Emulsified Microemulsions and Oil-Containing Liquid Crystalline

When adding tetradecane (TC) to the MLO−water−F127 system at constant temperature, the internal ..... Accounts of Chemical Research 0 (proofing),...
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Langmuir 2005, 21, 569-577

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Emulsified Microemulsions and Oil-Containing Liquid Crystalline Phases Anan Yaghmur,† Liliana de Campo,† Laurent Sagalowicz,‡ Martin E. Leser,‡ and Otto Glatter*,† Institute of Chemistry, University of Graz, Graz, Austria, and Nestle´ Research Center, Lausanne, Switzerland Received July 9, 2004. In Final Form: October 21, 2004 Self-assembled nanostructures, such as inverted type mesophases of the cubic or hexagonal geometry or reverse microemulsion phases, can be dispersed using a polymeric stabilizer, such as the PEO-PPOPEO triblock copolymer Pluronic F127. The particles, which are described in the present study, are based on monolinolein (MLO)-water mixtures. When adding tetradecane (TC) to the MLO-water-F127 system at constant temperature, the internal nanostructure of the kinetically stabilized particles transforms from a Pn3m (cubosomes) to a H2 (hexosomes) and to a water-in-oil (W/O, L2) microemulsion phase (emulsified microemulsion (EME)). To our knowledge, this is the first time that the formation of stable emulsified microemulsion (EME) systems has been described and proven to exist even at room temperature. The same structural transitions can also be induced by increasing temperature at constant tetradecane content. The internal nanostructure of the emulsified particles is probed using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). At each investigated composition and temperature, the internal structure of the dispersions is observed to be identical to the corresponding structure of the nondispersed, fully hydrated bulk phase. This is clear evidence for the fact that the self-assembled inner particle nanostructure is preserved during the dispersion procedure. In addition, the internal structure of the particles is in thermodynamic equilibrium with the surrounding water phase. The internal structure of the dispersed, kinetically stabilized particles is a “real” and stable self-assembled nanostructure. To emphasize this fact, we denoted this new family of colloidal particles (cubosomes, hexosomes, and EMEs) as “ISASOMES” (internally self-assembled particles or “somes”).

Introduction Lyotropic liquid crystalline phases of binary surfactantlike lipid-water systems have attracted significant attention in the literature due to their possible role in the structure and function of biological membranes and living cells.1-6 Moreover, their potential applications as matrixes that mimic biological systems in composite material synthesis,7 for the solubilization of active molecules8-11 (vitamins, enzymes, proteins, and so on), as a base for drug-delivery systems,12-14 for the synthesis of flavors,15 and for the crystallization of membrane proteins16-19 have been recently discussed. * Corresponding author. Phone: +43 316 380 5433. Fax: +43 316 380 9850. E-mail: [email protected]. † University of Graz. ‡ Nestle ´ Research Center. (1) Hyde, S. T. Curr. Opin. Solid State Mater. Sci. 1996, 1, 653-662. (2) Rizvi, T. Z. J. Mol. Liq. 2003, 106, 43-53. (3) Seddon, J. M. Curr. Opin. Colloid Interface Sci. 2001, 6, 242243. (4) Luzzati, V.; Vargas, R.; Mariani, P.; Gulik, A.; Delacroix, H. J. Mol. Biol. 1993, 229, 540-551. (5) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (6) Luzzati, V. Curr. Opin. Struct. Biol. 1997, 7, 661-668. (7) Zhao, D.; Huo, Q.; Feng, J.; Chmelka, B. F.; Stucky, G. D. J. Am. Chem. Soc. 1998, 120, 6024-6036. (8) Caboi, F.; Nylander, T.; Razumas, V.; Talaikyte, Z.; Monduzzi, M.; Larsson, K. Langmuir 1997, 13, 5476-5483. (9) Caboi, F.; Amico, G. S.; Pitzalis, P.; Monduzzi, M.; Nylander, T.; Larsson, K. Chem. Phys. Lipids 2001, 109, 47-62. (10) Caboi, F.; Murgia, S.; Monduzzi, M.; Lazzari, P. Langmuir 2002, 18, 7916-7922. (11) Nylander, T.; Mattisson, C.; Razumas, V.; Miezis, Y.; Håkansson, B. Colloids Surf., A 1996, 114, 311-320. (12) Drummond, C.; Fong, C. Curr. Opin. Colloid Interface Sci. 2000, 4, 449-456. (13) Pouton, C. W. Adv. Drug Delivery Rev. 1997, 25, 47-58. (14) Engstro¨m, S.; Norde´n, T. P.; Nyquist, H. J. Pharm. Sci. 1999, 8, 243-254. (15) Vauthey, S.; Milo, Ch.; Frossard, Ph.; Garti, N.; Leser, M. E.; Watzke, H. J. J. Agric. Food Chem. 2000, 48, 4808-4816.

