From the Microscopic to the Mesoscopic Properties of Lyotropic

Jan 16, 2008 - Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew UniVersity of Jerusalem,. Jerusalem 91904, Israel, Depart...
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Langmuir 2008, 24, 2118-2127

From the Microscopic to the Mesoscopic Properties of Lyotropic Reverse Hexagonal Liquid Crystals Dima Libster,†,£ Paul Ben Ishai,‡ Abraham Aserin,† Gil Shoham,§ and Nissim Garti*,† Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel, Department of Applied Physics, The Hebrew UniVersity of Jerusalem, GiVat Ram, Jerusalem 91904, Israel, and Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel ReceiVed August 20, 2007. In Final Form: NoVember 7, 2007 In the present study we aimed to explore a correlation between the microstructural properties of the lyotropic reverse hexagonal phase (HII) of the GMO/tricaprylin/phosphatidylcholine/water system and its mesoscopic structure. The mesoscopic organization of discontinuous and anisotropic domains was examined, in the native state, using environmental scanning electron microscopy. The topography of the HII mesophases was imaged directly in their hydrated state, as a function of aqueous-phase concentration and composition, when a proline amino acid was solubilized into the systems as a kosmotropic (water-structure maker) guest molecule. The domain structures of several dozen micrometers in size, visualized in the environmental scanning electron microscopy, were found to possess fractal characteristics, indicating a discontinuous and disordered alignment of the corresponding internal water rods on the mesoscale. On the microstructural level, SAXS measurements revealed that as water content (Cw) increases the characteristic lattice parameter of the mesophases increases as well. Using the water concentration as the mass measure of the mixtures, a scaling relationship between the lattice parameter and the concentration was found to obey a power law whereby the derived fractal dimension was the relevant exponent, confirming the causal link between the microscopic and mesoscopic organizations. The topography of the HII mesophase was found to be affected by the microstructural parameters and the composition of the samples. Thermal analysis experiments involving these systems further confirmed that the behavior of water underpins both microscopical and mesoscopic features of the systems. It was shown that both the swelling of the lattice parameter and the mesoscopic domains is correlated to the bulk water concentration in the water rods.

1. Introduction The structural features of lyotropic liquid crystals (LLC) have in recent years made them one of the more promising avenues for research into applications such as drug delivery1-3 and as a template for self-assembly systems.4 The three- and twodimensional symmetry of the cubic and hexagonal LLC phases, combined with their large interfacial area and balanced content of hydrophobic and hydrophilic domains, make them very promising universal carriers, with numerous advantages over most of the other systems used at the present time. A critical aspect for such applications is a comprehensive understanding of mesoscale structure and its dependence on experimentally measurable microscopic parameters, such as lattice constants. LLC can be formed by polar lipids and certain surfactants upon interaction with water.5-7 Among the mesophases formed * To whom correspondence should be addressed. Tel: 972-2-658-6574/ 5. Fax: 972-2-652-0262. E-mail: [email protected]. † Casali Institute of Applied Chemistry. ‡ Department of Applied Physics. § Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology. £ The results presented in this manuscript will appear in the Ph.D. dissertation of D.L. in partial fulfillment of the requirements for the degree of Doctor in Applied Chemistry, The Hebrew University of Jerusalem, Israel. (1) Barauskas, J.; Misiunas, A.; Gunnarsson, T.; Tiberg, F.; Johnsson, M. Langmuir 2006, 22, 6328-6334. (2) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223-234. (3) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 1999, 4, 449-456. (4) Lagerwall, J.; Scalia, G.; Haluska, M.; Dettlaff-Weglikowska, U.; Roth, S.; Giesselmann, F. AdV. Mat. 2007, 19, 359-364. (5) Imura, T.; Hikosaka, Y.; Worakitkanchanakul, W.; Sakai, H.; Abe, M.; Konishi, M.; Minamikawa, H.; Kitamoto, D. Langmuir 2007, 23, 1659-1663. (6) Angius, R.; Murgia, S.; Berti, D.; Baglioni, P.; Monduzzi, M. J. Phys. Condens. Matter 2006, 18, S2203-S2220.

by glycerol mono-fatty acids (monoglycerides) and water are lamellar (LR), reverse hexagonal (HII), and cubic liquid crystalline structures (QG or QD). The formation and phase behavior of glycerol monooleate/water (GMO/water) systems have been extensively studied.8-10 Monoolein-based LLC are regarded as promising systems for many applications, including their utilization as pharmaceutical vehicles, food systems, and chemical reaction media.11-13 It was demonstrated that peptides of pharmaceutical importance can be incorporated into an inverted bulk cubic phase of GMO/water and be protected from enzymatic degradation.14 The structural features of reverse hexagonal liquid crystals (HII) are less studied compared with those of cubic and lamellar phases.15-17 The reverse hexagonal mesophase (P6mm) is characterized by dense packing of infinitely long and straight water-filled rods, exhibiting two-dimensional ordering (Figure 1a). Each cylinder is surrounded by a layer of surfactant molecules (7) Shearman, G. C.; Ces, O.; Templer, R. H.; Seddon, J. M. J. Phys. Condens. Matter 2006, 18, S1105-S1124. (8) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (9) Kaasgaard, T.; Drummond, C. J. Phys. Chem. Chem. Phys. 2006, 8, 49574975. (10) Misquitta, Y.; Caffrey, M. Biophys. J. 2001, 81, 1047-1058. (11) Leser, M. E.; Sagalowicz, L.; Michel, M.; Watzke H. J. AdV. Colloid Interface Sci. 2006, 123-126, 125-136. (12) Sagalowicz, L.; Mezzenga, R.; Leser, M. E. Curr. Opin. Colloid Interface Sci. 2006, 11, 224-229. (13) Vauthey, S.; Milo, C.; Frossard, P.; Garti, N.; Leser, M. E.; Watzke, H. J. J. Agric. Food Chem. 2000, 48, 4808-4816. (14) Ericsson, B.; Eriksson P. O.; Lo¨froth J. E.; Engstro¨m, S. Am. Chem. Soc., Washington, DC 1991, 251-265. (15) Montalvo, G.; Valiente, M.; Rodenas, E. Langmuir 1996, 12, 52025208. (16) Mezzenga, R.; Meyer, C.; Servais, C.; Romoscanu, A. I.; Sagalowicz, L.; Hayward, R. C. Langmuir 2005, 21, 3322-3333. (17) Shearman, G. C.; Khoo, B. J.; Motherwell, M. L.; Brakke, K. A.; Ces, O.; Conn, C. E.; Seddon, J. M.; Templer, R. H. Langmuir 2007, 23, 7276-7285.

