A Reverse Micellar Mesophase of Face-Centered Cubic Fm3̅m

Article pubs.acs.org/Langmuir

A Reverse Micellar Mesophase of Face-Centered Cubic Fm3̅m Symmetry in Phosphatidylcholine/Water/Organic Solvent Ternary Systems Isabelle Martiel,† Laurent Sagalowicz,‡ and Raffaele Mezzenga*,† †

Food and Soft Materials Science, Institute of Food, Nutrition & Health, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland ‡ Nestlé Research Center, Vers-Chez-Les-Blanc, CH-1000 Lausanne 26, Switzerland S Supporting Information *

ABSTRACT: We report the formation of a reverse micellar cubic mesophase of symmetry Fm3̅m (Q225) in ternary mixtures of soy bean phosphatidylcholine (PC), water, and an organic solvent, including cyclohexane, (R)-(+)-limonene, and isooctane, studied by small-angle X-ray scattering (SAXS) and oscillatory shear rheology at room temperature. The mesophase structure consists of a compact packing of remarkably large reverse micelles in a face-centered cubic (fcc) lattice, a type of micellar packing not yet reported for reverse micellar mesophases. Form factor fitting in the pure L2 phase and in the Fm3̅m−L2 coexistence region yields quantitative estimations of the PC interface rigidity. The compact Fm3̅m structure results mainly from release of lipid tail frustration and hard-sphere interactions between monodisperse micelles, as suggested by a comparison with the Fd3̅m structure found in the PC/water/α-tocopherol system.

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INTRODUCTION Phospholipid-based nonlamellar mesophases1,2 offer a promising alternative to monoglycerides for delivery system applications in pharmaceutical, food, and personal care products. Phosphatidylcholine (PC), which forms only lamellar mesophases in water, 3 can be driven to self-assemble spontaneously into nonlamellar LLC mesophases by the addition of a third apolar component (oil modifier), for instance vitamin E,4 fatty acids,5 isooctane,6 decane,6 or cyclohexane,7 by increasing the temperature or the negative spontaneous curvature of the surfactant interface. Angelico et al.7 have investigated in detail the PC/water/ cyclohexane full ternary diagram, reproduced in Figure 1A. This diagram shows a great variety of nonlamellar mesophases: reverse hexagonal H2, reverse micellar cubic I2, nematic arrangement of reverse micellar rods N2 (equivalent to a positionally disordered hexagonal), reverse micellar L2. All these phases, except the N2, have a stability domain in equilibrium with excess water. In this initial report about the PC/water/cyclohexane ternary diagram, Angelico et al.7 described samples from the small area of cubic mesophase I2 as stiff and isotropic between crossed polarizers. The reverse type was established from the logical position within the phase sequence, between reverse hexagonal H2 and reverse micellar mesophases L2, and from the observation that the phase coexists with excess water, without being diluted to a liquid phase. It was proved that the mesophase consists of closed © 2013 American Chemical Society

reverse micellar aggregates by measuring the water and oil diffusion coefficient by NRM self-diffusion measurements. On the basis of limited SAXS data, the space group symmetry was tentatively assigned to Im3̅m (bcc packing). We propose here a different structure identification, a fcc close-packing (Fm3̅m space group). This is to the best of our knowledge the first report of the formation of a face-centered cubic micellar mesophase of symmetry Fm3̅m in reverse configuration by a small molecular surfactant.8 We also show that the exact same structure is formed by replacing cyclohexane with (R)-(+)-limonene, a food-compatible solvent, which has been used as an efficient phase modifier for monoglyceride-based mesophases.9 We also discovered the reverse Fm3̅m mesophase in the PC/water/isooctane ternary diagram, in which a reverse cubic micellar mesophase had been reported but not identified.6 To support our hypothesis, we studied in detail the structural parameters throughout the ordered micellar mesophase stability range and in coexistence with the adjacent disordered L2 phase and hexagonal H2 mesophase. The rheological behavior of the cubic mesophase was also investigated. Finally, we compare the fcc structure induced by isooctane, cyclohexane, or limonene with the Fd3m ̅ and L2 phases formed in presence of α-tocopherol.4 Received: November 7, 2013 Revised: December 2, 2013 Published: December 2, 2013 15805

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several times. The samples were then left in the dark for equilibration during a few days, up to several weeks in some cases. Small Angle X-ray Scattering (SAXS). SAXS measurements were performed with a MicroMax-002+ microfocused X-ray machine (Rigaku), operating at 4 kW, 45 kV, and 88 mA. The Kα X-ray radiation of wavelength λ = 1.5418 Å emitted at the Cu anode is collimated through three pinholes of respective sizes 0.4, 0.3, and 0.8 mm. The scattered intensity was collected on a two-dimensional Triton-200 X-ray detector (20 cm diameter, 200 μm resolution) for at least 30 min. The scattering wave vector is defined as q = 4π sin(θ)/λ, where 2θ is the scattering angle. The SAXS machine is equipped with two sample chambers with different sample-to-detector distances, giving access to q ranges of 0.005 to 0.22 Å−1 and 0.01 to 0.44 Å−1, respectively. The lowest angle chamber provides higher resolution in scattering curves. Unless stated otherwise, data was acquired from the higher angle, lower resolution chamber. Silver behenate was used for q vector calibration. Scattered intensity data were azimuthally averaged using SAXSgui software (Rigaku). Solid samples were loaded in a Linkam hot stage with temperature control in a cell formed by two thin mica sheets and a rubber O-ring 1 mm spacer. Liquid samples were filled into 1.5 mm diameter quartz capillaries, sealed with epoxy glue (UHU). The X-ray machine is thermostatted at 20 ± 0.1 °C, taken as room temperature. Measurements were conducted at room temperature, unless stated otherwise. Form-factor fits were performed using IGOR Pro 6 (Wavemetrics Inc., Lake Oswego, OR) with NIST package for SANS data analysis (version 3.00). Peak deconvolution was done using OriginPro 8.5 (OriginLab, Morthampton, MA).