Lipids, such as monoglycerides or phospholipids, are known for their ability to form various mesophases, that is, complex lyotropic liquid crystalline phases, when put into water. Depending on the temperature, water content, and lipid molecular structure (single or double chained, saturation degree, and length of fatty acyl chain), either a fluid isotropic phase (the so-called L2 phase) or a lamellar, hexagonal, or cubic mesophase is formed.20-28 The monoolein (MO)-water system, for example, is a widely investigated binary system.20-25,28-30 MO has a rich phase diagram that includes a fluid isotropic phase (L2), a lamellar phase (LR), an inverted hexagonal phase (H2), and a reversed bicontinuous cubic liquid crystalline phase (V2). It has been shown that the structure of this binary system can be modulated by adding a third component.9,31,32 Molecules leading to the formation of a layer with a positive spontaneous curvature (H0 > 0), such as (16) Rummel, G.; Hardmeyer, A.; Widmer, C.; Chiu, M. L.; Nollert, P.; Locher, K. P.; Pedruzzi, I.; Landau, E. M.; Rosenbuch, J. P. J. Struct. Biol. 1998, 121, 82-91. (17) Landau, E. M.; Rummel, G.; Rosenbusch, J. P.; Cowan-Jacob, S. W. J. Phys. Chem. B 1997, 101, 1935-1937. (18) Caffrey, M. Curr. Opin. Struct. Biol. 2000, 10, 486-497. (19) Caffrey, M. J. Struct. Biol. 2003, 142, 108-132. (20) Chernik, G. G. Curr. Opin. Colloid Interface Sci. 2000, 4, 381390. (21) Larsson, K. Nature 1983, 304, 664. (22) Larsson, K. Chem. Phys. Lipids 1972, 9, 181-195. (23) Larsson, K.; Fontell, K.; Krog, N. Chem. Phys. Lipids 1980, 27, 321-328. (24) Lutton, E. S. J. Am. Oil Chem. Soc. 1965, 42, 1068-1070. (25) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223-234. (26) Qiu, H.; Caffrey, M. J. Phys. Chem. B 1998, 102, 4819-4829. (27) Qiu, H.; Caffrey, M. Chem. Phys. Lipids 1999, 100, 55-79. (28) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II 1996, 6, 723-751. (29) Chung, H.; Caffrey, M. Nature 1994, 368, 224-226. (30) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213-219. (31) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2000, 16, 1004410054.

10.1021/la0482711 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/17/2004

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sodium oleate,32 induce a transition from V2 to LR, while the addition of an oil, such as oleic acid, induces a structural transition from V2 to H2, that is, leading to the formation of a film layer with negative spontaneous curvature32 (H0 < 0). The MO-water system has been investigated in the presence of a wide range of different hydrophilic and lipophilic guest molecules of biological and pharmaceutical relevance.8-10 Examples are hydrophobic vitamins such as vitamins K1 and A, aspirin, tea tree oil, and 1-adamantanamine hydrochloride (a water soluble drug against A-influenza and Parkinson’s disease). It was found that the nature of these guest molecules significantly influences the phase behavior of this system.8-10 This effect is dependent on the amount of the solubilized guest molecule and on the water content. The amphiphilic triblock copolymer Pluronic F127 was used33-40 to stabilize colloidal aqueous dispersions of V2 (cubosomes) and H2 (hexosomes). Recently, the inner periodicity and the outer shape of these MO-based soft particles have been investigated by different techniques: 34-38,41-47 small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and 13 C NMR. It was found that the addition of a small amount of oleic acid, triolein, or retinyl palmitate to the MOwater system induces the transformation from cubosomes to hexosomes.34,37,45,46 Due to their possible biological relevance, their high interfacial area, their low viscosity, and their capability to solubilize both hydrophilic and hydrophobic active molecules, there is great interest in the utilization of these dispersions for the administration of drugs, for the formulation of delivery systems,12,48,49 and for the stability enhancement of incorporated enzymes and proteins. Recently, we reported on the temperature behavior of aqueous submicron-sized dispersions of the binary monolinolein (MLO)-water system.50 It was found that the internal nanostructure of the dispersions is in thermodynamic equilibrium with the surrounding aqueous phase, since, upon heating, the dispersions change from the (32) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2001, 17, 77427751. (33) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611-4613. (34) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964-6971. (35) Larsson, K. J. Dispersion Sci. Technol. 1999, 20, 27-34. (36) Spicer, P. T.; Hayden, K. L.; Lynch, M. L.; Ofori-Boateng, A.; Burns, J. L. Langmuir 2001, 17, 5748-5756. (37) Nakano, M.; Teshigawara, T.; Sugita, A.; Leesajakul, W.; Taniguchi, A.; Kamo, T.; Matsuoka, H.; Handa, T. Langmuir 2002, 18, 9283-9288. (38) Landh, T. J. Phys. Chem. 1994, 98, 8453-8467. (39) Patton, J. S.; Carrey, M. C. Science 1979, 204, 145-148. (40) Lindsto¨rm, M.; Ljusberg-Wahren, H.; Larsson, K.; Borgsto¨rm, B. Lipids 1981, 16, 749-754. (41) Almgren, M.; Edwards, K.; Karlsson, G. Colloids Surf., A 2000, 174, 3-21. (42) Borne, J.; Nylander, T.; Khan, A. J. Phys. Chem. B 2002, 106, 10492-10500. (43) Spicer, P. T.; Small, W. B.; Lynch, M. L.; Burns, J. L. J. Nanopart. Res. 2002, 4, 297-311. (44) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2001, 17, 3917-3922. (45) Monduzzi, M.; Ljusberg-Wahren, H.; Larsson, K. Langmuir 2000, 16, 7355-7358. (46) Neto, C.; Aloisi, G.; Baglioni, P.; Larsson, K. J. Phys. Chem. B 1999, 103, 3896-3899. (47) Larsson, K. Curr. Opin. Colloid Interface Sci. 2000, 4, 449-456. (48) Boyd, B. J. Int. J. Pharm. 2003, 260, 239-247. (49) Siekmann, B.; Bunjes, H.; Koch, M. H. J.; Kirsten, W. Int. J. Pharm. 2002, 244, 33-43. (50) de Campo, L.; Yaghmur, A.; Sagalowicz, L.; Leser, M. E.; Watzke, H.; Glatter, O. Langmuir 2004, 20, 5254-5261.