10.1021/la702570v CCC: $40.75 © 2008 American Chemical Society Published on Web 01/16/2008

Lyotropic ReVerse Hexagonal Liquid Crystals

Figure 1. (a) Schematic presentation of HII mesophase, showing the packing of infinitely long water-filled rods, surrounded by lipid layers. (b) The ternary phase diagram of GMO/tricaprylin/water at 25 °C. The dilution lines represent the surfactant/oil weight ratio.19

that are perpendicular to the cylindrical interface such that their hydrophobic moieties point outward the water rods. The HII phase of GMO/water binary system exists only at elevated temperatures (the cubic phase is transformed into the HII mesophase only at ca. 85 °C). However, it was shown that an additional component can be included in the preparation to induce the mesophase to be stable at room temperature.18 Recently, the ability of triacylglycerols (triglycerides, TAG) to promote formation of HII phases was investigated.19 It was found that TAGs with medium-chain fatty acids, in particular tricaprylin, can solvate the GMO tails, affecting their critical packing parameters (CPP) and transforming the lamellar and cubic phases into a hexagonal mesophase that can remain stable at room temperature. A large area of HII mesophase was identified in the corresponding triangular phase diagram, starting from a minimum tricaprylin concentration of 3 wt % on dilution line 96:4 between 20-25 wt % water and up to a maximum concentration of 13.5 wt % tricaprylin on dilution line 85:15, between 10 and 20 wt % water (Figure 1b). At higher water concentrations, a stable reverse hexagonal phase was formed in equilibrium with excess water. In our previous studies it was also demonstrated that phosphatidylcholine (PC) can be incorporated into the ternary GMO/ tricaprylin/water hexagonal system.20 Solubilization of PC, which is known to act as a transdermal drug permeation enhancer, improved the elastic properties of the HII mesophase and enhanced its thermal stability. The combined positive effects of PC enabled the solubilization of a cyclic undecapeptide, cyclosporin A (CSA), which is an efficient immunosuppressive agent and two skin (18) Borne, J.; Nylander, T.; Khan, A. Langmuir 2001, 17, 7742-7751. (19) Amar-Yuli, I.; Garti, N. Colloids Surf. B 2005, 43, 72-82. (20) Libster, D.; Aserin, A.; Wachtel, E.; Shoham, G.; Garti, N. J. Colloid Interface Sci. 2007, 308, 514-524.

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penetration enhancers (Labrasol and ethanol), obtaining stable mesophases at physiological temperature. This system showed a moderate viscoelastic response, which is an advantage over the highly viscous cubic phases that are very difficult to handle, and their practical utilizations are therefore relatively limited. Recently, the structure of the hexagonal phase at the molecular level has been investigated by several authors using small-angle X-ray scattering (SAXS) and its dynamic aspects have been explored by NMR relaxation and self-diffusion techniques.21-25 Nevertheless, very few studies have been conducted to study the mesoscopic structure of these systems.26,27 Such studies are especially important since the properties of soft materials at mesoscale lengths usually cannot be directly correlated with their microscopic molecular structure. The ability to comprehend and predict the mesoscopic properties of materials such as liquid crystals requires a detailed characterization of each of the different structure levels involved. This type of study should then establish the correlation between the molecular level and mesoscopic organizations of these systems, if any.28 The classical model of the macroscopic hexagonal mesophase assumes a polycrystalline structure of randomly oriented microdomains. The issue of polydomain structure has recently attracted increasing scientific interest, mainly since it seems to link the microstructure and the mesostructure properties of liquid crystalline materials. Coppola et al.27 confirmed the existence of large lyotropic domains using PGSE NMR on a system consisting of pentaethylene glycol dodecyl ether (C12E5), sodium dodecyl sulfate, decane, and water. Moreover, shear-induced domain orientation changes in the rigid reverse hexagonal phase of anionic surfactant AOT (bis-(2-ethylhexyl) sodium sulfosuccinate) mixed with the zwitterionic lecithin (R-phosphatidylcholine) were followed by a 31P NMR chemical shift in anisotropy characteristics and complementary 1H NMR.26 In these experiments the authors found that the initially disordered samples with polydomain structures became macroscopically aligned after Couette shear. The main purpose of the current study was, therefore, to examine a potential correlation between the microstrucrural properties and the mesoscopic structural properties of the reverse hexagonal LLC phase. Such correlation, if present, was assumed to be reflected by polycrystalline alignment of microdomains of this phase. The microscopic structure was characterized by SAXS measurements, while the mesoscopic structural characterization was performed by environmental scanning electron microscopy (ESEM) and differential scanning calorimetry (DSC). ESEM is a powerful technique for the investigation of hydrated and insulating samples in their native (e.g., undried) state and at low pressures.29-31 In contrast to the conventional scanning electron microscope (SEM) which operates under a high vacuum, in ESEM (21) Marques, E.F.; Edlund, H.; La Mesa, C.; Khan, A. Langmuir 2000, 16, 5178-5186. (22) Caboi, F.; Amico, G. S.; Pitzalis, P.; Monduzzi, M.; Nylander, T.; Larsson, K. Chem. Phys. Lipids 2001, 109, 47-62. (23) Popescu, G.; Barauskas, J.; Nylander, T.; Tiberg, F. Langmuir 2007, 23, 496-503. (24) Angelico, R.; Ceglie, A.; Olsson, U.; Palazzo, G. Langmuir 2000, 16, 2124-2132. (25) Kamo, T.; Nakano, M.; Leesajakul, W.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2003, 19, 9191-9195. (26) Liu, L.; John, V. T.; McPherson, G.; Maskos, K.; Bose, A. Langmuir 2005, 21, 3795-3801. (27) Coppola, L.; Oliviero, C.; Olsson, U.; Ranieri, G. A. Langmuir 2000, 16, 4180-4184. (28) Tang, D.; Marangoni, A. G. AdV. Coll. Interface Sci. 2006, 128-130, 257-265. (29) Stokes, D. J.; Thiel, B. L.; Donald, A. M. Langmuir 1998, 14, 44024408. (30) Stelmashenko, N. A.; Craven, J. P.; Donald, A. M.; Terentjev, E. M.; Thiel, B. L. J. Microsc. 2001, 204, 172-183. (31) D’Emanuele, A.; Dinarvand, R. Int. J. Pharm. 1995, 118, 237-242.