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RESULTS AND DISCUSSION Mesophase Equilibration. As underlined by Angelico et al.,6,7 sample preparation in PC/water/oil ternary systems requires relatively long equilibration times. Contrary to the more studied monoglyceride-based LLC mesophases, it is not appropriate here to homogenize the sample by heating to the isotropic fluid state, because there is no transition to isotropic fluid below 200 °C.3 Consequently, we equilibrated the samples by mixing them thoroughly and let them equilibrate until the diffraction pattern no longer evolved, i.e., from a few days to several weeks depending on samples. The diffraction patterns remained essentially unchanged through heating−cooling (up to about 60 °C) and freezing−thaw events (down to about −18 °C), as well as over several months equilibration. The time and temperature stability of the mesophase are discussed separately in the Supporting Information. Space Group Identification. Figure 2A, C shows the SAXS pattern of PC/water/cyclohexane and PC/water/ limonene micellar cubic mesophase samples in excess water. The diffraction patterns present three unusually large but intense and defined peaks. To identify the mesophase structure, we checked the observed spacing ratio sequence against various LLC mesophase structure hypotheses, according to Table 1. The agreement of the spacing ratios is evidenced by the quality of the linear regression in the plot q vs (h2 + k2 + l2)1/2. Despite the uncertainty on the high-q peak positions, we found an excellent agreement with a face-centered cubic (fcc) crystal mode (F---), as shown in Figure 2B, D, F (diamond symbols). A body-centered cubic (bcc) Im3̅m structure is definitively ruled out by the poor agreement with observed spacing ratios (Figure 2B, D). The two space groups of mode F--- encountered for micellar LLC, Fd3̅m and Fm3̅m, differ essentially by (i) extra reflections ({200}, {420}, etc.) in the Fm3m ̅ structure, and (ii) the characteristic intensity of the reflections. The most intense reflection is {311} in Fd3̅m, whereas it is {111} in Fm3̅m (see Table 1). In our diffractograms, the latter point clearly indicates

Figure 1. (A) Phase diagram PC/water/cyclohexane, reproduced with permission from ref 7. Copyright 2000, American Chemical Society. Lα stands for the liquid crystalline lamellar phase, H2 for reverse hexagonal phase, N2 for nematic phase, and I2 for micellar cubic phase, identified in the present report as face-centered reverse micellar cubic Fm3̅m. W indicates a coexistence with excess of water. (B) Chemical structure of compounds used in this work.

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EXPERIMENTAL SECTION

Materials. Soy bean phosphatidylcholine (Epikuron 200) was purchased from Cargill, Germany. Composition details are given in the Supporting Information. Cyclohexane was purchased from Fluka. (R)(+)-Limonene and isooctane were purchased from Sigma-Aldrich. Tocopherol (α-DL-tocopherol) was purchased from DSM. Milli-Q water was used for all sample and solution preparation. Sample Preparation. The αoil value of a given sample is defined as the mass fraction of oil in the lipid mixture: moil αoil = mPC + moil (1) where moil is the mass of organic oil and mPC is the mass of Epikuron 200. For all oils except tocopherol, no cosolvent was required, as Epikuron 200 is soluble in these solvents. Epikuron 200 and the oil were weighed together in a Teflon-capped glass vial or tube. The vial was tightly closed to prevent any evaporation of solvent. Epikuron 200 was thoroughly mixed with the organic solvent by vortexing vigorously and allowing for a few hours equilibration, with vortexing steps at regular time intervals. In the case of tocopherol, ethanol was used as a cosolvent. When a clear, homogeneous solution was obtained by complete solubilization of Epikuron 200 and tocopherol, ethanol was evaporated in a rotary evaporator, set at 140 rpm, water bath temperature 60 °C, during about 30 min. Total ethanol evaporation was controlled by weighing. The PC-tocopherol mixture was then weighed into a Teflon-capped vial or tube. After preparation of the homogeneous PC−oil mixture, water was added. The vial was tightly closed, and the sample was homogenized by vortexing and centrifuging 15806

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Figure 2. Upper panels A, C, E: Typical SAXS patterns of representative PC/water/oil samples: (A) PC/water/cyclohexane, with α = 0.55 in excess water. (C) PC/water/limonene, with α = 0.53 in excess water. Inset: data from higher resolution chamber. (E) PC/water/isooctane sample, with α = 0.50 in excess water, in high-resolution chamber. Inset: azimuthal plot along the first reflection ring. Missing or hardly identifiable reflections are omitted. The reflections {200} and {222} were positioned by a deconvolution procedure. Lower panels B, D, F: phase indexations of the measured peaks in space group Fm3̅m (diamonds) and Im3̅m (crosses), corresponding to closed or open symbols marking the peaks in the panel directly above. For Fm3̅m, linear regressions coefficients of determination are all above R2 = 0.9995 (B, closed symbols); for Im3̅m, R2 ≃ 0.97.

