Influence of Vitamin E Acetate and Other Lipids on the Phase Behavior

Article pubs.acs.org/Langmuir

Influence of Vitamin E Acetate and Other Lipids on the Phase Behavior of Mesophases Based on Unsaturated Monoglycerides L. Sagalowicz,*,† S. Guillot,‡,§ S. Acquistapace,† B. Schmitt,† M. Maurer,‡ A. Yaghmur,‡,∥ L. de Campo,‡,⊥ M. Rouvet,† M. Leser,† and O. Glatter*,‡ †

Nestlé Research Center, Vers-Chez-Les-Blanc, CH-1000 Lausanne 26, Switzerland Department of Chemistry, University of Graz, Heinrichstraße 28, A-8010 Graz, Austria § Centre de Recherche sur la Matière Divisée, 1B rue de la Férollerie, 45071 Orléans Cedex 2, France ∥ Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark ⊥ Department of Applied Mathematics, Research School of Physical Sciences and Engineering, Australian National University, Canberra, Australia ‡

ABSTRACT: The phase behavior of the ternary unsaturated monoglycerides (UMG)−DL-α-tocopheryl acetate−water system has been studied. The effects of lipid composition in both bulk and dispersed lyotropic liquid crystalline phases and microemulsions were investigated. In excess water, progressive addition of DL-α-tocopheryl acetate to a binary UMG mixture results in the following phase sequence: reversed bicontinuous cubic phase, reversed hexagonal (HII) phase, and a reversed microemulsion. The action of DL-α-tocopheryl acetate is then compared to that of other lipids such as triolein, limonene, tetradecane, and DL-α-tocopherol. The impact of solubilizing these hydrophobic molecules on the UMG−water phase behavior shows some common features. However, the solubilization of certain molecules, like DL-α-tocopherol, leads to the presence of the reversed micellar cubic phase (space group number 227 and symmetry Fd3̅m) while the solubilization of others does not. These differences in phase behavior are discussed in terms of physical−chemical characteristics of the added lipid molecule and its interaction with UMG and water. From an applications point of view, phase behavior as a function of the solubilized content of guest molecules (lipid additive in our case) is crucial since macroscopic properties such as molecular release depend strongly on the phase present. The effect of two hydrophilic emulsifiers, used to stabilize the aqueous dispersions of UMG, was studied and compared. Those were Pluronic F127, which is the most commonly used stabilizer for these kinds of inverted type structures, and the partially hydrolyzed emulsifier lecithin (Emultop EP), which is a well accepted food-grade emulsifier. The phase behavior of particles stabilized by the partially hydrolyzed lecithin is similar to that of bulk sample at full hydration, but this emulsifier interacts significantly with the internal structure and affects it much more than F127.

1. INTRODUCTION There is an increasing interest in the pharmaceutical, cosmetic, and food industry of using self-assembled structures to solubilize hydrophilic, amphiphilic and lipophilic molecules.1−7 These include normal micelles, vesicles, reversed microemulsions, and lyotropic liquid crystalline phases including the inverted micellar cubic, the inverted hexagonal (HII), and the inverted bicontinuous cubic phases.8−11 The advantages of using such mesophases are that they can improve certain functionalities.12−15 Proven ones are, for example, an increase in the solubilization of lipophilic and crystalline materials, a control of membrane protein crystallization, an increased bioavailability or bioefficacy of drugs and nutrients, and a controlled or sustained release of solubilized bioactive materials.4,13,16−21 Unsaturated monoglycerides (UMG) such as glycerol monooleate (GMO) or glycerol monolinolein (MLO), when mixed with water and optionally in the presence of other additives, give rise to the presence of a zoo of self© XXXX American Chemical Society

assembled structures. GMO in excess water exhibits a reversed bicontinuous cubic phase over a wide temperature range.22 Addition of amphiphilic molecules, like polyglycerol esters or phosphatidylcholine, stabilizes at high content the fluid lamellar (Lα) liquid crystalline phase23 while progressive addition of lipophilic compounds such as tetradecane or lipophilic aromas leads to the structural transition to HII, reversed micellar cubic phase, and reversed microemulsions.9,24 Vitamin E is the major and most potent lipid-soluble antioxidant.25−27 It acts as radical scavenging antioxidant in lipoproteins and efficiently interrupts the chain propagation of lipid oxidation, thus protecting low-density lipoproteins from oxidation.28 Vitamin E is also associated with lowered risk of coronary heart disease and atherosclerosis,29,30 and ischemic Received: December 21, 2012 Revised: May 19, 2013

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dx.doi.org/10.1021/la305052q | Langmuir XXXX, XXX, XXX−XXX

