J . Phys. Chem. 1990, 94, 796-801
796
Vesicles versus Membrane Fragments in DODAC Suspensions Robert B. Pansu,*vt Bernard Arrio,: Jacques Roncin,+ and Jean Fauret Physicochimie des Rayonnements, CNRS URA75. Universite Paris Sud Bat 350, 91 405 Orsay Cedex. France, and Bioenergetique Membranaire, CNRS URA 1128, Universite Paris Sud Bat 330, 91405 Orsay Cedex, France (Received: February 27, 1989; In Final Form: July 13, 1989)
We report on the organization properties of DODAC molecules in water solutions. We have measured a value of 8.3 i 0.5% for the encapsulation efficiency of DODAC for 5NS, a membrane adsorbed probe. This value, which is consistent with most published ones, implies that vesicles are only a minor fraction in DODAC suspensions. Light-scatteringexperiments show that most of the DODAC molecules are organized in small open structures: flat disklike bilayers. These membrane fragments are formed by destruction of the vesicles during the sonication process. Upon annealing or after addition of Fe(CN),&, fragments gather to form a population of vesicles with a narrow size distribution. This size monodispersity has been rationalized by geometric models, assuming thermodynamic equilibrium. But our studies show that, as phospholipid vesicles, DODAC suspensions are not in thermodynamic equilibrium. Models that consider both the energetics and the kinetics of the vesicles formation have been proposed by Lasic and by Fromherz. The formation of a size paucidisperse population of vesicles from a size polydisperse population of membrane fragments, that we observe, is the first experimental evidence supporting such models.
Introduction The organization properties of a surfactant in water are well described by considering the shape of the surfactant molecules. Geometric models have been successful in explaining that, depending on the ratio of the surface of the polar head to the hydrophobic core, micelles, bilayers (spherical vesicles), or microemulsions are formed. They can also predict the size and the aggregation number of the surfactants.' Extensions of these models have been madeZto account for the narrow size distribution that is observed for vesicles obtained by the sonication protocol. But geometric models are based on an assumption of thermodynamic equilibrium and cannot explain that it is now possible, with use of various protocols, to produce vesicles of various sizes. The assumption, that vesicle suspensions are in thermodynamic equilibrium, is not generally true. While most studies on vesicles have been done on phospholipid ones, we have been interested in a more e ~ o t i cvesicle-forming ,~ surfactant: DODAC (N,N-dimethyl-N-octadecyl-1-octadecanaminium chloride). It can be expected that the singular properties of this molecule will be helpful to elucidate some aspects of the organization properties of bilayers. DODAC suspensions have been extensively used as microheterogeneous media for solar energy conversion$-7 but the organization properties of DODAC are still a subject of debate.* Kunitake was the first to discover DODAC aggregate formation in 1 977.9 From his observations by electron microscopylo as well as from the very low encapsulation efficiency of DODAC suspension,l' he concludes that DODAC molecules form flat disklike bilayers. This is in disagreement with work of Fendler12who, using quasi-elastic light scattering (QELS), observed a size monodisperse population of vesicles as predicted by geometric models and concluded that DODAC suspensions are composed of closed vesicles, similar to ones formed by phospholipids. We have developed both encapsulation and light-scattering measurements on DODAC suspensions, and this enables us to measure that, in the samples prepared according to the standard protocol, vesicles are only a minor component of the suspension. Both vesicles and disks coexist in DODC suspensions. The relative concentration of each aggregate depends on the preparation protocol. As for phospholipid vesicles, the assumption of thermodynamic equilibrium cannot be made in the case of DODAC suspensions. We show that the metastability of the DODAC membrane fragment is due to electrostatic interactions. We observe the formation of a population of DODAC vesicles with a narrow size distribution from a polydisperse population f
Physicochimie des Rayonnements. Bioenergetique Membranaire.