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techniques a gas is circulated around the sample during the imaging. By means of differential pumping, while the electron gun is kept at the low pressure of around 10-6 Torr, the pressure is increased down the column, finally reaching ∼10 Torr at the sample chamber. The pressure and temperature of the samples can be controlled by a Peltier cooling stage and, using water vapor as the circulating gas, a saturated water vapor pressure can be obtained. As a result, evaporation of water from the sample is prevented, thereby allowing direct imaging of hydrated specimens.32,33 Despite the great potential of ESEM for imaging colloidal systems, surprisingly relatively little scientific research has been done using this methodology in colloid science.34-36 To the best of our knowledge, the present study is the first such application of ESEM experiments for the characterization of lyotropic liquid crystals. 2. Experimental Section 2.1. Materials. Monoolein, GMO, distilled glycerol monooleate (min. 97 wt % monoglyceride), and 2.5 wt % diglyceride (acid value 1.2, iodine value 68.0, melting point 37.5 °C, and free glycerol 0.4 wt %) were obtained from Riken Vitamin Co. (Tokyo, Japan). Tricaprylin (97-98 wt %) was purchased from Sigma Chemical Co. (St. Louis, MO). Phosphatidylcholine of soybean origin (Epikuron 200, min. 92% PC) was obtained from Degussa (Hamburg, Germany). L-Proline was purchased from Shanghai Kyowa Amino Acid Co. (Shanghai, P. R. China). Water was double distilled. All ingredients were used without further purification. 2.2. Preparation of HII Mesophases. The phase diagram of GMO/ tricaprylin/water is presented in Figure 1b. Each phase diagram can be divided into dilution lines that represent a constant weight ratio between the surfactant and oil, i.e., dilution line 90:10 means 90 wt % GMO and 10 wt % tricaprylin. PC (10 wt %) was incorporated into the binary GMO/tricaprylin mixture (9:1 weight ratio) and pure water or aqueous phase, containing proline (1:1 water/proline weight ratio) was added in various concentrations (8-35 wt %). It should be noted that while the water phase concentration was varied in the samples, the PC concentration (10 wt %) and GMO/tricaprylin weight ratio (9:1) were kept constant. The GMO/PC/tricaprylin/water hexagonal liquid crystals were prepared by mixing weighed quantities of GMO, tricaprylin, and PC while heating to 80 °C. This was done in sealed tubes under nitrogen atmosphere to avoid oxidation of the GMO and PC. An appropriate quantity of preheated water was added at the same temperature and the samples were stirred and cooled to 25 °C. In the case of proline solubilization, it was first dissolved in water (1:1 weight ratio) prior to incorporation to GMO/PC/tricaprylin mixtures. The samples were allowed to equilibrate for 24 h before examination. 2.3. Small-Angle X-ray Scattering (SAXS). Scattering experiments were performed using Ni-filtered Cu KR radiation (0.154 nm) from an Elliott rotating anode X-ray generator that operated at a power rating of 1.2 kW. The X-ray radiation was further monochromated and collimated by a single Franks mirror and a series of slits and height limiters and measured by a linear position-sensitive detector. The samples were held in 1.5 mm quartz X-ray capillaries inserted into a copper block sample holder. The temperature was maintained at T ( 0.5 °C with a recirculating water bath. The camera constants were calibrated using anhydrous cholesterol. The scattering patterns were desmeared using the Lake procedure implemented in home-written software.37 (32) Stokes, D. J. AdV. Eng. Mater. 2001, 3, 126-130. (33) Donald, A. M.; He, C.; Royall, C. P.; Sferrazza, M.; Stelmashenko, N. A.; Thiel, B. L. Colloids Surf. A 2000, 174, 37-53. (34) Sui, G.; Micic, M.; Huo, Q.; Leblanc, R. M. Colloids Surf. A 2000, 171, 185-197. (35) Mohammed, A. R.; Weston, N.; Coombes, A. G. A.; Fitzgerald, M.; Perrie, Y. Int. J. Pharm. 2004, 285, 23-34. (36) Hashimoto, M.; Ujiie, S.; Mori, A. AdV. Mat. 2003, 15, 797-800. (37) Lake, J. A. Acta Crystallogr. 1967, 23, 191-194.

