pubs.acs.org/Langmuir © 2009 American Chemical Society
Structural Rearrangements and Interaction within HII Mesophase Induced by Cosolubilization of Vitamin E and Ascorbic Acid Liron Bitan-Cherbakovsky,† Idit Yuli-Amar, Abraham Aserin, and Nissim Garti* Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. † The results presented in this manuscript will appear in the MSc thesis of L.B.-C. in partial fulfillment of the requirements for the degree of Magister in Applied Chemistry, The Hebrew University of Jerusalem, Israel. Received April 5, 2009. Revised Manuscript Received May 25, 2009 We investigated the effect of ascorbic acid (AA) cosolubilized with vitamin E (VE) on reverse hexagonal (HII) mesophase. The HII phase comprises monoolein (GMO)/D-R-tocopherol (VE) in a ratio of 90/10 by weight and 12.5 wt % water. The macrostructural characteristics of this system were determined by polarized light microscopy and small-angle X-ray scattering measurements. We used differential scanning calorimetry and attenuated total reflectance Fourier transform infrared to characterize the microstructure, the vibration of the functional groups, and the location of the AA guest molecule. AA was incorporated to the system in two steps: 1-4 wt % AA and 5-6 wt % AA. We compared this system to one containing tricaprylin as the oil phase, as previously reported. These measurements revealed that AA is localized first in the water rich-core and in the interface, and acts as a chaotropic molecule that decreases the water melting point. When a larger quantity of AA (5-6 wt %) is added, the system is saturated, and the AA is located in the inner cylinder and manifested by more moderate distortion. The addition of AA also causes alteration in the behavior of the GMO hydrocarbon chains and makes them more flexible. Further addition of AA caused the GMO hydrocarbon chain to be more solvated by the VE hydrocarbon chain and enabled additional migration of VE; hence a decrease in the hydrophobic melting temperature occurred (similar to tricaprylin). Increasing the amount of AA weakened the bonding between the GMO and water and created new bonds between AA and GMO and AA with water.
Introduction Certain surfactants in aqueous media can be self-associated to form various types of lyotropic liquid crystals (LLCs) in which the lamellar, hexagonal, and cubic phases are the most common structures. LLCs are used as model matrices to imitate biological processes where the phase behavior of lipids plays a mediating role. Furthermore, these systems have the potential to incorporate biologically active molecules with various physicochemical properties, protect them from degradation, and, when needed, release them in a controlled manner. Therefore, LLCs are utilized as host systems for drugs, enzymes, vitamins, and other active molecules in pharmaceutical, biotechnical, and food applications.1-7 Reverse hexagonal (HII) LLCs are promising candidates as delivery vehicles for nutraceuticals as well as pharmaceutical substrates due to their structural characteristics. These structures can incorporate hydrophilic, lipophilic, and amphiphilic compounds *Author whom correspondence should be addressed. Address: Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew University of Jerusalem, Givat Ram Campus, Jerusalem 91904, Israel. Tel: 972-2-658-6574/5. Fax: 972-2-652-0262. E-mail:
[email protected]. (1) Caboi, F.; Lazzari, P.; Pani, L.; Monduzzi, M. Chem. Phys. Lipids 2005, 135, 147–156. (2) Caboi, F.; Nylander, T.; Razumas, V.; Talaikyte, Z.; Monduzzi, M.; Larsson, K. Langmuir 1997, 13, 5476–5483. (3) Pouzot, M.; Mezzenga, R.; Leser, M.; Sagalowicz, L.; Guillot, S.; Glatter, O. Langmuir 2007, 23, 9618–9628. (4) Sagalowicz, L.; Mezzenga, R.; Leser, M. E. Curr. Opin. Colloid Interface Sci. 2006, 11, 224–229. (5) Tan, G.; Xu, P.; John, V. T.; He, J.; McPherson, G. L.; Agarwal, V.; Bose, A. Langmuir 2008, 24, 10621–10624. (6) Dong, Y.-D.; Dong, A. W.; Larson, I.; Rappolt, M.; Amenitsch, H.; Hanley, T.; Boyd, B. J. Langmuir 2008, 24, 6998–7003. (7) Libster, D.; Ben Ishai, P.; Aserin, A.; Shoham, G.; Garti, N. Langmuir 2008, 24, 2118–2127.