of membrane fragments. This is in agreement with the predictions of the model for the formation process of vesicles proposed independently by Lasid3 and Fromherz.14
Materials and Method N,N-Dimeth yl-N-octadecyl- 1-0ctadecanaminium chloride (DODAC) (Tokyo Kasei) is purified by recrystallization from acetone/water mixtures (95/5 volume ratio) according to Li and Kevana5 Ascorbic acid, dithiothreitol, and ferrous chloride are used as received. 5NS 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy was purchased from Molecular Probe Inc. and used as received. VesiclePreparation. DODAC is added to the aqueous solution as a dry powder at a concentration of mol/L. DODAC powder is allowed to swell overnight forming a gel. The 5-mL aliquots are dispersed by soniation with a Sonimass sonicator for 4 min using a 5-mm sonication tip. The sample is placed in a water bath at 40 OC (the temperature in the sample during sonication is 60 "C). Samples are filtrated through 0.8-pm Millipore filters to remove sonication-tip debris. Chloride titration shows that less that 5% of the DODAC is lost during filtration. Vesicle Characterization. The size monodispersity of the vesicles is routinely checked by quasi-elastic light scattering (QELS). Typical QELS spectra are exemplified in Figures 4 and 5. Dots correspond to experimental points. The solid line is the theoretical fit to the curve assuming the size monodisperse sample. In the upper panel of Figure 5, weighted residues of the fit, given by R , = (Zexp/Ith) - 1, are represented. ( I ) Tartar, H. V. J . Phys. Chem. 1955, 59, 1195-9. Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; Wiley: New York, 1980. Lianos, P.; a n a , R.J. Colloid Interface Sei. 1981, 84, 100. (2) Israelachvili, J. N.; Marcelja, S.; Horn, R.G. Q.Reu. Biophys. 1980, 13, 121-200. (3) Miller, D. D.; Evans, D. F. J. Phys. Chem. 1989, 93, 323-33. (4) Fendler, J. H. Acc. Chem. Res. 1980, 13, 7. (5) Li, A. S. W.; Kevan, L. J. Am. Chem. SOC.1983, 105, 5752-5. (6) Henglein, A.; Proske, TH.; Schnecke, W. Ber. Bunsen-Ges. Phys. Chem. 1972.82, 956-62. (7) Suddaby, B. R.;Brown, P. E.; Russel, J. C.; Whitten, D. G. J . Am. Chem. Sac. 1985, 107, 5609-17. (8) McNeil, R.; Thomas, J. K. J. Colloid Interface Sei. 1980, 522-27. (9) Kunitake, T.; Okahata, Y . J. Am. Chem. Sac. 1977, 99, 3860-1. (10) Kunitake, T.; Okahata. Y.;Shimomura, M.; Yasunami, S.; Takarabe, K. J . Am. Chem. Sac. 1981, 103, 5401-13. (11) Kunitake, T.; Okahata, Y.; Yasunami, S. Chem. Lett. 1981, 1397-400. (12) Tran, C. D.; Klahn, P. L.; Romero,A,; Fendler, J. H. J . Am. Chem. Sac. 1978, 100, 1622-24. (1 3) Lasic, D. D. Biochim. Biophys. Acta 1982,692,501-2. Lasic, D. D. J. Theor. Biol. 1987,124,3541. Lasic, D. D. Biochem. J . 1988,256, 1-1 1. (14) (a) Fromherz, P. Chem. Phys. Letr. 1983,94,259-66. (b) Fromherz, P.;Roecker, C.; Rueppel, D. Faraday Discuss. Chem. Sac. 1986,81, 39-48.
0022-3654/90/2094-0796$02.50/00 1990 American Chemical Society
The Journal of Physical Chemistry, Vol. 94, No. 2, 1990 191
Vesicles vs Membrane Fragments in DODAC Suspensions The QELS spectrum of the DODAC vesicles can be reasonably fitted by a Lorenzian curve as shown in Figure 5. This shows that DODAC vesicles are paucidisperse in size. From the width of the Lorenzian, the diffusion coefficient D and the hydrodynamic radius Rh of the vesicles can be measuredIs vi12 = 2q2D/2ir
(1)
where 4irn
q = - sin (0/2)
x
kT and Rh = 6irqD
(2)
and where v l i 2 = half-width at half-height of the adjusted theoretical curve; D = mutual diffusion coefficient; n = solvent index of refraction: X = laser wavelength; 0 = observation angle; q = solvent viscosity; T = temperature; and k = Boltzmann constant. The hydrodynamic radius of DODAC vesicles as measured by QELS is 80 f 5 nm, depending on the sample. The intensity of the scattering light is proportional to the concentration of the scatterers for dilute solutions.1618 The area under the QELS signal is proportional to the square of the scattered intensity. Thus, the concentration of the scattering particles can be obtained from QELS under the same conditions as for static scattering measurement^.^^^^ In the case of DODAC suspensions, the QELS spectrum is dominated in the low-frequency domain by the vesicle signal (Figures 4 and 5 ) . From the area under the QELS spectrum, the concentration of vesicles can be deduced. Encapsulation Measurements. Vesicles have the particular capability of enclosing inner water pools whose solute composition can be different from the external water phase. The encapsulation efficiency of a probe molecule by a vesicle suspension is the ratio of the number of probe molecules inside the vesicles to the total number in the solution. We have measured the encapsulation efficiency by DODAC vesiclesm of paramagnetic probe. We report here results obtained for a membrane-adsorbed anionic probe: the 5-doxylstearic acid (5NS). The EPR spectra where recorded on a Bruker Model ER 200D EPR spectrometer equipped with a cavity dedicated for measurements in water solutions. The fraction of probe molecules dissolved inside the vesicles is obtained by first measuring the total population of 5NS and then the population remaining after selectively reducing the outer population by an externally added reducing agent. We are currently using two types of reducing solutions to check if some specific chemical interactions occur between the reducer and the probe. The reducing solutions are as follows: 0.1 mol/L in ascorbic acid neutralized by 0.05 mol/L of TRIS;210.9 M dithiothreitol and 0.1 M ferrous chloride in anhydrous methanol. A tubing and syringe setup is used in order to introduce samples and add and mix the reducer without disturbing the geometry and gain of the EPR cavity. The reproducibility of the intensity measurements is better than 3%. The volume of the added reducing solution is 3% or less of the aliquot volume. For encapsulation measurements, the aqueous solution in which DODAC suspensions are prepared contains the following: 5NS ( mol/L; the molar fraction of the probe in the membrane is saccharose (0.1 mol/L to prevent osmotic shocks from (1 5) Photon Correlation Spectroscopy; Cummins, H. Z., Pike, G. R., Eds.; Plenum Press: London, 1974. Chu, B., Laser Light Scattering Academic Press: 1974. Selser, J. C.; Yeh, H.; Baskin, R. J. Biophys. J . 1976, 16, 337-56. Light Scattering in Liquids and Macromolecular Solutions; Degiorgo, V., Corti, M., Gilio, M., Eds.; Plenum Press: London, 1980. (16) Tanford, C. Physical Chemistry of Macromolecules; Wiley: New York, 1961. (17) Cabane, B.; Duplessix, R.; Zemb, T. Surfactant in Solution; Mittal, K. L., Ed.; Plenum: London, 1984, Vol. 1, p 373-402. (18) Gaylor. K.; Snook, I.; Van Megen, W. J . Chem. Phys. 1980, 75, 1682-9. (19) Kinetic versus thermodynamic control of DODAC vesicles formation. Pansu, R. B. To be published. (20) Pansu, R. B.; Arrio, B.; Bear, S.; Faure, J.; Roncin, J. Absorption, Diffusibility and Reduction of Paramagnetic Probes on DODAC Membranes. To be published. (21) Castle, J. D.; Hubbell, W. L. Biochemistry 1976, 15, 4818-30.
