Mesoscopic Structures of Triglyceride Nanosuspensions Studied by

Article pubs.acs.org/JPCB

Mesoscopic Structures of Triglyceride Nanosuspensions Studied by Small-Angle X‑ray and Neutron Scattering and Computer Simulations Martin Schmiele,† Torben Schindler,† Martin Westermann,‡ Frank Steiniger,‡ Aurel Radulescu,§ Armin Kriele,∥ Ralph Gilles,⊥ and Tobias Unruh*,† †

Friedrich-Alexander-Universität ErlangenNürnberg, Physik Department, Staudtstrasse 3, 91058 Erlangen, Germany Center for Electron Microscopy of the Jena University Hospital, Ziegelmühlenweg 1, 07743 Jena, Germany § Jülich Centre for Neutron Science (JCNS), Lichtenbergstrasse 1, 85747 Garching, Germany ∥ Outstation at Heinz Maier-Leibnitz Zentrum (MLZ), Helmholtz-Zentrum Geesthacht (HZG), Lichtenbergstrasse 1, 85747 Garching, Germany ⊥ Heinz Maier-Leibnitz Zentrum (MLZ), Technische Universität München, Lichtenbergstrasse 1, 85747 Garching, Germany ‡

S Supporting Information *

ABSTRACT: Aqueous suspensions of platelet-like shaped tripalmitin nanocrystals are studied here at high tripalmitin concentrations (10 wt % tripalmitin) for the first time by a combination of small-angle X-ray and neutron scattering (SAXS and SANS). The suspensions are stabilized by different lecithins, namely, DLPC, DOPC, and the lecithin blend S100. At such high concentrations the platelets start to self-assemble in stacks, which causes interference maxima at low Q-values in the SAXS and SANS patterns, respectively. It is found that the stack-related interference maxima are more pronounced for the suspension stabilized with DOPC and in particular DLPC, compared to suspensions stabilized by S100. By use of the X-ray and neutron powder pattern simulation analysis (XNPPSA), the SAXS and SANS patterns of the native tripalmitin suspensions could only be reproduced simultaneously when assuming the presence of both isolated nanocrystals and stacks of nanocrystals of different size in the simulation model of the dispersions. By a fit of the simulated SAXS and SANS patterns to the experimental data, a distribution of the stack sizes and their volume fractions is determined. The volume fraction of stacklike platelet assemblies is found to rise from 70% for S100-stabilized suspensions to almost 100% for the DLPC-stabilized suspensions. The distribution of the platelet thicknesses could be determined with molecular resolution from a combined analysis of the SAXS and SANS patterns of the corresponding diluted tripalmitin (3 wt %) suspensions. In accordance with microcalorimetric data, it could be concluded that the platelets in the suspensions stabilized with DOPC, and in particular DLPC, are significantly thinner than those stabilized with S100. The DLPC-stabilized suspensions exhibit a significantly narrower platelet thickness distribution compared to DOPC- and S100-stabilized suspensions. The smaller thicknesses for the DLPC- and DOPCstabilized platelets explain their higher tendency to self-assemble in stacks. The finding that the nanoparticles of the suspension stabilized by the saturated lecithin DLPC crystallize in the stable β-tripalmitin modification with its characteristic platelet-like shape is surprising and can be explained by the fact that the main phase transformation temperature for DLPC is, as for unsaturated lecithins like DOPC and S100, well below the crystallization temperature of the supercooled tripalmitin emulsion droplets.

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INTRODUCTION

In a previous purely SAXS-based study the X-ray powder pattern simulation analysis (XPPSA) method has been developed to study the structures of such nanocrystalline triglyceride suspensions ranging from small length scales (stabilizer layer, platelet thicknesses) to larger scales (interparticle distances, platelet diameters).3 This was done by a fit of a linear combination of the scattering intensities originating from different structural elements, namely, from ensembles of

Suspensions of lecithin stabilized triglyceride nanoparticles prepared by high pressure melt homogenization have been investigated thoroughly as potential drug delivery systems over the past 2 decades.1,2 Upon cooling, the emulsion droplets crystallize predominantly into the stable β-modification or thermodynamically unstable α-modification. For the β-modification thin platelet-like crystals with diameters in the colloidal range are characteristic. Because of the small thickness of the platelets of only a few unit cells, in small-angle scattering patterns a strongly broadened 001 Bragg reflection can be observed.3 © 2014 American Chemical Society

Received: March 14, 2014 Revised: June 18, 2014 Published: June 20, 2014 8808

dx.doi.org/10.1021/jp502580a | J. Phys. Chem. B 2014, 118, 8808−8818

The Journal of Physical Chemistry B

Article

and DLPC, respectively, which show different strengths of the stack-related interference maxima in the small-angle scattering patterns. A native tripalmitin suspension stabilized with the lecithin blend S100 has been taken as a reference, since S100stabilized triglyceride suspensions have been frequently used in previous pharmaceutical studies.7−9 Differences in the amount of stacks in the three dispersions are discussed with regard to the different geometry of their nanoparticles.

platelets sharing a common platelet thickness to the SAXS data. By this, it is possible to obtain a distribution for the thicknesses of the platelets from the fitted linear coefficients. Furthermore, in the SAXS study it turned out that the shape and intensity of the broadened 001 Bragg peak are sensitive to properties of the lecithin stabilizer layer, and by this, the Bragg peak can be utilized to study different models for the structural arrangement of the lecithin molecules covering the platelets.3 In a recent study a revised version of the XPPSA method has been presented.4 The new version, hereinafter referred to as the X-ray and neutron powder pattern simulation analysis (XNPPSA), allows now the simultaneous analysis of X-ray and neutron scattering data, fitting of the scattering contrasts of the stabilizer layer, and a computation of the scattering patterns on an absolute scale. At the same time the method has been tremendously improved in performance. Several structural models for the arrangement of the lecithin molecules in the stabilizer layer have been tested by simultaneously fitting the SAXS and SANS patterns of dilute tripalmitin suspensions. It turned out that the obtained thickness of the stabilizer monolayer of 12 Å is significantly thinner than the 21 Å obtained from the analysis of SAXS data only.3 From the best matching model it was concluded that the hydrophobic tails of the lecithin molecules are arranged rather flatly and are densely packed in an inner shell on the particles surfaces and the hydrophilic headgroups with additional water molecules in an outer shell. For higher triglyceride concentrations it has been demonstrated by SAXS and TEM that the isolated platelets start to self-assemble spontaneously in stacks.5,6 In SAXS patterns the stack formation is established by the appearance of additional lamellar interference maxima in the SAXS patterns. In the stacks the large (001) faces of the crystalline platelets arrange in parallel with typical repeat distances in the range between 30 and 60 nm, as it has been observed for lecithin stabilized tripalmitin nanoparticles that were costabilized either by the cationic surfactant cetylpyridinium chloride (CPCl) or with the anionic bile salt sodium glycocholate (NaGC, used in this study).6 For the suspensions costabilized by CPCl it was observed that the platelets start to self-assemble in stacks for tripalmitin concentrations larger than 4 wt % with a decreasing repeat distance upon rising tripalmitin concentration. The origin of the stack formation was attributed to an entropic effect that can be described in a simplified way by the overlap of the platelet exclusion volumes at a critical concentration.6 However, electrostatic interactions might also be an issue because of the ionic costabilizers that are necessary to prevent the gelation of the suspensions upon crystallization. It turned out that at constant tripalmitin concentrations the mean repeat distance decreases with increasing amounts of the ionic costabilizer.6 The stack formation is a reversible process. Upon dilution the self-assemblies disintegrate, which can be inferred from the disappearance of the stack-related interferences maxima in the small-angle scattering patterns. From a pharmaceutical point of view the formation of such platelet stacks can be relevant for the long-term stability and the viscosity of tripalmitin suspensions. The main aim of this study is to get for the first time a reliable and quantitative description of the complex structure of native tripalmitin (10 wt % tripalmitin) nanosuspensions by simultaneously analyzing the SAXS and SANS patterns with the XNPPSA method on an absolute scale. Therefore, we study native tripalmitin suspensions stabilized by the lecithins DOPC