Libster et al. 2.4. Differential Scanning Calorimetry (DSC). A Mettler Toledo DSC822 (Greifensee, Switzerland) calorimeter was used. The DSC measurements were carried out as follows. LLC samples (5-15 mg) were weighed, using a Mettler M3 microbalance, in standard 40 µL aluminum pans and immediately sealed by a press. The samples were rapidly cooled in liquid nitrogen from 30 to -40 °C, at a rate of 10 °C min-1. The samples remained at this temperature for 30 min and then were heated at 1 °C min-1 to 60 °C. An empty pan was used as a reference. The instrument determined the fusion temperatures of the solid components and the total heat transferred in any of the observed thermal processes. The enthalpy change associated with each thermal transition was obtained by integrating the area of the relevant DSC peak. DSC temperatures reported here were reproducible to (0.5 °C. 2.5. ESEM. The ESEM work was performed using an Environmental SEM (FEI company), equipped with a Peltier-controlled cooling stage. The accelerating voltage used was 15 kV. The samples were viewed in water vapor at a pressure of 8 Torr. Special attention was given to keep the samples in their native hydrated state. Therefore, the samples were examined at various temperatures in the range of 5-30 °C to determine the thermal stability of the mesophases at 8 Torr. Most samples were imaged at 12-15 °C in order to avoid water evaporation. We are aware that these are borderline conditions considering the saturated vapor pressure curve of water,33 which is used to fit the conditions necessary for liquid water to be stabilized. However, it should be noted that we worked in the range of 8-35 wt % water content in the LLC, when the water is bound to the surfactant hydrophilic heads (as shown and discussed below). This is supposed to increase the evaporation temperature of the water, providing slightly more flexible experimental conditions. As the experimental temperature was increased (at a fixed pressure of 8 Torr), structural rearrangements were detected in the samples starting from ∼18 °C. Direct water evaporation was detected above 22 °C.

3. Results 3.1. SAXS Measurements. The SAXS measurements, presented here, were conducted to elucidate the effect of water concentration and proline solubilization on the changes observed in the lattice parameters of the corresponding GMO/tricaprylin/ PC/water mixtures. The SAXS patterns obtained for these mixtures included Bragg peaks with ratios of d-spacings of 1:(1/x3):(1/x4) correlating to the first three diffraction maxima (hk ) 10, 11, 20) which are expected from close packed cylindrical micelles arranged with 2D hexagonal symmetry (Figure 2a). Such SAXS experiments revealed that, with increasing water concentration, a significant increase in the lattice parameter of the mesophases was observed. The results indicated a relatively wide variation range of the lattice parameter, from 47 Å in the presence of 8 wt % water phase to 66 Å at 29 wt % water phase concentration (Figure 3). These changes seem to reflect the structural evolution of the hexagonal phase as a function of water content. To demonstrate the influence of a guest molecule on both the microscopic and mesoscopic organization of the mesophases, a proline amino acid was added to the water (at 50 wt %) as a kosmotropic solute.38 The kosmotropic effect of proline on the reverse hexagonal phases can be clearly observed in Figure 3. Although some swelling of the HII mesophases also takes place in the presence of proline, a shrinkage in lattice parameter was monitored both at very low (8-11 wt %) and high water phase concentrations (>23 wt %), compared with the system without the amino acid. This suggests dehydration of the GMO and PC polar heads within the mesophase, probably due to competition for water between the solvated proline and the surfactants. Another observation from the X-ray profiles is that higher order peaks (38) Koynova, R.; Brankov, J.; Tenchov, B. Eur. Biophys. J. 1997, 25, 261274.

Lyotropic ReVerse Hexagonal Liquid Crystals

Figure 2. Small-angle X-ray scattering pattern of HII mesophase consisting of GMO/tricaprylin/PC (57.6, 6.4, 10 wt %, respectively) at 26 wt % aqueous phase. (a) The system contains pure water. (b) The system contains an aqueous phase of 50 wt % proline. The d-spacings of the three Bragg peaks are observed to be related by a ratio of 1:(1/x3):(1/x4), consistent with two-dimensional hexagonal symmetry. d ) 2π/q, where q is the amplitude of the scattering vector.

Figure 3. Lattice parameter (r) (0.6 Å of HII mesophases containing GMO/tricaprylin with a weight ratio of 9:1 and 10 wt % PC as a function of the aqueous phase concentration, as measured by SAXS. (4) The system containing pure water and (0) the system containing 50 wt % proline.

(11 and 20) have higher scattering intensities in the water-based systems compared to the systems containing proline (Figure 2), indicating a significantly more organized and swelled structure in the blank mesophases. 3.2. ESEM. In the present study we wanted to characterize the topography of the surface of a bulk HII phase by ESEM and to link it to the underlying mesoscopic polydomain structure,27,39 reflected in structural analysis performed by SAXS and thermal analysis (DSC). Taking advantage of the practical potential of the ESEM methodology and refining the relevant experimental conditions to be most suitable for our systems (see Experimental Section above) we were able to visualize the structural features of the surface of the bulk HII phase. A typical ESEM micrograph of hexagonal phase (Figure 4) clearly reveals the mesoscopic polydomain arrangement, reflected by multilayer alignment. The inspected surface organization consists of a random alignment of polycrystalline domains, each several tens of micrometers in size. These domains can be imaged due to their contrast differences, as further discussed below. Controlling the pressure and temperature of the samples via a Peltier cooling stage, a (39) Ramos, L. Langmuir 2004, 20, 2215-2219.