Figure 1. Spectrum of 5 N S in DODAC suspensions (trace B). For comparison, the spectrum of 5NS in ethanol is presented (trace A). This illustrates the influence of the environment on the shape of the EPR spectrum. The spectrum of 5 N S in DODAC is typical of membraneadsorbed probes. The high viscosity of the membrane freezes the movements of the probes with respect to the external magnetic field. Both isotropic and anisotropic contributions to the spectrum are observed, leading to a broad spectrum. From the E P R spectrum of 5 N S in DODAC suspensions, it can be concluded that there is no 5NS molecule in the aqueous phase.
destroying the membrane; TRIS (tris(hydroxymethy1)aminomethane) (4 X mol/L to fix pH). The viscosity of the solution is obtained from tables by assuming that it is set by saccharose concentration and temperature.
Results Encapsulation Efficiency of DODAC Vesicles. Kunitake observed disks and Fendler observed vesticles. The measurement of the encapsulation of 5NS gives direct information on the fraction of molecules that actually forms vesicles. Open disks do not contribute to encapsulation, whereas the expected encapsulation efficiency of 5NS by vesicles is close to 0.5, regardless to their exact shape or size. 5NS is a membrane-adsorbed probe because of its charged and hydrophobic nature.*O This localization is confirmed by the shape of the EPR spectrum displayed on Figure 1. The spectrum observed in DODAC suspensions is close to the one observed in phospholipid vesicles.22 The encapsulation efficiency of a mixture of open disks and closed vesicles is a function of the respective area of the vesicle inner and outer surfaces and of the disk surface. But as 5NS are also charged molecules, their affinity for each type of location also depends on the electrostatic potential at each interface. The encapsulation value f is given by mi,&iv Niv
E = Ni,
+ No, + Nf
-
+ m,,@@ov + m&.,f mive@@ir
Niv,ov,f are population density numbers; are DODAC mass fractions; $iv,ov,f are surface potentials; /3 = e / k T where e is the charge of the electron; and T i s the temperature. Subscripts ov, iv, and f stand for outside vesicle, inside vesicle, and fragments, respectively. Let's define A 4 the transmembrane potential given by
A 4 = div - 40" Since 4," = 4f23and mi, = m0:4 can be assumed, one gets
(22) Seelig, J. In Spin Labeling, Berliner, L. J., Ed.; Academic Press: New York, 1976. Griffith, 0. H.; Jost, P. C. In Spin Labeling, Berliner, L. J., Ed.; Academic Press: New York, 1976; pp 469-92. (23) Pansu, R. B.; Arrio, B.; Lan, L.; Faure, J.; Roncin, J. Determination of DODAC Vesicle versus Open Membrane Fragment Ratio Using Paramagnetic Probe Entrapment. To be published. (24) In the case of DODAC vesicles, the radius (Rh= 80 nm) is large comuared to the thickness of the membrane ( a = 5 nm). The exact surface ratid is given by miv/mov= [(l - a/2R# - (i - a / R # j / [ 1 - ( 1 - a/2Rh))] = 1 - 3 / 2 ( a / R )= 0.91.
798 The Journal of Physical Chemistry, Vol. 94, No. 2, 1990
Pansu et al.
j
:l
,
,
,
,
1
r1
,
,
,
h
, s
.
,
,
,
1
,
I?