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MATERIALS AND METHODS Materials. Tripalmitin (Dynasan 116, 96% triglycerides and 3% diglycerides of palmitic acid10) was kindly provided by Sasol GmbH, Witten, Germany. 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC, cf. Figure 1a) and 1,2-dioleoyl-sn-glycero-3-

Figure 1. Chemical structures of (co)stabilizers used in this study: (a) saturated lecithin DLPC (12:0), (b) monounsaturated lecithin DOPC (18:1), and (c) the bile salt sodium glycocholate (NaGC).

phosphocholine (DOPC, cf. Figure 1b) (purity of ≥98%) were purchased from Lipoid GmbH, Ludwigshafen, Germany. Sodium glycocholate (NaGC, cf. Figure 1c) was purchased from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany. D2O (D214H, isotopic purity of 99.9% atom D) was obtained from Euriso-Top GmbH, Saarbrücken, Germany. The purity specifications of all substances were provided from the manufacturers, and all substances were used without any further purification. S100, a purified soybean lecithin (≥94% phosphatidylcholine), was provided by Lipoid GmbH. According to the manufacturer’s specification, S100 is rich in doubly unsaturated 18:2 fatty acids (about 65% of all fatty acids). Besides its content of 18:1 (∼13%) and 18:3 (∼5%) fatty acids, S100 has a total fraction of about 80% of unsaturated C18 fatty acids (structurally similar to DOPC in Figure 1b). The remaining hydrocarbon moiety is dominated by 18:0 and 16:0 chains. Sample Preparation. The suspensions were prepared in a similar way as previously reported.4 The native tripalmitin suspensions consist of 10% tripalmitin, 2.4% lecithin (DLPC, DOPC, or S100), and 0.6% sodium glycocholate (NaGC) in D2O (here and in the following all chemical concentrations are given in wt % for an equivalent fully protiated suspension if not explicitely mentioned). Small amounts of the anionic costabilizer NaGC are crucial to prevent gelation of the 8809



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nanoemulsions upon crystallization.1 The lecithin and tripalmitin were heated to 80 °C. The NaGC was dissolved in D2O and also heated to 80 °C. After the two liquids were mixed, the hot mixture was dispersed for 3 min with an UltraTurrax T25 Basic disperser (IKA-Werke GmbH & Co. KG, Staufen, Germany) at 22.000 rpm. The hot preemulsion was then passed into a preheated (>65 °C) continuously working APV-2000 high-pressure melt homogenizer (APV Deutschland GmbH, Unna, Germany). The dispersion was homogenized at successively increasing pressure between 1 and 2 kbar for 4 min. The obtained nanoemulsion was allowed to cool to room temperature and finally stored at 6 °C. Dilute suspensions were prepared by diluting the native suspensions with D2O to a final concentration of 3% tripalmitin. Small- and Wide-Angle X-ray Scattering (SAXS, WAXS). SAXS and WAXS patterns were collected from the native and dilute suspensions about 1 week after their preparation on a Kratky-type camera (S3-MICROpix, Hecus X-ray Systems GmbH, Graz, Austria) at FRM II in Garching, Germany. Pure D2O was measured as reference. The X-ray microfocus source (Xenocs, Sassenage, France) of the camera, equipped with a focusing single bounce mirror optics, provides a microfocused beam at the Cu Kα wavelength of λ = 1.5418 Å. The Kratky block collimator was adjusted to a final beam size of about 0.25 × 0.25 mm2 at the sample position. For this setting an X-ray flux in the primary beam of about 107 photons per second was achieved. Two-dimensional SAXS and onedimensional WAXS patterns were recorded with a 2D Pilatus 100 K detector and a Mythen line detector, respectively (Dectris AG, Baden, Switzerland). The liquids were measured in a reusable quartz capillary with about 0.01 mm wall thickness (Hilgenberg GmbH, Malsfeld, Germany) at a temperature of 30 °C. The sample−detector distance was calibrated to 289.5 mm using a silver behenate standard (Eastman Kodak Company).11 The inner diameter of the capillary was calculated from transmission runs of the empty and D2O filled capillary (cf. ref 12) to be 0.76 mm. By use of the measured sample transmission, the thickness of the capillary, and a measurement of a secondary standard (glassy carbon,13 kindly provided by the 15ID-D USAXS beamline at the Advanced Photon Source, Argonne, IL, USA), the scattered intensity was converted to an absolute scale (1/cm/sr).14 The one-dimensional scattering function was obtained by azimuthally averaging the twodimensional images with the program fit2dcorr, an extension for the program fit2d.15 By use of the sample−detector distance and the pixel size of the detector, the data were put on an sscale (s = Q/(2π) = (2/λ)sin(2θ/2), where 2θ is the scattering angle). The scattering curve of the D2O background was subtracted from the patterns of the suspensions. The 1D WAXS patterns were calibrated with β-tripalmitin powder and corrected for the scattering of the D2O background. Small-Angle Neutron Scattering (SANS). The SANS measurements were carried out at the KWS-2 beamline16 of JCNS at FRM II. Neutrons with a wavelength of λ = 4.5 Å and a wavelength spread of Δλ/λ = 20% were used. Taking measurements at three different sample−detector distances, namely, 2, 8, and 20 m, an s-range between 0.004 and 0.54 nm−1 is covered. By use of a collimation length of 20 m, entrance and sample apertures of 30 × 30 mm2 and 8 × 8 mm2, respectively, exposures with good statistics were taken for 10, 5, and 10 min at the sample detector distances of 2, 8, and 20 m, respectively. The samples were measured in Hellma QX 404

