperature TOas a function of the lipid molar fraction x , along with the estimation of y, enables us to determine the parameters AN and C that determine the average micellar aggregation number g and the osmotic compressibility of the solution. The coexistence curve of the diCE-PC/water system is a highly asymmetric curve with an upper consolute point. The magnitude of Ap controls x,, the product $2 affects the value of T,,and y regulates the skewness of the whole curve. The shape and the position of the coexistencecurve are quite sensitive to the effective values of Ap, y, and C. In fact keeping two parameters fixed, the third cannot be changed more than 107% without worsening the fit to the coexistence d a h 8 The addition of urea affects the diCE-PC/water system by bringing down the miscibility gap, decreasing the parameters A p and C, and increasing y. These effects have been related to the change induced by urea on the lipid-water interactions, and to the increment of the water dielectric constant produced by urea. Finally, the increment of y was ascribed to the effectiveincreasing molecular volume of the lipid, due to urea adsorption at the micellar interface.8 The purpose of the present paper is to study the effect induced by heavy water (DzO) on the thermodynamic parameters of dioctanoylphosphatidylcholine dispersions in water. It is well-known that D20 has stronger hydrogen bonds, compared to HzO at the same temperature, which make it amore structured solvent than regular water. Thus, all physicochemical parameters that are strictly related to the fluid structure will show large differences between DzO and HzO. This is the case for the viscosity, heat capacity, and temperature of maximum density, which all indicate that heavy water is a more structured solvent than HzO at the same temperature. The stronger hydrogen bonding occurring in DzO also produces the lowering of cmc values as was shown by quasi elastic light scattering (QELS) studies on sodium dodecyl sulfate solutions in HzO/DzO.’l Materials and Methods DiCs-PC was purchased from Avanti Polar Lipids Inc. (Birmingham, AL)in a powder form. The purity was stated to be greater than 99% in diCgPC content, and the lipid was used ~~~~
~
(11) Chang, N. J.; Kaler, E. W. J. Phys. Chem. 1986,89, 2296. (12) Oakenfull, D.; Fenwick, D. E. Aust. J . Chem. 1975,2%, 715. (13) Sheu, E.; Chen, S. H. J. Phys. Chem. 1988,92,4466.
Lo Nostro et al.
1042 Langmuir, Vol. 10, No. 4, 1994 340
, , , , , , , , , , , , , ,
,,,,,,,,, ,,,,,
3
,
3
5
.
0
0
7
18 I O 4
4
e, , ,o, , , , O.Oo0 0.001 0.002 0.003 0.004 0.005 0.006 lipid molar fraction, x
Figure 2. Coexistence curves of diC~-PC/D20/H20solutions for different D20/H20 ratios.
For T < TOthe dispersion separates into an upper, transparent and low-viscosity phase, and a lower phase that is more turbid and more viscous. Reganlingthe HzO/DIO mixtures,we assumedthat heavywater mixes perfectly with water in the whole range 0-100% ;therefore, we supposethat D2O is uniformly distributed and that ita partition coefficientin the two regions after phase separation is 60%. Such a hypothesis has not been experimentally checked out.
34 5
Ar/KB1-8600
185
270
0 0
experimental data fitting curve
Figure 3. Fitting curve of the diCe-PC/DaO coexistence curve showing the statistical limit of the A d k B parameter. Table 1 a 0.00 0.25 0.50 0.75 1.00
TcW) 319.20 323.20 326.30 329.50 332.10
Figure 4. Tc and xc versus the D2O/H20 ratio (a).
AP/kB(K) 8900* 5% 8900 5% 8800 5% 8600 5%
** 8600*5%
C/kB(K) 13.25 13.55 13.75 14.05 14.30
xc
6.16 X 6.60 X 6.95 X 7.32 X 7.64X
Bc
l(r
lo-' lo-' l(r
1O-C
56340 49080 37 920 25225 23270
without any further purification. Bidistilled water was fitered through a Millipore water purification system to remove ionic impurities. Heavy water was purchased from Cambridge Isotope Laboratories (Cambridge, MA), having 99.8% isotopic purity. Samples were prepared by weighing the lipid, H20, and D2O directly in a glass tube. The lipid concentration was varied by diluting the sample with the same solvent. After the preparation, the sample was kept in a refrigerator at 4 OC for at least 10 h, until a clear and foam-free solution was formed. The tube was then placed in a temperature-controlled bath (i0.03"C). The phase separation waa induced by heating the sample and then cooling it to near the transition temperature TO.Above TO the sample contained an optically clear, monophase region. The temperature was slowly decreased until the solution became visually slightly cloudy, and then it was raised up until the lipid dispersion was clear again. The up-and-down temperature cycle was repeated several times in order to obtain a good average value. We found that the cloud point (cooling cycle) was always about 0.5 "C below the reclarification point (heating cycle), and the true TOvalue was assumed to be the average of the cloud point and of the reclearification temperature. Once TOhad been determined for a specific sample, the lipid molar fraction was changed by dilution and the heating-cooling cycle was then repeated on the new sample.