The {200} and {222} reflections are better visible at higher resolution in the best equilibrated samples (inset in Figure 2C). In the neighboring I2+H2 coexistence region, the first and most intense reflection of the H2 phase {10} generally appears in the region where we identified the weak {200} reflection of the Fm3̅m structure (see Supporting Information). However, as the cubic samples appear completely isotropic by crosspolarized optical light microscopy, a coexistence with hexagonal phase is unlikely. A strong argument to exclude the coexistence with a hexagonal phase is that the cubic lattice parameter varies with sample composition in this region (see below), which would not be the case in a phase coexistence according to the Gibbs phase rule. Note that in diffraction patterns from direct micellar Fm3̅m mesophases, the {200} reflection is sometimes missing11 or partially merged with the intense and broad {111} reflection.12−14 The unusual peak broadness in the cyclohexane- and limonene-based cubic mesophases is to be interpreted, along with the sharp decrease of the peaks intensity with q, as a true indication of poor long-range ordering, rather than a metastability effect or poor mesophase equilibration. Neglecting the instrumental resolution of the laboratory SAXS equipment in this case of extreme peak broadness, in the cyclohexane and limonene systems we get for the first peak of broadness w1 a correlation length l ≈ 2π/w1 of about 80 nm. The micellar cubic mesophase formed in the presence of isooctane is also of symmetry Fm3̅m (Figure 2E). More reflections are visible at higher q, and the peaks are sharper than in the case of cyclohexane and limonene, which indicates a

Table 1. Indexation of the Bragg Reflections with Spacing Ratios, Packing Fraction, and Lipid Tail Stretching Coefficient for Some Reverse Micellar Mesophase Symmetriesa spacing ratio

Im3m ̅ (bcc)

√2 √3 2 √6 √8 √10 √11 √12 √14 4 √18 √19 √20 packing fractionb stretching coefficientb

110

211 220 310 222 321 400 411 420 0.68 29%

Fd3̅m

Fm3m ̅ (fcc)

111

111 200

220

220

311 222

311 222

400

400

311

311 420 0.74 41%

0.71 18−27%

P63/mmc (3D hcp) 100 002 101 102 110 103 200 112 201 004 202 104

(1) (6/√32) (√41/√32) (√68/√32) (√96/√32) (√113/√32) (2) (√132/√3) (√137/√32) (12/√32) (√164/√32) (√176/√32)

0.74 41%

a The reflections observed generally as most intense in micellar systems are printed in bold. For the P63/mmc (3D hcp), the c/a ratio is fixed at (8/3)1/2 and spacing ratios are between brackets. bValues from ref 10.

a Fm3̅m structure, although the Fm3̅m structure has not yet been reported for micellar cubic mesophases of reverse type.8 15807

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longer-range order. The size of the micelles is significantly larger than for limonene and cyclohexane. In some isooctanebased Fm3̅m mesophase samples, we observed a nonisotropic distribution of the intensity along the rings, with six more intense spots arranged in a hexagon (Figure S1 in Supporting Information and azimuthal intensity plot in the inset in Figure 2E). This typical anisotropic pattern strongly supports that the presently reported mesophase consists of micelles organized in a 3D fcc packing. Structural Parameters. Figure 3A shows a schematic representation of a face-centered cubic cell of reverse micelles.

N×

4 3 πR c = (1 − ϕlip)a3 3

(2)

where ϕlip is the lipid volume fraction (oil and PC tails) in the mesophase, and a is the lattice parameter. In a fcc close-packing of spheres, the closest contact between two adjacent spheres is along the diagonal of the side of the cubic cell. It follows that the characteristic thickness of the apolar layer between two adjacent micelles Llip obeys: a 2 = 4R c + 2L lip

(3)

Considering the half of the lipid characteristic length, Llip/2, is convenient because it can be directly compared to the length of the PC tails. The packing volume f raction, calculated as the fraction occupied by the polar core (water and PC heads) and the oil-swollen tail corona, is given by: 4

ϕυ =

N × 3 π (R c + ltail)3 a3

(4)