quartz cuvettes with a 0.5 mm path length (Hellma GmbH, Müllheim, Germany) at a temperature of 30 °C. For data reduction the QtiKWS program was used. The two-dimensional raw data were corrected for dark current (measured while blocking the beam with a boron carbide slab), transmission, and detector sensitivity (measured with a 1.5 mm plexiglass sample). Measurements with the plexiglass standard were also used to put the data on an absolute scale. Since D2O scatters completely flat in the small-angle scattering range, no D2O background was subtracted from the samples. The D2O and all further flat incoherent backgrounds are later considered by an additive constant in the fits. Photon Correlation Spectroscopy (PCS). The particle size (z-average) and polydispersity index of the nanoparticles were determined by photon correlation spectroscopy (PCS). Cumulant method was used for the analysis of the PCS spectra collected using a spectrometer (Brookhaven Instruments Corporation, Holtsville, NY, USA), comprising a BI-200 SM goniometer, a Mini-L 30 compact diode laser (30 mW, 637 nm), and a photo multiplier tube at a scattering angle of 90°. For the measurements a few droplets of the sample were dispersed in purified water inside a glass cuvette and measured for 1 min at 22 °C. Freeze−Fracture Transmission Electron Microscopy (TEM). Freeze−fracture TEM preparations were performed at the Center for Electron Microscopy of the Jena University Hospital, Germany. A small droplet of the tripalmitin suspension is embedded between two copper sandwich profiles. The sandwiches are rapidly frozen in a liquid propane/ethane mixture (1:1) cooled by liquid nitrogen, placed in a freeze− fracture unit (BAF 400D, BAL-TEC, Liechtenstein), and fractured in a vacuum chamber at −140 °C and 10−6 mbar. The fractured samples are shadowed under an angle of 35° with platinum/carbon covering the fracture with a 2 nm layer of Pt/ C. Subsequently 15−20 nm of carbon is deposited from top in order to stabilize the replica. The replica were detached from the copper profiles in deionized water, cleaned for 1 h in a mixture of methanol and chloroform (1:1), fished up on copper grids, and transferred into a transmission electron microscope (Zeiss CEM 902A, Carl Zeiss AG, Oberkochen, Germany) operated at 80 kV. Micro Differential Scanning Calorimetry (μDSC). μDSC heating and cooling runs were recorded with a Micro DSC III instrument (Setaram, Caluire-et-Cuire, France). The samples were heated from about 5 to 70 °C and subsequently cooled to 5 °C at scan rates of 0.1 K/min. About 250 mg of the native suspension and the same amount of water were filled in the sample and the reference cell, respectively. For the measurement of pure tripalmitin 7.6 ± 0.2 mg of tripalmitin were filled into the sample cell. An empty cell was used as reference. For this measurement a heating run from 2 to 85 °C and a subsequent cooling run back to 2 °C were conducted at scan rates of 0.04 K/min. All sample and reference cells were tightly sealed. XNPPSA Method. Simulation Model and Computational Aspects. The XNPPSA method allows the computation of SAXS and SANS patterns for different structural elements in a dispersion and can determine their frequencies by a (simultaneous) fit of a linear combination of their macroscopic scattering cross sections (MSCS) dΣ/dΩ to experimental SAXS and SANS patterns.3,4 In the previous SAXS and SANS study on dilute triglyceride suspensions it was sufficient to consider only isolated platelets 8810



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atomic positions of the β-tripalmitin molecules and the parameters for β-tripalmitin′s triclinic unit cell were taken from ref 17. The platelets are covered by a stabilizer layer. A monolayer is sufficient to model the stabilizer layer because of the low concentration of the lecithin and NaGC stabilizers.4 Furthermore, because of the amphiphilic character of the lecithin molecules, the stabilizer layer is subdivided into two shells. The outer shell of thickness dosl represents the hydrophilic phosphocholine headgroups, and the inner shell of thickness disl represents the hydrophobic acyl chains. The electron densities (EDs) and neutron scattering length densities (NSLDs) for the inner (isl) and outer (osl) shell of the stabilizer layer and for the dispersion medium (dm) are denoted by ρXisl, ρXosl, ρXdm and ρnisl, ρnosl, ρndm, respectively. Together with the shell thicknesses these parameters can be used to characterize the structural arrangement of the lecithin molecules in the stabilizer layer. For the simulation of platelet stacks it is necessary to know the distribution of the platelet thicknesses. The less complex SAXS and SANS patterns of a diluted suspension (3% tripalmitin), lacking of any stack-related interferences, are fitted by the simulated MSCS of ensembles of isolated platelets in order to determine the thickness distribution function of the nanoparticles. In a second step this distribution function is used for the simulation and analysis of the native suspensions to determine the properties of the formed stacks. For the dilute suspensions in each simulation run the MSCSs (dΣ/dΩ)isp were computed for five ensembles. All 500 platelets in ensemble i share the same thickness of i unit cells of tripalmitin, where the thickness d001 of one unit cell with respect to the crystallographic direction perpendicular to the (001) lattice planes is 4.02 nm for β-tripalmitin. The two lateral diameters of the parallelepipeds are subject to Gaussian distributions with mean values of 120 and 100 nm, respectively, and variances of 20 nm. The shell thicknesses disl and dosl were scanned in steps of 0.5 Å in the ranges from 4 to 10 Å and from 3 to 9 Å, respectively, adding up to 169 simulation runs. For the native suspensions the MSCS (dΣ/dΩ)ist for two ensembles (i = 2, 3) are simulated in each simulation run. All 100 stacks in ensemble i consist of i platelets. In each ensemble the platelet thicknesses are subject to the fitted thickness distribution obtained from the analysis of the corresponding dilute suspension. The repeat distances of the platelets within the stacks, dst, are subject to a narrow Gaussian distribution with mean distances of 36 nm (native DOPC and S100 suspensions) and 37.9 nm (DLPC), respectively. The values for dst can be with good accuracy read off directly from the lamellar peaks in the small s range in the experimental SAXS and SANS data. Similar as for the dilute suspensions, parameter scans were carried out in the simulations for the platelet stacks for disl, dosl, and additionally σst. disl and dosl are scanned in steps of 0.5 Å in the ranges between 5 and 10 Å and between 3 and 7 Å, respectively. For σst three different values, namely, 2, 3, and 4 nm, are used. Thus, in total 297 simulation runs were performed for the DLPC- and DOPC(S100)-stabilized suspensions, respectively. The simulations were performed using the C++ program XNDiff on the high performance computing cluster Woodcrest of Regionales Rechenzentrum Erlangen (RRZE). Each simulation run for a particular combination of the stabilizer layer shell thicknesses disl and dosl (for native suspensions additionally the variance of the repeat distances σst) lasts about

of different thicknesses and diameters as structural elements in the computer simulations.4 In this study we have to include in addition platelet stacks as structural elements to reproduce the much more complex experimental SAXS and SANS patterns of the native suspensions and to refine the structure and amount of these stacks in the dispersions. In the computation of the MSCSs, for each isolated platelet or platelet stack a powder average is carried out, where the orientations of the platelet or stack are equally distributed over the unit sphere. Because of the experimentally limited coherence volumes in SAXS and SANS, the total MSCS can be calculated as the sum over all MSCSs of the single platelets and stacks, interparticle scattering needs not to be considered except for the stacks. Figure 2a the arrangement of the platelets in the suspensions is schematically shown for sufficiently high platelet concen-