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Figure 4. ESEM micrographs of HII mesophase of GMO/tricaprylin/ PC (63, 7, 10 wt %, respectively) containing 20 wt % water at 14 °C and magnification of 500×.

formation of pure hexagonal phase was followed by heating the samples from 5 to 12 °C (Figure 5a and b). According to the SAXS measurements, at a relatively low temperature (5 °C), mixed crystalline Lc + HII phases exist,40 while the formation of pure HII phases occurs above 12 °C. These changes are clearly demonstrated in Figure 5a-c when a structure that lacks an organized pattern transforms into a structure consisting of polydomain alignments of pure hexagonal phase. To confirm that the samples observed were in their native hydrated state, we increased the temperature in the measurement chamber to cause gradual dehydration of the surfactants and eventual evaporation of the water. Around 20 °C the water started evaporating, leading to the gradual destruction of the sample, and finally, an almost flat surface was obtained (26-30 °C), indicating complete evaporation of the water (Figure 5d). To quantify the structural information obtained using ESEM, a fractal analysis of the micrographs was applied. A fractal analysis is a powerful approach for quantifying the structure of complex systems which exhibit a measure of self-similarity. Such similarity is usually reflected at different length scales, as was discussed in detail by Marangoni.41-44 Formally this kind of relationship can be expressed by eq 1

M(ar) ) af(D)M(r)

(1)

where M is any measurable quantity of the system, r is length, a is the scaling factor, and f(D) is a linear function of the fractal geometry, D.45 The fractal dimension of the image was established using the sandbox method.46 The image size was set to 1024 × 1024 pixels, with a total of N ) 220, and the grayscale histogram was obtained. The number of pixels with a value above 244 in the gray scale (40) Amar-Yuli, I.; Wachtel, E.; Shalev, D. E.; Moshe, H.; Aserin, A.; Garti, N. J. Phys. Chem. B 2007, 111, 13544-13553. (41) Marangoni, A. G. Trends Food Sci. Technol. 2002, 13, 37-47. (42) Narine, S. S.; Marangoni, A. G. Phys. ReV. E 1999, 59, 1908-1920. (43) Tang, D.; Marangoni, A. G. Chem. Phys. Let. 2006, 433, 248-252. (44) Tang, D.; Marangoni, A. G. J. Am. Oil Chem. Soc. 2006, 83, 377-388. (45) Muniandy, S. V.; Kan, C. S.; Lim, S. C.; Radiman, S. Physica A 2003, 323, 107-123. (46) Bunde, A.; Havlin, S. A Brief Introduction to Fractal Geometry. Fractals in Science; Bunde, A., Havlin, S., Eds.; Springer-Verlag: New York 1995; pp 1-24.

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Figure 5. ESEM micrographs of HII mesophase of GMO/tricaprylin/PC (63, 7, 10 wt %, respectively) containing 20 wt % water at magnification of 1000× and different temperatures. (a) 5, (b) 12, (c) 22, (d) 30 °C.

(white is 255), S(N), were counted. The image pixel size was recursively coarsened to Nj ) 22(10-j) (j ) 1-9) and a histogram obtained each time. The respective S(Nj) were subsequently counted. The fractal dimension (Dg) was obtained from linear regression of the log plot according to eq 2

log(S(Nj)) ∝ Dg log(Nj)

(2)

The results of such calculations are illustrated in Figure 6, plotted as a function of water and water/proline concentration, respectively. The procedure was repeated for a number of samples at the same concentration in order to establish a statistically significant estimate. The obtained fractal dimension illustrates the effect of the aqueous phase concentration and proline solubilization on LLC. Even by a visual observation of the ESEM micrographs, it can be seen that the structure of the LLC surface changes according to the water content (Figure 7). At low water

content, the structures are characterized by relatively small domains (Figure 7a). At high water content, significantly larger domains are observed (Figure 7b). The fractal dimensions obtained from the application of the procedure above on the systems examined were in the region of 0.65-0.9. Since ESEM is sensitive to the interface between domains and the fractal dimensionsit reflects not shape but mesoscopic structure of the domains46sthe reasonable way to interpret the results would be as follows. If the sample is perfectly ordered, then no mesoscopic interface would be evident and the obtained dimension, Dg, would be 0. If a complete disorder is present in the samples, then the ESEM image is expected to present a uniform white picture and Dg ) 1. For 0 < Dg < 1, there exists a competition between order and disorder in the overall structure of the interface. The source of ordering, be it molecular or otherwise, will be reflected in the concentration dependences of Dg. The results obtained in the present study thus

Lyotropic ReVerse Hexagonal Liquid Crystals

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Figure 6. Fractal dimension derived from the ESEM images as a function of the water concentration (top panel) and as a function of the aqueous phase of water/proline (weight ratio 1:1) concentration (bottom panel). It is seen that in both cases the fractal dimensions are less than 1 and that in both cases a change in behavior is observed around 14% water concentration.

demonstrate a large measure of disorder within the systems examined, with a discontinuous and disordered alignment of the water rods. 3.3. DSC Experiments. DSC experiments were utilized to examine the impact of water concentration and proline “salting out” effect on the hexagonal systems of GMO/tricaprylin/PC/ water. Since the interface of the surfactants and water strongly depends on hydrogen bonds, both the microscopic and mesoscopic behavior of the LLC should be affected by the state of water. Typical DSC scans of the hexagonal phase are shown in Figure 8a. As reported in detail in one of our previous works by YuliAmar et al.,47 two endothermic events were detected for the HII phase at relatively high water concentration (20 wt %). Peak A (at ca. 0 °C) was confirmed to present the fusion of the water phase and peak B (at ca. 6 °C) was due to the melting of the tricaprylin + GMO (eutectic) mixture. In a later work from our laboratory,20 it was found that PC, embedded into the ternary GMO/tricaprylin/water system, strongly interacts with GMO/tricaprylin molecules through hydrophobic interactions. Such interactions may lead to a decrease in the number of GMO/tricaprylin molecules that participate in the melting transition of peak B. Monitoring the thermal behavior of the mesophase as a function of water phase content at 8-29 wt % (Figure 8b), we found that at relatively low water concentrations (8-11 wt %), melting of the tricaprylin + GMO (eutectic) mixture splits into a doublestep endothermic event (peaks B and C). However, at higher water concentrations, the melting of the lipids (peak B) seems to switch into a one-step process, as described above (Figure 8a). Moreover, following the water behavior in these systems (Figure 9), one can see that the water fusion peak (peak A) moves progressively to higher temperatures as the water concentration increases (from -3.5 °C at 8 and 11 wt % of water to 0 °C at 29 wt % of water). With increasing aqueous phase concentration, the water activity is increased, transforming from the slightly bound to the free state. This can be confirmed by following the changes in the enthalpy of peak A (Figure 9), which increases (47) Amar-Yuli, I.; Wachtel, E.; Ben Shoshan, E.; Danino, D.; Aserin, A.; Garti, N. Langmuir 2007, 23, 3637-3645.

