Article pubs.acs.org/JPCB
Structural Characterization of Lecithin-Stabilized Tetracosane Lipid Nanoparticles. Part II: Suspensions M. Schmiele,† S. Busch,‡ H. Morhenn,§ T. Schindler,† T. Schmutzler,† R. Schweins,∥ P. Lindner,∥ P. Boesecke,⊥ M. Westermann,# F. Steiniger,# Sérgio S. Funari,▽ and T. Unruh*,† †
Professur für Nanomaterialcharakterisierung (Streumethoden), Friedrich−Alexander−Universität Erlangen−Nürnberg, Staudtstr. 3, 91058 Erlangen, Germany ‡ German Engineering Materials Science Centre (GEMS) at Heinz Maier-Leibnitz Zentrum (MLZ), Helmholtz-Zentrum Geesthacht GmbH, Lichtenbergstr. 1, 85747 Garching, Germany § Heinz Maier-Leibnitz Zentrum (MLZ), Technische Universität München, Lichtenbergstr. 1, 85747 Garching, Germany ∥ DS/LSS, Institut Laue-Langevin (ILL), 71 Avenue des Martyrs, CS20156, 38042 Grenoble CEDEX 9, France ⊥ European Synchrotron Radiation Facility (ESRF), 71 Avenue des Martyrs, CS40220, 38042 Grenoble CEDEX 9, France # Center for Electron Microscopy of the Jena University Hospital, Ziegelmühlenweg 1, 07743 Jena, Germany ▽ HASYLAB, Notkestr. 85, 22603 Hamburg, Germany S Supporting Information *
ABSTRACT: Using photon correlation spectroscopy, transmission electron microscopy, microcalorimetry, wide-angle X-ray scattering (WAXS), and smallangle X-ray and neutron scattering (SAXS, SANS), the structure of 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-stabilized colloidal tetracosane suspensions was studied from the molecular level to the microscopic scale as a function of the temperature. The platelike nanocrystals exhibit for tetracosane an unusual orthorhombic low-temperature crystal structure. The corresponding WAXS pattern can be reproduced with a predicted orthorhombic unit cell (space group Pca21), which usually occurs only for much longer even-numbered n-alkanes. Special emphasis was placed on the structure of the DMPC stabilizer layer covering the nanocrystals. Their structure was investigated by SAXS and SANS, using suspensions with different neutron scattering contrasts. As for the emulsions in Part I, the crystallized nanoparticles are covered by a DMPC monolayer. Their significant smaller thickness of 10.5 Å (for the emulsions in Part I: 16 Å) could be related to a more tilted orientation of the DMPC molecules to cover the expanded surface of the crystallized nanoparticles.
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scanning calorimetry (DSC) cooling runs.3,5,8,10 These pretransitions were assigned to a crystallization of the HM emulsifiers (and small amounts of the oil) at the oil−water interface prior to the crystallization of the full droplet. The formation of such a solidified emulsifier shell can induce an interfacial heterogeneous nucleation of the droplets. In the case of triglyceride nanodispersions, it was observed that HM lecithins can significantly slow down the polymorphic transformation of the crystallized nanoparticles from the metastable α-modification (spherical or barrel-shaped nanoparticles) to the stable β-modification (platelets).9,10,14 Because such an α−β polymorphic transformation is accompanied by a vigorous surface expansion, this effect was attributed to the formation of such a rigid shell by the HM lecithins. Also, the amount of the emulsifier and the presence of other cosurfactants are important parameters because the surface coverage of the nanoparticles by surfactants affects their