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emulsified cubic phase (denoted as ECP) via the emulsified hexagonal phase (EHP) to the emulsified L2 phase (ELP) in a reversible way, in analogy to the binary nondispersed bulk MLO-water mesophases, which are in equilibrium with excess water. The aim of the present study is to investigate the effect of the solubilization of tetradecane (TC) on the internal nanostructure of the MLO-based ECP, EHP, and ELP aqueous dispersions.50 Of special interest in the present investigation, from both a scientific and an industrial point of view, is to study the possibility of the formation of what we denote as an emulsified microemulsion (EME) system. It will be shown that it is possible to form a water-in-oil microemulsionin-emulsion system which is different from the well-known double emulsion (emulsion-in-emulsion system) due to the fact that the kinetically stablilized internal W/O emulsion in the double emulsion system is replaced by the thermodynamically stable W/O microemulsion as the “inner” emulsion in the EME. Stable emulsified microemulsions are superior over double emulsion systems, since they consist of stabilized droplets in an aqueous continuous phase, which contain in the inner part an entrapped equilibrium nanostructure. The present study is organized in the following way: in the first part, the impact of the solubilized tetradecane oil content on the internal nanostructure of the MLObased aqueous dispersions is discussed, and in the second part, the effect of temperature on these confined structures is described. It will be shown that the addition of tetradecane opens up the possibility of forming reversible and tunable self-assembled dispersed nanostructured systems. Materials and Methods Materials. Monolinolein (emulsifier TS-PH 039, glycerol monolinoleate, MLO) was supplied by Danisco A/S (Brabrand, Denmark). This distillated monoglyceride consists of 93.8% monoglyceride and 4.1% diglycerides. The fatty acids are composed of 91.8% linoleate, 6.8% oleate, and ∼1% saturated fatty acids. Tetradecane (TC) was obtained from Sigma Chemical Co. (St. Louis, MO). Pluronic F127 (PEO99-PPO67-PEO99) was a gift from BASF Corporation (Mount Olive, NJ). These ingredients were used without further purification. The water was double distilled. Preparation of Ternary MLO-Oil-Water Nondispersed Systems (Bulk Phase). The nondispersed samples were prepared in Pyrex tubes by weighing the appropriate amounts of MLO, oil, and water, heating them by using an air gun, and homogenizing them by vigorous agitation with a Vortex. They were then left to cool to room temperature. Formulation of MLO-Based Oil-Loaded Aqueous Dispersions. A mixture of MLO (viscous liquid) and tetradecane (TC) was weighed into a 20 mL vial. F127 and then water were added to give a sample of a total weight of 10 g. Then, the sample containing all four ingredients was treated by ultrasonication for 20 min, resulting in a milky dispersion. The typical composition of the prepared dispersions was 95 wt % water and 5 wt % MLO-oil-F127 mixture, whereby the concentration of the F127 was constant (0.375 wt %) and the weight ratio, R (R ) ((mass of TC)/(mass of MLO)) × 100) was varied. The value of R was in a range of 0-110. Ultrasonication was carried out using a high intensity ultrasonic processor (SY-LAB G.m.b.H, Pukersdorf, Austria), at 30% of the maximum power, and 0.5 s pulses interrupted by 0.5 s breaks. There was no external sample cooling applied. Small-Angle X-ray Scattering (SAXS) Measurements. The used SAXS equipment consisted of a SAXSess camera51 (Anton-Paar, Graz, Austria), which is connected to an X-ray (51) Bergmann, A.; Orthaber, D.; Scherf, G.; Glatter, O. J. Appl. Crystallogr. 2000, 33, 869-875.