10
o
I1
Reduction Time (min)
Figure 2. Relative amplitude of the EPR signal as a function of the time after mixing with the reducing agent. Data obtained with use of IFe,D-
TT) and ascorbate as reducing agents are compared. In both cases, a rapid decay is first observed corresponding to the reduction of the probes on the outside of the vesicles. After a while, only the inner probes remain, which are reduced slowly. The difference between {Fe,DTT]and ascorbate in the reduction rate of the inner population is due to the progressive destruction of the vesicle by {Fe,DTT).The encapsulated population is obtained after extrapolation of the slowly decaying population to time 0. TABLE I: Measured and Expected Values of Encapsulation by DODAC Suspensions"
probe molecules ZW I4C sucrose glucosamine BIS-TRIS
measured Z values ref Water-Soluble Probes 0 0 0 0
0.165 0.13 f 0.2 0.25 0.2
23 32 11 33
expected valuesb a
b
3.2 3.2 3.2 3.2
0.38 0.38 0.38 0.38
Membrane-Adsorbed Probes tempate
-I
CAT I6 Cd2+
+I +2
5.8 5.76 4.5 f 2 2.7
23 34 23 35
58 58 42 35
8.1 8.1 4.5 3.8
"The encapsulation efficiencies of DODAC suspensions are expressed in [mole]-' for water-soluble probes (liter of inner volume per mole/liter of DODAC) and in percent for membrane-adsorbed probes. *The values for the entrapment by DODAC solution were calculated by first assuming that the sample is a population of spherical vesicles with an hydrodynamic radius of 80 nm (column a) and then assuming that 12% of the sample is constituted by vesicles (column b). In both cases, a value of 8.2 mV is assumed for the potential difference between the two surfaces of the vesicles. Expected values for spherical vesicle mixtures have been obtained assuming a hydrodynamic radius of 80 nm for the vesicles and a polar head surface of 0.4 nm2. Thus, .$ is directly related to the fraction of DODAC molecules that are forming vesicles. The value of the transmembrane potential for DODAC is 8.2 mV,23which is introduced as a correction of 1.37. The encapsulation of 5NS is measured after factor, eA@, the reduction of the outer population. The reduction kinetics provide evidence for two types of environments for the probe molecules characterized by a rapid ( VR > s-I) and a slow s-I) reduction rate. These two populations can be ( VR < identified, respectively, as the outer and inner ones by simple experiments.2s The fraction of inner population is obtained by extrapolating the slow decay to time zero as shown in Figure 2. The encapsulation value of 5NS in DODAC prepared by the standard protocol is 8.3 f 0.5%. This value is small compared to the 50% that is the expected value for a neutral membrane-adsorbed molecule and even smaller compared to 61% expected for anionic ones. But this value is in (25) Pansu, R. B. Photochimie dans les Membranes Synthetiques de DODAC. Thesis, Llniversite Paris Sud France. 1988.
i
60
120
180
240
300
Sonication Time ( s )
Figure 3. Scattered light intensity and encapsulation efficiency of DODAC suspensions as a function of the sonication time. The scattered light intensity is measured from the part of the QELS signal corresponding to the size paucidisperse population of 80 nm. The correlation between the two values confirms the attribution of the QELS signal to that of
vesicles. The decrease of the encapsulation efficiency as the sonication time is increased shows the destruction of the vesicles by ultrasound. agreement with already published values of encapsulation by DODAC suspensions (Table I). The probe can be shared in two ensembles: membrane adsorbed ones that measure surface area and water solubles ones that measure the inner volume of vesicles. Expected values for the encapsulation, assuming that DODAC molecules are organized in vesicles of 80 nm, are given for comparison in the table. It can be seen that there is 1 order of magnitude discrepancy between expected values and the measured one. On the contrary, encapsulation values of both water-soluble and membrane-adsorbed probes can be rationalized, assuming that only 12% of the DODAC molecules are organized in vesicles. Discussion of Encapsulation Results. Various interpretations have been proposed for these low encapsulation values. First of all, destruction or loss of vesicles during the measurement procedure has been suggested.35 This may be true when the inner population is measured after removing the outer one by gel chromatography. But with our reduction technique, no loss of matter can occur. The vesicle destruction is monitored during the encapsulation measurement itself. The reduction rate of the inner population is the sum of the rate of destruction of the vesicles by the reducing solution and of the rate of diffusion of the reducing agent through the membrane. The decay rate of the slow component gives an upper value to the destruction rate of vesicles. Destruction, if any, is slow compared to the duration of the experiment as seen in Figure 2. To explain the low encapsulation efficiency of membrane-adsorbed probe molecules (lower part of Table I), it may be assumed that a fraction of the probe is in fact slightly soluble in the water phase. But EPR spectroscopy has the advantage over other measurement techniques in that the spectral shape is very sensitive to the environment. The shape of the spectrum displayed in Figure 1 confirms the location of the probe as specifically absorbed in the membrane. To explain data obtained from water-soluble probe molecules (upper part of Table I), it has been proposed that DODAC vesicles may not be spheres but rather very flat ellipsoids.26 In addition to the fact that such a shape would be difficult to justify from the point of view of molecular packing, this interpretation fails to explain the low encapsulation of membrane-absorbed probe molecules. The encapsulation of membrane-adsorbed probes is expected to be close to 50% whether the vesicles are spheres or flat ellipsoids. The encapsulation values of both water-soluble and membrane-adsorbed probe molecules can only be rationalized by the existence of open structures in DODAC preparations in (26) Herman, U.: Fendler, J. H. Chem. Phys. Left. 1979, 64, 270-4.