INTRODUCTION The structure of the lipid−water interface in lipid nanodispersions is important in many respects in pharmaceutical and food science. The amount and type of emulsifier and further additives can have a significant impact on the crystallization behavior, polymorphic transitions, and stability of the lipid nanoparticles as well as the release and protection of encapsulated active substances. Emulsifiers with a low melting point or main phase-transition temperature (LM emulsifiers, typically with short or unsaturated acyl chains) with respect to the crystallization temperature, Tc, of supercooled oil droplets do not exhibit a pronounced effect on Tc. In contrast, emulsifiers with a high melting point/main phase-transition temperature (HM emulsifiers, typically with long acyl chains and, in particular, those with chain lengths close to that of the oil) at the oil−water interface of alkane1−5 and pure or mixed triglyceride emulsion droplets6−13 can considerably accelerate their nucleation, that is, leading to much higher Tc. Emulsions stabilized by HM emulsifiers often exhibit characteristic precrystallization events prior to the main crystallization of the droplets in differential © 2016 American Chemical Society
Received: March 10, 2016 Revised: May 19, 2016 Published: May 27, 2016 5513
DOI: 10.1021/acs.jpcb.6b02520 J. Phys. Chem. B 2016, 120, 5513−5526
Article
The Journal of Physical Chemistry B Table 1. Chemical Composition of the TCS Dispersions (given in wt %) sample
composition
H−H−D H−D−D D−H−D D−D-D D−HD−HD
2.71% 2.75% 3.11% 3.10% 3.20%
TCS TCS TCS-d50 TCS-d50 TCS-d50
H−H−D (15%) H−H−H (10%)
13.78% TCS 10.00% TCS
0.92% 0.99% 0.92% 0.98% 0.71% 0.26% 4.66% 3.33%
DMPC DMPC-d54 DMPC DMPC-d54 DMPC DMPC-d54 DMPC DMPC
0.092% 0.094% 0.095% 0.093% 0.095%
NaGC NaGC NaGC NaGC NaGC
96.28% 96.17% 95.87% 95.82% 29.79% 65.94% 81.10% 86.33%
0.46% NaGC 0.33% NaGC
D2O D2O D2O D2O H2O D2O D2O H2O
scanning calorimetry (μDSC) and temperature-resolved wideangle X-ray scattering (WAXS) for that purpose. After that we focus on the structure of the interfacial DMPC stabilizer layer of the tetracosane nanocrystals and discuss structural changes with respect to the stabilizer layer of the corresponding emulsion droplets from Part I. The structure of the stabilizer layer is analyzed by a combined analysis of smallangle X-ray and neutron scattering (SAXS, SANS) data of the nanosuspensions.
crystallization and polymorphic transitions and by this, in the case of an insufficient stabilization, promotes particle aggregation and a gelation of the dispersions.15−18 The confined space in nanoparticles and the influence of emulsifiers can not only stabilize a polymorph in the nanoparticles, which is only metastable in the bulk lipid, but may also lead to crystal structures that are not adopted by the bulk lipid.19−21 In food science, the structure of the interfacial layer can be important with regard to the oxidation of substances with potential health-promoting properties such as β-carotene and lipids rich in polyunsaturated fatty acids (PUFAs, e.g., ω-3 fatty acids). 22−26 While both LM and HM lecithins were approximately equally good in preventing the oxidation of βcarotene in triglyceride nanoemulsions, in solid lipid nanoparticles, LM lecithin was much less efficient than HM lecithin.27 This was attributed to the HM lecithin’s ability to form a rigid emulsifier shell and the hindered polymorphic α−β transformation. (The less ordered α modification is believed to better accommodate foreign molecules than the β-modification.9,14) Recently, Salminen et al. demonstrated that lipid nanoparticles prepared from a mixture of fish oil and the highmelting triglyceride tristearin and a HM lecithin stabilizer are physically and oxidatively stable carriers for ω-3-rich fish oil.28,29 They argued that the good oxidative protection is due to a formation of a protective shell of crystallized tristearin around a liquid fish oil core when the crystallization of the tristearin shell is induced by the solidified interfacial layer of the HM lecithin via interfacial heterogeneous nucleation. For pharmaceutical applications the long-term stability and biodegradability of lipid drug carriers and the release or protection of encapsulated drugs are important parameters, which can be affected by the structure of the interfacial stabilizer layer and the associated polymorphic transformations of the carrier lipid.8−10,14,30,31 Despite their importance, little is known about the structure of the