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Figure 1. Effect of solubilized tetradecane (TC) content on the scattering curves of MLO-based emulsified systems at 25 °C (black lines) and those of the nondispersed bulk sample with excess water (red lines). The R ratio [((mass of oil)/(mass of MLO)) × 100] varies from 0 (a), to 19 (b), 75 (c), and 110 (d). The intensities were normalized by the respective MLO plus TC concentration. F127 was used to stabilize the dispersed particles. generator (Philips, PW 1730/10) operating at 40 kV and 50 mA with a sealed-tube Cu anode. A Go¨bel mirror is used to convert the divergent polychromatic X-ray beam into a focused lineshaped beam of Cu KR radiation (λ ) 0.154 nm). The 2D scattering pattern is recorded by an imaging-plate detector (model Fuji BAS1800 from Raytest, Straubenhardt, Germany) and integrated to the 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)sinθ/2, with λ being the wavelength and θ the scattering angle. For indexing the different mesophases, we used the reflection laws summarized in the Supporting Information of our recent work.50 These comprise the space groups that have been determined for cubic and hexagonal phases containing membrane lipids.52 The interplanar distances, 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 only one broad correlation 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 the generalized indirect Fourier transformation (GIFT) method.53 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 (52) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 998, 221256. (53) Bergmann, A.; Fritz, G.; Glatter, O. J. Appl. Crystallogr. 2000, 33, 1212-1216.

Peltier element, (0.1 °C) and equilibrated at each experimental temperature for at least 10 min before measurement. All temperature scans were performed in the heating direction. Cryogenic Transmission Electron Microscopy (cryoTEM). A laboratory-built controlled environment vitrification system for Cryo-TEM (very similar to the one described by Egelhaaf et al.54) was used. The humidity in the environment chamber was about 95% for cubosomes and 100% for the other dispersions. The environmental chamber temperature was maintained at 25 °C. A droplet of 5 mL of dispersion was deposited onto a 200 mesh copper grid (25 °C, Quantifoil R2/2 or S7/2; both Jena, Germany) that was covered with a carbon film containing holes. It was left between two filter papers 595 (Schleicher & Schuell, Dassel, Germany) for ∼2 s before being propelled into liquid ethane. Frozen grids were stored in liquid nitrogen and transferred into a cryo-holder (Gatan 626, Pleasanton, CA) that was kept at -180 °C. Sample analysis was performed in a Philips CM12 transmission electron microscope (Philips, Eindhoven, The Netherlands) at a voltage of 80 kV. Low dose procedures were applied to minimize beam damage. The images were recorded with a Gatan 794 slow scan digital camera (Pleasanton, CA). Dynamic Light Scattering (DLS). The used DLS instrument is a laboratory-built goniometer, equipped with a diode laser (Coherent Verdi V5, l ) 532 nm, Pmax ) 5 W) with single mode fiber detection optics (OZ from GMP, Zu¨rich, Switzerland), an ALV/SO-SIPD/DUAL photomultiplier with pseudo-cross-cor(54) Egelhaaf, S. U.; Schurtenberger, P.; Mu¨ller, M. J. Microsc. 2000, 200, 128-139.

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relation, and an ALV 5000/E correlator with fast expansion (ALV, Langen, Germany). The measurements were carried out at a scattering angle of q ) 90° at different temperatures, using an effective laser power in the sample of P ) 200 mW. Data were collected in repeated measurements (approximately 10 to 25) of 10 s each, until a total of 10 000 000 counts were detected. The intensity autocorrelation functions, which were affected least by intrinsically appearing dust particles, were averaged. From these functions, the average diffusion coefficient, D, was obtained by means of a second-order cumulant analysis of the intensity autocorrelation function.55 The hydrodynamic radius, RH, was calculated using the Stokes-Einstein relation:

RH )

kBT 6πηD

(1)

where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the continuous medium at a given temperature.