Figure 2. (a) Visualization of isolated and stacked platelet-like nanocrystals as the structural elements the tripalmitin suspensions consist of at sufficiently high concentration. (b) Structural model for a stack of nanocrystals used in the computation of SAXS and SANS patterns. Because of the amphiphilic character of the lecithin molecules, the stabilizer layer is subdivided into two shells for the fatty acid chains (inner shell) and the phosphocholine headgroups (outer shell), respectively (lower inset). The molecular arrangement of the tripalmitin molecules in the triclinic unit cell for β-tripalmitin is indicated in the upper inset.

trations. The suspensions consist of platelet stacks with different numbers of platelets per stack and a fraction of remaining isolated platelets. In Figure 2b the structure for a particle stack consisting of three crystalline β-tripalmitin platelets is visualized in detail. Isolated platelets can be considered as a special case of a stack consisting of only one particle. Each platelet assumes a parallelepipedal shape. The 8811



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Table 1. Molecular Volumes and Scattering Length Densities for DLPC, DOPC, and Water DLPC -head -chains DOPC -head -chains D2O

chemical formula

V (Å3)

bcoh (fm)

ρn (10−6 Å−2)

Z (eu)

ρX (nm−3)

C32H64NO8P C10H18NO8P C22H46 C44H84NO8P C10H18NO8P C34H66 D2O

986 319 667 1303 319 984 29.9

34.3 60.1 −25.8 39.3 60.1 −20.8 19.2

0.35 1.88 −0.39 0.30 1.88 −0.21 6.36

342 164 178 434 164 270 10

347 514 267 333 514 274 333

NSLDs in each shell and that the number of phosphocholine headgroups is correlated to the number of acyl chains between both shells. The constraints involve the EDs and NSLDs of the phosphocholine headgroups and acyl chains as well their molecular volumes. The molecular volumes V for DLPC and DOPC in their liquid crystalline Lα-phase and water at 30 °C19−21 and the EDs ρX = Z/V and NSLDs ρn = bcoh/V are listed in Table 1 (Z and bcoh denote the number of electrons and the coherent neutron scattering length of the compounds). Because of their chemical resemblance to DOPC, the lecithins in S100 (mixed acyl chains of mostly mono- and polyunsaturated C18 chains) are modeled with DOPC (18:1) molecules. From the fitted coefficients ci, which represent a volume weighted distribution for the platelet thicknesses, a number weighted distribution ni for the platelet thicknesses can be obtained. The ni can be estimated from the ci via

12 h on a node with two dual core Xeon 5160 Woodcrest CPUs. The parameter scans for geometric parameters like disl, dosl, and σst are necessary, since they cannot be fitted later in contrast to the EDs and NSLDs of the stabilizer layer shells and the dispersion medium. Fits. The simultaneous fits for the SAXS and SANS data using eq 1 were carried out with the Mathematica program BatchMultiFit whose source code is together with the source code of the simulation program XNDiff available on sourceforge.net.18 With the XNPPSA method the MSCSs dΣ/dΩ for SAXS and SANS can be expressed as a linear combination of the simulated macroscopic scattering cross sections for the ensembles of isolated platelets with different thicknesses, (dΣ/dΩ)ist, and stacks with different numbers of platelets per stack, (dΣ/dΩ)ist, as N

N

sp st ⎛ dΣ ⎞ ⎛ dΣ ⎞i ⎛ dΣ ⎞i ⎜ ⎟ = ψ ∑ c⎜ ⎟ ⎟ + A + (1 − ψ ) ∑ di⎜ i ⎝ dΩ ⎠ ⎝ dΩ ⎠sp ⎝ dΩ ⎠st

i=1

−1 N ci ⎛⎜ sp ci ⎞⎟ ni = ⎜∑ ⎟ i ⎝ i=1 i ⎠

i=2

(1)

Here, Nsp ≥ 1 denotes the maximum thickness of isolated platelets given in numbers of the thickness of one β-tripalmitin unit cell along the crystallographic direction perpendicular to the (001) lattice planes, d001 = 4.02 nm. Nst ≥ 2 describes the maximum number of platelets per stack. The linear coefficients ψci can be interpreted as the volume fraction of an ensemble of isolated platelets sharing a common thickness of i unit cells.4 Likewise (1−ψ)di can be interpreted as the volume fraction of an ensemble of stacks consisting of i platelets. In this context ψ is the volume fraction of all isolated platelets. The completeness relation for the volume fractions, Nsp i=1

where it is assumed that no correlation between the thickness and diameter of the platelets exists.4 With these two thickness distributions, volume and number-averaged mean platelet thicknesses (in numbers of d001 = 4.02 nm), ⟨i⟩V and ⟨i⟩N, respectively, can be calculated for each suspension as ⟨i⟩V =

i=2

∑i cii ∑i ci

,

⟨i⟩N =

∑i nii ∑i ni

=

∑i ci ∑i

ci i

(4)

For the native suspensions stacks with up to three platelets and platelet thicknesses of up to five unit cells were used in the full form of eq 1 (Nsp = 5 and Nst = 3). As in the simulations, the thickness distribution of the remaining isolated platelets, ci, was fixed to the values obtained from the best fit of the corresponding dilute suspension. Equation 2 was included as a fit constraint, and instead of the ci, the di and ψ (0 ≤ ψ ≤ 1) were fitted. For all fits in this study the completeness relation eq 2 was relaxed such that the sum may vary within 20% bounds around 1. Furthermore, a scaling factor χ (0.8 ≤ χ ≤ 1.2) for the SAXS curve and constants A > 0 (cf. eq 1) to match the backgrounds at higher scattering angles were permitted in the fits. The simulated SANS patterns were smeared prior to the fitting using the experimental wavelength spread Δλ/λ = 0.2.4 For dilute suspensions the fits were done for all simulated combinations of the stabilizer thicknesses disl and dosl, thus providing the best values for them with 0.5 Å resolution. Similarly, for the native suspensions, disl, dosl, and the variance of the stack repeat distance, σst, were optimized by selecting the best fit for all simulated combinations of disl, dosl, and σst.