stabilizer layer of lipid nanoparticles. In Part I (10.1021/acs.jpcb.6b02519), the structure of the stabilizer layer was analyzed in lipid nanoemulsions, using tetracosane (C24H50) nanodispersions, stabilized by the lecithin DMPC, as a model system. Here in Part II we use the same nanodispersions. At first we study their melting and crystallization behavior. Because a reliable assignment of structural transformations between known (or unknown) modifications of the matrix lipid upon thermal treatment by differential scanning calorimetry (DSC) and ultrasonic velocity and attenuation measurements can be difficult, 4,5,32,33 such measurements should be complemented by other techniques, such as temperatureresolved X-ray diffraction.8,10,19,20 Here we use microdifferential
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MATERIALS AND METHODS
Materials, Samples, and Notation. The same DMPCstabilized tetracosane (TCS) nanodispersions as in Part I were used in this study. The sample notation follows the one from Part I: Three letters indicate the levels of deuteration of TCS (core), DMPC (stabilizer layer), and the aqueous dispersion medium, respectively. The first letter can be H (TCS) or D (fully deuterated TCS-d50), the second can be H (DMPC), D (chain deuterated DMPC-d54), or HD (molar ratio DMPC/ DMPC-d54 = 3:1), and the last can be H (H2O), D (D2O), or HD (volume ratio H2O/D2O = 2:1). The different levels of deuteration for TCS, DMPC, and water in the SANS experiments allow a more reliable investigation of the structure of the DMPC-stabilizer layer as with SAXS measurements alone. Five dispersions, namely, H−H−D, H−D−D, D−H−D, D− D−D, and D−HD−HD, were prepared (each, if fully protiated, with 3% TCS, 1% DMPC, and 0.1% of the bile salt sodium glycocholate (NaGC) in water). Additionally, two dispersions with higher TCS concentrations, H−H−D (15%) and H−H− H (10%), were prepared. The composition of all dispersions is listed in detail in Table 1. Photon Correlation Spectroscopy (PCS). PCS measurements of the TCS suspensions were done as described in Part I. Microdifferential Scanning Calorimetry (μDSC). A Micro DSC III (Setaram, Caluire-et-Cuire, France) was used for the microcalorimetric heating and cooling scans. 150−500 mg of sample were heated in a sealed stainless-steel cell from 6 to 70 °C and subsequently cooled back down to 6 °C at a scan rate of 0.1 K/min. The reference cell was filled with the same amount of water. The enthalpies of fusion and crystallization can be obtained by integration within a region of interest of the heating and cooling curves, respectively. For suspensions prepared with deuterated substances (TCS-d50, DMPC-d54, D2O), the enthalpies were corrected for the differences in weight density between the deuterated and protiated substances. Cryo- and Freeze-Fracture Transmission Electron Microscopy (TEM). Cryo- and freeze-fracture TEM preparations were performed at the Center for Electron Microscopy of the Jena University Hospital. For cryo-TEM, 2 μL of the H− 5514
DOI: 10.1021/acs.jpcb.6b02520 J. Phys. Chem. B 2016, 120, 5513−5526
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sizes of 0.9 × 0.9 mm2 (tungsten blades) and 0.7 × 0.7 mm2 (scatterless Si-blades) at a distance of Lc = 1.5 m, provides an X-ray photon flux of about 2.09 × 107 ph/s at the Cu Kα wavelength of λ = 1.5418 Å at the sample position. The sample holder (the same as at the ID02 beamline) was mounted in air directly behind the evacuated collimation system and in front of the evacuated detector tube. The patterns were recorded with a 2D Pilatus3 300 K detector (Dectris AG, Baden, Switzerland) at a sample−detector distance of 145.7 mm. The scattering patterns were corrected for sample transmission (measured with a PIN-diode) and thickness and exposure time (300 s for powders and 900−1800 s for the dispersions). Modeling the SAXS and SANS Data of the Suspensions (XNPPSA Method). The X-ray and neutron powder pattern