where ltail is the equilibrium length of a PC lipid tail. Note that the packing volume fraction ϕυ is larger than (1 − ϕlip) because the PC tails and the oil penetrating into the tails corona are taken into account. Figure 3B presents the variation of the limonene-based Fm3̅m (fcc) mesophase structural parameters at water saturation as a function of the α ratio, throughout its range of stability, i.e., as pure mesophase and in coexistence with the adjacent mesophases H2 and L2. The boundary with the Fm3̅m +L2 phase was determined visually by the presence of isotropic liquid phase on top of the equilibrated sample, as illustrated in Figure 5A. Within the pure Fm3m ̅ phase domain, the lattice parameter increases with α, while the calculated micellar core radius Rc shows almost no variation. The variation of lattice parameter is compensated to a certain extent by the variation of composition in the mesophase. Conversely, the lipid layer halfthickness Llip/2 increases steadily from 1.35 nm to over 1.7 nm, showing that the lattice parameter increase arises mainly from micelles moving apart from each other in the oil-swelling mesophase. At α ≤ 0.55 in the pure Fm3m ̅ phase, Llip/2 is larger than the maximal tail extension, 1.6 nm, implying that the micelles cannot be in direct contact at these compositions. Taking the equilibrium tail length equal to the thickness of the tails corona at the transition to coexistence with H2 (ltail = 1.35 nm), we calculate that the packing unit volume fraction drops from 0.74 to 0.63 within the pure Fm3̅m phase domain. When further decreasing the oil fraction above the maximal packing volume fraction for spheres (ϕυ ≃ 0.74), a transition to hexagonal is necessary to reach higher packing fractions (up to ϕυ ≃ 0.91 for cylinders7). The fcc lattice parameter in coexistence with H2 remains, however, constant at the same value as at the maximal packing volume fraction of spheres. A pure Fm3̅m mesophase is formed at packing volume fractions that are below the maximum packing fraction of a noncompact bcc packing (ϕυ ≃ 0.68, see Figure 3B). Yet we found no trace of bcc packing, even in the Fm3̅m−L2 coexistence region. The exclusive formation of a fcc structure is a strong indication that the micelles behave rather as rigid hard spheres than as soft spheres. With ltail/Rc ≃ 0.16, the micelles are clearly situated in the fcc-forming “crew-cut” micelles regime.12−14 The dense coverage of short PC tails does not create any long-range repulsion potential or steric hindrance.

Figure 3. (A) Structure of the face-centered cubic (fcc) cell, of symmetry Fm3̅m. The lattice parameter a, radius of micellar core Rc, and characteristic lipid length Llip are indicated. Surfactant and micelles are not to scale. (B) Variation of lattice parameters, radius of Fm3̅m micellar core Rc, half of characteristic lipid length Llip/2, and packing volume fraction ϕυ with the α ratio in the PC/water/limonene system, in excess water. Rc,fcc, Llip and ϕυ are calculated from eqs 2, 3, and 4 respectively, using ltail = 1.35 nm in eq 4. Rc,L in the L2 subphase, with polydispersity (error bar), was obtained by form factor fitting in the L2 phase (see text). Plain and dotted lines are only a guide for the eye. Vertical dashed lines indicate the phase transitions (±0.05 on α scale).

This fcc cell contains N = 4 micelles in total, at each corner and in the center of each face. The lattice parameter measured by SAXS is the length of the side of the cubic cell. The dimension of the micelles polar core Rc is derived by equating the volume of polar material (water and PC head groups) in the fcc cell from its structure and from the experimental mesophase composition: 15808

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Table 2. Comparison of the Structural Parameters of the Pure Reverse Micellar Cubic Mesophases in Ternary Systems PC/ Water/Oil for Various Oilsa oil weight fraction in lipid phase α max. water capacityc (%) oil volume fraction space group lattice parameter (nm) radius of polar core of micelles (nm) correlation length (nm; no. of unit cells) plateau modulus (Pa)

α-tocopherol

cyclohexane

limonene

isooctane

0.68−0.70d 20 0.61−0.63 Fd3̅md 14.4 (α = 0.70) 1.9b 318;d 20 5.0 × 105

0.52−0.57e 26e 0.45−0.51 Fm3̅m 19.3 (α = 0.55) 5.0 ∼80; ∼4 ND

0.52−0.57 33 0.43−0.49 Fm3̅m 19.9 (α = 0.55) 5.2 ∼80; ∼4 3.2 × 104

0.51 50 0.32 Fm3̅m 31.5 9.8 ∼200; ∼7 1.5 × 104

a

Shear rheology data is presented in the Supporting Information. ND = not determined. bFor Fd3̅m, radius calculated assuming a single micellar size.26 cVaries with α by ±2%. dFrom ref 4. eFrom ref 7.

An apparent deviation from the classical hard sphere behavior could be seen in the fact that the L2 phase appears in coexistence with the fcc at ϕυ ≃ 0.63, a higher packing volume fraction than expected for monodisperse hard spheres (ϕυ ≃ 0.545).34 We attribute the higher concentration of the fluid−crystal transition to the presence of micellar polydispersity, as observed in the L2 phase. In model hard spheres, a slight polydispersity has been shown to push this transition to higher ϕυ.15 Our simple geometrical analysis of the filling of the fcc cell suggests that most of the oil resides in the large “voids” between the micelles. It is possible to coarsely evaluate the degree of penetration of the organic solvent in the lipid tails corona, from the mesophase geometry and composition. Assuming that the PC lipid tails have a length ltail = 1.35 nm, for cyclohexane at α = 0.55 we found a volume fraction of about 20% oil in the tails corona, i.e., about two oil molecules per PC molecule. This approximate result in the Fm3̅m mesophase remains consistent with the findings of Eastoe at al.16 in the highly dilute L2 phase. In the case of isooctane, we found a degree of penetration of about 10%. Comparison with the Fd3̅m and 3D-hcp Structures. While direct micellar mesophases are mainly governed by micellar packing compactness and intermicellar interactions, lipid tail frustration comes as a significant additional parameter determining the structure of reverse micellar phases. To minimize lipid tail frustration, micelles may partially adopt the shape of the polyhedral cage by faceting at the expense of surface energy10 and create two populations of micelles with different sizes. This leads to the formation of the Fd3̅m structure,1,4,26 which appears as the best compromise in terms of free energy considering micellar packing, lipid tail frustration, and interface spontaneous curvature for most reverse mesophase-forming systems (Table 1). Many reverse, but only one direct27 Fd3̅m, structures have been reported, which shows how influential lipid frustration can be. In ternary systems, the presence of a third apolar component may completely release the lipid tail frustration by partitioning in the polyhedral cage corners, in which case the micelles may freely adopt a spherical shape inside the mesophase17 and arrange in the most compact packing available, in the present case the fcc packing. Contrary to cyclohexane, isooctane, and limonene, αtocopherol added in large enough proportions in the PC/ water systems leads to the formation of the Fd3̅m mesophase.4 The tocopherol-based Fd3m ̅ displays totally different characteristics compared to the new reverse Fm3̅m phases (Table 2 and Figure 4A). The plateau modulus is significantly higher, and the lattice parameter and micelles are smaller. The volume fraction