Figure 7. ESEM micrographs of HII mesophase containing various water contents (a) 8, (b) 29 wt % (magnification ×1000).

from 10 J g-1 at 8 wt % aqueous phase to 56 J g-1 at 29 wt % aqueous phase, indicating a significant increase in the bulk water content. Simultaneously, with increasing aqueous phase concentration, the enthalpy of GMO/tricaprylin mixture (peak B) dropped from 29 to 3 J g-1 for the higher and lower water concentrations in the LLC, respectively (Figure 9), while the melting temperatures of these peaks did not exhibit any change. Attempts were also made to investigate the impact of proline solubilization on the mesophases by DSC analysis. A typical thermogram of LLC that contains proline is presented in Figure 8c. The most obvious difference in the thermal behavior observed when proline is added to the LLC is the disappearance of peak A in all examined samples (in the aqueous phase concentration range of 8-35 wt %). As indicated above, proline tends to bind water molecules, thereby interacting with the tetrahedral network of water, and as a result leads to the salting-out effect. Consequently, the activity of water should decrease and it can be classified as bound or nonfreezable type of water, below the

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Figure 8. DSC scans of the GMO/tricaprylin/PC/water systems with weight ratio of GMO/tricaprylin 9:1 and 10 wt % PC, and different water concentrations. (a) 23 wt % water, (b) 11 wt % water, (c) 23 wt % aqueous phase with weight ratio of water/proline 1:1. Peak A is the water fusion peak, and peaks B and C are due to GMO/tricaprylin melting.

Figure 9. Fusion temperature (Tm) (0.5 °C of water (0) (peak A) and melting enthalpies of water fusion (9) (peak A) and GMO/ tricaprilyn (4) (peaks B and C) as a function of the water concentration in HII mesophases of GMO/tricaprylin/PC/water with weight ratio of GMO/tricaprylin 9:1 and 10 wt % PC. The onset of critical behavior (14 wt %water) is shown by an arrow.

detection capability of the DSC experiments. In the case of proline solubilization, the melting temperatures and enthalpies of peaks B and C were found to be insensitive to the variation in water content at all examined compositions.

4. Discussion On the microstructural level, the observed swelling of the HII mesophases seems to be logical and can be accounted for by two different effects: an increase in the distance between adjacent water cylinders or increase in the inner radius of the cylinders. According to the literature, while the first effect could be mostly attributed to the loss of positional ordering of the water cylinders, the second effect could be mostly attributed to the swelling of the water channels themselves and is therefore highly dependent

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on the relative concentration of water, oil, and surfactants.48 An increase in the lattice parameter as a function of water concentration of the hexagonal phase (ternary mixtures of GMO/ tricaprylin/water) was also reported by Amar-Yuli and Garti.19 The authors of this work classified the hexagonal phases according the degree of order (as determined by diffraction peak broadening), categorizing them as less ordered and more well-ordered structures at relatively low (20 wt %) water content, respectively.47 In the present systems that are stabilized by 10 wt % PC, the values measured for the lattice parameter are considerably higher compared to the systems without the phospholipids.47 While in a less ordered region (20 wt %), the lattice parameter increases by a much higher factor (up to10 Å). The tendency of PC to increase the lattice parameter can probably be attributed to the larger size of the hydrated headgroup compared to GMO. In other words, PC decreases the curvature of the system, thereby increasing the radius of the water channels and hence the lattice parameter of the HII phase. Diffraction peak broadening at low water content was also observed in our systems (data not shown); however, it was less pronounced compared to the system without PC.19 Perhaps, it is due to the described decrease of the curvature, imposing more hydrated and more well-ordered structures already at low water concentrations. It should be noted that the increase in the lattice parameter is not linear, as observed in Figure 3, indicating relatively slow hydration at low water phase concentration (8-14 wt %) and much faster hydration of the surfactant headgroups at a region of more well ordered structures (>20 wt %). In order to follow both the microscopic and mesoscopic structural variations, proline was solubilized in the system. As shown above, the kosmotropic effect of the solute on the HII phase microstructure was clearly demonstrated in this case (Figure 3). In general, kosmotropic (water-structure makers) and chaotropic (water-structure breakers) solutes have a very significant impact on the properties of liquid crystalline mesophases by indirect (Hofmeister) interactions with these structures. It was demonstrated that kosmotropic solutes stabilize the structure of bulk water.38,49,50 They are likely to be solubilized within the bulk water and be excluded from the interfacial areas. Waterstructures (kosmotropes) interfere with the tetrahedral network of water and, as a result, dehydration of the surfactant polar heads takes place (“salting-out” effect) and the amount of interfacial water is decreased. Hence, proline promotes higher curvature of the reversed mesophases and the lattice parameter decrease in the presence of the guest molecule, as compared to the proline-free system. Also, the fact that higher order peaks (11 and 20) have significantly lower scattering intensities in all examined samples containing proline, compared to the empty systems (Figure 2), can be attributed to less hydrated interface in the presence of the solute. Interestingly, at intermediate aqueous phase content the salting out effect was not noticed. Starting from 26 wt % up to 35 wt % of water phase (corresponding to the lattice parameters of 60.1-60.9 Å), no additional swelling could be observed, indicating that the solubilized proline slows down and eventually prevents further hydration of the hydrophilic heads of the surfactants (Figure 3). It is also important to note that solubilization of proline into the system enhanced the (48) Ramos, L.; Fabre P. Langmuir 1997, 13, 682-686. (49) Tsvetkova, N.; Koynova, R.; Tsonev, L.; Quinn, P.; Tenchov, B. Chem. Phys. Lipids 1991, 60, 51-59. (50) Saturni, L.; Rustichelli, F.; Di Gregorio, G. M.; Cordone, L.; Mariani, P. Phys. ReV. E 2001, 64, 040902(R).