Nst

ψ ∑ ci + (1 − ψ ) ∑ di = 1

(3)

(2)

must be fulfilled. For the dilute suspensions only single platelets with thicknesses of up to five unit cells (Nsp = 5) are used in the simulations and fits. By setting ψ to 1, in eqs 1 and 2 only the first sum involving the ci for the isolated platelets is considered. Besides the ci in eq 1 also the EDs ρXisl, ρXosl, ρXdm and the NSLDs ρnisl, ρnosl which are included in the mathematical expressions for (dΣ/dΩ)isp (and (dΣ/dΩ)ist) can be fitted.4 ρndm is fixed to the NSLD of D2O. For the structural arrangement of the lecithin molecules in the stabilizer layer we use the model that turned out to provide the best simultaneous SAXS and SANS fits for the dilute suspensions in the previous study.4 The model assumes that the headgroups are situated only in the outer stabilizer layer shell and the acyl chains only in the inner shell. Water penetration into both shells is allowed. Three fit constraints for ρXisl, ρXosl, ρnisl, ρnosl ensure the same water penetration levels for the EDs and 8812



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Figure 3. Freeze−fracture electron micrograph taken from tripalmitin suspensions stabilized by (a) S100 (15% tripalmitin, 3.6% S100, 0.9% NaGC, in H2O) (see ref 4 for related image), (b) DOPC (10% tripalmitin, 2.4% DOPC, 0.6% NaGC, in H2O, and (c) DLPC (10% tripalmitin, 2.4% DLPC, 0.6% NaGC, in D2O). The black bars correspond to a length scale of 200 nm.

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RESULTS AND DISCUSSION Characterization of the Native Suspensions. PCS. Monomodal particle size distributions with diameters (zaverage) of about 120 nm for S100 and about 135 nm for DOPC and DLPC are obtained from the measured correlation functions (cf. Figures S1−S3 in the Supporting Information). The polydispersity indices are close to 0.2 for all suspensions where the suspension stabilized by DLPC shows a slightly smaller value as for DOPC and S100. The particle sizes remained stable over 1 month, and also no macroscopic particles could be detected by visual control. WAXS. The WAXS patterns (Figure S4 in the Supporting Information) of all three native suspensions are dominated by three characteristic peaks at s = 2.19, 2.61, and 2.72 nm−1 which can be attributed to the stable β-modification of tripalmitin.22 Furthermore, two small reflections at about s = 2.31 and 2.43 nm−1 are observed. These reflections do not fit any of the known modifications of tripalmitin but have been observed already in previous studies22,23 at very similar positions. For similar lecithin stabilized triglyceride suspensions these reflections were found to appear for mass ratios of lecithin/ triglyceride above 20%.23 This is in full agreement with our observation of the additional reflections for suspensions with a 24% ratio. The quantification of the unknown modification (plus possible traces of the thermodynamically unstable αmodification) is difficult from the available WAXS data. A more accurate estimation of the amount of the unknown modification can be achieved later by an analysis of the μDSC melting curves of the dispersed nanoparticles. Freeze−Fracture TEM. Freeze−fracture electron micrographs taken from native tripalmitin suspensions stabilized by S100, DOPC, and DLPC, respectively, are shown in Figure 3. All three suspensions are dominated by platelet-like nanoparticles with diameters in the range between 50 and 400 nm. The platelet-like shape is characteristic for the stable βmodification of tripalmitin9 which supports the findings from the WAXS data. No spherical nanoparticles, which are characteristic for the thermodynamically instable α-modification,9 were observed in the micrographs. μDSC. The μDSC heating and cooling runs for the native suspensions are shown in Figure 4. While the cooling runs exhibit only one main crystallization event, in the heating runs a fractionated melting behavior can be observed. Here, the

Figure 4. μDSC heating and cooling runs for the native tripalmitin suspensions (10%) stabilized by the lecithins DLPC (12:0), DOPC (18:1) and the purified soybean lecithin S100.

partially overlapping melting peaks can be interpreted as a sequence of size-dependent melting events with each peak corresponding to an ensemble of platelets sharing a specific thickness.24,25 The small melting peaks below 45 °C (at 40 °C for S100, 41.5 °C for DLPC, and 43.5 °C for DOPC) can be most probably attributed to small nanocrystals of the unknown modification or traces of the α-modification (mp for bulk αtripalmitin of ∼45 °C 26) as it was demonstrated by Bunjes et al.22 The enthalpies of fusion for these events amount to about 1−1.2 J/g, and since the total enthalpies of fusion ΔHfus for the suspensions are on the order of 15−16 J/g (cf. Table 2), the fraction of the unknown or α-modification with respect to the total amount of tripalmitin is thus well below 10%. Table 2. PCS z-Averages (D), Polydispersity Indices (σD), and Enthalpies of Fusion ΔHfus and Crystallization ΔHcry μDSC

PCS

8813

sample

D (nm)

σD

ΔHfus (J/g)

DLPC DOPC S100

135.3 135.5 120.5

0.18 0.22 0.22

15.1 15.2 16.0

ΔH*fus (J/g)

ΔHcry (J/g)

ΔH*cry (J/g)

16.5 16.6 17.5

−14.6 −15.3 −16.4

−15.9 −16.7 −17.9



Article

instead of H2O, the enthalpies ΔH were corrected for the D2O − H2O mass difference. The corrected enthalpies ΔH*fus and * in Table 2 are for all suspensions slightly lower than the ΔHcry 18.8 J/g one would expect for 10% bulk β-tripalmitin but well above the 12−13 J/g for 10% of bulk α-modification. This supports the finding from the WAXS measurements and the TEM micrographs that the nanoparticles in all three suspensions predominantly crystallized in the stable βmodification. The finding that the DLPC-stabilized suspension crystallizes in the β-modification is remarkable, since saturated lecithins usually promote a crystallization of the emulsion droplets into more spherical nanoparticles in the α-modification.8 For example, for the lecithins DMPC (14:0) and DPPC (16:0) this was explained by precrystallization events in DSC cooling curves (prior to the crystallization of the liquid triglyceride core), which were attributed to a solidification of the saturated acyl chains at temperatures roughly correlated to the main phase transition of the corresponding lecithin.8 The precrystallized acyl chains on the liquid triglyceride nanodroplets hinder at a later stage the crystallization of the nanodroplets into platelet-like crystals with the β-modification and promote by this template effect a crystallization into the α-modification.8 For the DLPC-stabilized suspension no precrystallization events prior to the crystallization of the nanodroplets are found in the cooling run. The reason why this does not happen is most probably that the main phase transition temperature of −2 °C for the shorter DLPC molecules is as for unsaturated lecithins like DOPC (18:1) and S100 well below the crystallization temperature of the supercooled nanodroplets, and thus, the acyl chains of the DLPC remain fluid which facilitates the morphological change of the nanodroplets into thin β-tripalmitin platelets. Simulations and Fits: Dilute Tripalmitin Suspensions (3%). The measured SAXS and SANS patterns for the three dilute tripalmitin suspensions (3%) are plotted in Figure 5a. Besides the diffuse small-angle scattering signal arising from the shape of the platelets, the scattering patterns feature a 001 Bragg peak stemming from the crystalline structure of βtripalmitin. The 001 Bragg peaks are strongly broadened because of the small thickness of the platelets along the direction perpendicular to the (001) lattice planes. In Figure 5a also the corresponding best simultaneous fits to the SAXS and SANS patterns are shown which describe the data very well. The shell thicknesses disl and dosl, the EDs and NSLDs for the shells, and the dispersion medium for the best fits can be found in Supporting Information Table S1. The total stabilizer thicknesses (dtot = disl + dosl) of 10.5 Å (DLPC), 11.0 Å (DOPC), and 10.0 Å (S100) are in good agreement with the 12 Å found in the previous study on dilute S100 and DOPCstabilized tripalmitin suspensions and indicate again a rather flat arrangement of the lecithin molecules in the stabilizer layer. The inner shell is fully occupied by the acyl chains, since the fitted EDs and NSLDs from Table S1 for the inner shell agree with those of the pure acyl chains from Table 1. Since the EDs (NSLDs) for the outer shell are lower (higher) than the ED (NSLD) of the pure phosphocholine headgroup, additional water molecules are present in the outer shell. For DLPC the lowest water penetration level is found. The dependence of the target function values of the fits for all simulated combinations of disl and dosl is visualized in Figure 6a exemplary for the dilute DLPC-stabilized suspension. A white dot marks the best fit for all combinations of disl and dosl.