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heart disease in cross-cultural epidemiology.31 Recently, tocopherol (vitamin E covers 8 molecules including 4 tocopherol molecules) and tocopheryl acetate were solubilized into reversed bicontinuous cubic phases.32,33 These mesophases were made mainly with monoglycerides and dispersed into aqueous buffer. Absorption of the tocopherol from this liquid crystalline phase was not significantly different from absorption from normal micelles based on polysorbate 20 (Tween 20) when ingested by patients suffering from pancreatic insufficiency.33 The average absorption of vitamin E from selfassembled structures was also estimated to be higher than from supplements taken with standard breakfast. Therefore, it is crucial to design self-assembled delivery systems for solubilizing vitamin E. Dong et al. used phytantriol to solubilize vitamin E acetate in various liquid crystalline phases.34 Phytantriol is an alternative to the unsaturated monoglycerides to form reversed bicontinuous cubic phases. Up to about 10% of vitamin E acetate was added to phytantriol. When adding this tocopherol derivative, it was found that the structure changes from the reversed bicontinuous cubic phase to the reversed hexagonal phase.34 Bitan-Cherbakovsky et al.35,36 reported on the formation of vitamin E loaded in mesophases made of glycerol monooleate with vitamin E and water contents up to 35 and 12.5%, respectively. In a series of samples, ascorbic acid was present in addition to vitamin E. They found that vitamin E enhances the formation of the HII phase, and for one composition, where ascorbic acid was incorporated, the reversed micellar cubic phase was observed.35,36 The precise knowledge of the fine self-assembled structures and the full understanding of their phase behavior under different experimental conditions are of prime importance in the formation of either food or pharmaceutical delivery systems. For example, it was demonstrated that the rate of molecule release depends on the type of internal structure.19,37 The diffusion coefficient of solubilized sugar is about ten times larger in the reversed bicontinuous cubic structure than in the reversed hexagonal phase.38 The release properties of solubilized molecule are important for medical and pharmaceutical applications as well as for aroma impact.1,8 It is the aim of the present paper to study which phases can be formed when solubilizing DL-α-tocopheryl acetate (particular form of vitamin E acetate) and other oil-soluble lipids into unsaturated monoglyceride−water systems. The examined quantity of the tocopherol derivative ranges from 0 to 95%. Vitamin E acetate is solubilized in both dispersed and nondispersed mesophases. Small-angle X-ray scattering (SAXS), polarized microscopy, and visual appearance were used to determine the nature of the phase present. The formed mesophases were then dispersed into excess water using either partially hydrolyzed lecithin (Emultop), which is a wellaccepted food grade emulsifier, or Pluronic F127 as stabilizers. The hydrolyzed lecithin contains a relatively large quantity of lysophospholipids. Thus, it is a hydrophilic emulsifier suited to obtain stable oil-in-water (O/W) emulsions. Therefore, it can be thought as a good emulsifying candidate for obtaining stable aqueous dispersions of UMG mesophases. The nature of the dispersed phase was determined using SAXS and cryogenic transmission electron microscopy (cryo-TEM). We also compare the influence of other oil-soluble lipids such as vitamin E (non-acetated form) and triolein on the phase behavior of the unsaturated monoglyceride−water system. Finally we compare the results obtained with the investigated

monoglyceride to what was done in the literature for the phytantriol−water system.

2. MATERIALS AND METHODS 2.1. Materials. The used unsaturated monoglyceride is the commercial lipid Dimodan U/J (Danisco, Denmark). It contains about 96 wt % monoglycerides. The hydrocarbon tail consists predominantly of C18 chains (91%). The lipophilic chains are distributed as follows: C18:2 (61.9%), C18:1 (24.9%), C18:0 (4.2%), and C16:0 chains (6.8%). About 2 wt % diglycerides are also present. DL-α-Tocopheryl acetate and DL-α-tocopherol are from DSM (USA). Triolein ([cis]-9 glyceryl trioleate) was obtained from Sigma (USA).The used partially hydrolyzed lecithin is Emultop EP from Cargill (USA). It is a deoiled lecithin, and the phospholipids are partially hydrolyzed. In the particular batch used, the weight ratio of lysophosphatidylcholine to phosphatidylcholine was determined by the supplier to be 1:1.75. Pluronic F127 is a gift from BASF (Florham Park, NJ, USA). The water used was Milli-Q A10 from Millipore (France). 2.2. Sample Preparation of Bulk Samples. The nondispersed (bulk) samples were prepared in Pyrex tubes by mixing the appropriate amounts of lipid materials (DL-α-tocopheryl acetate, DLα-tocopherol, triolein) and Dimodan U/J and water, heating them using an air gun, and then homogenizing by vigorous agitation with a vortexer. They were then let cool down to room temperature and equilibrated at least one day before subsequent examination. 2.3. Macroscopic and Microscopic Phase Examination. Bulk samples were examined through cross polarizers. Birefringency, visual inspection, and viscosity were used to determine the formed phases. The L2 phase is not birefringent, is completely clear, and has a very low viscosity. The lamellar phase is birefringent and turbid and has a low viscosity. The hexagonal phase is birefringent and relatively turbid and has a medium viscosity. The cubic phase is not birefringent, is completely clear, and has a high viscosity. Additional checking of the phases was carried out using polarized microscopy (cross Nichols) on a Zeiss Axioplan microscope (Zeiss, Germany). Identification of the various phases was carried out according to Rosevear.39 2.4. Small-Angle X-ray Scattering (SAXS) Measurements. SAXS experiments were performed on a SAXSess camera40 (AntonPaar, Graz, Austria) using an X-ray generator (Philips, PW1730/10) operated at 40 kV and 50 mA with a sealed-tube Cu anode. A Göbel mirror is used to convert the divergent polychromatic X-ray beam into a focused line-shaped beam of Cu Kα radiation (wavelength, λ = 0.154 nm). The 2D scattering pattern is recorded by an imaging-plate detector (model Fuji BAS1800 from Raytest, Straubenhardt,Germany) and then integrated to one-dimensional scattering function I(q) using SAXSQuant software (Anton Paar, Graz, Austria), where q is the length of the scattering vector, defined by q = (4π/λ) sin θ/2, λ being the wavelength and θ the scattering angle. For indexing the peaks of the different liquid crystalline phases, the reflection laws of the space groups that have been determined for cubic and hexagonal phases containing membrane lipids are used. The interplanar distance, d, between two reflecting planes is given by d = 2π/q, which enables us to calculate the corresponding mean lattice parameter a. The scattering profiles of the so-called L2 phase show typically only one broad peak, for which the position of the observed maximum is shifted to lower q-values due to the “smearing effects” of the line-shaped primary beam. These profiles were desmeared by fitting these data with GIFT (generalized indirect Fourier transformation method).41 For the L2 phase, d is called the characteristic distance. The sample was filled at room temperature into the sample holder (capillary in a metal block), temperature controlled by a Peltier element (±0.1 °C), and equilibrated at each experimental temperature for at least 10 min before performing the measurement. All temperature scans were performed in the heating direction. 2.5. Dispersion Preparation. Dispersions consisting of 5 wt % dispersed materials that include 4.625 wt % lipids (Dimodan U and vitamin E acetate), and 0.375 wt % of an emulsifier (Emultop or B