simulation analysis (XNPPSA) method allows the computation of SAXS and SANS patterns of nanocrystalline dispersions. Typically, structural elements like individual nanocrystals of different thicknesses and assemblies of nanocrystals with different numbers of nanocrystals are simulated. The frequencies of such different structural elements can be determined by fitting a linear combination of their macroscopic scattering cross sections (MSCSs,dΣ/dΩ) to the experimental SAXS and SANS patterns.34−38 In previous studies on aqueous suspensions of lecithin-stabilized triglyceride nanocrystals, the XNPPSA method has been applied successfully to determine (1) a volume-weighted distribution of the nanocrystal thicknesses,34−38 (2) the structure of their lecithin stabilizer35−37 and, at higher concentrations, (3) the size and amount of self-assembled nanocrystal structures in the suspensions.34,37 Here we use the XNPPSA method for points (1) and (2), that is, to determine a distribution of the nanocrystal thicknesses and the structural arrangement of the lecithin molecules at the interface between the TCS nanocrystals and the aqueous dispersion medium. Again, as in the case of the emulsions in Part I, we use suspensions with low TCS concentrations. The fits are restricted to the s range between 0.01 and 0.5 nm−1. This makes it possible to neglect interparticle interactions, significantly simplifying the simulations. Later in the Results section it is demonstrated by TEM that the nanocrystals possess a platelike shape. In the simulations, eight ensembles, each consisting of 200 individual nanocrystals, were used. While all nanocrystals in ensemble i share a common thickness of i molecular layers of TCS, their two lateral dimensions l1 and l2 are subject to Gaussian normal distributions with a mean and variance of μ1,2 ± σ1,2 = 50 ± 20 nm (μ1,2 ± σ1,2 = 75 ± 25 was also tested). Later in the Results section it is also shown by WAXS experiments that the TCS nanocrystals do not crystallize in the triclinic crystal structure (space group P-1, Z = 1 molecule per unit cell) of bulk TCS39,40 but in an orthorhombic structure (space group Pca21, Z = 4), whose cell parameters and atomic coordinates are calculated in Appendix B in the Supporting Information (SI). The crystal structure is visualized in Figure S4. The cell consists of two molecular layers of TCS, which would allow with the present simulation method only simulations of nanocrystals with an even number of molecular layers of TCS; however, in the suspensions also crystals with an odd number of molecular layers can be present (cf. Figure 1). For that purpose also crystals built up by one, three, five, and seven molecular layers of TCS are calculated using the orthorhombic cells from Figure S5.
H−H (10%) and D−H−D suspensions was applied to a copper grid covered by a holey carbon film (1.2/1.3 Quantifoil Micro Tools, Jena, Germany), and excess liquid was blotted automatically for 2 s between two strips of filter paper. Subsequently, the sample was rapidly plunged into liquid ethane (cooled to −180 °C) in a cryobox (Carl Zeiss NTS, Oberkochen, Germany). Excess ethane was removed with a piece of filter paper. The sample was transferred with a tiltable cryo-holder (Gatan 626-DH, Pleasanton, CA) into the precooled cryo-electron microscope (Philips CM 120, The Netherlands) operated at 120 kV and viewed under low-dose conditions. The images were recorded with a 1k CCD Camera (FastScan F114, TVIPS, Gauting, Germany). The observation of the internal structure of the TCS plates was difficult because at high magnifications the samples degraded quickly. For a tilt series a sequence of six micrographs was taken (0−25° in steps of 5°). The image shift was calculated by cross-correlation using the software EMMENU V3.0 (TVIPS). For freeze-fracture a small droplet of the H−H−H (10%) suspension was embedded between two copper sandwich profiles. The sandwiches were 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 