Figure 4. (A) Comparison between PC/water/cyclohexane (top curve) and PC/water/tocopherol (bottom curve) reverse micellar cubic mesophases. The peak indexations correspond respectively to space groups Fm3̅m (fcc) and Fd3̅m. The cells are represented schematically. The expected positions of all reflections have been calculated from the positions measured for well-defined reflections. (B) SAXS curves (open symbols) and form factor fits (lines) for samples in the pure L2 phase, with αoil = 0.85 in excess water: from bottom to top, isooctane (circles), cyclohexane (triangles), limonene (squares), and tocopherol (diamonds). The fitting curves are showed only on the fitted ranges. Inset shows the corresponding SLD profiles. Parameters of the fits are given in Table 3 and in the text. Curves have been shifted for clarity.

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Table 3. Fit Parameters for SAXS Data from PC/Water/Oil Samples in L2 Phase with a Polydisperse Core−Shell Spheres Model with Hard Spheres Interactionsa oil

α

phase

Rw (nm)

σ/Rw

ts (nm)

Rc (nm)

ϕ

2k + k (kBT)

cyclohexane cyclohexane cyclohexane limonene limonene isooctane isooctane

0.97 0.85 0.60 0.85 0.60 0.85 0.59

L2 L2 L2+Fm3̅m L2 L2+Fm3̅m L2 L2+Fm3̅m

(3.35)b 2.49 2.76 2.92 4.24 8.17 8.52

0.18b 0.160 0.142 0.157 0.146 0.165 0.152

(1.43)b 1.96 2.46 1.44 1.07 1.48 1.08

(4.78)b 4.45 5.22 4.36 5.31 9.65 9.60

0.066 0.18 0.48 0.19 0.50 0.23 0.48

1.63b 1.77 1.94 1.82 1.98 1.65 1.83

a

See text for details. Rc = Rw + ts. bFrom SANS measurements in ref 16. Radius and shell size are between brackets because the SLD profiles slightly differ between SANS and SAXS.

interactions. The only fixed parameters were the X-ray scattering length density (SLD) of the water core (9.4691 × 10−6 Å−2) and the solvent, calculated from the respective chemical structures: 7.9723 × 10−6 Å−2 for limonene, 7.6646 × 10−6 Å−2 for cyclohexane, and 6.7812 × 10−6 Å−2 for isooctane. The corresponding SLD profiles are represented in the inset of Figure 4B. As the SAXS data were measured in arbitrary units and not normalized, the curves were shifted by multiplication by a constant factor as necessary in the fitting procedure. The core radius Rw, core polydispersity p = σ/Rw (where σ is the root mean squared deviation), shell thickness ts, and shell SLD were used as variables for the form factor, and the particle volume fraction ϕ for the hard sphere structure factor. The background scattering was also included in the fit parameters. The shell SLD obtained by fitting was always between 1.0 × 10−6 and 1.1 × 10−6 Å−2, which is a reasonable value for a phosphorus-containing shell. The micellar volume fractions obtained by fitting were consistent with the experimental composition of the samples. The fitting parameters are given in Table 3, as well as interface rigidities from eq 6. Values of interface rigidity are in good agreement with those of Eastoe et al.16 in dilute L2 phase. For comparison, a SAXS curve from a tocopherol-based sample of comparable composition is also shown in Figure 4B. This curve is characterized by a single broad peak at q = 0.166 Å−1, which could not be reliably fitted but shows that the micelle distribution is significantly polydisperse. The position of the scattering maximum corresponds to a characteristic length scale of d = 2π/q = 3.79 nm, which is fully consistent with the characteristic diameter of the micelles observed in the PC/ tocopherol/water Fd3̅m mesophase (Table 2). The rigidity of the interface 2k + k̅ was found to be of comparable magnitude for all Fm3̅m-forming oils (Table 3). The low values of 2k + k̅ obtained here suggest that the bending properties of the PC interface are influenced by the addition of oil. In pure egg yolk PC vesicles, the measured bending modulus of the PC monolayer k is 8 kBT.30 Low values of 2k + k̅ can be reached only by decreasing the bending modulus k or by assuming a very high saddle-splay modulus k̅. However, at water content lower than that of the saturated Fm3̅m and L2 phases, the micelles adopt a cylindrical morphology,7,31 which is only possible if the bending modulus k strongly dominates the saddle-splay modulus k̅.32 Consequently, the bending modulus k controls the interface properties. One of the effects of oil addition is therefore probably to decrease the bending modulus, which is reflected in the low values of 2k + k̅. Similarly, with the H2-forming phospholipid phosphatidylethanolamine (PE), it was measured that addition of only 16% tetradecane decreased the bending modulus k from 13 to 11 kBT in the H2 phase.33