The Journal of Physical Chemistry, Vol. 94, No. 2, 1990 799
Vesicles vs Membrane Fragments in DODAC Suspensions I
I
Sonication1 Scattered I Hvdrodvnamic ,
4.6
89.4
Power [ Relative]
400
800
1200
1600
Frequency [Hz]
Figure 4. Destruction of the vesicles by the sonication process. The power spectrum of the scattered intensity is represented at various intervals during sonication. The signal is due to the size paucidisperse population of the vesicles (see Discussion in the text). The amount of vesicles decreases as the sonication proceeds. Note that the average Rh of the vesicle population decreases only slightly with sonication. The vesicle population is destroyed, but the vesicle size remains constant. The small decrease of the average f?h is due to the increasing contribution of the small membrane disks. The sample marked with an asterisk has been submitted to a 12-h annealing (60 "C). The recovery of the vesicle population can be observed.
agreement with the observation of disks of Kunitake. Destruction of Vesicles by Sonication. The coexistence of disks and vesicles is not in agreement with thermodynamic predictions. But indeed it can be shown that DODAC suspensions are not in thermodynamic equilibrium. We have reported the encapsulation mol of DODAC solution as a function of values of 5NS in the sonication time on Figure 3. It can be observed that encapsulation diminishes as sonication time is increased. This shows that the sonication destroys the vesicles to produce a mixture of vesicles and disks in a metastable state. From the encapsulation efficiency of DODAC, it can be concluded that only 12% of the surfactant molecules are organized in vesicles. This is confirmed by the study of the sonication process using QELS intensity measurements. In the same figure, the intensity of light scattered by the sample is shown as a function of the sonication time. The scattered intensity originates from both the vesicles and disks, but as vesicles are bigger than disks, they give a stronger ~ i g n a 1 . IThe ~ scattered intensity is a good indicator of the amount of vesicles; the decrease of the scattered intensity with sonication time confirms the destruction of the vesicles. More information on the destruction process can be obtained from QELS data, where the size of the vesicles can be monitored at the same time as their concentration. QELS spectra obtained for increasing sonication times are represented in Figure 4. The decrease of the scattered intensity due to the disappearance of vesicles can be observed again. The QELS spectrum is dominated by a size monodisperse population of vesicles regardless of the sonication time. The size of the vesicles can be measured from the Lorenzian fit and is reported in Figure 4. One has to note that while the vesicle population is destroyed, the average radius of the remaining vesicles is stable. When DODAC vesicles have a radius of 80 nm, they are not cut into smaller vesicles, rather they are destroyed into very small parts. There is a critical radius under which vesicles do not exist. The fragments are not observed in the QELS spectrum due to their size, which is small compared to that of vesicles. The scattered intensity is proportional to the mass fraction of the scattering particles I , = mi = [CJM, where mi is the mass fraction of the scatterer; ci is the concentration of scatterer; and M I is the molecular weight of the scatterer. Even if there are more fragments, the scattered intensity is proportional to their total mass. Second, the intensity of the
CREQJENCY ir nz
Figure 5. Recovery of the vesicle population upon addition of Fe(CN)6'. QELS spectra are shown at three stages of the recovery: (A) before the addition of Fe(CN)6e; (B) after addition of lo4 mol/L of Fe(CN),' (vesicle recovery); (c)after addition of 2.2 X lo4 mol/L of Fe(CN)6e (vesicle flocculation). The scattered intensity by the sample is represented in the inset. A 10-fold increase of the scattered intensity can be observed upon addition of Fe(CN)t-. This increase of intensity is due
to the fact that at identical mass fraction big particles scatter more intensity than smaller ones. u p to 10" mo:/L of Fe(CN)6', vesicles are formed with a narrow size distribution and an average hydrodynamic radius of 81 nm. After addition of 2.2 X lo4 mol/L in Fe(CN)64-, vesicle flocculation occurs and vesicles clusters can be observed. Conditions: DODAC, mol/L; saccharose, 5%, TRIS, 4 X IO-' mol/L, 12 min of sonication of a 5-mL sample with a 12-mm sonication tip. fragment QELS spectrum spreads over a much wider frequency domain than that for vesicles so that their contribution cannot be distinguished from the solvent background. Formation of a Size Monodisperse Population of Vesicles. Metastability of the Membrane Fragments. After sonication, the sample is kept at a temperature of 60 OC for 12 h. The spectrum after annealing is shown in Figure 4. The recovery of the vesicle population is observed. The new vesicles have the same hydrodynamic radius as before sonication. Thus, vesicles are formed with a radius that equals their critical destruction radius. This recovery of vesicles from disks during annealing indicates that fragments are thermodynamically unstable compared to vesicles. During the same 12 h, a sample kept at room temperature does not show any substantial recovery of the vesicle population. It is a common observation that DODAC suspensions are much more stable than phospholipid ones and that DODAC samples can be kept for months4 without observable macroscopic changes. Thus, fragments are unstable with respect to vesicles though they can be metastable for weeks. Salt Effects on the Stability of Fragments. The origin of this metastability, which is specific to DODAC assemblies, can be revealed by the study of the effect of added salts. The influence of added salts on the turbidity of DODAC suspensions has been studied in detail by A. M. Carmona-Ribeiro et aL2' They show that DODAC suspensions follow nicely the predictions of the DLVO for the stability of hydrophobic colloidal suspensions. We have studied the effect of added salts on DODAC suspension by QELS. In Figure 5, successivealiquots of Fe(CN)6e have been added to a suspension of DODAC disks. The insert shows the scattered intensity as a function of added salts. A 10-fold increase of the scattered intensity is observed as the salt content is increased. Up to lo4 mol/L of Fe(CN),", this increase in the scattered intensity is due to the formation of vesicles as shown by the QELS spectrum. At higher concentrations of Fe(CN):-, flocculation of vesicles occurs, leading to big aggregates. The clusters give rise to the low-frequency peak observed on the QELS spectrum. The metastability of DODAC disks can be (27) Carmona-Ribeiro, A. M.; Yoshida, L. S.; Chaimovich, H. J . Phys. Chem. 1985, 89, 2928-33. (28) Overbeek, J. T. G. J . Colloid Interface Sci. 1978, 58, 408-22.