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F127). This raw mixture was treated by ultrasonication for 20 min at 30% of the maximum power in pulse mode and at cycle 0.5. Two ultrasonication devices (SY-LAB GmbH, Pukersdorf, Austria) for Figures 2 and 8 and Dr. Hielscher Ultraschallprozessor (model 400, Germany) for Figures 3−7 were used. 2.6. Cryogenic Transmission Electron Microscopy (CryoTEM). The vitrification of the investigated samples was performed using a controlled-environment vitrification system for cryo-transmission electron microscopy (cryo-TEM) built in house, with the chamber humidity set to 100% at a temperature of 23 °C. Copper grids (Quantifiol Multi A or S7/2, Germany) covered with carbon films containing holes were used. 5 μL drops of solution were deposited onto the grids. Before being propelled into liquid ethane, the specimens were blotted for 3 s between two filter papers. Frozen grids were conserved in liquid nitrogen. For observation they were transferred into a cryo-holder (Gatan 626-DH), kept at −180 °C. The investigations were performed on a FEI Tecnai Spirit BioTWIN (FEI, The Netherlands) at 120 kV, with the images being recorded with a Quemesa camera (Olympus, Japan).

content higher than 35−40%, the cubic phase is fully hydrated and a biphasic region is present where the inverted bicontinuous cubic Pn3̅m phase coexists with excess of water. As shown in Figure 1, the phase behavior gets much simpler when the solubilized amount of vitamin E is larger than about 5%, and the lamellar liquid crystalline phase and the two reversed bicontinuous cubic phases are no longer present. Progressive addition of water leads to the presence of the reversed microemulsion (also called L2 phase), followed by the formation of the HII phase. The HII phase coexists with water under full hydration conditions. At a vitamin E content higher than 30%, only the L2 phase is present and can coexist with excess of water (Winsor II type micellar solution). It can also be seen that the maximum water solubilization strongly decreases with increasing vitamin E content. Maximum water solubilization is in the range of 35−40% in the reversed bicontinuous cubic phase and decreases to less than 25% in the reversed hexagonal phase. Within the HII domain, the water solubilization capacity decreases strongly when increasing D-αtocopherol amount. At higher vitamin E content, water solubilization in the L2 domains remains first relatively constant (at vitamin E content in the range of 30−50%), before decreasing smoothly when the vitamin E content is higher than 50%. It should also be noticed that at high vitamin E concentrations crystals coexist with the mesophase in some samples. This crystallization does not take place immediately at 23 °C, but is triggered by agitation and lowering temperature. This indicates that the monophasic system (neat mesophase) can be in a metastable equilibrium and that the crystal nucleation takes place via inhomogeneous nucleation. The presence of a low amount of saturated monoglycerides present within the used emulsifier (Dimodan U/J) is likely to be responsible for this behavior. 3.2. Dispersions Made with Vitamin E Acetate. For many applications, the bulk structures present in the ternary phase diagram shown in Figure 1 cannot be used as such. Therefore, it is necessary to disperse these phases into excess water in the presence of a secondary emulsifier.8,44,45 This emulsifier should be relatively hydrophilic as is the case for obtaining an oil-in-water (O/W) emulsion. In addition, it should be efficient as stabilizer without having a significant interaction with the primary emulsifier (unsaturated monoglycerides in our case) that could disrupt or even destroy the internal structure. In the present report, two secondary emulsifiers were used: partially hydrolyzed lecithin and Pluronic F127. Hydrolyzed lecithin is a food-grade emulsifier, and Pluronic F127 is commonly used for processing mesophase dispersions since it provides good steric stabilization and it does not significantly influence the particle internal structures.46 The formation and characterization of dispersions of the various internal structures are discussed in the present work. 3.2.1. Dispersions Stabilized by Pluronic F127. Figure 2 shows the temperature-dependent phase behavior of the particle internal structure in the aqueous dispersions based on UMG at different δ ratios of UMG relative to the total lipid (UMG + vitamin E acetate) phase content as determined by SAXS. The dispersions were prepared at constant concentration of Pluronic F127 (0.375 wt %) and a constant total lipid content of 4.625 wt %, consisting of different Dimodan U/J:DLα-tocopheryl acetate ratios, and 95 wt % water. The phase diagram of these internally structured dispersions (Figure 2) is in relatively good agreement at ambient