Results and Discussion For the investigations described in the present study, we used the monoglyceride monolinolein (MLO) that is less studied in the literature than the well-known monoolein (MO). The MLO allows the formation of all three major phases in the binary system MLO-water, that is, the reversed types of the bicontinuous cubic phase (V2), the hexagonal phase (H2), and the L2 phase, within a feasible experimental temperature range (between 20 and 95 °C). To better characterize the internal nanostructures of the oil-loaded MLO-based aqueous dispersions, the corresponding nondispersed bulk systems (mesophases coexisting with excess water) are also investigated. This allows a direct comparison of the confined nanoscaled internal structure of the aqueous dispersions with those of the corresponding nondispersed bulk systems and, as a consequence, a better understanding of the observed structural changes in the dispersed systems. 1. Effect of Tetradecane (TC) on the Confined Internal Structure of MLO-Based Aqueous Dispersions. Figure 1 shows the effect of tetradecane (TC) solubilization on the internal structure of the MLO-based aqueous dispersions, stabilized by F127, in comparison with the structure of the corresponding TC-MLO-water nondispersed, fully hydrated bulk systems (Pn3m, H2, and W/O microemulsion coexisting with excess water). The SAXS scattering curves with various R values (in the range 0-110) at 25 °C are shown: in the absence of oil (see Figure 1a), the scattering curve of the dispersion shows six peaks in the characteristic ratio for a cubic structure of the type Pn3m (cubosomes, emulsified cubic phase (ECP)). As soon as tetradecane is present (at R ) 19, in Figure 1b), the scattering curve of the dispersion shows three peaks in the characteristic ratio for a hexagonal phase (hexosomes, EHP). A further increase of the TC concentration in the dispersion (R ) 75, in Figure 1c, and R ) 110, in Figure 1d) leads to a scattering curve which shows only one broad peak, which is typical for a concentrated microemulsion. The scattering curves of each of these dispersions at increasing TC content correspond very well to those of the nondispersed fully hydrated bulk phases with the same R value (see Figure 1). This means that the internal structure of the particles is very similar to that of the nondispersed fully hydrated bulk phase. This implies that, upon increasing the TC content, we actually observe a transition from an emulsified cubic (55) Pecora, R. In Dynamic Light Scattering. Applications of Photon Correlation Spectroscopy; Pecora, R., Ed.; Plenum Press: New York, 1985; p 420.

Figure 2. Comparison of scattering curves from the MLObased dispersions (black lines) with those from the nondispersed bulk sample with different water content (open symbols) at 25 °C. The intensities were normalized first by the respective MLO plus TC concentration and then shifted by a constant arbitrary factor for better visibility. The R ratio varies from 19 (a) to 110 (b).

phase (ECP, cubosomes) via an emulsified H2 phase (EHP, hexosomes) to an emulsified W/O microemulsion (EME). The proof for this finding will be discussed below in greater detail. It is worth noting that the dispersed internal structures as well as the structures of the nondispersed bulk samples depend not only on the TC content (R value) but also on the water content in the respective phase, of course. Figure 2 shows exemplarily how the structures of TC-MLO mixtures change with increasing water content: at R ) 19 (Figure 2a), an inverse microemulsion system is formed at low water content (0 and 5% water; the scattering curves of these systems have a single broad peak), and a H2 phase, at higher water content (from 10 to 25% water). Thereby, with increasing water content in the single phase regions, the observed peaks move to lower angles, corresponding to larger structures (“swelling” with water). At 25% water, the water solubilization capacity of this sample is exceeded, and the H2 phase is in equilibrium with excess water. Above this fully hydrated condition, no more change is observed in the peak positions and in the corresponding structure parameters (the mean lattice parameter, a, for the hexagonal phases and the d spacing that is called the characteristic distance for the L2 phase, which are presented in Table 1). Figure 2b shows the same water solubilization trend for a sample with a higher TC/MLO mixing ratio (R ) 110), but the water solubilization capacity is already exceeded at 15% water. At this high TC content, only an inverse microemulsion system is formed. With increasing water content, the scattering

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Table 1. Structure Parameters (the Mean Lattice Parameter, a, for the Cubic and Hexagonal Phases and the d Spacing that Is Called the Characteristic Distance for the L2 Phase) as Derived from SAXS Investigations that Were Carried out on the Dispersed and Nondispersed MLO-Based Bulk Systems as a Function of r (for Both Systems) and Water Content (for Nondispersed Systems)a investigated temp water system no. (°C) content (wt %) 1b 2a 2b 2c 2d 2e 2fb 2gb 2hb 2ib 3b 4a 4b 4c 4db 4eb 4fb 4gb

25 25 25 25 25 25 25 39 58 76 25 25 25 25 25 39 58 76

40 (95)c 0 5 10 15 20 25 (95)c 25 (95)c 25 (95)c 25 (25)c 20 (95)c 0 5 10 15 (95)c 15 (95)c 15 (95)c 15 (95)c

R 0 19 19 19 19 19 19 19 19 19 75 110 110 110 110 110 110 110

space mean a d group (nm) (nm) Pn3m L2 L2 H2 H2 H2 H2 H2 H2 L2 L2 L2 L2 L2 L2 L2 L2 L2

8.6 3.2 3.8 4.5 4.8 5.9 6.2 5.9 5.6 4.8 6.7 4.3 5.6 6.7 7.2 6.8 6.4 5.9

a The aqueous dispersions contain 95 wt % water and 5 wt % of a mixture of MLO, TC, and F127, whereby the concentration of the F127 was constant (0.375 wt %). b Sample nos. 1, 2f-i, 3, and 4d-g represent SAXS data for both systems (the dispersed and the nondispersed mesophases with excess water). They have the same space group and the same structure parameters. c The water content in the emulsified samples (95 wt %).