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The Journal of Physical Chemistry, Vol. 94, No. 2, 1990
described in the following way. DODAC suspensions are generally prepared in pure water, at low ionic strengths. Under these conditions, high electrostatic repulsions exist between fragments of the same charge, preventing collisions and gathering of membrane disks. Reduction of the electrostatic repulsions by an increase in the ionic strength or by partial neutralization of the surface charge density by added salts allows gathering of fragments to form vesicles.
Discussion We have shown that the low encapsulation efficiency of DODAC suspension is due to the presence of open structures. The light-scattering experiments have shown that these open structures are very small aggregates: probably flat disks composed of a bilayer of surfactant. Kinetic Control. The coexistence of fragments and vesicles is not in agreement with thermodynamic expectations. The samples in Figure 5 have the same composition and have been measured under the same conditions of temperature and pressure. The annealing experiment described in Figure 4 confirms that no chemical destruction of the samples occurs during the sonication process. As these samples differ from one another, we can conclude that, as phospholipid vesicles, DODAC suspensions are not in thermodynamic equilibrium. This kinetic control is not completely unexpected. Two processes could have allowed surfactant aggregates to reach their equilibrium state: monomer exchange between aggregates and fusion of aggregates. The exchange rate is proportional to the critical micellar concentration (cmc) of the surfactant molecule. As the cmc of double-chained surfactants is very low, the rate of the exchange of monomers between aggregates is slow for surfactants organized in bilayers. The second process by which equilibrium can be reached is the fusion and fission of aggregates. But it is observed that the fragments are metastable for weeks. Salt effects show that this is because electrostatic repulsions prevent fusion of small membrane fragments, which may lead to the formation of vesicles. The two processes that enable micelles to reach their equilibrium state are not efficient in the case of vesicles. Thermodynamics does not describe the state of the solution, but gives a good framework. For example, the existence of a critical radius under which vesicles cannot exist is an assumption common to most models; Figure 4 is a rather spectacular illustration of this idea. In the same way, annealing experiments show that fragments are unstable with respect to vesicles. This is in agreement with thermodynamic predictions and with the hypothesis of the geometric models that assume that membrane fragments cannot exist because of the unfavorable interaction between the core of the membrane and the water phase at the edge of the fragments. Kinetic Models. The finest prediction of the thermodynamic models is the size paucidispersity of the vesicles obtained by sonication. This size paucidispersity is also observed for DODAC vesicles. Two kinetic models have been proposed to explain the size monodispersity of vesicles. Tenchov describes the sonication process as a stochastic scission of large vesicles into smaller ones down to a minimal radius. This leads to a paucidisperse population of vesicles with a Wiebull d i ~ t r i b u t i o n . ~But ~ this model assumes that fragments, if formed, rapidly fuse with vesicles. This hypothesis does not agree with the stability of DODAC membrane fragments. The second model has been proposed independently by D. D. LasicI3 and P. Fromherz.I4 This model assumes that small fragments are initially produced. First disks gather to form larger ones, then the large disks close into vesicles, and finally vesicles can grow by fusion with fragments or other vesicles. The vesicles are formed with a size that is defined by the balance between two antagonistic interactions that dominate the enthalpy of the disks. The edge tension at the edge of the disk, where the hydrophobic core of the membrane is in contact with the water phase, tends (29) Tenchov, B. G.;Yanev, T. K.; Tihova, M. G.; Koynova, R. D. Eiochim Eiophys Acta 1985, 816, 122-30.