3. RESULTS AND DISCUSSION 3.1. Ternary Phase Diagram of Unsaturated Monoglycerides−Vitamin E Acetate−Water. The ternary phase diagram of unsaturated monoglycerides−DL-α-tocopheryl acetate−water at 23 °C, which was obtained mainly by polarized light microscopy and macroscopic phase examination, is presented in Figure 1. For simplicity, in the rest of the text,

Figure 1. The ternary vitamin E acetate−Dimodan U/J−water phase diagram at 23 °C. Lines indicate the phase boundaries according to the phase behavior determined for individual points. Dashed lines are tentative phase boundaries for samples where the mesophases coexist with water.

we will use the term vitamin E acetate (or vitamin E), but all the experiments were performed using DL-α-tocopheryl acetate (or DL-α-tocopherol). At a vitamin content lower than 5% (here and in the rest of the text, % always corresponds to weight ratio), a large variety of mesophases can be formed, in analogy to the binary unsaturated monoglycerides−water system. Upon increasing the water content, first a reversed microemulsion phase is formed, which is followed by a lamellar liquid crystalline phase, and then two different reversed bicontinuous cubic phases. With an amount of water larger than about 15%, the reversed bicontinuous cubic phase of the gyroid type (space group symmetry: Ia3̅d) is present first,42,43 followed by the reversed bicontinuous cubic structure of the diamond type at a higher water content (space group with symmetry Pn3̅m). At a water C


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remark may explain why the liquid crystalline phase HII could be more destabilized in the dispersed state than in bulk phase. In the dispersed state, the transition to the reversed microemulsion (L2), which shows much less order than the HII, may be favored. 3.2.2. Dispersions with Partially Hydrolyzed Lecithin: Phase Behavior and Comparison with F127. Dispersions of mesophases having various internal structures were processed. The weight ratios between the investigated commercial monoglyceride and the stabilizers (hydrolyzed lecithin or F127) were chosen according to the phase diagram of Figure 1. The ratio between monoglyceride and vitamin E actetate was 98.9:1.1 for the reversed bicontinuous cubic phase, 90:10 for the reversed hexagonal (HII) phase, and 45:55 for the reversed microemulsion. These samples were characterized by SAXS and cryo-TEM analysis (Figures 3−7). SAXS patterns display much Figure 2. Phase diagram obtained by SAXS for aqueous dispersions containing 95 wt % water, 0.375 wt % Pluronic F127, and 4.625 wt % lipid. The lipid is a mixture of Dimodan U/J (unsaturated monoglycerides) and vitamin E acetate. The diagram shows the phase present, within the dispersed oil droplets, as a function of δ the percentage of Dimodan U/J in the lipid (Dimodan U/J + vitamin E acetate) and the investigated temperature. Black squares correspond to the reversed bicontinuous cubic phase. Empty red squares correspond to a mixture between reversed hexagonal (HII) and reversed bicontinuous cubic phase. Red triangles correspond to the internal HII phase. Empty blue triangles correspond to mixture between the reversed hexagonal phase (HII) and the reversed microemulsion. Filled blue circles correspond to the reversed microemulsion.

temperatures with that of the phase diagram of the corresponding nondispersed bulk ternary system shown in Figure 1. The sequence of the phase transitions seen at 25 °C, when increasing DL-α-tocopheryl acetate content, is the same for the dispersion (Figure 2) as for the corresponding fully hydrated bulk phases. The transition from the reversed bicontinuous cubic phase to the reversed hexagonal phase takes place for both systems at about 5% vitamin E in the binary vitamin E/Dimodan U/J mixture. The most noticeable difference is the composition at which the transition between the reversed hexagonal phase and the microemulsion takes place. It takes place in the nondispersed phase in excess water at a vitamin E acetate content in the range of 30−35 wt %, while in the dispersion with Pluronic F127, it takes place between 23 and 28 wt %. In another study, it was also found that this transition from HII to the reversed microemulsion phase appears at a lower temperature in the dispersed state compared to the bulk phase.46 This indicates that the transition from the HII phase to the L2 phase is favored in the dispersed state compared to the bulk. This stabilization of the L2 phase, in reference to the HII phase, cannot be attributed only to the presence of Pluronic F127 since F127, being more hydrophilic than the monoglyceride and vitamin E acetate, would favor structures with positive curvatures and would stabilize the HII phase. This result is attributed to the dispersion and the small particle size. The particle size was determined to be about 200 nm for HII dispersions made with a similar experimental procedure and limonene.47 This small droplet size corresponds to less than 50 unit cells since, for the HII phase, the unit cell size is about 6 nm. In the dispersed state, these dispersed hexagonal structures are more defective than the bulk ones due to the interface with the aqueous phase and the mechanism of particle stabilization leading to a higher free energy. This