The melting peaks at temperatures in the range between 45 °C and the bulk mp of β-tripalmitin (about 65 °C) can be attributed to the known thickness dependent melting of platelets in the β-modification, with thicker platelets melting at higher temperatures than thinner ones.24 As a trend, the heating curves for the DOPC- and S100-stabilized suspensions follow roughly he same course whereby the curve for S100 is shifted by about 3 K to higher temperatures. The heating curve for the DLPC-stabilized suspension follows a similar trend at the beginning and at the end of the considered temperature range as the curve of the DOPC-stabilized suspension. However, with the appearance of a very strong melting peak in the range between 52 and 55 °C (maximum at 53.9 °C), a pronounced difference to the curves from the DOPC and S100stabilized suspensions can be observed. The sharp peak and a distribution of the enthalpy of fusion being focused on a smaller temperature range compared to the suspensions stabilized with S100 and DOPC give reason to expect both thinner platelets and a narrower distribution of the platelet thicknesses of the DLPC-stabilized suspension. By comparison of the distributions of the enthalpy of fusion of the DOPC- and S100stabilized suspensions, a thinner mean thickness can be inferred for the DOPC-stabilized suspension, too. A prediction of the thickness for those platelets linked to the strong melting peak in the DLPC suspension from the μDSC data alone is rather difficult. In a previous study, where the μDSC melting curve of a S100-stabilized tripalmitin suspension was compared with the temperature dependent evolution of the peak width of the 001 Bragg peak in SAXS patterns, the temperature ranges for the melting of platelets with a thickness of 2 and 3 unit cells were 50−54 and 54−56 °C, respectively.5 By this, one might suppose that the large peak is caused by platelets with thicknesses of either 2 or 3 unit cells or both of it. A precise determination of the enthalpy of fusion for the sharp melting peak is difficult because of the overlap with the neighboring peaks. However, this enthalpy can be estimated to not exceed 45% of the total enthalpy of fusion in the temperature range that is attributed to the melting of βtripalmitin nanocrystals. The enthalpy of fusion in the range from 45 °C to about 56 °C, covering the range of thicknesses up to 3 unit cells, corresponds to about 80% of the enthalpy of fusion that is attributed to the melting of β-tripalmitin. The cooling curves exhibit sharp crystallization peaks at 24.8, 23.0, and 22.2 °C for the DLPC-, DOPC- and S100-stabilized suspensions, respectively, with corresponding onset temperatures of 26.4, 27.5, and 24.7 °C. The higher crystallization temperature for the DLPC- and DOPC-stabilized suspensions, compared with the S100-stabilized suspension, agrees with the observation that lecithins with a higher degree of unsaturation in their acyl chains lead to lower crystallization temperatures.8 For bulk tripalmitin the reported enthalpies of fusion ΔHβfus for the β-modification are 188 J/g,7 188 ± 5 J/g (this study), 205 J/g,27 and 219.9 J/g.28 For the slow cooling rate of −0.04 K/min used in this study the crystallization enthalpy for bulk tripalmitin of −180.8 ± 4.8 J/g indicates a direct crystallization into the stable β-modification. Fast cooling rates promote a crystallization into the unstable α-modification. Crystallization enthalpies for the α-modification, ΔHαcry, were found to be −120 J/g,7 −124 J/g,27 and −134.5 J/g.28 The total enthalpies of fusion and crystallization, ΔHfus and ΔHcry, respectively, for the native suspensions are listed in Table 2. Since the suspensions were prepared using D2O 8814



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Black dots indicate “good” fits where the target function values deviate by less than 10% from the one of the best fit. From the distribution of the black dots it is apparent that the thickness of the inner shell is better defined than the outer shell thickness. The fits with the black and white dots are used to calculate mean values, standard deviations, and maximum deviations from the mean value for the fit parameters, in particular for the stabilizer shell thicknesses, EDs and NSLDs of the inner and outer stabilizer shell, and the linear coefficients (volume fractions) ci for the distribution of the platelet thicknesses. The mean values and standard deviations are also listed in Table S1. The parameters from the best fit most often agree within the standard deviation with the mean values; only for the DOPC-stabilized suspension the parameters for the outer shell exhibit small deviations beyond. In Figure 5b−d the distributions of the best fitted parameters ci are shown for the three diluted suspensions. For DLPC the fitted EDs and NSLDs from Table S1 for the inner and outer stabilizer layer and the dispersion medium are also visualized in Figure 5e and Figure 5f, respectively. The black points and the error bars mark the mean values and the maximum deviations of the corresponding parameters, respectively, which were obtained by the statistical analysis of all reasonable fits (black dots in Figure 6). The values for the best fitted volume fractions ci for platelets with thicknesses of i unit cells, together with the volume and number weighted mean platelet thicknesses ⟨i⟩V and ⟨i⟩N (calculated from eq 4 and given in units of d001 = 4.02 nm), respectively, are listed in Table 3. Table 3. Volume Fractions ci of Platelets with Thicknesses of i Unit Cells Figure 5. (a) Best simultaneous fits for the SAXS and SANS data for the dilute tripalmitin (3%) suspensions stabilized by DLPC, DOPC, and S100, respectively. The position of the 001 Bragg peak is indicated by the vertical dashed line. The curves were scaled with the factors next to the curves for a better representation. (b)−(d) show the fitted platelet thickness distributions for the three suspensions. Hereby the coefficients ci (cf. Table 3) represent the volume fractions of isolated platelets with thicknesses of i unit cells. The fitted (e) electron densities ρX and (f) neutron SLD ρn for the inner (isl) and outer (osl) stabilizer layer as well as for the dispersion medium (dm) are plotted for DLPC. All fit parameters for the dilute suspensions can be found in Supporting Information Table S1.

