J. Phys. Chem. 1992, 96, 8653-8661
Organization of Phosphatidylcholine and Bile Salt in Rodlike Mixed Micelles Rex P.Hjelm, Jr.,* Los Alamos Neutron Scattering Center, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
P. Thiyagarajan, Intense Pulsed Neutron Source Division, Argonne National Laboratory, Argonne, Illinois 60439
and Hayat Alkan-Onyuksel Department of Pharmaceutics, University of Illinois at Chicago, Chicago, Illinois 6061 2 (Received: June 9, 1992; In Final Form: July 16, 1992)
Small-angle neutron scattering (SANS) of aqueous mixed colloids of the bile salts glycocholate or glycochenodeoxycholate with egg yolk phosphatidylcholine and mixtures of taurocholate with dipalmatoylphosphatidylcholine (DPPC) shows that all of these systems form particles with rodlike morphology in parts of the concentration-compositionphase map. The formation of rqdlike mixed micelles thus is a general feature of the phosphatidylcholine-bile salt systems. The orientation of the phosphatidylcholine and bile salts in the rod-shaped mixed micelles is addressed by SANS measurements of the aqueous mixtures of taurocholate with DPPC and with DPPC with the choline methyls deuterated. The results are consistent with the phosphatidylcholine being arranged in the rod such that the long axis is perpendicular to the axis of the rodlike particle. The amount of bile salt in the rods calculated from the small-angle neutron scattering assuming radial arrangement of phosphatidylcholine suggests that the bile salt is inserted with its long axis parallel to the rod axis. Comparison of the cross-sectional radius of gyration and contrast-weighted mass per unit length of the egg yolk phosphatidylcholine-bile salt mixtures with those from the DPPC-bile salt mixtures indicates that this arrangement of the two components is a general feature of these systems.
Introduction The study of self-assembly of particles in mixed aqueous colloids of bile salt (Figure 1) with long-chain fatty acid esters of phosphatidylcholine is important in understanding the physiology of bile, in the physical chemistry of mixed micelle systems, and has potential applications in the design of drugdelivery systems. These mixtures have been used as models for the structure of bile for some time now. The morphology of the particles found in these systems is thought to be analogous to the types of particles found in native bile,' even though bile is a more complex system than the simple mixed The physical chemical principles governing the self-assembly of these particles may help in understanding the structure and action of native bile in emulsifying fatty materials in the bile duct, gall bladder, and intestine. The potential applications of these mixtures are as drug-delivery systems for poorly soluble drugs4 and in oral formulations. Thus, understanding self-assembly in these systems will be important in realizing and evaluating these pharmaceutical uses. Understanding this system is a problem in the physical chemical principles governing self-assembly of mixed micelle systems. Presently, there is little in the way of general principles to guide our understanding of this type of system. This particular case relates to other issues in lipid biophysics, as well. Determination of particle morphology and the organization of components in the particles is an important first step in understanding the principles of self-organization in these systems. The complex nature of the water-egg phosphatidylcholinebile salt phase map was first explored by polarization microscopy and X-ray scattering by Small and collaborator^.^ Their work established the existence of isotropic phases in dilute mixtures where sufficient bile salt detergent was present to solubilize the phosphatidylcholine, and it is these phases that interest us here. The presence of mixed micelles in the isotropic phases was inferred from this early effort.63' Later work using dynamic light scattering ( D E ) further characterized the particles in the isotropic by showing that the mixed micelles become larger as the total lipid concentration is lowered. Further work using nuclear magnetic resonancelo (NMR) and DLS8.9demonstrated the presence of a second isotropic phase containing vesicles. The transition between mixed micelles and vesicles occurs when the total lipid concen-
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OH
I1
OH /;-SO3.