were 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 was deposited from the top 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. Small-Angle Neutron Scattering (SANS). SANS measurements of the TCS suspensions were performed as described in Part I at the D11 instrument at ILL in Grenoble at a temperature of 30 °C. Small- and Wide-Angle X-ray Scattering (SAXS, WAXS). SAXS measurements of the TCS suspensions were recorded as described in Part I at the ID02 beamline at ESRF in Grenoble at a temperature of 30 °C. WAXS patterns of the TCS suspensions (30 °C) were simultaneously recorded with the SAXS patterns at the ID02 beamline. The data reduction of the WAXS patterns follows one of the SAXS patterns. WAXS patterns of TCS and β-tripalmitin powder were measured at 25 °C at the former A2 beamline at HASYLAB (DESY, Hamburg, Germany) for 10 s with a line detector (sample−detector distance 135 mm, wavelength 1.5 Å). The 2θ scale of the WAXS patterns was calibrated with the recorded pattern of β-tripalmitin, and reference values for the peak positions were provided by the A2 beamline. To allow a comparison with the WAXS patterns of the suspensions measured at the ID02 beamline, we converted the 2θ scale to angles corresponding to a wavelength of 1 Å. Temperature-resolved WAXS patterns for TCS and TCS-d50 powder, the dispersions H−H−D (15%) and D−H−D, and D2O were recorded with a lab-based microfocus SWAXS camera (Ganesha 300 XL+ instrument, SAXSLAB ApS/JJ X-ray Systems ApS, Skovlunde, Denmark).34 A 30 W X-ray microfocus tube with a parallel beam multilayer optics (GeniX 3D, Xenocs, Sassenage, France), followed by a collimation system consisting of two pinholes with aperture 5515
DOI: 10.1021/acs.jpcb.6b02520 J. Phys. Chem. B 2016, 120, 5513−5526
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The MSCS dΣ/dΩ of the suspensions with 3% TCS can be expressed as a linear combination of the simulated (dΣ/dΩ)i of the eight ensembles dΣ = dΩ
8
8
⎛ dΣ ⎞ ∑ ci⎜⎝ ⎠⎟ + A with dΩ i
∑ ci = 1
i=1
(1)
i=1
The constant A > 0 is used in the fits to match the (incoherent) backgrounds at large s values. The fitted linear coefficients, ci, can be interpreted as the volume fraction of the structural elements in the ith ensemble, which must fulfill the completeness relation on the right side of eq 1. Thus, in the present case the ci provides a volume-weighted distribution of the nanocrystal thicknesses.36 Assuming that the nanocrystal diameters and thicknesses are uncorrelated, number weights, ni, can be estimated from the ci36 as
Figure 1. Simulation model for the nanocrystalline TCS suspensions.
Because of the orthorhombic unit cells the nanocrystals possess a cuboid shape in the simulations. The thickness of one molecular layer TCS along the direction of the reciprocal lattice vector G001 is given by d002 = d001/2 = c/2 = 6.53/2 = 3.27 nm (cf. Figure 1). As in the case of the emulsions in Part I, the DMPC stabilizer layer covering the TCS nanocrystals is modeled with two shells (lower inset in Figure 1). Simulations with different inner and outer shell thicknesses in the ranges 4 ≤ di ≤ 10 Å and 2 ≤ d0 ≤ 10 Å were carried out in steps of 1 Å. The electron densities (EDs) and neutron scattering length densities (NSLDs) of the X/n inner and outer shells and the dispersion medium (ρX/n i , ρo , X/n and ρm ), which are included in the mathematical expressions of the simulated MSCSs (dΣ/dΩ)i of the ensembles, can be determined later in the fits to the SAXS and SANS patterns.36,37 For the structural arrangement of the DMPC molecules in the stabilizer layer, model I from Part I (mixture of the DMPC headgroups and water in the outer shell and mixture of the DMPC acyl chains and water in the inner shell) was applied, using the same fit constraints as in the case of the emulsions (cf. Appendix C.1 (supporting information in Part I)) with the EDs and NSLDs of water and DMPC (Table 2). Because the nanocrystals can assume all orientations in the dispersions, a powder average is carried out for each nanocrystal in the computations of their MSCSs.36