of oil in the cell is notably high, given the theoretical micellar packing volume fraction of the Fd3̅m structure. This last point suggests that the slightly polar tocopherol molecules participate in the interface. The insertion of tocopherol in the interface structure, almost as a cosurfactant, would deeply modify the interface bending properties. In particular, the negative spontaneous curvature would be increased due to the bulky apolar moiety, leading to small Fd3̅m micelles. The theoretical most compact packing parameter of monodisperse hard spheres (ϕυ ∼ 0.74) is reached both in the face-centered cubic (fcc) and three-dimensional hexagonal close-packing (3D hcp) structures (Table 1). The 3D hcp packing has been encountered only on rare occasions as a bulk micellar mesophase,18−22 and only once so far with reverse micelles, in a dioleoylphosphatidylcholine (DOPC)−dioleoylglycerol mixture in excess water.23 This reverse phase is stabilized by the addition of 25−40% cholesterol in the lipid mixture.23 The authors stated that the phase formed also without cholesterol.8 The structural determination mechanism between fcc and 3D hcp packings remains to this date elusive. The energy difference between the two close-packed structures seems to be very low. So-called random hexagonally close packed structures (random mixture of 3D hcp and fcc) are also found.24,25 Disordered Micellar Phase L2 and Interface Rigidity. The free energy of curvature of a spherical micelle of radius R in the flexible surface model7 is given by ⎛1 ⎞2 k̅ fc = 2k ⎜ − H0⎟ + 2 ⎝R ⎠ R

(5)

where H0 is the spontaneous curvature, and k and k̅ are the bending and saddle-spay moduli (with k > 0 and −2k < k̅ < 0) of the interface. The rigidity 2k + k̅ of the interface is linked to the polydispersity p of the micelles:16,28 2k + k ̅ =

kBT 8πp2

−

kBT f (ϕ) 4π

(6)

where f(ϕ) = (1/ϕ){ϕ ln(ϕ) + (1 − ϕ)ln(1−ϕ)} represents the entropy of mixing of the micelles in the random mixing approximation at the volume fraction ϕ.28 In the case of cyclohexane, limonene, and isooctane, it was possible to recover the polydispersity of the micelles by fitting the form factor16 (Figure 4B). In cases where the L2 phase is in coexistence with the micellar cubic mesophase Fm3̅m, the L2 phase was isolated after equilibration (see next section) and analyzed separately. The fitting was performed according to Eastoe et al.,16 using a core−shell polydisperse spheres model with a Schultz size distribution,29 coupled with hard spheres 15810

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correspond to the border of the pure Fm3m ̅ phase (α = 0.57), we find a micellar radius Rc,F = 5.1 nm in the Fm3̅m gel subphase. The calculated micellar radii in the two subphases, obtained independently, are in remarkable agreement (see Figure 3B). This strongly supports the hypothesis that the structure of the Fm3̅m phase consists of a packing of single-size spherical micelles in a cubic lattice, in this case in equilibrium with a fluid of disordered micelles of same size, similar to fluid− crystal coexistences observed at intermediate volume fractions in phase diagrams of hard spheres.34 The coexistence of direct micelles in fluid and solid state have been reported for block copolymer micelles.35

The interface rigidity seemed slightly higher in L2 + Fm3m ̅ coexistences than in pure L2 phases. It is likely that the formation of the Fm3̅m mesophase is made possible in such systems through the release of lipid tail frustration by the small and mobile apolar molecules. The efficient release of frustration by the oil enables the selfassembled structure to reach packing fractions higher than that of the two-sized micellar Fd3̅m (Table 1). Coexistence of Ordered and Disordered Micelles. Samples in three-phase coexistence Fm3̅m+L2+excess water were equilibrated for several weeks to allow separation of the three subphases in the order of density (Figure 5, picture in

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CONCLUSION

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ASSOCIATED CONTENT

We identified a reverse face-centered cubic micellar mesophase of symmetry Fm3̅m in simple ternary systems PC/water/ organic solvents, including cyclohexane, limonene, and isooctane. This mesophase structure is reported for the first time in reverse configuration. Detailed investigation of the mesophase structural parameters reveals the similarity of the micellar phase behavior with that of hard spheres at high volume fractions. We propose that the formation of compact micellar mesophases of oil-continuous type is possible in PC/ water/oil ternary systems because of a 2-fold effect of the apolar oil: it releases the frustration of surfactant tails by filling the large geometric space between micelles in the fcc cell, but it also subtly modifies the interface bending properties by penetrating the surfactant tail corona, which leads to monodisperse micelles. The newly reported reverse micellar mesophases present a number of promising features for applications in lyotropic liquid crystalline delivery systems. They are stable in equilibrium with excess water, therefore in principle dispersible into particles for use as a delivery system. Finally, on the basis of exceptionally large micelles, their maximal water content is notably larger than in the Fd3̅m micellar mesophase, leading to higher loading capacities.

