J. Phys. Chem. 1995, 99, 1299-1305
1299
Formation of Polymerlike Mixed Micelles and Vesicles in Lecithin-Bile Salt Solutions: A Small-Angle Neutron-Scattering Study Jan Skov Pedersen Department of Solid State Physics, Ris@National Laboratory, DK-4000 Roskilde, Denmark
Stefan U. Egelhaaf Labor f u r Elektronenmikroskopie I , ETH Zurich, CH-8092 Zurich, Switzerland
Peter Schurtenberger* Institut fiir Polymere, ETH Zurich, CH-8092 Zurich, Switzerland Received: July 25, 1994; In Final Form: October 31, 1994@
We report a small-angle neutron-scattering (SANS) study of the structural properties of mixed bile saltlecithin micelles and vesicles. The SANS data from a mixed micellar sample are in agreement with the presence of flexible cylindrical micelles. We present an expression for the scattering cross section of polydisperse wormlike micelles which is capable of quantitatively describing the scattering intensity Z(q) over an extended range of scattering vectors and which allows us to incorporate overall size, polydispersity, flexibility, and local structure of the micelles in a self-consistent way. At concentrations below the micellar phase limit, where a spontaneous micelle-to-vesicle transition can be observed, we find that the scattering intensity at higher values of q is not consistent with a population of vesicles only and that a coexistence of vesicles and wormlike micelles occurs.
an increase of the mixed micellar size with increasing dilution in the mixed micellar region is generally observed until a The structure and properties of mixed lecithin-bile salt maximum is reached near the micellar phase limit. Upon micelles is still a controversial issue despite theoretical and dilution beyond the phase limit, the spontaneous formation of experimental efforts devoted to their physicochemical characmixed vesicles occurs. After a small transition region, the terization. In the literature two models are found which both vesicle size decreases upon further dilution. From the combinaare based on scattering experiments. In their pioneering article, tion of static and dynamic light scattering experiments we were Mazer et al. proposed the so-called "mixed disk" model for the able to self-consistently interpret the obtained data using an structure of the bile salt-lecithin mixed micelles present in these iterative fitting procedure. We showed that in the mixed solutions.' The mixed micelles were assumed to be disklike, micellar region of the phase diagram, the data are consistent consisting of a lecithin bilayer with bile salt molecules with the presence of flexible cylindrical (wormlike) mixed incorporated within the disk in a fixed stoichiometry and forming micelles, whereas the mixed disk model' was clearly not a "ribbon" around the perimeter, thus preventing contact between consistent with the experimental data. Using modem concepts water and the lecithin hydrocarbon chains. However, a subsefrom polymer and colloid physics, it was also possible to quent small-angle neutron-scattering (SANS) study presented incorporate micellar flexibility and interparticle interactions, and data which seemed in clear disagreement with the existence of to include the polydispersity of the aggregates inherent in the disklike mixed micelles but were consistent with a rodlike self-assembly process responsible for the micelle formation. m ~ r p h o l o g y . ~Several -~ light-scattering studies presented furOur study" indicated that the molecular weight and the ther evidence for flexible cylindrical mixed micelle^,^^^ and overall size (Le., the contour length L ) of these micelles experiments based on cryo-TEM even provided direct visualizadramatically increases upon dilution, in relation to the cortion of cylindrical structures in these systems.*s9 Although there responding decrease of the lecithin-to-bile salt ratio in the exists an increasing number of experimental reports supporting aggregates due to the very different free monomer concentrations a cylindrical structure for the mixed micelles, there is still limited of NaTCDC ( ~ 0 . 7x M)'OsL4and egg PC (5 M).15 quantitative information available on the dependence of the micellar size, polydispersity, and flexibility as a function of the The growth of flexible cylindrical micelles could therefore be solution composition, data which would be vital for any attempt interpreted as a result of the decreased spontaneous curvature to derive a thermodynamic model for these systems. of the mixed micelles and the avoidance of energetically unfavorable endcaps.16-1s Concomitantly to the increase of the This recently led us to reanalyze the concentration dependence micellar size, the persistence length was found to decrease from of the mixed micellar size and the previously postulated micelle~ 2 5 08, at higher concentrations to Z l O O 8, at the micellar to-vesicle transitionlo in bile salt-lecithin solutions using static phase limit. Using explicit expressions for the influence of and dynamic light scattering measurements over a wide range of scattering angles." The effect of dilution on mixed micellar intermicellar interactions on the light-scattering intensity on the stock solutions of bile salt and lecithin has already been level of a vinal expansion, we were also able to obtain described in a number of previous p u b l i ~ a t i o n s . ' ~ ' ~First ~ ' ~ ~ ' ~ quantitative agreement between the structural parameters of the micelles and the measured apparent molar mass on absolute scale. However, in this comparison we had to use the linear Abstract published in Advance ACS Abstracts, December 15, 1994.