Results and Discussion In Figure 2 we report the coexistence curve of the dicePC/D20/H20 system as a function of the D20/H20 ratio (a), which ranges between 0 and 1 (0.00, 0.25,0.50, 0.75, 1.00). By increasing the amount of heavy water, the upper consolute temperature T, rises from 319 K in the case of pure H2O (a = 0) to 332 K for pure D2O (a = 1). The asymmetry of the curve also changes. As a increases the curve becomes more broadened and shifts toward larger values of x,. Figure 3 reports the diCe-PC/D20 coexistence curve fitting where the f5% limit of statistical variation of Ap/kB is illustrated. The coexistence curve can be used to determine the values of Ap and C as described by the BlankschteinThurston-Benedek (BTB) theory. In fact x c can be estimated from the following equation:
x p = 16079 - 212 exp( A) kBTc
(4)
where kg is the Boltzmann constant. The average aggregation number at the critical point, g,, can then be calculated with the aid of the ladder model theory of micellar gr0wth4J0
where NO= 55 f 5?O K, = exp(-Ap/kBT,), and xmC is the lipid molar fraction at the cmc at T,. Since xcmc = 5.5 X lV 2~10 and x , = 1-10-3, we can rewrite eq 5 as
The values of the parameters derived from experiment are reported in Table 1 as a function of a. Ap and C have been calculated by means of least-squares fitting to the coexistence curve. For y we used the value of 26 as estimated in a previous papefl and we assumed that it does not change with different amounts of D20 in solution. We note that Ap/kB slightly decreases as a function of cy, showing that the free energy of micellar growth is slightly smaller in DzO than in H20, implying that the polydispersity is less. In Figure 4, T, and x, are plotted versus a. They both increase linearly with the content of D20.
Phase Separation of DiCs-PCIHpO Micellar Solutions
assuming that DzO produces stronger hydrophobic interactions (HI) than water, as already reported in the literature. l4
14.50
13.001 0.0
Langmuir, Vol. 10, No. 4,1994 1043
'
'
0.25
'
'
0.50
'
'
0.75
'
'
1.0
D,ORI,O (a)
Figure 5. Mean field parameter ratio (u).
(Clk~) versus the DzO/HZO
Figure 5 plots C/kB versus CY. It shows that the interaction parameter depends linearly on the amount of heavy water in the solvent. Finally, the effect of heavy water on the average aggregation number at the critical point is given in Table 1,whereg, is shown to decrease as a function of D2O content in the system. This effect is mainly due to the fact that Tc is rising as a function of CY. Summarizing, we observe that as CY increases Ap slightly decreases, while T,,x,, and C increase linearly in such a way that g, significantly decreases. In a previous papel.8 the depression of T, in the dice-PC/water/urea system was explained by the weakening of the effective interactions between lipid monomers. In this work, for the dicePC/DzO/H20 system we detect a substantial increment of T,and C which is probably due to stronger effective lipidlipid interactions that result from the stronger hydrogen bonding in D2O. The effects induced by heavy water on the diCa-PC/HzO system can then be explained by
Conclusions The solvent isotope effect in the dioctanoylphosphatidylcholine/water system is quite pronounced. It manifests itself by up-shifting the coexistence curve upon addition of DzO. The phase separation of dic8-PC/D~o/HzO solutions can be well accounted for by the BTB theory, and the corresponding thermodynamic parameters show a significant effect induced by DzO. It has been demonstrated that while DzO affects less than 10%the value of the free energy advantage of micellar growth, the value of the mean field interaction parameter (0 increaseswith the amount of heavy water. The average aggregation number at the critical point decreases sharply as a function of the relative amount of D20. Previous experiments pointed toward the presence of stronger hydrogen bonds in D2O solutions and indicated that heavy water is a more structured solvent than HzO at the same temperature. In the same way, a consistent explanation of our results can be made by invoking the capability of D20 in increasing the hydrophobic interactions in dicePC dispersions. Acknowledgment. P.L.N. and N.S. appreciate an appointment as visiting scientists at the Center for Materials Science and Engineering of MIT, where this research was carried out. This research is supported by a grant from the Basic Energy Sciences Division of the U.S.Department of Energy. (14)Chou, S. I.; Shah, D.0.J. Colloid Interface Sci. 1981, 80, 49.