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formation of the stable HII phase at very low pure water concentration, 4 wt %, compared with the proline-free system (8 wt %) (note that 8 wt % aqueous phase in the presence of proline corresponds to a value of 4 wt % of pure water). It is suggested that such behavior occurred due to the reduction of the interfacial area at the water/surfactants interface. Under these conditions, the formation of the pure HII mesophase (possessing low surface area) was speeded up. Using ESEM techniques, an alignment of polycrystalline domains was observed on a mesoscale. It was demonstrated that the topography and mesoscopic organization of reverse hexagonal LLC can be directly imaged in their hydrated state (Figure 4). Dynamic structural rearrangements, including formation and destruction of the HII mesophases, were observed (Figure 5). Variations in the structural properties were followed as a function of the aqueous phase concentration (Figure 7). It is evident from the results of these experiments that contrast difference effects between the domains present a good and sufficient tool for the analysis of the liquid crystalline mesostructures by the ESEM. We surmise that the source of contrast in our system depends on the surface topography of the examined LLC samples. It was noticed that the edges of the samples resulted in micrographs with higher contrast than the flat surfaces. This is attributed to the well-recognized phenomenon that the probability of secondary electron escape is higher from sloping areas, such as edges, leading to signals of higher intensity. Our results for the topography of the surface structure of the HII phase (demonstrating large domains of several tens of micrometers) are well correlated with the findings of Coppola et al.27 These investigators, using the PGSE NMR method, showed that the reverse hexagonal phase can be characterized by very large lyotropic domains which do not restrict the free motion of water, on a space length of at least 20 µm. In the present work we also found that as the water content of the hexagonal phase increases, both the microstructural lattice parameter and the mesoscopic domains swell. The fractal dimensions, Dg, obtained from the ESEM experiments reflect the interface between differently aligned domains. An alternative approach for the interpretation of this data would be to estimate the two-point correlation function between nonaligned rods, defined by eq 3

P(r) )

1 N

∫V 〈F(r′)F(r′ + r)〉d3r

(3)

which presents a measure of rod density inside the hexagonal phase. r represents the distance between rods, F(r) is the spatial density, N is the number of units, and V is the volume of the sample. For self-similar structures, it is expected (eq 4) to be scaled by the relationship presented in eq 4:

P(r) ∝ rDs

(4)

where Ds is the spatial fractal dimension.45 If the fractality of the system is in fact manifested in the boundary between regions over an Euclidean space, then Ds is related51 to Dg by eq 5:

Ds ) 3 - Dg

(5)

Using eqs 3-5, the normalized two-point density correlation functions are plotted against aqueous phase concentration of these systems (Figure 10). The values of Ds are in the region of 2.1-2.4. A direct conclusion from these results is that the mesophase is loosely packed. When the aqueous phase consists of pure water, the graph tends toward a plateau starting from 20 (51) Wong, P.; Cao, Q. Phys. ReV. B 1992, 45, 7627-7632.

Figure 10. Normalized density correlation function of GMO/ tricaprylin/PC/water with weight ratio of GMO/tricaprylin 9:1 and 10 wt % PC, as a function of (a) water concentration and (b) aqueous phase with of water:proline weight ratio of 1:1. Trend lines of the correlation function behaviors are shown on both datasets.

wt % water concentration, indicating that the interface is stabilized and invariant to further increase of water content. However, with proline added, P(r) continues a monotonic increase up to an aqueous phase concentration of 35 wt %. Since proline impairs the formation of interfacial water, the implication is that the interface is stabilized by the interfacial water. This conclusion is further confirmed by the scaling of the lattice parameter with water or water/proline concentration by the relationship (eq 6):

r ∝ C Dg

(6)

where r is the lattice parameter and C is the water content. The validity of the statement is demonstrated in Figure 11. On the basis of eqs 4-6, we can propose the following dependence for P(r) on the water concentrations (eq 7):

P(r) ∝ C3Dg-Dg

2

(7)

In the proline-free systems, a slow hydration at low water phase concentration (8-14 wt %) and a much faster hydration of the surfactant headgroups at the more well-ordered structure region (>20 wt %) are reflected both by the similar trends of the microstructural lattice parameter and the mesoscopic CDg. The onset of critical behavior seems to be shifted to higher values of the aqueous-phase concentrations (23-35 wt %) in the case of proline solubilization, indicating a less hydrated interface in the presence of the amino acid, as discussed above. This means

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Figure 11. Lattice parameter r(A) (0) and the exponent of the fractal geometry C(Dg) (O) of the GMO/tricaprylin/PC/water system with weight ratio of GMO/tricaprylin 9:1 and 10 wt % PC. These parameters are plotted as a function of (a) the water concentration and (b) water/proline concentration (aqueous phase with water/proline ratio of 1:1) The onset of critical behavior is related in both data sets.

