J. Phys. Chem. 1995, 99,
1299-1305
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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, Ris0 National Laboratory, DK-4000 Roskilde, Denmark
Stefan U. Egelhaaf Labor fur Elektronenmikroskopie
1,
ETH Zurich, CH-8092 Zurich, Switzerland
Peter Schurtenberger* Instituí für Polymere, ETH Zurich, CH-8092 Zurich, Switzerland
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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 I(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 maximum is reached near the micellar phase limit. Upon dilution beyond the phase limit, the spontaneous formation of mixed vesicles occurs. After a small transition region, the vesicle size decreases upon further dilution. From the combination of static and dynamic light scattering experiments we were able to self-consistently interpret the obtained data using an iterative fitting procedure. We showed that in the mixed micellar region of the phase diagram, the data are consistent with the presence of flexible cylindrical (wormlike) mixed micelles, whereas the mixed disk model1 was clearly not consistent with the experimental data. Using modem concepts from polymer and colloid physics, it was also possible to incorporate micellar flexibility and interparticle interactions, and to include the polydispersity of the aggregates inherent in the self-assembly process responsible for the micelle formation. Our study11 indicated that the molecular weight and the overall size (i.e., the contour length L) of these micelles dramatically increases upon dilution, in relation to the corresponding decrease of the lecithin-to-bile salt ratio in the aggregates due to the very different free monomer concentrations of NaTCDC («0.7 x 10-3 M)10·14 and egg PC (< 10-10 M).15 The growth of flexible cylindrical micelles could therefore be interpreted as a result of the decreased spontaneous curvature of the mixed micelles and the avoidance of energetically unfavorable endcaps.16-18 Concomitantly to the increase of the micellar size, the persistence length was found to decrease from «250 Á at higher concentrations to «100 Á at the micellar phase limit. Using explicit expressions for the influence of intermicellar interactions on the light-scattering intensity on the level of a virial expansion, we were also able to obtain 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
Introduction The structure and properties of mixed lecithin—bile salt micelles is still a controversial issue despite theoretical and experimental efforts devoted to their physicochemical characterization. In the literature two models are found which both are based on scattering experiments. In their pioneering article, Mazer et al. proposed the so-called “mixed disk” model for the structure of the bile salt—lecithin mixed micelles present in these solutions.1 The mixed micelles were assumed to be disklike, consisting of a lecithin bilayer with bile salt molecules incorporated within the disk in a fixed stoichiometry and forming a “ribbon” around the perimeter, thus preventing contact between water and the lecithin hydrocarbon chains. However, a subsequent small-angle neutron-scattering (SANS) study presented data which seemed in clear disagreement with the existence of disklike mixed micelles but were consistent with a rodlike morphology.2-5 Several light-scattering studies presented further evidence for flexible cylindrical mixed micelles,6·7 and experiments based on cryo-TEM even provided direct visualization of cylindrical structures in these systems.8·9 Although there exists an increasing number of experimental reports supporting a cylindrical structure for the mixed micelles, there is still limited quantitative information available on the dependence of the micellar size, polydispersity, and flexibility as a function of the solution composition, data which would be vital for any attempt to derive a thermodynamic model for these systems. This recently led us to reanalyze the concentration dependence of the mixed micellar size and the previously postulated micelleto-vesicle transition10 in bile salt—lecithin solutions using static and dynamic light scattering measurements over a wide range of scattering angles.11 The effect of dilution on mixed micellar stock solutions of bile salt and lecithin has already been described in a number of previous publications.1·10·12·13 First ®
Abstract published in Advance ACS Abstracts, December 15, 1994.
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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.11 However, both the estimate of the micellar flexibility and the local packing density in the micelles as well as the characterization 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.
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dilution of dilution or total Figure lipid concentration ctot (where ctot = 50/dilution mg/mL) for NaTCDC 0.9. (O) Results for and egg yolk lecithin at a molar ratio L/BS H2O previously reported.11 (·) Results for D2O. The phase limit for the onset of vesicle formation is shown as a dashed line for H2O and as a solid line for D2O. The two samples characterized by SANS are indicated with arrows. 1. Apparent molar mass Mapp as a function =
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 previously.11·19 Buffer (0.15 M NaCl/Tris, 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. D2O was chosen in order to minimize the incoherent background from hydrogen and obtain 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 (SANS) experiments were performed at the SANS instrument at the DR3 reactor at Ris0 National Laboratory, Denmark.20 A range of scattering vectors q from 0.004 to 0.5 Á-1 was covered by four combinations of neutron wavelength (3.5 and 10 Á) 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 mm. 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 («0.70 mM)10·14 and of the lecithin (< 10~7 mM).15 The average excess scattering length density per unit mass , of the micelles and vesicles was calculated from the known chemical composition of the bile salt and egg yolk lecithin using in the the appropriate molecular volume of the monomers a
aggregates and the aggregate composition (corrected for the free monomer concentrations) and taking into account exchange of deuterium and hydrogen atoms. Throughout the data analysis corrections were made for instrumental smearing.20·23 For each instrumental setting the ideal model scattering curves were smeared by the appropriate resolution function when the model scattering intensity was compared to the measured one by means of least-squares methods. The parameters in the models were optimized by conventional least-squares analysis, and the errors of the parameters were calculated by conventional methods.24
Results and Discussion Whereas our previous light-scattering studies have been conducted in aqueous buffer, we now have to work with D2O in order to optimize the scattering contrast and to limit the incoherent background. While the general aggregation behavior is unchanged, the use of D2O instead of H2O can lead to a small shift of the micellar phase limit. Figure 1 summarizes the effect of dilution of a mixed micellar stock solution on the apparent molar mass Mm of the aggregates present in these solutions as measured by means of static light scattering and compares the data with those obtained previously in H2O.11 The micellar phase boundary in D2O has indeed shifted by approximately 30% to lower concentrations, but the general features of a dilution-induced micellar growth and a micelle-to-vesicle transition are present in both solvents. To quantitatively test the
previously used light-scattering data analysis procedure, we have chosen a sample in the mixed micellar region close to the phase limit, where the micellar size is largest (dilution 1:29, sample 29), and a sample in the micelle—vesicle coexistence region (dilution 1:40, sample 40) for our SANS experiments. The two samples are indicated with arrows in Figure 1. The experimental data from the SANS measurements with samples 29 and 40 are summarized in Figure 2. A first and qualitative inspection of Figure 2 reveals distinct differences between the two samples. Whereas the micellar sample exhibits a monotonic decrease of the intensity I(q) with increasing q as expected for cylindrical micelles (Figure 2A), a nonmonotonic
Polymerlike Mixed Micelles and Vesicles
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Figure 3. “Holtzer plot” ql(q) versus q for sample 29. Also shown are the fit results for polydisperse wormlike cylinders with persistence length Zp = 150 Á using the two-shell model (see Table 1 and text for details). The solid lines correspond to the intensity smeared by the instrumental resolution, and the dashed line corresponds to the ideal intensity.