Figure 5. SAXS curve from a PC/water/limonene sample (α = 0.60, 25% water), showing coexistence of reverse micellar L2 and reverse micellar cubic Fm3̅m (reflections marked by blue triangles). The dotted red line shows an approximate fitting of the form factor over the reflection-free region (double arrow), using a core−shell polydisperse spheres model with a Schultz size distribution. Bottomleft inset: picture of a PC/water/limonene sample tilted, showing three-phase coexistence Fm3̅m+L2+excess water, with α = 0.60 and 32% water. The excess water is present as small droplets entrapped at the bottom of the vials. Top-right inset: schematics of the L2−Fm3̅m equilibrium.

S Supporting Information *

Anisotropic 2D-scattering pattern in PC/water/isooctane Fm3̅m sample, data from H2-Fm3̅m coexistences, study of time and temperature stability of the reverse Fm3̅m mesophase, shear rheology data from limonene- and isooctane-based Fm3̅m samples, and composition of Epikuron 200. This material is available free of charge via the Internet at http://pubs.acs.org.

inset). The samples were completely isotropic under observation between two crossed polarizers. A sample with only a small excess water component was gently stirred before filling the capillary for SAXS measurement to collect both L2 and Fm3m ̅ subphases. An approximate fit of the form factor over the high-q region (Figure 5) without Fm3̅m reflections was performed using a core−shell polydisperse spheres model with a Schultz size distribution, as presented in previous section, but taking S(q) = 1 in the fit, that is, assuming an indefinitely diluted systems of noninteracting spheres. The approximate fitting procedure yielded the following parameters: average core radius Rw = 3.92 nm, core polydispersity p = 0.118, and shell thickness ts = 1.22 nm. The radius of the micellar polar region (core and shell) in the L2 liquid subphase is thus Rc,L = 5.14 ± 0.46 nm. Note that the precise fitting of the isolated L2 subphase (Table 3) yielded slightly different but compatible values. From the positions of the reverse micellar cubic reflections, the lattice parameter in the Fm3̅m symmetry was calculated to be 19.1 nm. Considering that in the cubic subphase the α ratio and the water content

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AUTHOR INFORMATION

Corresponding Author

*E-mail: raff[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

The authors thank Daniel Peters for measuring the initial water content of Epikuron 200 and Nicole Baumann for preparing one of the mesophase samples. This work was funded by NESTEC Ltd. 15811

dx.doi.org/10.1021/la404307x | Langmuir 2013, 29, 15805−15812

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(22) Besson, S.; Ricolleau, C.; Gacoin, T.; Jacquiod, C.; Boilot, J.-P. A new 3D organization of mesopores in oriented CTAB silica films. J. Phys. Chem B 2000, 104, 12095−12097. (23) Shearman, G. C.; Tyler, A. I. I.; Brooks, N. J.; Templer, R. H.; Ces, O.; Law, R. V.; Seddon, J. M. A 3-D hexagonal inverse micellar lyotropic phase. J. Am. Chem. Soc. 2009, 131, 1678−1679. (24) Byelov, D. V.; Hilhorst, J.; Leferink op Reinink, A. B.; Snigireva, I.; Snigirev, A.; Vaughan, G. B.; Portale, G.; Petukhov, A. V. Diffuse scattering in random-stacking hexagonal close-packed crystals of colloidal hard spheres. Phase Transitions 2010, 83, 107−114. (25) Meijer, J.-M.; de Villeneuve, V. W. A.; Petukhov, A. V. In-plane stacking disorder in polydisperse hard sphere crystals. Langmuir 2007, 23, 3554−3560. (26) Pouzot, M.; Mezzenga, R.; Leser, M.; Sagalowicz, L.; Guillot, S.; Glatter, O. Structural and rheological investigation of Fd3m inverse micellar cubic phases. Langmuir 2007, 23, 9618−9628. (27) de Geyer, A.; Guillermo, A.; Rodriguez, V.; Molle, B. Evidence for spontaneous formation of three-dimensionally periodic cellular structures in a water/oil/surfactant/alcohol system. J. Phys. Chem B 2000, 104, 6610−6617. (28) Gradzielski, M.; Langevin, D.; Farago, B. Experimental investigation of the structure of nonionic microemulsions and their relation to the bending elasticity of the amphiphilic film. Phys. Rev. E 1996, 53, 3900−3919. (29) Kotlarchyk, M.; Chen, S.-H. Analysis of small angle neutron scattering spectra from polydisperse interacting colloids. J. Chem. Phys. 1983, 79, 2461−2469. (30) Méléard, P.; Gerbeaud, C.; Bardusco, P.; Jeandaine, N.; Mitov, M.; Fernandez-Puente, L. Mechanical properties of model membranes studied from shape transformations of giant vesicles. Biochimie 1998, 80, 401−413. (31) Cavaco, C. Structural and dynamic properties of giant polymerlike lecithin reverse micelles; Ph.D. Thesis, Swiss Federal Institute of Technology Zurich, Zurich, 1994. (32) Safran, S.; Turkevich, L.; Pincus, P. Cylindrical microemulsions: a polymer-like phase? J. Phys. Lett. 1984, 45, 69−74. (33) Chen, Z.; Rand, R. The influence of cholesterol on phospholipid membrane curvature and bending elasticity. Biophys. J. 1997, 73, 267− 276. (34) Anderson, V. J.; Lekkerkerker, H. N. W. Insights into phase transition kinetics from colloid science. Nature 2002, 416, 811−815. (35) Castelletto, V.; Caillet, C.; Fundin, J.; Hamley, I. W.; Yang, Z.; Kelarakis, A. The liquid-solid transition in a micellar solution of a diblock copolymer in water. J. Chem. Phys. 2002, 116, 10947−10958.