curves show a shoulder in addition to the first broad peak. This can be explained by the fact that the solubilized water increases the order in the inverse microemulsion system. This leads in the scattering curves to the appearance of a side maximum, which is directly related to the main maximum and therefore does not indicate the presence of any additional phase in the system. It is not feasible, or at least experimentally difficult, to determine directly the water content in the internal phase of the droplets for the different dispersions. However, from the very good agreement of the scattering curves of the dispersions with those of the respective nondispersed bulk samples coexisting with excess water, it is evident that the structures are practically the same, which implies also that they have the same water content. Thus, the water content in the dispersed droplets can be determined from the maximum water solubilization capacity of the bulk samples. Without oil (R ) 0), the water content50 was determined to be ∼32%. Upon increasing the TC content to R ) 19, 75, and 110, the water content decreases to less than 25, 20, and 15%, respectively. It is very important to note that this good structural agreement between the dispersed phases and the fully hydrated bulk phases (see all typical examples in Figure 1 and Table 1) implies also that the TC/MLO mixing ratio is maintained (the TC does not separate from MLO to form normal emulsion droplets in addition to the EME), despite the high energy input of the dispersion procedure, and moreover, the polymer that is used to stabilize the particles does not disturb the internal structure. Stability tests of the dispersions that are presented in Figure 1 show, interestingly enough, no change in the internal nanostructure of the dispersions after 4 months of storage at room temperature. Figure 3 shows the control of stability against aging for two examples of oil-loaded

Figure 3. Control of stability against aging. The scattering curves of two different MLO-based dispersions having R ratios of 19 and 75 are shown in parts a and b, respectively. The measurements were carried out at 25 °C: after preparation (black line), after 2 months (red line), and after 4 months (green line). The curves are shifted by a constant arbitrary factor for better visibility. F127 was used to stabilize the dispersed particles.

dispersions (hexosomes and EME) in more detail. Clearly, no change in the internal structure is detected. This means that the addition of TC to monoglyceride-water mixtures leads to the ability to form confined nanoscaled tunable hierarchical structures. The internal structure of the dispersed particles can be easily tuned (accessibility of diverse internal structures, such as Pn3m, H2, and L2, is possible) by adding a certain amount of tetradecane at a certain temperature. These findings encouraged us to denote this family of colloidal particles as “ISASOMES” (internally self-assembled particles or “somes”) in order to emphasize the fact that the internal nanostructure of the kinetically stabilized particles is formed through selfassembly principles and that the internal self-assembled structure of the particles can be easily tuned by adjusting the tetradecane content in the system and/or temperature. Note also that the data described in this work show for the first time, to our knowledge, that it is principally possible to disperse also reversed microemulsion systems without losing the microemulsion structure in the interior of the particles. This means that the internal microemulsified water droplets are thermodynamically stable and not lost with time to the continuous phase (see Figure 1c and d). The impact of hydrophilic or hydrophobic additives on phase transitions in liquid crystalline mesophases is known to be related to the degree of their penetration into the interfacial film region and, as a consequence, to their

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Figure 5. Cryo-TEM observations for R ) 19; hexosomes are observed; notice in parts a and b the presence of curved striation (stars) and hexagonal internal symmetry (arrows). FFT (part c) of the particle on the bottom left leads to a lattice parameter of a ) 6.5 nm.

Figure 4. Cryo-TEM images for R ) 0 (parts a and b are taken from ref 50); nanostructured particles together with some vesicles were observed. The Fourier transform of the internal structure of the particle (inset in part b) shows a hexagonal arrangement with interplanar distances, d, of ∼6 nm. The internal structure is observed along the [111] axis, and the crystallographic planes observed are of the {110} type. This is compatible with a cubic structure of Pn3m symmetry with a lattice parameter, a, of 8.5 nm.

effect on the spontaneous curvature of interfacial films.8-10,32,56 The localization of the solubilized material, either in the domain of the polar part of the lipids or in the apolar part of the hydrocarbon chain of the surfactant, depends on the molecular structure of the solubilized molecules and their polarity.8-10,32 In the systems discussed in the present work, the increase of the R ratio stands for the induction of a negative spontaneous curvature of the polar-apolar interface of the MLO membrane as a result of the attractions of TC with the hydrophobic tails of the MLO molecules. This effect induces the transition from Pn3m to H2 and at higher TC content to L2. This is an indication that the addition of TC increases the flexibility of the surfactant film, since the H2 phase structure is destabilized in favor of a microemulsion phase structure. A simple approach for the prediction of the tendency of a particular lipid (or surfactant) to form mesophases (such as micellar solutions and hexagonal and cubic phases) is described in the literature.5 The approach takes into account an “effective” molecular geometry of the lipids.5 This geometric effect is described by the well-known critical packing parameter CPP ) vs/a0l, where vs is the hydrophobic chain volume, a0 is the headgroup area, and l is the hydrophobic chain length.57 The CPP is affected by the temperature, lipid polarity, water content, addition

of hydrophobic or hydrophilic additives, and other tuning parameters.8-10,25,31,32,37,50 Inverted type mesophases are favored if the effective CPP is increased. Concerning the systems discussed in the present work, the addition of tetradecane leads to an increase in the effective volume of the surfactant chains and, as a consequence, to an increase of the value of the effective CPP, making the transition to H2 and with a higher TC content to L2 plausible. Nakano et al.37 found that the addition of oleic acid to MO-based aqueous dispersions that are stabilized by F127 leads to the transformation of the internal particle structure from Pn3m via H2 to an inverted micellar cubic phase (CMIC) with the Fd3m space group. However, in the

(56) Koynova, R.; Tenchov, B. Curr. Opin. Colloid Interface Sci. 2001, 6, 277-286.