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separately and simultaneously, while the hydrophilic/hydrophobic nature of the guest molecule will determine the molecule’s preferable location. Generally, a polar hydrophilic guest molecule will accommodate within the aqueous domains, composed of dense packed, straight water-filled rods, and a hydrophobic one, may interact within the lipid hydrophobic tails, thereby being oriented radially outward from the centers of the water rods.8,9 A diverse group of bioactive compounds, once delivered without a protecting carrier, show either a water-poor solubility or cleavage susceptibility in oral/topical delivery conditions, and therefore display poor bioavailability.10,11 During the past decade, several reports were published regarding solubilization of water-insoluble bioactive molecules, such as vitamin K and tocopherol acetate, into liquid crystalline phases.2,12,13 Furthermore, in vitro and in vivo studies demonstrated enhancement in various bioactive substrate release profiles and bioavailability based on their accommodation in liquid crystalline systems.2,12,13 Nevertheless, the solubilization capacities were limited, and, above a critical level, phase transition occurred.14 The molecular and structural behavior of different guest molecules has not yet been explained. (8) Amar-Yuli, I.; Wachtel, E.; Ben-Shoshan, E.; Danino, D.; Aserin, A.; Garti, N. Langmuir 2007, 23, 3637–3645. (9) Libster, D.; Ben Ishai, P.; Aserin, A.; Shoham, G.; Garti, N. Int. J. Pharm. 2009, 367, 115–126. (10) Gabizon, A.; Shmeeda, H.; Barenholz, Y. Clin. Pharmacokinet. 2003, 42(5), 419–436. (11) Getie, M.; Wohlrab, J.; Neubert, R. H. H. J. Pharm. Pharmacol. 2005, 57 (6), 423–428. (12) Dong, Y. D.; Larson, I.; Hanley, T.; Boyd, B. J. Langmuir 2006, 22, 9512– 9518. (13) Rowinski, P.; Rowinska, M.; Heller, A. Anal. Chem. 2008, 80, 1746–1755. (14) Lopes, L. B.; Speretta, F. F. F.; Vitoria, M.; Bentley, L. B. Eur. J. Pharm. Sci. 2007, 32, 209–215.
Published on Web 10/23/2009
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Recently, we reported on the solubilization of ascorbic acid (AA), ascorbyl palmitate, R-tocopherol, and R-tocopherol acetate in HII systems composed of monoolein (glycerol monooleate, GMO), tricaprylin (TC), and water.15 To study their influence and understand their interactions at the molecular level, we used attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, combined with polarized light microscopy, differential scanning calorimetry (DSC), and small-angle X-ray scattering (SAXS). The four bioactive molecules with diverse polarity and solubility properties interacted differently with each component and were positioned in unique locations in the HII structure. In the current research we focus on the solubilization impact of AA on a ternary mixture of GMO/R-tocopherol/water (90/10 wt % ratio and 12.5 wt % water or 78.75, 8.75, 12.50 wt % of GMO, R-tocopherol, and water, respectively). Previously we reported on the feasibility of solubilizing R-tocopherol (vitamin E (VE)) as the oil phase (VE replacing the TC) and producing an HII phase at room temperature.15 Here we explore the ability of this new HII phase, already containing the essential bioactive molecule VE, which produces the liquid crystal structure, to incorporate additional bioactive molecules, including a hydrophilic compound such as AA. R-Tocopherol, commonly known as vitamin E (VE), acts as a very efficient radical-chain-breaking antioxidant in tissues.16 VE, thanks to its van der Waals interactions with the membrane phospholipids, can also be used as a membrane-stabilizing agent.12,17 It is also the main natural lipid-soluble antioxidant present in the cell antioxidant defense system, including the cell membranes of the intestine and stomach.15,18 It is well-known that AA, usually known as vitamin C (VC), is an essential nutraceutical commonly used in