Figure 3. SAXS patterns of dispersed reversed bicontinuous cubic phases containing 4.574% Dimodan U/J, 0.051 wt % vitamin E acetate, 0.375 wt % stabilizer, and 95 wt % water. The ratio between the investigated monoglyceride and vitamin E acetate is fixed at 98.9:1.1. Spectrum at the bottom was obtained using a dispersion stabilized by partially hydrolyzed lecithin. The position of the weak peaks (arrowed) corresponds to the primitive reversed bicontinuous cubic phase (symmetry of the space group Im3̅m). Spectrum at the top was obtained using a dispersion stabilized by Pluronic F127. The position of the peaks (arrowed) corresponds to the reversed diamond bicontinuous cubic phase (symmetry of the space group Pn3m ̅ ).

sharper peaks for the aqueous dispersions stabilized by F127 as compared to those stabilized by the hydrolyzed lecithin. This different effect is clearly shown in Figures 3 and 5 for the internal reversed bicontinuous cubic and internal reversed hexagonal phases. This result strongly suggests that the use of the hydrolyzed lecithin significantly lowers the degree of structural order. The SAXS findings were confirmed by cryoTEM observations of the internal reversed bicontinuous cubic phase (Figure 4). For the dispersions stabilized by F127, periodic motifs compatible with an internal reversed diamond bicontinuous cubic phase are clearly observed; whereas for the dispersions prepared by the partially hydrolyzed lecithin, no clear motif (highly disordered internal structure) is observed. For the internal HII phase, both curved striations and hexagonal periodicity are observed for both stabilizers (Figure 6). These motifs are characteristic of dispersed HII phases. Again, the motif is more evident for the F127 emulsifier than for the partially hydrolyzed lecithin, suggesting a higher degree D


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Figure 4. Cryo-TEM image of the dispersed reversed bicontinuous cubic structures. The SAXS spectra and composition corresponding to this dispersion are given in Figure 3. The picture on the left has been obtained using a dispersion stabilized by the partially hydrolyzed lecithin while the picture on the right has been obtained using a dispersion stabilized by Pluronic F127. It should be noted that the periodic motif for the partially hydrolyzed lecithin (left picture) is not clearly visible; whereas it is clearly seen for F127 (right picture). This confirms a better formed structure when using Pluronic F127. The small star indicates an ice crystal.

ratio between the phospholipid and the lysophospholipid of about 1.75:1 forms vesicles48,49 when dispersed in water may explain this finding. F127 forms micelles and needs to be associated with other components of the dispersion such as the unsaturated monoglyceride to form vesicles when solubilized into water.50 More detailed structural information including the lattice parameter of the internal structure of the aqueous dispersions can be inferred by the analysis of the SAXS patterns. Nondispersed samples of monoglycerides (Dimodan U) in excess water at 23 °C display a diamond cubic structure (space group of symmetry Pn3̅m) with a lattice parameter of 9.25 nm (data not shown). Dispersion made with Pluronic F127 and Dimodan U without vitamin displays a diamond cubic structure (symmetry Pn3̅m) with a lattice parameter of 9.55 nm (data not shown). Additional peaks or humps are present, and their positions correspond to the primitive cubic structure (symmetry Im3̅m). Those findings are in very good agreement with the previous work of Salonen et al.42 The likely explanation for this phase behavior is that the stabilizer F127, which is more hydrophilic than monoglycerides, slightly influences the internal structure and favors cubic phases with higher amount of solubilized water and larger lattice parameter. Incorporation of F127 into cubic phases with sufficiently large water channels is also known to induce a structural transformation from the diamond cubic phase (symmetry Pn3̅m) to the primitive cubic phase (symmetry Im3̅m).23,50,51 Addition of 1.1% vitamin E acetate, which is lipophilic, to the aqueous dispersion (Figure 3), has a back tuning structural effect compared to F127, since SAXS indicates only the presence of the diamond cubic phase (symmetry Pn3̅m) with a lattice parameter of 9.25 nm, which is lower than for the internal structure of vitamin-free cubosomes.

Figure 5. SAXS patterns of dispersed reversed hexagonal phases (HII) containing 4.1625 wt % Dimodan U/J, 0.4625 wt % vitamin E acetate, 0.375 wt % stabilizer, and 95 wt % water. The ratio of the investigated monoglyceride to vitamin E acetate is fixed at 90:10. Spectra at the bottom and at the top were obtained using a dispersion stabilized by the partially hydrolyzed lecithin and F127, respectively. For F127 (top spectrum), three peaks are clearly observed and correspond to the HII phase, while for the partially hydrolyzed lecithin (bottom spectrum), only the first peak is visible. However, the position and the shape of the detected peak and the performed cryo-TEM analysis (Figure 6) demonstrate that a HII structure is present also for the partially hydrolyzed lecithin.

of ordering for the former. An interesting observation is that the number of observed vesicles seems larger for the partially hydrolyzed lecithin compared to the polymeric stabilizer F127 (Figure 6). The fact that the partially hydrolyzed lecithin with a E