(57) Isrealachvilli, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525-1568.

Figure 6. Cryo-TEM image for R ) 75; emulsified microemulsion particles are observed. Notice the more circular shape (than that of cubosomes (R ) 0) and hexosomes (R ) 19)) and the contrast inside the particles. The top inset is a FFT of the arrowed particle showing a diffuse peak of brightness and revealing the internal structure of the particle. The bottom inset is a FFT of the background showing no peak of brightness in the water matrix.

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Figure 7. Temperature dependence of scattering curves for the MLO-based dispersions (red lines) with those from the nondispersed bulk sample (black lines). The temperature effect in the range 25-76 °C was carried out with two systems with different R values of 19 (a) and 110 (b). The intensities were normalized by the respective MLO plus TC concentration. The curves are shifted by a constant arbitrary factor for better visibility. F127 was used to stabilize the dispersed particles.

Figure 8. Reversibility: scattering curves of two dispersions with different R values of 19 (a) and 110 (b). These samples were measured while heating (at 25, 39, 58, and 76 °C (black lines)) and while cooling (at 58, 39, and 25 °C (red lines)). The curves are shifted by a constant arbitrary factor for better visibility. F127 was used to stabilize the dispersed particles.

present study in which tetradecane was used to tune the internal particle structure, instead of CMIC, a reverse microemulsion was formed. The complete replacement of MLO by TC (MLO-free sample) leads to “structureless” particles (normal emulsion), while Nakano et al.37 found that the replacement of MO by oleic acid leads to the formation of a dispersed sample with an internal CMIC structure. This difference is not surprising because oleic acid possesses a clear amphiphilic character and can localize at the interface, while TC is an oil and therefore has no surface activity. In addition to the SAXS analysis, the submicron-sized dispersed particles were investigated also by cryo-TEM, as shown in Figures 4-6. For the unloaded particles (R ) 0), fast Fourier transforms (FFTs) of the internal structures of the dispersed particles (Figure 4 is taken from ref 50) are compatible with the cubic Pn3m symmetry and render a lattice parameter of ∼8.5 nm, which is in very good agreement with the SAXS analysis. The obtained images show that almost all formed ECPs have attached vesicles (Figure 4a). The vesicles are large, but taking their thin shell into account, only a small amount of material is used for their formation. This cubosomevesicle coexistence was observed also in previous cryoTEM studies on dispersed monoglycerides, in which F127 was used as a stabilizer.34,36 As already found by SAXS, a low amount of added TC (R ) 19) induces the formation of a H2 internal particle structure (hexosomes, EHP) (Figure 5). The particles show internal arrangements of a hexagonal symmetry and/or curved striations. The

presence of curved striations and/or hexagonal symmetry indicates the formation of hexosomes.34 As observed earlier for hexosomes,34,50 and in contrast to cubosomes, no or only very few vesicles coexist with the dispersed particles. FFT of the internal H2 phase reveals a characteristic distance of ∼5.6 nm, leading to a lattice parameter of ∼6.5 nm, which is in good agreement with the SAXS analysis. In the presence of a high TC content (R ) 75), SAXS revealed that the particles have the internal structure of the L2 phase. This is confirmed by the obtained cryo-TEM images (Figure 6) which show structures that are clearly different from those obtained for hexosomes and cubosomes. No long range order is observed in the case of EMEs. As shown in Figure 6, the projected shape of the particles is almost circular, which is usually not the case for hexosomes and cubosomes. The outer shape of the dispersed droplets is more similar to that found in oil-in-water or water-in-oil emulsion systems. However, in contrast to normal oil (or water) droplets in ordinary emulsion systems, the brightness inside the particles is not uniform but shows an internal arrangement. The FFTs of the EME particle reveals also the presence of an internal structure. The position of the diffuse peak of brightness in the FFT (top inset in Figure 6) is in a fair agreement with the characteristic distance determined by SAXS (∼7.5 nm). The overall size of the oil-loaded dispersed particles was determined by DLS. It was found that the average hydrodynamic radius of these particles is in the range 120-200 nm.