medicine, food, and biochemistry, thus it has been considerably investigated.19 The antioxidant AA efficiently protects important organic and biological molecules against oxidative degradation by its property of strong hydrophility.20 Another use of AA is in cosmetic and dermatological products, since it has many favorable effects on the skin as a reducing agent.21 It also enhances the elasticity of the skin by promoting the formation of collagen.20,22 A detailed molecular examination of the interactions between AA and each of the HII phase’s components was made to understand the nature of this complementary solubilization system for delivery of synergistic antioxidants - one working in aqueous phase and the other in lipidic interphase. The present manuscript emphasizes the gradual (two-step) solubilization action of AA in the presence of an additional essential compound, VE, and their impact on structural aspects related to the HII mesophase. The outcomes of this study will be compared to the analysis of the GMO/TC/water mixture as a reservoir system, regarding the nature of the component that affects the phase behavior, cylinder swelling, and location of the solubilizate. This study utilized a combination of polarized light microscopy, DSC, SAXS, and ATR-FTIR spectroscopy. (15) Amar-Yuli, I.; Aserin, A.; Garti, N. J. Phys. Chem. 2008, 112(33), 10171– 10180. (16) Liu, K.; Chougnet, A.; Woggon, W.-D. Angew. Chem., Int. Ed. 2008, 47, 5827–5829. (17) Bradford, A; Atkinson, J.; Fuller, N.; Rand, R. P. J. Lipid Res. 2003, 44, 1940–1945. (18) Granger, D. N.; Hernandez, L. A.; Grisham, M. B. Viewpoints Dig. Dis. 1986, 18, 13–17. (19) Weili, Y.; Rong, G. J. Dispersion Sci. Technol. 1999, 20, 1359–1387. (20) Palma, S.; Manzo, R.; Lo Nostro, P.; Allemandi, D. Int. J. Pharm. 2007, 345, 26–34. (21) Silva, G. M.; Maia Campos, P.M. B. G. Int. J. Cosmet. Sci. 2000, 22, 169– 179. (22) Haftek, M.; Mac-Mary, S.; Le Bitoux, M. A.; Creidi, P.; Seite, S.; Rougier, A.; Humbert, P. Exp. Dermatol. 2008, 17, 946–952.
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Experimental Section Monoolein (distilled glycerol monooleate, GMO) that consists of 97.1 wt % monoglyceride, 2.5 wt % diglyceride and 0.4 wt % free glycerol (acid value 1.2, iodine value 68.0, melting point 37.5 °C) were purchased from Riken (Tokyo, Japan). D-R-Tocopherol, vitamin E 5-96 (containing 1430 international units of vitamin E per gram), was obtained from ADM (Decatur, IL). L-(þ)-AA was purchased from Baker Chemical Co. (Phillipsburg, NJ). Water was double distilled. All ingredients were used without further purification. Sample Preparation. The GMO/R-tocopherol/water (and different concentrations of AA (1-6 wt %)) hexagonal liquid crystalline samples were formed by mixing all the components (GMO and R-tocopherol in 9:1 weight ratio) while heating to ∼70 °C in sealed tubes under nitrogen (to avoid oxidation of the GMO) for ca. 15 min. The samples were stirred and cooled to 25 °C. It should be noted that as a result of AA solubilization the concentrations of GMO and R-tocopherol were decreased, keeping their weight ratio of GMO/R-tocopherol (9:1) and water content constant. For example, the empty system was composed of 78.75, 8.75, and 12.50 wt % of GMO, R-tocopherol, and water, respectively, whereas the system loaded with AA (e.g., 4 wt %) contained 75.15, 8.35, 12.50, and 4.00 wt % of GMO, R-tocopherol, water, and AA, respectively. D2O was used for ATRFTIR measurements instead of water. Light Microscopy. The samples were inserted between two glass microscope slides and observed with a Nikon light microscope Eclipse 80i model (Nikon, Tokyo, Japan) equipped with cross-polarizers and attached to a digital camera Nikon DXM 1200C and PC-monitor. The samples were analyzed at room temperature. SAXS. Scattering experiments were performed using Ni-filtered Cu KR radiation (0.154) from an Elliott rotating anode X-ray generator that operated at 1.2 kW. X-radiation was further monochromated and collimated by a single Franks mirror and a series of slits and height limiters, and measured by a linear positionsensitive detector. The samples were held in 1.5 mm quartz X-ray capillaries inserted into a copper block sample holder. 