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Figure 6. Cryo-TEM image of the dispersed reversed hexagonal structures. The SAXS spectra and composition, corresponding to this dispersion, are given in Figure 5.The picture on the left has been obtained using a dispersion stabilized by the partially hydrolyzed lecithin while the picture on the right has been obtained using a dispersion stabilized by Pluronic F127. The hexagonal phase can be identified by the presence of curved striations (small arrow) or hexagonal periodicity (long arrow).60

The effect of the partially hydrolyzed lecithin on the internal structure of the aqueous dispersion containing 1.1% vitamin E is much stronger than for F127 since the diamond cubic structure (symmetry Pn3̅m) is not observed. Instead, only weak peaks, indicating a rather disordered phase, are present. Their indexation is compatible with the presence of the Im3̅m structure. This result is a direct consequence of the penetration of the partially hydrolyzed lecithin into the particle internal structure. These low molecular weight emulsifiers can be accommodated into the internal structure of the particles and modify it while this effect is less pronounced for the macromolecular stabilizer F127, which mostly acts as an efficient stabilizer adhering to the outer surface of the dispersed particles.23 In addition, the position of the first peak in the SAXS HII phase spectra indicates a higher lattice parameter for dispersion using partially hydrolyzed lecithin, compared to Pluronic F127 (Figure 5). This strongly suggests that the partially hydrolyzed lecithin favors structures with more positive curvature compared to F127. The partially hydrolyzed lecithin is more hydrophilic than the monoglycerides and the vitamin E acetate used, and it penetrates more into the structure than F127. Figure 7 shows the SAXS spectrum of the particles having a ratio between Dimodan U and vitamin E acetate of 45:55 and containing partially hydrolyzed lecithin as a stabilizer. A large broad peak is present, while it is absent for the structureless vitamin E acetate-in-water (O/W) emulsion. It is characteristic of a reversed microemulsion structure (L2 phase) that is formed due to the solubilization of relatively high concentration of the hydrophobic additive vitamin E acetate.52 In conclusion, the phase behavior of particles stabilized by the partially hydrolyzed lecithin is similar to that of the bulk sample at full hydration. However, it lowers the long-range

Figure 7. SAXS patterns of an emulsified reversed microemulsion (EME) containing 2.544 wt % Dimodan U/J, 2.081 wt % vitamin E acetate, 0.375 wt % partially hydrolyzed lecithin, and 95 wt % water (top spectrum) and the reference emulsion containing 4.625 wt % vitamin E acetate, 0.375 wt % partially hydrolyzed lecithin, and 95 wt % water (bottom spectrum). It should be noted that there is a large peak for the EME, which is absent for the reference structureless emulsion.

order of the internal structures to a much greater extent than F127. 3.3. Comparison between the Ternary Phytantriol− Vitamin E Acetate−Water System and the Ternary Unsaturated Monoglyceride−Vitamin E Acetate−Water System. Dong et al. studied the effect of vitamin E on the phytantriol−water system.34 It is not possible to precisely compare between the vitamin E−Dimodan U/J water system investigated in the present work and the vitamin E− F


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phytantriol−water system since Dong et al. have studied in detail the phase behavior of the ternary system up to a weight ratio of vitamin E acetate:(vitamin E acetate + phytantriol) less than about 11%; whereas we report on the full vitamin E acetate−Dimodan U/J phase diagram but with less detail on the vitamin E acetate:Dimodan U/J small ratio. However, it can be stated that generally the effect of vitamin E acetate is qualitatively similar for both systems. At temperatures lower than 40 °C, the solubilization of this hydrophobic additive leads to the transition from a reversed bicontinuous cubic Pn3̅m phase to a reversed hexagonal (HII) phase in both systems. One difference is that the temperature domain of the HII phase seems to be more restricted in the vitamin E acetate−phytantriol−water system as compared to the vitamin E acetate−Dimodan U/J−water system. This finding may simply result from the fact that the temperature domain of the HII phase in the binary system phytantriol with excess water is restricted to an interval of about 5 °C in the bulk phase and does not exist in dispersion obtained with F127 in the work of Dong et al.34 while this HII phase is more stable and exists in a temperature range of about 40 K (between 45 and 90 °C) in the binary Dimodan U/J−water system as determined previously by Mezzenga et al.43 3.4. Effects of Various Lipids on the Phase Behavior of Unsaturated Monoglyceride−Water System. In order to determine if the phase diagram (Figures 1 and 2) obtained for the unsaturated monoglycerides (Dimodan U/J)−vitamin E acetate−water system is general, the addition of other lipids was also studied. Table 1 shows the phase behavior obtained when vitamin E (non-acetated form) or (R)-(+)-limonene is solubilized in the system Dimodan U/J−water at or close to the hydration limit. Results from Table 1 were obtained using a combination of SAXS (Figure 8), polarized microscopy, and visual appearance. It can be seen that the reversed HII phase is observed when the weight ratio of vitamin E:Dimodan U/J is less than 37.5:62.5; whereas the reversed micellar cubic phase is observed when this ratio is between 37.5:62.5 and 55:45, and the reversed microemulsion structure is observed at a weight ratio higher than 60:40. The phase behavior of dispersions based on Dimodan U/J− triolein−water, using Pluronic F127 as a secondary emulsifier, was also studied. The phase behavior obtained with triolein (Figure 9) is very similar to the one obtained with vitamin E acetate (Figure 2). In particular, for both added lipids the reversed micellar cubic phase is absent. There were several studies dealing with the effect of the oils tetradecane and (R)(+)-limonene on the phase behavior of the systems monolinolein−water and Dimodan U/J−water.9,42,53,54 Figure 10 represents the phase diagram of dispersions of the tetradecane-loaded Dimodan U/J-based dispersion (using Pluronic F127 as stabilizer). When the ratio of the added oil in reference to oil + unsaturated monoglyceride content is between 5 and 20% (at room temperature), the initial reversed bicontinuous cubic phase is transformed to the reversed hexagonal phase. Between 25% and about 35%, the reversed micellar cubic phase forms, and finally at an oil content higher than 37%, only the reversed microemulsion phase is present. Similar behavior is observed when replacing tetradecane by limonene. In particular, for both oils, there is a relatively wide composition range, in which the lipid particles have an internal structure corresponding to the reversed micellar cubic phase.53 It can be observed that there are some similarities between the various systems studied or discussed. In particular