REFERENCES

(1) Seddon, J. M.; Robins, J.; Gulik-Krzywicki, T.; Delacroix, H. Inverse micellar phases of phospholipids and glycolipids. Phys. Chem. Chem. Phys. 2000, 2, 4485−4493. (2) Shearman, G. C.; Ces, O.; Templer, R. H.; Seddon, J. M. Inverse lyotropic phases of lipids and membrane curvature. J. Phys.: Condens. Matter 2006, 18, S1105−S1124. (3) Fontell, K. In Frontiers in Colloid Science In Memoriam Professor Dr. Bun-ichi Tamamushi; Nakagaski, M., Shinoda, K., Matijević, E., Eds.; Steinkopff: Darmstadt, Germany, 1983; Vol. 68; pp 48−52. (4) Barauskas, J.; Cervin, C.; Tiberg, F.; Johnsson, M. Structure of lyotropic self-assembled lipid nonlamellar liquid crystals and their nanoparticles in mixtures of phosphatidyl choline and α-tocopherol (vitamin E). Phys. Chem. Chem. Phys. 2008, 10, 6483−6485. (5) Templer, R. H.; Seddon, J. M.; Warrender, N. A.; Syrykh, A.; Huang, Z.; Winter, R.; Erbes, J. Inverse bicontinuous cubic phases in 2:1 fatty acid/phosphatidylcholine mixtures. The effects of chain length, hydration, and temperature. J. Phys. Chem. B 1998, 102, 7251− 7261. (6) Angelico, R.; Ceglie, A.; Colafemmina, G.; Delfine, F.; Olsson, U.; Palazzo, G. Phase behavior of the lecithin/water/isooctane and lecithin/water/decane systems. Langmuir 2004, 20, 619−631. (7) Angelico, R.; Ceglie, A.; Olsson, U.; Palazzo, G. Phase diagram and phase properties of the system lecithin/water/cyclohexane. Langmuir 2000, 16, 2124−2132. (8) Shearman, G.; Tyler, A.; Brooks, N.; Templer, R.; Ces, O.; Law, R.; Seddon, J. Ordered micellar and inverse micellar lyotropic phases. Liq. Cryst. 2010, 37, 679−694. (9) Salonen, A.; Guillot, S.; Glatter, O. Determination of water content in internally self-assembled monoglyceride-based dispersions from the bulk phase. Langmuir 2007, 23, 9151−9154. (10) Rappolt, M.; Cacho-Nerin, F.; Morello, C.; Yaghmur, A. How the chain configuration governs the packing of inverted micelles in the cubic Fd3m-phase. Soft Matter 2013, 9, 6291−6300. (11) Hamley, I. W.; Pople, J. A.; Diat, O. A thermally induced transition from a body-centred to a face-centred cubic lattice in a diblock copolymer gel. Colloid Polym. Sci. 1998, 276, 446−450. (12) McConnell, G. A.; Gast, A. P.; Huang, J. S.; Smith, S. D. Disorder-order transitions in soft sphere polymer micelles. Phys. Rev. Lett. 1993, 71, 2102−2105. (13) Hamley, I. W.; Daniel, C.; Mingvanish, W.; Mai, S.-M.; Booth, C.; Messe, L.; Ryan, A. J. From hard spheres to soft spheres: The effect of copolymer composition on the structure of micellar cubic phases formed by diblock copolymers in aqueous solution. Langmuir 2000, 16, 2508−2514. (14) Lodge, T. P.; Pudil, B.; Hanley, K. J. The full phase behavior for block copolymers in solvents of varying selectivity. Macromolecules 2002, 35, 4707−4717. (15) Colombo, J.; Dijkstra, M. Effect of quenched size polydispersity on the fluid-solid transition in charged colloidal suspensions. J. Chem. Phys. 2011, 134, 154504. (16) Eastoe, J.; Hetherington, K. J.; Sharpe, D.; Steytler, D. C.; Egelhaaf, S.; Heenan, R. K. Droplet structure in phosphocholine microemulsions. Langmuir 1997, 13, 2490−2493. (17) Vacklin, H.; Khoo, B. J.; Madan, K. H.; Seddon, J. M.; Templer, R. H. The bending elasticity of 1-monoolein upon relief of packing stress. Langmuir 2000, 16, 4741−4748. (18) Clerc, M. A new symmetry for the packing of amphiphilic direct micelles. J. Phys. II 1996, 6, 961−968. (19) Zeng, X.; Liu, Y.; Impéror-Clerc, M. Hexagonal close packing of nonionic surfactant micelles in water. J. Phys. Chem. B 2007, 111, 5174−5179. (20) Berret, J.-F.; Molino, F.; Porte, G.; Diat, O.; Lindner, P. The shear-induced transition between oriented textures and layer-slidingmediated flows in a micellar cubic crystal. J. Phys.: Condens. Matter 1996, 8, 9513−9517. (21) Soni, S. S.; Brotons, G.; Bellour, M.; Narayanan, T.; Gibaud, A. Quantitative SAXS analysis of the P123/water/ethanol ternary phase diagram. J. Phys. Chem B 2006, 110, 15157−15165. 15812

dx.doi.org/10.1021/la404307x | Langmuir 2013, 29, 15805−15812