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.23 DSC. A Mettler Toledo DSC822 (Greifensee, Switzerland) measuring model system was used. The DSC measurements were carried out as follows: 5-15 mg hexagonal liquid crystalline samples 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 þ30 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 40 °C. An empty pan was used as a reference. The instrument determined the fusion temperatures of the 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 (0.2 °C. ATR-FTIR. An Alpha P model spectrophotometer, equipped with a single reflection diamond ATR sampling module, manufactured by Bruker (Ettlingen, Germany), was used to record the FTIR spectra (GMO/VE/water with AA in different concentrations). The spectra were recorded with 50 scans, with spectral resolution of 2 cm-1, at room temperature. The absorbance intensities reported here were reproducible to (0.005. ATR-FTIR Data Analysis. Multi-Gaussian fitting was utilized to resolve individual bands in the spectra. The peaks were analyzed in terms of peak frequencies, width at half-height, and areas. (23) Lake, J. A. Acta Crystallogr. 1967, 23, 191–194.
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Figure 1. Polarized optical microscope images of the HII crystalline phase at 25 °C. (a) Birefringent colorful texture of GMO/VE þ12.5 wt % water without AA. (b) Birefringent colorful texture of GMO/VE þ12.5 wt % water þ 3% AA. (c) Birefringent colorful texture of GMO/VE þ12.5 wt % water þ 6% AA. Table 1. Melting Temperatures (°C ( 0.2°C) of the HII Systems (GMO/VE/Water), Empty and Loaded with 1-6 wt % AA
Figure 2. DSC thermograms of GMO/VE/water HII mesophase
with two endothermic events at -2.1 ( 0.2 °C (peak A) and 11.9 ( 0.2 °C (peak B) of 0 wt % AA and thermograms of GMO/VE/ water þ AA up to 6 wt % with one endothermic event of water with intention of decrease and two endothermic events of the tails that decrease and gather to one peak.
Results and Discussion The Bulk Properties of the HII Phase Containing VE and Solubilizing AA. We studied the impact of AA guest molecules on the macrostructure of the HII mesophases formed by the mixture of GMO/VE/water with a GMO/VE weight ratio of 90/10, and 12.5 wt % water. Up to 6 wt % AA was solubilized in this new HII system. Each sample containing both VE and AA in variable amounts was identified by a light microscope with crossed polarizers. Polarized light microscope images at room temperature of the 0, 3, and 6 wt % AA loadings in GMO/VE/ water system are shown in Figure 1a-c, respectively, representing the empty system and moderate and maximal solubilization capacity. All three images, in the absence and presence of AA, display birefringent colorful textures that can be attributed to the hexagonal symmetry. It should be noted that both images representing the loaded HII systems exhibit clear texture, implying that the hexagonal phase enabled the AA accommodation without any precipitation. 13108 DOI: 10.1021/la901195t
AA (wt%)
water peak A (°C)
0 1 2 3 4 5 6
-2.1 -2.5 -3.8 -5.5 -6.0 -6.5 -6.8
tail peak B (°C) 11.9 8.3 8.6 8.7 9.2
4.8 4.9 5.5 5.9 5.6 5.4