Figure 2. Normalized scattering intensity as a function of scattering vector q for the mixed micellar sample 29 (A) and sample 40 with coexisting micelles and vesicles (B). Individual symbols represent data obtained with different combinations of neutron wavelength and sample—detector distance. (Note that these curves are slightly shifted due to resolution effects.) The lines represent the results from a fit with a two-shell model (see text for details). The solid lines correspond to the intensity smeared by the instrumental resolution, and the dashed lines correspond to the ideal intensity for the given structural model.
^-dependence with clear minima and maxima can be observed for sample 40 as expected for shelllike vesicles (Figure 2B).4 On the basis of our previous light-scattering work, we can now try to analyze the data shown in Figure 2 and try to extract additional and quantitative information on the flexibility and local structure of the wormlike micelles and on the possible coexistence of micelles and vesicles at concentrations beyond the micellar phase limit.
Flexibility and Local Structure of Lecithin—Bile Salt Mixed Micelles. While light-scattering experiments are ideally suited for a determination of the overall size and apparent molar mass of large cylindrical micelles due to the very low q range, the flexibility (i.e., the persistence length Zp) and the local structure (i.e., the mass per unit length, Mu and the crosssectional radius of gyration, Rq,Cs) of the micelles can be determined in an indirect way only from a simultaneous analysis of static and dynamic light scattering data.11,25 The SANS measurements provide data at much higher values of q and thus
allow for a direct verification of these quantities. The starting point for such an analysis are the predictions from polymer physics for the q dependence of l(q) for flexible cylindrical particles based on the wormlike chain model commonly used to describe the chain conformation of “realistic” semiflexible polymer chains.26-28 For semiflexible polymers and polymerlike cylindrical micelles, I(q) has several distinct regimes which permit a quantitative study of the different length scales characterizing overall dimension, flexibility and local cylindrical cross section:28-30 For very low values of q (1/q < Rq, where Rq is the radius of gyration of the micelles) accessible for example in lightscattering experiments, the scattered intensity I(q) becomes insensitive to structural details and is dominated by the finite overall length of the particles. At intermediate q (Rcs « IIq « Rq, where Rcs is the cross-sectional radius of the cylinder), l(q) becomes much more sensitive to the local aggregate structure, and polymer theory predicts for flexible polymer coils that I(q) should decay with a power law of the form I(q) q~2. At large values of q, I(q) is controlled by distances over which polymers are rodlike rather than flexible, and at Qlp «s 1.9 one observes a crossover to an asymptotic q~l dependence for I(q), which is typical for locally cylindrical structures. A particularly sensitive way to test the agreement between theoretical and experimental I(q) for semiflexible chains is the so-called “Holtzer plot” (or “bending rod” plot) shown in Figure 3, in which ql{q) is plotted versus q.31·32 We see a clear indication of the expected crossover at q ^ 0.013, which leads to an estimate of Zp 145 Á. A more quantitative characterization of the scattering data can be made using the following expression for the scattering cross section da(g)/dQ: ~
áo(q)!áQ,
=
cKCÁQm2S.Jq)SJq)(M)K
(1)
where cwc is the total concentration of bile salt and phospholipid in the micelles, Apm is the average excess scattering length density per unit mass, Swc(q) and Sa(q) are the normalized scattering functions of the infinitely thin wormlike chains and of the cross section, respectively, and (M)w is the weight average molar mass. In eq 1 we have assumed that the scattering from
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wormlike chain can be written as the product of the scattering function of the infinitely thin wormlike chain and of the cross section, which is only valid if the length of the chains is significantly larger than the cross-sectional radius Rcs. The scattering function Sm(q) of polydisperse wormlike chains can be written as a
TABLE 1: Results from Nonlinear Least-Squares Analysis of the SANS Data for the Mixed Micellar Sample 29 ( „, = 5.61 1010 cm/g, /„ 150 A, aL =
1.87