Pansu et al. to be reduced to the length of the border and to bend the disk. The elasticity of the membrane opposes such a movement. For small disks, the elastic interaction prevails and the fragments are flat disks. Above a critical size, bending and closing occur and vesicles are formed. The size dispersity of the vesicles depends on the relative rate of the three steps: gathering of fragments, closing into vesicles, and growth of vesicles. A monodisperse population is obtained when the rate of closure is rapid compared to the rate of gathering. For all vesicles to be formed with the same size, fragments have to close when they reach the critical radius, before collisions with other fragments occur. For the population to remain monodisperse, vesicle growth by fusion has to be slow compared to the lifetime of the sample. In the case of DODAC, we have shown that the membrane fragments that have been postulated as intermediates in the process of vesicle formation are formed during the sonication process. Disk gathering is slow compared to disk closure because gathering is a multibcdy process limited by the electrostatic repulsions whereas closing is only slightly influenced by ionic strength. The growth of DODAC vesicles with time is not observed during the lifetime of the samples. Thus, the conditions required by the model are fulfilled in the case of DODAC suspensions. In agreement with predictions, we have observed the formation of a size paucidisperse population of vesicles (Figures 4 and 5). DODAC suspensions provide the first experimental evidence supporting the model that describes the formation of a size monodisperse population of vesicles from a size polydisperse population of membrane disks. Extensions. Let us try to generalize the results obtained with DODAC. These conditions on the existence of fragments and on the rate constants can be expected to be fulfilled in systems other than DODAC. The phospholipid family of vesicles are very important and have been extensively studied because of their occurrence in nature. We have shown that the sonication process is strong enough to produce DODAC membrane disks. This is probably true also for phospholipids even if membrane fragments have never been observed for phospholipid vesicles. But, we have shown that DODAC membrane disks are metastable only because of their high surface charge density. Most phospholipid vesicles have low surface charge density and are prepared under physiological conditions, Le., high ionic strength. In the case of phospholipids, no electrostatic interaction can avoid a rapid gathering of membrane disks. It can be assumed that phospholipid membrane disks are produced during sonication but that they quickly gather to form vesicles. If the gathering process remains slow compared to the closing process, the observed size monodispersity can be justified without the assumption of thermodynamic equilibrium. Fragments should be observed with other charged surfactants. We have explained the particularity of DODAC suspension, the stability of membrane disks, to be due to electrostatic interactions. Dihexadecylphosphate (DHP) is an anionic surfactant in basic media and a neutral one in acidic media. In both cases, vesicles can be observed with QELS30 But, Tricot3' has shown that the encapsulation efficiency of DHP vesicles depends critically on preparation conditions and particularly on pH. The encapsulation efficiency of Ru( bpy)32+by DHP suspensions has been measured as a function of pH. The probe, due to its charge, is probably adsorbed on the membrane. Indeed, the encapsulation value (30) Tricot, Y. M.; Furlong, D. N.; Mau, A. W.-H.;Sasse, W. H. F. Aust. J . Chem. 1985, 38, 527-35. (31) (a) Tricot, Y. M.; Manassen, J. J . Phys. Chem. 1988, 92, 5239-44. (b) Tricot, Y. M.; Furlong, D. N.; Sasse, W. H. F.; Daivis, P.; Snook,I.; Van Megan, W. J . Colloid Interface Sci. 1984.97, 380. (c) Tricot, Y . M.; Fendler, J. H. J . Am. Chem. Sac. 1984, 106, 7359. (d) Tricot, Y. M.; Fendler, J. H. J . Phys. Chem. 1986, 90, 3369. (32) Carmona-Ribeiro, A. M.; Chaimovich, H. Eiochem. Eiophys. Acta 1983, 733, 172-9. (33) Pansu, R.; Faure, J.; Johannin, G.; Arrio, B. N o w . J . Chim. 1986, 10, 285-8. (34) Lim, Y. Y. Fendler, J. H. J . Am. Chem. Sac. 1979, 101, 4023-9. (35) Watzke, H. J.; Fendler, J . H. J . Phys. Chem 1987, 91, 854-61.
J. Phys. Chem. 1990, 94, 801-809 decreases from 50% at pH 6 where DHP vesicles are only slightly charged to 0% in basic media where DHP molecules are charged. As for DODAC, this is indicative of the presence of charged fragments.
Conclusions Our encapsulation measurements show that DODAC suspensions obtained by sonication are essentially composed of open fragments. The light-scattering experiments show that vesicles are destroyed into fragments by the sonication process. These fragments are metastable. This metastability is due to the electrostatic repulsions between these charged colloidal particles. We observed that 12%of the DODAC molecules is organized in
80 1
a size paucidisperse population of vesicles. This size monodispersity has been rationalized by geometric models assuming thermodynamic equilibrium. But, our studies show that, as phospholipid vesicles, DODAC suspensions are not in thermodynamic equilibrium. Models including also considerations on the kinetics of the vesicles formations have been proposed by D. D. Lasic and by P. Fromherz. We show that the hypotheses of these models are fulfilled in the case of DODAC. In agreement with the predictions of the model, we have observed the formation of a size paucidisperse population of vesicles from a size polydisperse population of membrane fragments. Registry No. DODAC, 107-64-2;5NS, 29545-48-0;Fe(CN),&, 13408-63-4.