−1 8 ci ⎛ ci ⎞ ni = ·⎜⎜∑ ⎟⎟ i ⎝ i=1 i ⎠
(2)
A volume- and number-averaged thickness of the nanocrystals, ⟨i⟩V and ⟨i⟩N (in units of d002 = 3.27 nm), can be calculated as36 8
⟨i⟩V =
∑i = 1 ci·i 8
∑i = 1 ci
8
⟨i⟩N =
∑i = 1 ni ·i 8
∑i = 1 ni
8
=
∑i = 1 ci 8
∑i = 1
ci i
(3)
The computer simulations were performed with the C++ program XNDiff on the high-performance computing clusters Lima and Woodcrest at Regionales Rechenzentrum Erlangen (RRZE). The fits were carried out using the Mathematica program BatchMultiFit. The source codes of XNDiff and BatchMultiFit are available as free software.43 In contrast with the fits of the emulsions in Part I, where different size distributions were allowed for the five emulsions, we use for the sake of simplicity the same distributions for the lateral sizes and thicknesses of the nanocrystals for all five suspensions. Allowing individual lateral size distributions for each suspension would significantly increase the extent of the computer simulations. Different platelet diameters become
Table 2. Molecular Volumes, V, Neutron Scattering Lengths, b, NSLDs, ρn = b/V, Number of Electrons, Z, and ED, ρX = Z/V, Used in the Fits of the TCS Suspensions (30 °C)a chem. formula TCS (Pca21) TCS-d50 (Pca21) TCS (Pca21, LD) TCS-d50 (Pca21, LD) DMPC DMPC-h DMPC-ch DMPC-cH-D54 water heavy water
C24H50 C24D50 C24H50 C24D50 C36H72NO8P C10H18NO8P C26H54 C26D54 H2O D2O
ρ (g/cm3) b
0.943 1.083b,c 0.915d 1.051d,c
0.996f 1.103g
V (Å3)
b (fm)
ρn (10−6 Å−2)
Z (−)
ρX (nm−3)
595 595 613 613 1101e 319e 782e 782 30.0 30.2
−27.4 493.1 −27.4 493.1 31.0 60.1 −29.1 533.0 −1.68 19.1
−0.46 8.29 −0.45 8.04 0.28 1.88 −0.37 6.82 −0.56 6.32
194 194 194 194 374 164 210 210 10 10
326.0 326.0 316.5 316.5 339.7 514.1 268.5 268.5 333.3 331.1
a For (heavy) water, V was calculated via V = M/(ρ·NA), where ρ denotes the weight density of the substance, M is the molar mass, and NA = 6.022 × 1023 mol−1 is the Avogadro constant. For DMPC(-d54) the values are given for the phosphocholine headgroup (h, including the two carbonyl groups) and the remaining molecular part of the two C13 acyl chains (ch). In the case of the orthorhombic Pca21 crystal structure of TCS(-d50), LD denotes the structure with a 3% lower density. bUsing the orthorhombic cell with a volume of 2379.6 Å3 from Tetracosane_Pca21_C24H50.cif in the SI (four molecules per cell). cObtained from ρ for TCS (Pca21) by respecting the different molar masses for TCS-d50 and TCS. dUsing the orthorhombic cell with a volume of 2451.8 Å3 from Tetracosane_Pca21_C24H50_LD.cif in the SI (four molecules per cell). eTaken for the Lα phase at 30 °C from ref 41. fTaken at 30 °C from http://antoine.frostburg.edu/chem/senese/javascript/water-density.html. gTaken at 30 °C from ref 42.
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The Journal of Physical Chemistry B apparent only at very small s values (cf. SAXS patterns in Figure S1 in the SI); however, in the considered fit range between 0.01 and 0.5 nm−1 the SAXS patterns of all five suspensions show only minor deviations. Therefore, again only the SAXS pattern of one suspension (H−H−D) is included in the simultaneous fits with the five SANS patterns. Allowing individual thickness distributions would tremendously increase the number of fit parameters because instead of only eight parameters for the simultaneously fitted linear coefficients ci, 5·8 = 40 linear coefficients would be required. The simulated SANS patterns of the suspensions were smeared prior to the fitting using the experimental wavelength spread of the D11 beamline. A scaling factor 0.8 < χ < 1.2 for the simulated SAXS pattern was permitted to account for possible errors in the determination of the absolute scale, and similarly the completeness relation in eq 1 was relaxed such that the sum may vary within 20% bounds around 1.