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Scheme 1. Possible Internal Nanostructures of MLO-Based Emulsified Droplets

2. Reversible Structural Transitions in the Entrapped Interior of the Emulsified Systems. Figure 7 shows the scattering curves of two MLO-based aqueous dispersions with R values of 19 and 110, respectively, as a function of temperature, and their corresponding nondispersed fully hydrated samples during heating in the temperature range 25-76 °C. The temperature increase induces changes in the symmetry from H2 to L2 of the confined nanostructures, as shown for R ) 19 in Figure 7a (see also ref 50). Increasing the temperature shifts the peaks to higher q values and reduces the structure parameters. In the presence of a higher TC content (R ) 110), as shown in Figure 7b, which shows a typical example of scattering data for all other TC-rich dispersions, the formation of EMEs at low temperatures is promoted. The d spacings of the confined L2 phase decrease with increasing temperature. Increasing temperature at constant oil content (and in the absence of oil, as discussed in ref 50) is expected to cause a decrease in the value of a0 due to the dehydration of the MLO headgroup and simultaneously an increase in the kink states in the lipid acyl chains and so their effective volume,58 which, as a consequence, increases the CPP (see Figure 7 and ref 50). The presented results show that, at each investigated temperature, the structure of the entrapped internal phase is the same as that of the nondispersed samples coexisting with excess water. This confirms recent results50 and shows that the internal structure of the dispersed particles at each temperature corresponds to an equilibrium mesophase. To elucidate the structural changes of the particle internal nanostructure that occur during heating-cooling processes in more details, a SAXS investigation on two aqueous dispersions (with R ) 19 and R ) 110, respectively) was carried out. The scattering curves of these two dispersions, which are presented in parts a and b of Figure 8, respectively, as a function of temperature (from 25 to 76 °C), reveal the structural reversibility of the confined nanostructures. In both samples, the peaks shift in (58) Geil, B.; Feiweier, T.; Pospiech, E. M.; Eisenblatter, J.; Fujara, F.; Winter, R. Chem. Phys. Lipids 2000, 106, 115-126.

dependence of temperature, and for R ) 19, there is additionally a reversible transition of H2 T L2 observed. Note that, at each investigated temperature, the scattering curves are identical and not dependent on whether the sample temperature was reached by heating or cooling. This indicates that the internal nanostructure in all samples, at a certain temperature, is independent of the thermal history: heating or cooling to the required temperature leads to the same structure. This fact is clear evidence that the formed nanostructures in the kinetically stabilized particles, in analogy to that in the nondispersed TC-rich bulk phases, are thermodynamic equilibrium structures. It is also worth noting that the emulsified particles show a swelling-deswelling behavior during heating-cooling cycles (denoted as “breathing mode”50), indicating that there is a reversible exchange of water inside-outside the confined internal particle structures during the cooling and heating cycles. Conclusions In this paper, we report on the effect of solubilizing tetradecane and/or altering temperature on the reversible structural transitions of the nanoscale interior domains of kinetically stabilized monolinolein (MLO)-based particles. The combined structural investigation of both the dispersed and the corresponding nondispersed bulk systems allowed investigating and evaluating the structural changes occurring in the interior of the emulsified particles. Scheme 1 summarizes the main findings of the present work: emulsified microemulsion (EME) particles can be formed through the addition of tetradecane to the MLOwater system. This is possible since the addition of tetradecane induces a transition of the internal particle nanostructure of our ISASOMES (internally self-assembled particles or somes) from Pn3m to H2 and L2 at a given temperature. The same phase transition can be induced by increasing temperature at an adjusted tetradecane level in the system. The tuning of the inner particle structures is easy to control by altering the system’s composition and/or varying the temperature. To

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our knowledge, this is the first time that the formation of stable emulsified microemulsion (EME) systems was described and proven to exist. The quantitative agreement between the structural parameters measured in the dispersed particle and the nondispersed bulk (mesophases coexisting with excess water) systems clearly demonstrates that the confined interior of the emulsified particles is neither destroyed by the preparation method nor influenced by the presence of the particle stabilizer F127. The presented data show that the oil molecules are incorporated into the internal nanostructure of the mesophase particles. There is no experimental evidence found for the formation of normal O/W emulsion droplets. Moreover, it is shown that also temperature can be used to control the symmetry of the internal nanostructure. The confined nanostructures in the emulsified particles are reversible structuressthey exist in thermodynamic equilibrium with the surrounding

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aqueous phasesand depend only on the actual temperature and oil content. With all likelihood, the features described in this work can also be induced by other hydrophobic molecules besides tetradecane. Acknowledgment. This work was financially supported by the Austrian Science Fund FWF (M711-N03) and the Nestle´ Research Center, Lausanne, Switzerland. We thank Martin Michel (Nestle´ Research Center, Lausanne, Switzerland) for helpful discussions and critical reading. We also thank Fred Chavannes, Olivier Girault, Jean-Albert Stucki, Dino Tomasi, and Laurent Peissard (CRN) for having built the environmental chamber for the cryo-TEM analysis. Thanks to Martine Rouvet (CRN) and Gu¨nther Scherf (IFC) for technical help. LA0482711