DLPC DOPC S100

c1

c2

c3

c4

c5

⟨i⟩V

⟨i⟩N

0.079 0.160 0.097

0.501 0.294 0.271

0.290 0.334 0.341

0.000 0.150 0.202

0.036 0.050 0.069

2.35 2.63 2.87

2.09 2.12 2.39

The curve progression of the distributions of the ci parameters displayed in Figures 5b−d and the mean volume weighted thicknesses ⟨i⟩V agree qualitatively very well with the results from the microcalorimetric measurements. First of all, the mean platelet thickness ⟨i⟩V for the DLPC-stabilized platelets is about 0.5 (0.3) unit cells thinner as for the suspension stabilized by S100 (DOPC). And interestingly, for the DLPC-stabilized suspension the thickness distribution is indeed narrower than the distributions for the DOPC- and S100-stabilized suspensions which also was concluded from the analysis of the DSC measurements. The high volume fraction c2 of platelets with a thickness of only 2 unit cells is very prominent for DLPC. This can be directly related to the small-angle scattering patterns that feature a bump in the SAXS and in particular in the SANS patterns at s = 0.135 nm−1 (marked with arrows). The form factor of platelets with a thickness of 2 unit cells in this range exhibits a broad maximum, and thus, these platelets contribute significantly more to the fits. For the other two suspensions the thickness distribution is broader, and thus, the oscillations for the form factors of platelets with different thicknesses average out in such a way that no bump can be observed. A direct quantitative comparison between the fitted thickness distributions and the melting curves is rather difficult. For the DLPC-stabilized suspension the contribution of platelets with

Figure 6. Map of the fit target function values obtained for the best simultaneous SAXS and SANS fits for each simulated combination of the inner (disl) and outer (dosl) stabilizer layer shell thicknesses for (a) the dilute (3%) and (b) the native (10%) tripalmitin suspension stabilized by DLPC. For the native suspension, for each pair (disl, dosl) the best target value from the scanned range of σst was chosen for the plot. 8815



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suspension. Remarkably, the fourth order and the fifth order of the stack-related interferences in the SAXS pattern of the DLPC-stabilized suspension possess almost the same scattered intensity as the third order, since they are enhanced by a constructive interference with the scattering from the underlying form factor maximum (marked with an arrow) of platelets with a thickness of two unit cells. The simultaneous fits can again reproduce the SAXS and SANS patterns with its stack-related interference maxima in the lower s-range very well. The best fitted shell thicknesses disl and dosl are exactly the same as for the dilute suspensions (cf. Supporting Information Tables S1 and S2). Also the fitted EDs and NSLDs for the stabilizer shells and the dispersion medium (cf. Supporting Information Table S2) are very similar to those from the dilute suspensions. From further reasonable fits, a similar range for the stabilizer shell thicknesses disl and dosl is found as for the dilute suspensions (cf. distribution of the black dots in Figure 6b). Again these fits were used for the calculation of mean values, standard deviations, and maximum deviations from the mean of the fit parameters. The best fitted values for the volume fractions d2 and d3 of stacks consisting of two and three platelets, respectively, the total volume fraction ψ of all remaining isolated platelets, and the variances σst for the repeat distances dst are listed in Table S2. In Figure 7b−d the distribution of the best fitted volume fractions ψci of the isolated platelets with different thicknesses and volume fractions (1−ψ)di of stacks with different numbers of platelets per stack is visualized for the three native suspensions. Again the black dots and error bars indicate mean values and maximum deviations obtained from the statistical analysis of all reasonable fits. It turns out that the native suspension stabilized by DLPC can be modeled solely with stacks consisting of three platelets (1−ψ = 1 and d2 = 0). For the DOPC-stabilized native suspension on average about 6 vol % of the platelets remain as single platelets in the dispersion (with the same platelet thickness distribution as for the dilute suspension in Figure 5c). 12 and 89 vol % of the platelets are self-assembled in stacks of two and three platelets, respectively (all volume fractions given without renormalization to 1). The SAXS and SANS patterns of the native S100-stabilized suspension which exhibit relatively weak stack-related interference maxima can be reproduced by a mixture of about 29 vol % of isolated platelets (ψ = 0.292) and stacks (about 69 vol %) consisting of two platelets. On average the sum over all volume fractions (cf. eq 2) in Table S2 deviates by less than 8% from 1. For the fitted scaling factor χ for the SAXS curves the situation is very similar as for the dilute suspensions; deviations of less than 15% from the ideal value of 1 are found. This also proves the strength of the XNPPSA method in reproducing simultaneously the rather complicated small-angle X-ray and neutron scattering patterns of self-assembled nanocrystal structures on an absolute scale and, in doing so, to refine their size and volume fraction. Thereby the small-angle scattering patterns are computed on the basis of a detailed structural model. There is no need to use any peak functions for the 001 Bragg peak and the stack-related maxima in conjunction with Scherrer’s equation, as is often done in other studies.29,30 Scherrer’s equation provides estimates for the mean platelet thickness (Bragg peak) and stack size (stack-related maxima) but no distribution functions and cannot make quantitative statements on the amount of the stacked-lamellae in the dispersions.

thicknesses of 1, 2, and 3 tripalmitin unit cells adds up to (c1 + c2 + c3)/∑ici = 96% where the largest contributions stems from platelets with thicknesses of 2 and 3 unit cells (87%). From the μDSC measurements a contribution of about 80% for the corresponding melting range of 45−56 °C was estimated which is a bit smaller but still fairly well agrees with the 87% found by the XNPPSA method. The scaling factor χ for the SAXS curve and the sum over all volume fractions ci was found to deviate less than 15% and 10% from the ideal value of 1, respectively. Such small scaling factors are necessary to take into account errors in the calibration of the absolute scale and sample concentration (SAXS and SANS), errors in the determination of the exact capillary thickness (SAXS), and small contaminations of the samples with H2O that reduce the coherent scattering from the particles (SANS). In the fits both SAXS and SANS data help to identify the distribution of the platelet thicknesses via the broadening of the 001 Bragg peak (in particular SAXS) and the form factor of the platelets (bump in the SANS pattern of the DLPC stabilized suspension). SAXS and in particular SANS are necessary for a proper characterization of the stabilizer layer. Simulations and Fits: Native Tripalmitin Suspensions (10%). The scattering patterns for the three native tripalmitin suspensions (10%) are shown in Figure 7a. Pronounced stackrelated interferences are found for the DOPC-stabilized suspension and in particular for the DLPC-stabilized