Partitioning of Nonpolar Solutes into Bilayers and Amorphous n-Alkanes Linda R. De Young and Ken A. Dill* Department of Pharmaceutical Chemistry, University of California, San Francisco, California 941 43 (Received: March 6, 1989; In Final Form: June 16. 1989)
We have measured the partition coefficients for hexane between an aqueous solvent and phospholipid bilayer membranes as a function of the surface density of the bilayer chains. The surface density was varied by temperature, phospholipid chain length, and the incorporation of cholesterol and was monitored by 2H NMR. We observe that increasing surface density leads to expulsion of the solute: hexane partitioning decreases by a factor of 9 as the surface density of the bilayer chains increases from 50%to 90% of its maximum value, a range readily accessible in biomembranes under physiological conditions. Benzene as solute shows similar behavior; thus, the dependence of partitioning on surface density appears to be a general property of the organization of the bilayer chains. In order to determine the relative contributions to partitioning of (i) chain ordering and (ii) contact interactions, we have also performed reference-state experiments for benzene transfer between water and amorphous n-alkanes of different chain lengths. The widely accepted value of 25 cal/mol per A* of surface area for the free energy of oil/water transfer per methylene group is appropriate only when solute and solvent molecules are of the same size. We find that Flory-Huggins theory satisfactorily corrects for the molecular size differences of solutes and solvents. Comparisons of the bilayer/water and oil/water temperature dependencies of benzene partitioning show that there is an additional entropic expulsion of solute from the bilayer, consistent with recent statistical thermodynamic theory suggesting the importance of the chain ordering in the bilayer.
Introduction Solute partitioning into lipid bilayers is important for many biological phenomena. Examples include drug and metabolite uptake, passive transport across membranes, and possibly the molecular mechanism of anesthetic drug action. In related interfacial phases, solute partitioning processes underlie micellar stability and catalysis and selectivity and retention in reverse-phase liquid chromatography.I Bulk thermodynamic models have often been used to characterize partitioning into bilayer membranes. However, lipid bilayers differ from bulk phases. They have high surface-tevolume ratios; they are interfacial phases. In interfacial phases physical properties vary with distance from the interface; in contrast, bulk phases are isotropic. For example, in the bilayer the surfactant chains are most highly aligned normal to the interface near the headgroups and the order diminishes near the chain ends2v3 In addition, the chain ordering of the bilayer phospholipids increases with surface density. In contrast, the properties of bulk phases are independent of interfacial properties such as the surface density. Bulk phases are therefore poor models of bilayers, and bulk thermodynamic treatments must be amended to account for the interfacial nature of the bilayer. The structural differences between bilayers and other interfacial phases, on the one hand, and bulk phases such as oil or octanol, on the other hand, will be manifested as differences in the nature of solute partitioning into them. Recent theory4 has predicted (1)Dill, K. A. J . Phys. Chem. 1987,91, 1980. (2) Hubbell, W. L.; McConnell, H. M. J . A m . Chem. SOC.1971,93,314. (3)Seelig, J. Q.Reo. Biophys. 1977, 10, 353.
0022-3654/90/2094-0801$02.50/0
that (i) there will be an equilibrium gradient of solute concentration in the bilayer in contrast to the uniform distribution expected in a bulk phase, which is consistent with neutron scattering experiments5 and experiments on planar bilayers;bs (ii) the partial chain ordering should disfavor solute retention in the bilayer relative to amorphous bulk phases; and (iii) the solute uptake should decrease significantly with increased surface density of the chains. Many previous studies have shown bilayer partitioning to be dependent on cholesterol concentration, temperature, or lipid t ~ p e . ~ - For ' ~ example, solute partitioning has been shown to (4)Marqusee, J. A.; Dill, K. A. J . Chem. Phys. 1986,85,434. ( 5 ) White, S.H.; King, G. I.; Cain, J. E. Nature 1981,290,161. (6)Andrews, D. M.; Manev, E. D.; Haydon, D. A. Spec. Discuss. Faraday SOC.1970. No. I . 46. (7)Brooks, D. E.;Levine, Y. K.; Rquera, J.; Haydon, D. A. Proc. R . SOC. London 1975,A347, 179. (8)White, S. H. Ann. N.Y. Acad. Sci. 1977,303, 243. (9)Simon. S. A.: Gutknecht. J. Biochim. Bioohvs. Acta 1980.596. . . 352. (10)Colley, C.; Metcalfe, J.'C. FEES Lett. i972,24,241. (11) Simon, S.A,; Stone, W. L.; Bennett, P. B. Biochim. Biophys. Acta 1979,550, 38. (12)Simon, S. A,; McDaniel, R. V.;McIntosh, T. J. J . Phys. Chem. 1982, 86, 1449. (13)Katz, Y.;Diamond, J. M. J . Membr. Biol. 1974,17, 101. (14)Antunes-Madeira, M. C.; Madeira, V. M. C. Biochim. Biophys. Acra 1985.820. 165.
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(17) Miller, K.W.; Hammond, L.; Porter, E. G . Chem. Phys. Lipids 1977, 20, 229. . . . , . . ...
0 1990 American Chemical Society