Table 1. Determination of the Phase Present in the System Vitamin E (or (R)-(+)-Limonene)−Dimodan U/J−Water at Room Temperature (23°C)a ratio

water (%)

monoglyceride:vitamin E 65/35

12.5

55/45 60/40 55/45 50/50 45/55 40/60 monoglyceride:limonene 60/40 50/50

phase(s) present

10 10

reversed hexagonal + small amount of reversed micellar cubic (symmetry of space group Fd3̅m) reversed hexagonal + micellar cubic reversed hexagonal + micellar cubic micellar cubic + water micellar cubic + L2 (reversed microemulsion) + water micellar cubic + L2 + water L2 + water

12.5 10

micellar cubic + L2 L2 + water

12.5 12.5 12.5 10

a

The phase determination was done based on SAXS analysis (Figure 8), polarized microscopy, and macroscopic phase examination. It is worth noting that there is a relatively large composition range where the reversed micellar cubic phase is present in the system vitamin E− Dimodan U/J−water, while it is absent for the system vitamin E acetate−Dimodan U/J−water. Notice also that transition from the reversed micellar cubic phase to the reversed microemulsion appears at higher lipid content for vitamin E than for (R)-(+)-limonene.

Figure 8. SAXS characterization of the system Dimodan U/J−DL-αtocopherol−Pluronic F127. Experiments were performed at room temperature (23 °C). From top to bottom, the weight ratio between Dimodan U/J and DL-α-tocopherol and the water content is as follows: 65/35 and 12.5 wt % water, 60/40 and 12.5 wt % water, 55/45 and 12.5 wt % water, 50/50 and 10 wt % water, 45/55 and 10 wt % water, 40/60 and 10 wt % water. Scattering curves are shifted vertically by arbitrary factors for better visibility.

progressive addition of oil, at room temperature, leads to the appearance of the reversed hexagonal phase and reversed microemulsion. However, there are still significant differences indicating that the solubilized oil molecular structure and its physicochemical properties have an important role in modulating the structure. When vitamin E acetate or triolein is used as an oil, the reversed micellar cubic phase is suppressed or only forms in a very narrow composition range (which would explain that it is not detected in the present study). Even among the lipids that tend to form the reversed micellar cubic phase (vitamin E, (R)G


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Figure 10. Phase diagram obtained by SAXS for dispersion containing 95 wt % water, 0.375 wt % Pluronic F127, and 4.625 wt % lipid. The lipid is a mixture of Dimodan U/J and tetradecane. The diagram shows the phase present, within the dispersed oil droplets, as a function of δ the percentage of Dimodan U/J in the lipid (tetradecane + Dimodan U/J), and investigated temperature. Black squares correspond to the reversed bicontinuous cubic phase (space group Im3̅m). Empty red squares correspond to a mixture between a reversed hexagonal (HII) and a reversed bicontinuous cubic phase. Red triangles correspond to the HII phase. Full blue circles correspond to a reversed microemulsion. Green diamonds correspond to the reversed micellar cubic structure. Blue open diamonds correspond to a mixture between the reversed micellar cubic structure and the reversed microemulsion. Green open triangles correspond to mixture between the HII and the reversed micellar cubic phase. Blue open triangles correspond to mixture between the HII phase and the reversed microemulsion. Reprinted with permission from ref 53. Copyright 2006 Elsevier.

Figure 9. Phase diagram obtained by SAXS for aqueous dispersions containing 95 wt % water, 0.375 wt % Pluronic F127, and 4.625 wt % lipid. The lipid is a mixture of Dimodan U/J (unsaturated monoglyceride) and triolein. The diagram shows the phase present, within the dispersed oil droplets, as a function of δ the percentage of Dimodan U/J in the lipid (Dimodan U/J + Triolein) and the investigated temperature. Black squares correspond to the reversed bicontinuous cubic phase (space group Im3̅m). Empty red squares correspond to a coexistence of two internal structures: a reversed hexagonal (HII) and a reversed bicontinuous cubic phase. Red triangles correspond to the HII phase. Empty blue triangles correspond to mixture between the reversed hexagonal phase (HII) and the reversed microemulsion. Blue circles correspond to the reversed microemulsion.