The thermotropic behavior of the GMO/VE/water was previously studied by DSC, yet without the additional incorporation of AA.15 In the absence of AA, during the heating scan from -40 to þ 40 °C of the GMO/VE/water mixture, two endothermic peaks were observed (Figure 2) with maxima at -2.1 ( 0.2 (peak A) and 11.9 ( 0.2 (peak B), as previously affirmed.15 Peak A was associated with the melting of ice, and peak B was attributed to the fusion of the hydrophobic moieties of the GMO solvated by the VE. When comparing the two systems containing VE or TC as the oil phase, one could notice that the water melting temperatures are equivalent; however, the hydrophobic tail fusion peaks are different and determined by the degree of hydrophobic interactions between the GMO and the oil, depending on the oil type. When TC was present, the melting peak appeared at a lower temperature, 5.9 ( 0.2 °C, but once the VE molecule replaced the TC, the melting of the hydrophobic moieties of the GMO occurred at higher temperature, 11.9 ( 0.2 °C (peak B). This result may represent the solvation level of GMO by the oil, implying that TC ore efficiently solvates the GMO tails, thereby inducing the formation of the HII phase at lower temperatures than if VE serves as the oil phase. On the other hand, with a view to eventual application as a reservoir vehicle for solubilization, higher solubilization levels of AA were obtained by this system, compared to the one containing TC. On the basis of the spatial structure of both molecules, it is reasonable to surmise that the TC would be easily accommodated between the GMO tails, while the VE accommodation is considered to be more difficult. ATRFTIR measurements will assist in verifying this argument. DSC outcomes show that the incorporation of AA molecules composed of five hydrogen-bond acceptor sites (four hydroxyl þ one ester group) had a significant effect on the thermal process of the mesophases, on both endothermic events reflected in peaks A and B (Figure 2, Table 1). As expected, an increase in content of the molecules capable of binding water (such as AA) gradually decreased the water thawing temperature, from -2.5 to -6.8 ( 0.2 °C in the presence of 1-6 wt % AA (Table 1). Unpredictably, embedment of 1-4 wt % AA also has a significant effect on the lipidic tail fusion temperatures. The addition Langmuir 2009, 25(22), 13106–13113
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Article Table 2. Lattice Parameters (a) of the HII Systems Empty and Loaded with AA at Different Concentrationsa AA (wt %)
Lattice Parameter (a) (A˚ ( 0.5 A˚)
0 1 2 3 4 5 6 a The measurements were carried out at 25°C.
Figure 3. Lattice parameters (a) of the HII system empty and loaded with AA up to 6 wt %. The measurements were carried out at 25 °C.
of even 1 wt % AA decreased the melting temperature of the GMO lipophilic moieties and divided the peak into two subpeaks with maxima at 8.3 and 4.8 ( 0.2 °C. Further incorporation of AA up to 4 wt %, although it did not affect the peak temperature, led to gradual decrease in the higher temperature peak area (correspondingly increasing the lower temperature peak region) up to its complete disappearance, once 5 wt % AA was added. In the presence of 5-6 wt % AA, solely a single peak was observed at ∼5.4 ( 0.2 °C. These findings may probably be attributed to the formation of additional hydrogen bonds in water upon incorporation of AA molecules and would also indicate that AA competes for water binding with GMO polar headgroups. At this point we cannot assign the lower temperature of ice melting solely to the water Langmuir 2009, 25(22), 13106–13113
48.0 50.0 50.6 51.0 52.4 52.7 53.0
binding by AA. The water behavior may be also affected indirectly by the presence of the AA guest molecules that results in reorganization and formation of additional hydrogen bonds in the HII structure. The split in the lipophilic melting peak together with its appearance at a lower temperature, upon entrapping AA molecules in the polar region, implies looser lipophilic assembly with two populations. This can be achieved indirectly by swelling the GMO polar head, thereby enabling additional penetration of VE and therefore more solvation of the GMO by VE molecules. It appears that, above a critical AA concentration (>4 wt %), all VE molecules sufficiently accommodate between the GMO tails; thus the degree of solvation is higher, and a single