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RESULTS AND DISCUSSION Sample Characterization by PCS, WAXS, TEM, and μDSC. PCS. The PCS-measured harmonic z -average of the hydrodynamic diameters, dPCS, and polydispersity indices, PdI, are listed for all suspensions in Table S1 in the SI and are visualized in Figure 2. The PCS-measured correlation functions
Figure 3. (a) WAXS patterns for TCS powder (black circles, A2 beamline) and the TCS suspension H−H−D (15%) (red squares, ID02 beamline). While the powder shows the characteristic peaks for TCS’s triclinic structure,21,44 the TCS nanoparticles in the suspension have crystallized in another modification. (b) WAXS pattern of the TCS suspension H−H−D (15%) Iexp (black circles, ID02 beamline)
Figure 2. PCS-measured harmonic z-averages of the hydrodynamic particle diameters (gray bars) and polydispersity indices (black dots) of the TCS suspensions studied.
and the fit Icalc (red curve) obtained with Maud for the orthorhombic Pca21 structure. The green line below shows the difference ΔI = Iexp − Icalc between the experimental and simulated pattern.
could be fitted with a monomodal size distribution. The dPCS values range between about 68 and 86 nm. The PdI values indicate size distributions with moderate polydispersities. For spherical particles like the emulsion droplets in Part I the harmonically z-averaged hydrodynamic diameters correspond approximately to the harmonic z-average of the distribution of their “real” diameters; however, as it is demonstrated later, the nanocrystals in the suspensions possess a more platelike shape. The measured dPCS values for the suspensions can therefore be regarded only as a crude approximation for the lateral diameters of the nanocrystals. WAXS. The WAXS pattern of TCS powder is visualized in Figure 3a. The powder diffractogram exhibits the characteristic peak positions of the triclinic low-temperature crystal structure of TCS.21,44 The triclinic structure is visualized in Figure S2 in the SI, using the CIF-file from Gerson et al.39,40 TCS-d50 powder shows the same characteristic peak positions as TCS powder and thus adopts a triclinic structure, too (cf. Figure S6). The pattern of the TCS suspension H−H−D (15%) measured at 30 °C is also shown in Figure 3a. Obviously, the nanocrystals do not crystallize in a triclinic structure. The suspensions with 3% TCS exhibited WAXS patterns with the same peak positions (data not shown). The positions of the
diffraction peaks can be fitted with an orthorhombic cell (a = 4.98 Å, b = 7.56 Å, c = 65.69 Å). The longest lattice constant c is in very good agreement with those of odd-numbered nalkanes (orthorhombic cell, space group Pbcm)45,46 and corresponds to the length of two TCS molecules standing upright. The orthorhombic crystal structure of the nanocrystals can be considered as their low-temperature structure because the μDSC data shown later do not exhibit any further crystallization events 6 °C below the onset of the crystallization of the nanoemulsions at >30 °C. The nanocrystals did not crystallize in one of the known rotator phases of TCS (RI, RII, RV47) because the WAXS patterns feature besides the intense 110 and 200 reflections also reflections at higher s, in particular, the “herringbone” peak 210 at s = 3.32 nm−1. The 210 peak is characteristic for the low-temperature orthorhombic crystal structures of odd-numbered n-alkanes (cf. figure 6 in ref 48 and figures 3 and 8 in ref 44) and absent in the rotator phases due to rotational disorder of the alkane molecules.49 5517
DOI: 10.1021/acs.jpcb.6b02520 J. Phys. Chem. B 2016, 120, 5513−5526
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Figure 4. (a) Cryo-TEM micrograph of the DMPC-stabilized suspension H−H−H (10%) showing TCS nanocrystals with diameters typically