(+)-limonene, and tetradecane), there are significant differences. First, the transition from the reversed micellar cubic phase to the reversed microemulsion appears in the presence of solubilized vitamin E at much higher lipid content than for limonene or tetradecane (Table 1 and Figure 10). This is most likely due to the fact that vitamin E contains a hydroxyl group (Figure 11) and is thus more amphiphilic and has less effect on structure and the curvature when compared to the more lipophilic compounds. There are also differences between reversed micellar phases containing (R)-(+)-limonene and tetradecane. In our previous work, we noticed that the values and the evolutions of the lattice parameter, in excess water, with oil addition, for the reversed micellar cubic phases containing (R)-(+)-limonene on one side and tetradecane on the other side, were very different.53 Lattice parameter was much larger (about 6 nm larger) for the tetradecane, and increasing oil content did not affect lattice parameter for tetradecane but corresponds to a decrease in lattice parameter for (R)(+)-limonene. This was attributed to a likely different localization of the lipophilic additive in the structure.53 Difference in water solubility for the reversed micellar phase, formed with different oils, may also contribute to the observed differences in lattice parameters. It is worth discussing briefly why some oils (such as vitamin E, limonene, and tetradecane) give rise to the presence of a reversed micellar cubic phase between the reversed hexagonal phase and the reversed microemulsion, while others (such as vitamin E acetate and triolein) do not. All these five molecules (Figure 11) have a very different molecular structure. Important aspects include molecular shape, polarity, localization of the most hydrophilic part(s), and molecular size. It may first appear surprising that vitamin E and vitamin E acetate lead to a

different phase behavior of the system lipid−unsaturated monoglyceride−water. However, vitamin E is slightly shorter than vitamin E acetate, and very likely more importantly, as mentioned above, it contains a hydroxyl group which gives some polarity and an interaction with the glycerol part of the monoglyceride and water. This explains that for the two molecules a different phase behavior is obtained. The phase behavior obtained with tetradecane and (R)-(+)-limonene on one side is also different from the one obtained with triolein. Triolein is large when compared to tetradecane and limonene. Its penetration into the self-assembled structure may not enable the formation of the reversed micellar cubic phase which consists of two different types of reversed micelles.55,56 This geometrical constraint may not be met with a large molecule like triolein. With smaller molecules like tetradecane and (R)(+)-limonene, this complex geometry might be easier to reach. The presence or absence of the reversed micellar cubic phase is often associated with the ease of molecules to relax frustration or in other words to fill the free volume left by the packing of reversed micelles.57−59 The fact that (R)-(+)-limonene, tetradecane, and vitamin E acetate act differently on the structure and lattice parameter and all three form the reversed micellar cubic phase suggests that a general rule for additives leading to the formation of this phase may be difficult to obtain and will require some dedicated studies in the future. H


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Figure 11. Molecular structure of the various lipids discussed in this work, and hydrophobic additives that influence the phase behavior. (a) Glycerol monooleate (cis form), (b) glycerol monoolinoleate (cis form), (c) DL-α-tocopheryl acetate, (d) DL-α-tocopherol, (e) tetradecane, (f) (R)(+)-limonene, and (g) triolein.

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(6) Phan, S.; Fong, W. K.; Kirby, N.; Hanley, T.; Boyd, B. J. Evaluating the link between self-assembled mesophase structure and drug release. Int. J. Pharm. 2011, 421 (1), 176−182. (7) Tiberg, F.; Johnsson, M. Drug delivery applications of nonlamellar liquid crystalline phases and nanoparticles. J. Drug Delivery Sci. Technol. 2011, 21 (1), 101−109. (8) Sagalowicz, L.; Leser, M. E.; Watzke, H. J.; Michel, M. Monoglyceride self-assembly structures as delivery vehicles. Trends Food Sci. Technol. 2006, 17, 204−214. (9) Yaghmur, A.; de Campo, L.; Salentinig, S.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Oil-loaded Monolinolein-based particles with confined inverse discontinuous cubic structure (Fd3m). Langmuir 2006, 22, 517−521. (10) Seddon, J. M. Structure of the inverted (HII) phase, and non lamellar phase transitions of lipids. Biochim. Biophys. Acta 1990, 1031, 1−69. (11) Sagalowicz, L.; Mezzenga, R.; Leser, M. E. Investigating reversed liquid crystalline mesophases. Curr. Opin. Colloid Interface Sci. 2006, 11, 224−229. (12) Rizwan, S. B.; Boyd, B. J.; Rades, T.; Hook, S. Bicontinuous cubic liquid crystals as sustained delivery systems for peptides and proteins. Expert Opin. Drug Delivery 2010, 7 (10), 1133−1144. (13) Sagalowicz, L.; Leser, M. E. Delivery systems for liquid food products. Curr. Opin. Colloid Interface Sci. 2010, 15, 61−72. (14) Amar-Yuli, I.; Azulay, D.; Mishraki, T.; Aserin, A.; Garti, N. The role of glycerol and phosphatidylcholine in solubilizing and enhancing insulin stability in reverse hexagonal mesophases. J. Colloid Interface Sci. 2011, 364 (2), 379−387. (15) Johnsson, M.; Barauskas, J.; Norlin, A.; Tiberg, F. Physicochemical and drug delivery aspects of lipid-based liquid

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the support of Günther Scherf in the SAXS experiments.

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REFERENCES

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