, on
3
On Figure 1. Chemical structures of bile salts used in this study. I: Gly-
cocholateconsists of cholate conjugated with glycine. The cholate moiety contains a cholesterol-likecore with hydroxyl groups at carbons 3,7,and 12. This gives one side of the core a hydrophilic character. 11: Glycochenodeoxycholate with the hydroxyl groups at positions 3 and 7. 111: Taurocholate is cholate conjugated with taurine. tration is sufficiently r e d ~ c e d . ~These J ~ studies together demonstrated the strong concentration and compositional dependence of particle morphology in these systems. Our SANS data"-13have given more detail on the morphology of the mixed micelles in egg yolk phosphatidylcholineglymholate aqueous mixed colloids. It was shown that at the highest total lipid concentrations, globular mixed micelles are present. The radius of gyration, Rg,of these was measured at 24.1 (0.3) A." If the particles are modeled as uniform spheres, the radius is 3 1.1 A. For a cylinder, the maximum likelihood dimensions are 27-A radius and 50-Aheight.13 When the total lipid concentration is lowered, the particles become elongated. Long rods are formed on further dilution. The cross-sectional radius of gyration, R,, measured for the rodlike particles is 19.1 A. A uniform rod with radius 27 A would give this value for R,. This radius appears
0022-3654/92/2096-8653!§03.00/0 0 1992 American Chemical Society
Hjelm et al.
8654 The Journal of Physical Chemistry, Vol. 96, No. 21, 1992
to be a constant characteristic of the elongated particles and rods formed in these mixture^.^^-^^ The generality of this particle morphology in other phosphatidylcholine-bile salt mixtures has yet to be demonstrated, however. To show the generality of rod formation in mixed micelles of phosphatidylcholine and bile salts, we have done measurements on mixtures of dipalmatoylphosphatidylcholine (DPPC) with taurocholate and on egg yolk phosphatidylcholine with glycochenodeoxycholate and show that the scattering is consistent with rodlike morphology. We show that the radii of the rods are nearly identical in these mixtures from measurements of R,. Measurements of the contrast-weighted mass per unit length, A&, show more variation but are consistent with a limited range of phosphatidylcholine packing densities in all systems studied. There appears to be a dependence of the rod radii on the total lipid concentration in the DPPC-taurocholate system, however. None of the studies carried out so far have given a definitive answer to the question of the arrangement of the phosphatidylcholine and bile salt components in the rodlike mixed micelles. Clearly, there are two ways in which rodlike phosphatidylcholine molecules can be assembled in the mixed micelle-either parallel to the rod axis or perpendicular to i t - a n d both models have been proposed for this system. Models with phosphatidylcholine assembled parallel to the rod axis picture the mixed micelle rods as being made up of stacked d i ~ k s . ~ ,The ~ J ~structure of each disk consists of a phosphatidylcholine bilayer surrounded by a ribbon of bile salt with the hydrophobic portions of the bile salt interacting with the fatty acid chains of the phosphatidylcholine (Figure 2A). This model, which was first proposed by Shankland,' uses the disklike structure of the mixed-disk model proposed by Small6 to describe the morphology of the basic repeating unit making up the rod. More recently, we showed, using maximum entropy techniques, that SANS data from phosphatidylcholine-glycocholatepreparations at high total lipid concentrations were consistent with the presence of a heterogeneous population of mixed micelles each consisting of particles of distinct lengths 50 and 100 A but with the same radius 25-27 A.I3 The discrete nature of the particle lengths at multiples of one lipid bilayer dimension suggested that the micelles consist of stacked disks. The alternativemodel with radially-oriented phosphatidylcholine was introduced by Ulmius and Lindblom et al.I4J5 as an interpretation of NMR data from the hexagonal phase in the phosphatidylcholine-cholate-water system. A similar model was put forward to explain the high-performance liquid chromatography (HPLC) elution behavior of mixed micelles in the isotropic phase by Nichols and Ozarowski.I6 In the models derived from the NMR and HPLC studies, the liquid mosaic tails of the phosphatidylcholine are more or less radially-arranged in the interior of the rod with the phospholipid headgroups in a shell on the surface (Figure 2B). The bile salt is arranged differently in the two models. In the Nichols-Ozarowski model,I6 the bile salts are arrayed on the surface between the headgroups with the cylinder axes of the molecules parallel to the micelle rod axis. The bile salt also serves to cap the ends of the rods in this model (Figure 2B). In the model of Ulmius and Lindblom et a1.,I6 some of the bile salt is inserted between the fatty acid tails, as well, with the axes perpendicular to the rod axis. In either case, the bile salt introduces a wedge, allowing the complex with phosphatidylcholine to associate into a rodlike particle. The questions are which of these two alternate arrangements-radial or axial-best describes the organization of the phosphatidylcholinein the rodlike micelles and how is the bile salt inserted into this structure? We will refer to the axial arrangement of phosphatidylcholine as the stacked-disk model (Figure 2A) and the radial arrangement as the radial-shell model (Figure 2B). We address the problem of the arrangement of the phosphatidylcholine and bile salt in the rodlike micelles by comparison of scattering from mixtures containing DPPC with the choline methyls perdeuterated with the scattering from the nondeuterated material. In principle, the large difference in neutron scattering
Pho
n
h
Figure 2. Schematic diagrams for the alternative models for the organization of phosphatidylcholine and bile salt in rodlike mixed micelles. A. The stacked-disk model. The figure in the box is one repeating unit of the stacked disk, the structure of which is explained in the text. B. The radial-shell model of Ulmius and Lindblom et al. and Nichols and Ozarowski. The figure in the box is a segment removed from the rod to show the radial arrangement of the phosphatidylcholine.