Figure 7. (a) Best simultaneous fits for the native tripalmitin dispersions (10%) stabilized by DLPC, DOPC, and S100, respectively. The curves where scaled with the factors next to the curves for a better representation. For the SAXS curve of DLPC the first 5 orders of the stack-related interferences are indicated. The fitted coefficients ψci and (1−ψ)di are shown in (b)−(d). The latter coefficients represent the volume fractions of stacks consisting of i particles. The remaining fit parameters including the electron densities ρX and neutron scattering length densities ρn for the inner and outer stabilizer layers are listed in Table S2. 8816



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suspension stabilized by the saturated lecithin DLPC this is surprising with regard to the results from Bunjes et al.8 but can be understood when realizing that the main phase transition temperature of DLPC is much lower than the crystallization temperature of the supercooled nanodroplets, as it is for the unsaturated lecithin DOPC. By use of the recently established XNPPSA method,4 it could be demonstrated that it is possible to fit the rather complicated SAXS and SANS patterns of the native suspensions simultaneously on an absolute scale. Thereby, the interference maxima in the small-angle patterns can be well reproduced when platelet stacks are included as new structural elements into the simulation model. The stack-related interference maxima in the scattering patterns of the DLPC- and DOPCstabilized suspensions are significantly enhanced with respect to those in S100-stabilized suspensions. It was quite surprising that the amount of platelets found to be self-assembled in stacks compared to the amount of remaining isolated platelets in the dispersions significantly depends on the stabilizing phospholipid. The fraction of isolated platelets not assembled in stacks drops from about 30% in native S100-stabilized suspensions to a few percent for DOPC-stabilized suspensions. The DLPC-stabilized suspension can be fully described when assuming that all particles are assembled in stacks. The distributions of the platelet thicknesses were obtained with molecular resolution by analyzing the SAXS and SANS patterns of the corresponding dilute dispersions. In accordance to the results from microcalorimetric measurements, it can be concluded that the platelets in suspensions stabilized by DLPC and DOPC are significantly thinner than those stabilized by S100 and that the distribution of the platelet thicknesses for DLPC-stabilized platelets is narrower than for DOPC- and S100-stabilized platelets. The higher diameter-to-thickness aspect ratio found for the DOPC-stabilized platelets and in particular for the DLPC-stabilized platelets explains their higher tendency to self-assemble in stacks compared to the S100stabilized platelets. The presented methodical approach for the detailed structural characterization of drug-free platelet stacks constitutes an essential prerequisite to study corresponding drugloaded stacks in the future. First very encouraging results could already be obtained in studies on the structure of DNA loaded dispersions where the DNA is sandwiched between cationically modified tripalmitin platelets. For these experiments it is in the same way essential to use a simultaneous SAXS and SANS evaluation as it could be demonstrated for the systems presented here. The XNPPSA method could also be useful for the analysis of SAXS and SANS patterns of other dispersions of stacked organic and inorganic (e.g., clay suspension30) platelets.

However, it should be noted that also for the XNPPSA method the fit parameters for the total volume fraction of stacks (1−ψ) and also the relative stack volume fractions di might be to a certain extent correlated to the variance of the repeat distances, σst. Within certain limits, higher variances σst might be compensated with stacks consisting of a larger number of platelets to generate sufficiently high interference maxima. For the three native suspensions studied here, the mean values for σst (cf. Table S2) are with about 3.0 nm virtually the same. Since the σst values are unknown, the fitted values for ψ and the distribution of the di should be considered as semiquantitative measures only. For the suspensions stabilized by DLPC and DOPC, fits were also carried out that included in addition stacks of four and five platelets (Nst = 5). However, no significant improvement in the fit quality was observed (data not shown). Finally, for the native suspensions, it can be concluded from the scattering patterns itself and the fitted values for ψ and the di that despite a similar composition of the sample the stack formation is significantly enhanced for the DOPC-stabilized suspension and in particular for the DLPC-stabilized suspension compared with the suspension stabilized by S100. This can be attributed to the different platelet geometries. Because of their larger platelet diameters (cf. PCS diameters in Table 2) and smaller mean platelet thicknesses (cf. ⟨i⟩V and ⟨i⟩N in Table 3) the platelets for the DLPC- and DOPCstabilized suspensions possess larger diameter-to-thickness aspect ratios than those stabilized with S100. This enhances the self-assembly of more platelets into stacks in the DLPCand DOPC-stabilized suspensions because of the higher exclusion volumes for platelets with larger aspect ratios. As for the dilute suspensions mostly the SANS data determine the stabilizer layer properties of the self-assembled platelets. Both SAXS and SANS help to estimate the fraction of stacks in the dispersion and the variance in the repeat distance σst. Contrast variation of the aqueous solvent or deuteration of the lecithins in SANS experiments would surely help in the structural characterization of the lecithin stabilizer layer. However, regarding that our main focus lies on the selfassembled structures, platelets made from protiated tripalmitin and stabilizers, dispersed in D2O, provide the best scattering contrast for the stacked lamellae and a low incoherent background. Using deuterated tripalmitin in H2O would provide a high coherent scattering contrast for the lamellae, too, but has the disadvantage of high incoherent backgrounds if not using neutron spin polarization analysis.31 Furthermore, deuterated tripalmitin-d98 has the drawback that it diminishes the 001 Bragg reflection, a key feature in the scattering patterns, due to a minimum of the structure factor of tripalmitin-d98 at the position of the 001 Bragg peak.4

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CONCLUSION Native tripalmitin suspensions (10 wt % tripalmitin) stabilized by the lecithins DLPC and DOPC, respectively, were structurally characterized by PCS, WAXS, μDSC, and freeze− fracture TEM and studied for the first time by a combination of SAXS and SANS to determine the size and amount of selfassembled platelet structures in the dispersions. A native tripalmitin suspension stabilized with the lecithin blend S100 has been taken as a reference. The scattering and microcalorimetric data as well as the electron micrographs prove that the triglyceride nanoparticles in all three suspensions crystallize predominantly in their stable β-modification. In the case of the

ASSOCIATED CONTENT

S Supporting Information *

Plots of the PCS measured correlation functions and WAXS patterns for the native suspensions as wells as tables of the fit parameters for the SAXS and SANS patterns in Figures 5a and 7a. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49-(0)9131-8525189. Fax: +49-(0)9131-8525182. 8817



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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Deutsche Forschungsgemeinschaft (DFG) through Project UN267/2-1 and the Cluster of Excellence Engineering of Advanced Materials and is based on SANS experiments performed at the Heinz Maier-Leibnitz Zentrum (MLZ) at the FRM II, Garching, Germany. We acknowledge the RRZE for use of the Woodcrest HPC cluster and Materials Science Lab of Heinz Maier-Leibnitz Centre (MLZ), Technische Universität München (TUM) in collaboration with Helmholtz-Zentrum Geesthacht (HZG), for granting the SAXS beamtime.

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