Introduction
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0022-365419512099- 1299$09.00/0
0 1995 American Chemical Society
Pedersen et al.
1300 J. Phys. Chem., Vol. 99, No. 4, 1995 number density of lecithin molecules in the micelles, AL, as a free parameter without a possibility of determining it independently. At concentrations below the micellar phase limit, mixed lecithin bile salt vesicles form spontaneously. Close to the micellar phase limit we found evidence for a coexistence of wormlike micelles and vesicles. At higher dilutions the data were consistent with a single phase of vesicles, whose size depends strongly upon the bilayer composition and decreases monotonically with increasing dilution.' However, both the estimate of the micellar flexibility and the local packing density in the micelles as well as the characterizaticn of the sizes and relative concentrations of micelles and vesicles in the coexistence region were done rather indirectly. It was not possible to test these properties more directly due to the limited spatial resolution of the light scattering experiment, which is related to the accessible range of values for the scattering vector q. Therefore we have performed the small-angle neutron-scattering experiments presented in this article on two samples in the micellar and coexistence region of the phase diagram. Due to the much more extended q range of a SANS experiment, we can thus directly test the consistency of the data with the proposed structural models for the mixed micelles and vesicles.
P,
dilution Figure 1. Apparent molar mass Mappas a function of dilution or total lipid concentration ctol(where cmt= 50/dilution mg/mL) for NaTCDC and egg yolk lecithin at a molar ratio L/BS = 0.9. (0)Results for H2O previously reported." ( 0 )Results for DzO. The phase limit for the onset of vesicle formation is shown as a dashed line for H20 and as a solid line for D2O. The two samples characterized by S A N S are
indicated with arrows. Materials and Methods Materials. Egg yolk lecithin (egg PC) and the sodium salt of taurochenodeoxycholic acid (NaTCDC) were obtained from Lipid Products (South Nutfield, Surrey, UK (grade I)) and Calbiochem, respectively, and used without further purification. D2O was from Dr. Glaser AG, Basel. Sample Preparation. Mixed micellar stock solutions with a lecithin-to-bile salt molar ratio of 0.9 were prepared by the method of coprecipitation as described Buffer (0.15 M NaCUTris, pH 8.15 for sample 40; 0.15 M NaCl for sample 29) was added to obtain a stock solution with a total lipid concentration of 50 mg/mL. D20 was chosen in order to minimize the incoherent background from hydrogen and obtain a high scattering contrast. The stock solution was equilibrated for approximately 24 h at 25 "C, and the final concentrations were subsequently prepared by a number of rapid dilution steps. Each sample was flushed with nitrogen, sealed, and "equilibrated" for at least 48 h at 25 "C. Methods. The small-angle neutron-scattering ( S A N S ) experiments were performed at the SANS instrument at the DR3 reactor at Risa National Laboratory, Denmark.20 A range of scattering vectors q from 0.004 to 0.5 A-' was covered by four combinations of neutron wavelength (3.5 and 10 A) and sampleto-detector distances (1-6 m). The wavelength resolution was 18% (full width at half-maximum value). The samples were kept in quartz cells (Hellma) with a path length of 2 111111. The raw spectra were corrected for background from the solvent, sample cell, and other sources by conventional procedures.21 The two-dimensional isotropic scattering spectra were azimuthally averaged, converted to an absolute scale, and corrected for detector efficiency by dividing by the incoherent scattering spectra of pure water.22 The scattering intensity was furthermore normalized by dividing by the concentration of lecithin and bile salt in the micelles and vesicles, taking into account the free monomer concentration (IMC)of the bile salt ( ~ 0 . 7 mh4)10-'4 0 and of the lecithin (