between hydrogen and deuterium leads to differences in scattering," which can be used to extract information on the positions of the headgroups. The changes in R,with headgroup deuteration are more consistent with the radial arrangement of DPPC than with the stacked-disk model. The measured values for A& are closer to the expected values for the radial-shell model (Figure 2B), given the measured &, than those for the stacked-disk model (Figure 2A). Further, evaluation of the molar ratio of bile salt to lecithin for the radial-shell model, using parameters implied by R,,is more in line with the values obtained from equilibrium dialysis measurementsI8 than when the stacked-disk model is used.
Theory Neutron scattering experiments measure the scattering intensity, I(Q), as the absolute differential cross section per unit scattering mass (cm2mg-l), as a function of the magnitude of the scattering vector, Q. The relationship between Q, the incident neutron wavelength X A), and the scattering angle 28 is given by Q = [4x/X] sin 8 (k-l). An alternate measure of the scattering intensity is the macroscopic differentialcross section per unit volume, dZQ/dQ (cm-I). We derive a theoretical description for the scattering from self-assembled mixed rodlike micelles having either of the two structures in order to analyze the results of the scattering measurements. The two models for the structure of the rodlike mixed micelle share common features of an inner shell of one average scattering contrast relative to the solvent, Aps, and a core at yet another average contrast relative to the solvent, Ap,. Ap = .fAp(r,z)dv/u
The Journal of Physical Chemistry, Vol. 96, No. 21, 1992 8655
Organization of Phosphatidylcholine and Bile Salt
= J[p(r,z) - ps] du/u (where ps is the scattering length density of the solvent), about the rod axis (here, defined as the z axis). The integral is evaluated over the volume of the rod, u. The contrast values depend on the chemical and isotope content of the respective core and shell regions of the rod. Thus, for the stacked-disk model, Ap, = nLApLVL/A, and Ap, = Y-'nLAPTcVTc/A,. For the Nichols-Ozarowski variant of the radial-shell model, Ap, = nLApTVT/A,and Ap, = nL(ApHVH + y-IApTCVTC)/As.In these expressions, the A's are the crosssectional areas of the core, c, and shell, s. The ApVs are the scattering lengths of the components minus the total scattering length of solvent which can occupy the molecular volume, V. The subscripts refer to the components: L is phosphatidylcholine; H refers to the headgroup of phosphatidylcholine; T makes reference to the tails of phosphatidylcholine; and TC here is taurocholate but can refer to any bile salt. y is the molar ratio of phosphatidylcholine to bile salt in the mixed micelle, and nL is the number of phosphatidylcholine molecules per angstrom along the rod. The scattering intensity (cm2/mg) for both models in the limit of infinite rod length, L,is given by 4r3NnL-'
'('I = 1 0 3 ~ ( r -
1; '[; ( : Aps
contrasts of the shell and core parts of the structure. At the lowest values of Q,the scattering function described by eq 1, or for any rod for that matter, over a domain in Q between Qmin and QmaX, defined by Qmin >> L-' and Qmax