melting peak with relatively low temperature is obtained. Moreover, the overall hydrophobic contribution that determines the fusion temperature of the GMO tails due to the combined AA and VE, at these concentrations (4-5 and 8.8 wt %, respectively), is equal to the role of the TC alone at the same content as the VE. The significant effects of even low guest molecule solubilization is in line with Caboi et al.’s observations when they incorporated vitamin K1 into a GMO/water mixture.2 The authors considered a varying lateral distribution of vitamin K1 in the lipid bilayer and assumed that the locally higher concentration of the vitamin triggered the phase transitions. SAXS was used to identify and characterize the structure of the phases. The SAXS patterns of the empty GMO/VE/water mixture and AA-loaded mixtures at 25 °C are shown in Figure 3. For each mixture, three peaks were observed, which were indexed as the [10], [11], and [20] reflections of a 2D HII phase (which is in line with the microscope images in Figure 1). From the three peak positions, we calculated the corresponding mean lattice parameter (a) of the hexagonal structures that are summarized in Table 2. SAXS analysis in the presence of AA molecules (Table 2) reveals an increase in the lattice parameter of the HII phases. Two stages of structural effect (i.e., cylinder swelling) can be detected while AA molecules are gradually incorporated. Upon addition of 1-3 wt % AA, the lattice parameter increases by ∼3 A˚, and further incorporation of 4-6 wt % AA results in an additional increase in a values (by 2 A˚). It should be noted that in the GMO/ VE/water system, once 3 wt % AA was added, the lattice parameter was increased by 4 A˚ and further increase in the solubilization loads did not further alter the a-values.15 Furthermore, previously we reported that AA performs as a chaotropic solute, which is known to exhibit significant impact on the properties of liquid crystalline mesophases by indirect (Hofmeister) interactions with the structures.24-26 The chaotrope (24) Koynova, R.; Brankov, J.; Tenchov, B. Eur. Biophys. J. 1997, 25, 261–274. (25) Tsvetkova, N.; Koynova, R.; Tsonev, L.; Quinn, P.; Tenchov, B. Chem. Phys. Lipids 1991, 60, 51–59. (26) Saturni, L.; Rustichelli, F.; Di Gregorio, G. M.; Cordone, L.; Mariani, P. Phys. Rev. E 2001, 64, 040902/1-4.
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Figure 5. (left) ATR-FTIR frequency (cm-1) as a function of AA concentrations (wt %) of the O-H bending absorption modes of the water. (right) ATR-FTIR frequency (cm-1) as a function of AA concentration (wt %) of the O-H stretching absorption mode of the GMO molecule.
Figure 4. ATR-FTIR frequency (cm-1) as a function of AA concentrations (wt %) of the O-D stretching absorption mode of the water.
AA was shown to destabilize the structure of bulk water and swelled the hexagonal-arranged cylinders (increased the area at the water/surfactants interface).24-26 Considering DSC outcomes, we can conclude that AA may increase the hydration level of the headgroups, thereby swelling the cylinders and increasing the lattice parameter. The two-step swelling profile (clearly seen in Table 2) is in line with the DSC results. It can be suggested that: (1) the AA accommodates in the polar region in two main positions within two stages, and/or (2) there are two steps of VE penetration upon incorporation of AA, which is exhibited by split hydrophobic peak in the first stage that is associated with melting of the GMO hydrophobic moieties at relatively higher temperatures. In the second step, upon entrapping AA molecules in the polar region, the two endothermic peaks converted into one lower temperature peak, which was associated with additional penetration of VE and higher solvation of the GMO by VE molecules. When comparing the two HII systems on the basis of either TC or VE, it can be surmised that, in the case of lower AA values (