that the mesoscopic polydomain alignment strongly depends on the lattice parameter value which in turn is a function of water concentration in the system. The current results suggest, therefore, that the topography of the HII phase is not random but likely to be dictated by the microstructural parameters and the composition of the samples. We would like to emphasize that what ESEM images is the domain wall structure and that this topography is linked to the curvature of the LLC rods. On the other hand, the curvature will influence the density of zwitterionic PC to nonionic GMO tails in the rod surface. As zwitterionic interaction will be governed by the dominant r-3 as opposed to r-6 of van der Waals interaction the free energy of the domain interface, and consequently its configurational entropy, will be governed mainly by this density. Therefore, it can be argued that Dg also reflects the effect of PC surface density (or rod curvature) on the configurational entropy of the domain interface. Hence, the experiments described above demonstrate that the fractal structure derived from ESEM analysis can provide the spatial fractal geometry of the system examined. This mesoscale property was demonstrated to correspond to the microscopic lattice parameter, as derived from the corresponding SAXS measurements. Furthermore, we were able to obtain the twopoint density correlation function for the systems involved. The resulting behavior points to the fundamental role of interfacial water in stabilizing the hexagonal structure. DSC measurements were conducted in order to link the water activity and behavior with both the microscopic and mesoscopic behavior of the systems. The obtained results revealed that the

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onset of critical behavior was observed around water content of 14 wt %, as reflected by the melting temperature increase, the increase of the enthalpy of the water fusion, and the decrease in the enthalpy of the GMO/tricaprilyn thermal transition (Figure 9). All these observations indicate a transformation from the slightly bound to the free state. On the basis of these results, we propose that the decrease in the enthalpy of peak B is due to the decrease of the number of unhydrated GMO/tricaprylin molecules that participate in the melting transition, as a consequence of the lipid hydration with an increase in the content of the water phase. The same onset of critical behavior (14 wt % water phase) was observed for the fractal dimension as a function of water content (Figure 6). Hence, the swelling of the mesoscopic domains and their fractal dimensions are shown to be directly linked to the water activity. The particular aqueous phase concentration of 14 wt % is probably required to stabilize the water/surfactants interface and start accumulating free water molecules in the tubes. When proline molecules are present in the aqueous phase, a much lower concentration of pure water is needed to stabilize the interface (14 wt % of aqueous phase corresponds to 7 wt % of pure water) due to the kosmotropic effect discussed above. However, in contrast to the proline-free systems, in the case of proline-containing systems no free water is observed by the DSC at higher water content compositions. Applying to our studied systems the equation used by Yaghmur et al.52 to quantify the free water concentration, it was found that the free water content increased from 3 wt % (of total water content in the sample) to 17 wt % for the lower and higher water contents in the empty LLC, respectively. Thus, it seems that the swelling observed for the lattice parameter (Figure 3) is proportional to the bulk water concentration in the water rods of the systems. In addition, it was noticed that the melting of the tricaprylin + GMO mixture splits into a double step endothermic event (peaks B and C, Figure 8c.), exhibiting the same behavior as the blank systems with low water concentrations (8-11 wt %). This trend can be explained by the presence of two polymorphic GMO/tricaprylin mixtures at low hydration levels of the lipids. While in the blank LLC the second polymorphic structure disappears due to increased hydration, in the presence of proline this polymorphic structure remains in the system, probably due to the low hydration level induced by the “salting-out” effect.

5. Conclusions In the current study we investigated the relationships between the microstructural and mesostructural properties of the reverse hexagonal LLC. Using ESEM techniques, it was shown that the mesoscopic organization of these systems is based on an alignment of polycrystalline domains, each of which is several tens of micrometers. It was also shown that the topography of HII mesophases can be imaged directly in their hydrated state, as a function of aqueous phase concentration. The domain structure visualized in the ESEM images was found to possess fractal characteristics (fractal dimensions of 0.65-0.9), indicating the considerably discontinuous and disordered alignment of the water cylinders on the mesoscale. To establish the role played by the interface in the stabilization of microscopic and mesoscopic structure, proline was solubilized into the systems. On the microstructural level, SAXS measurements demonstrated the significant effect of the aqueous phase concentration on the variation in the lattice parameter of the mesophases. It was found that as water content (Cw) increases, both the microstructural lattice parameter and the mesoscopic domains increase (swelling (52) Yaghmur, A.; Aserin, A.; Tiunova, I.; Garti, N. J. Therm. Anal. Cal. 2002, 69, 163-177.

Lyotropic ReVerse Hexagonal Liquid Crystals

effect). The fractal dimensions (Dg) extracted from the ESEM images reveal that the lattice parameter exhibits a power law dependence on the water concentration expressed by CDg, reinforcing the link between the microscopic and mesoscopic organizations. This correlation seems to be valid for both the proline-free systems and the proline-containing mesophases. These results suggest, therefore, that the topography of the HII phase is likely to be influenced by the microstructural parameters and the composition of the samples. Additionally, the density correlation function of the reverse hexagonal phase (and hence its packing) was shown to follow a power law dependence on the water concentration. DSC measurements linked the activity and general behavior of the water with both the microscopic and mesoscopic features of the systems. It was indicated that the swelling effect observed for the lattice parameter and the

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mesoscopic domains is very likely to be proportional to the bulk water concentration in the water rods. Furthermore, by the introduction of proline into the system, we were also able to demonstrate that it is mainly the interfacial water that stabilizes and establishes the microscopic and mesoscopic nature of the hexagonal phases studied. Acknowledgment. We would like to thank Professor Yuri Feldman of the Department of Applied Physics, The Hebrew University of Jerusalem, for helpful discussions on fractal structure of reverse hexagonal mesophases. We also thank Mrs. Evgenia Blayvas from the Unit for Nanocharacterization, The Hebrew University of Jerusalem, for technical assistance in conducting experiments by ESEM techniques. LA702570V