Effect of Sodium Diclofenac Loads on Mesophase Components and

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Langmuir 2008, 24, 7590-7595

Effect of Sodium Diclofenac Loads on Mesophase Components and Structure Rivka Efrat,*,† Deborah E. Shalev,‡ Roy E. Hoffman,§ Abraham Aserin,† and Nissim Garti*,† Casali Institute of Applied Chemistry, and NMR Unit, The Institute of Chemistry, and Wolfson Centre for Applied Structural Biology, The Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Safra Campus, GiVat Ram, Jerusalem 91904, Israel ReceiVed February 25, 2008. ReVised Manuscript ReceiVed April 23, 2008 We studied the effect of a model electrolytic drug on intermolecular interactions, conformational changes, and phase transitions in structured discontinuous cubic QL lyotropic liquid crystals. These changes were due to competition with hydration of the lipid headgroups. Structural changes of the phase induced by solubilization loads of sodium diclofenac (Na-DFC) were investigated by directly observing the water, ethanol, and Na-DFC components of the resulting phases using 2H and 23Na NMR. Na-DFC interacted with the surfactant glycerol monoolein (GMO) at the interface while interfering with the mesophase curvature and also competed with hydration of the surfactant headgroups. Increasing quantities of solubilized Na-DFC promoted phase transitions from cubic phase (discontinuous (QL) and bicontinuous (Q)) into lamellar structures and subsequently into a disordered lamellar phase. Quadrupolar coupling of deuterated ethanol by 2H NMR showed that it is located near the headgroups of the lipid and apparently is hydrogen bonded to the GMO headgroups. A phase transition between two lamellar phases (LR to LR*) was seen by 23Na NMR of Na-DFC at a concentration where the characteristics of the drug change from kosmotropic to chaotropic. These findings show that loads of solubilized drug may affect the structure of its vehicle and, as a result, its transport across skin–blood barriers. The structural changes of the mesophase may also aid controlled drug delivery.

Introduction Electrolytes are known to affect the structure of nonionic lyotropic liquid crystals (LLC).1–4 Their effect depends on the degree of hydration of the hydrophilic moiety of the surfactant, the possible conformational changes, and/or the nature of the anions and cations. Anions generally have a much stronger effect on the hydration of surfactants than do cations according to the Hofmeister series.2,5–10 This effect may be minor if small amounts of electrolytes are present in the system.6,7,9,10 However, increasing the electrolyte content can cause strong intermicellar interactions leading to conformational changes. Lyotropic or kosmotropic salts are usually small ions that impart strong electric fields at short distances and bind water molecules tightly, thereby extracting water from the interfacial regions.5–10 As a result, they reduce the interfacial area occupied by the headgroups of * To whom correspondence should be addressed. Phone: +972-2-6586574. Fax: +972-2-652-0262. E-mail: [email protected]. † Casali Institute of Applied Chemistry, The Institute of Chemistry. ‡ Wolfson Centre for Applied Structural Biology. § The NMR Unit, The Institute of Chemistry. (1) Takahashi, H.; Matsuob, A.; Hatta, I. Phys. Chem. Chem. Phys. 2002, 4, 2365–2370. (2) Morini, M. A.; Messina, P. V.; Schulz, P. C. Colloid Polym. Sci. 2005, 283, 1206–1218. (3) Harve, R. D.; Barlow, D. J.; Drake, A. F.; Kudsiov, L.; Lawrence, M. J.; Brain, A. P. R.; Heenan, R. K. J. Colloid Interface Sci. 2007, 315, 648–661. (4) Zou, A.; Hoffmann, H.; Freiberger, N.; Glatter, O. Langmuir 2007, 23, 2977–2984. (5) Miyagishi, S.; Okada, K.; Asakawa, T. J. Colloid Interface Sci. 2001, 238, 91–95. (6) Leontidis, E. Curr. Opin. Colloid Interface Sci. 2002, 7, 81–91. (7) Inoue, T.; Yokoyama, Y.; Zheng, L.-Q. J. Colloid Interface Sci. 2004, 274, 349–353. (8) Giner, I.; Pera, G.; Lafuente, C.; Lo´pez, M. C.; Cea, P. J. Colloid Interface Sci. 2007, 315, 588–596. (9) Alam, Md.; Naqvi, S.; Kabir-ud-Din, A. Z. J. Colloid Interface Sci. 2007, 306, 161–165. (10) Efrat, R.; Aserin, A.; Garti, N. J. Colloid Interface Sci. 2008, 321, 166– 176.

the surfactants, causing salting-out of the amphiphilic headgroups. Hydrotropic or chaotropic salts are usually large ions with weak electrical fields and weak hydration capabilities, which tend to destabilize the structure of the bulk water and increase the concentration of free water molecules that can hydrogen bond to the hydrophilic moiety of the surfactants, thereby increasing the amount of interfacial water and the amphiphilic interfacial area, causing salting-in.6,11,12 The major difficulty in predicting the behavior of Hofmeister ions is due to the simultaneous effects of cations and anions, which can sometimes act in opposing modes. The overall effect of adding ions is determined by the balance of the kosmotropic or chaotropic nature of the individual ions, making it essential to decouple the effects of the anions and cations.6,7 Lyotropic liquid crystals based on glycerol monooleate (GMO) have been used as delivery systems for a wide variety of drugs.13–15 Percutaneous absorption of some anti-inflammatory drugs is enhanced upon adding ethanol.16 The GMO/water/ethanol system has been shown to have the capacity to solubilize biomolecules.17 However, some drug molecules have been shown to affect the liquid crystalline mesophase array by disrupting the intermolecular forces. This can cause phase transitions within the array or separation into different phases.13–15 (11) Koynova, R.; Brankov, J.; Tenchov, B. Eur. Biophys. J. 1997, 25, 261– 274. (12) Lopez-Leon, T.; Gea-Jodar, P. M.; Bastos-Gonzalez, D.; Ortega-Vinuesa, J. L. Langmuir 2005, 21, 87–93. (13) Chang, C.-M.; Bodmeier, R. Int. J. Pharm. 1997, 147, 135–142. (14) Gabboun, N. H.; Najib, N. M.; Ibrahim, H. G.; Assaf, S. Int. J. Pharm. 2001, 212, 73–80. (15) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. AdV. Drug DeliVery ReV. 2001, 47, 229–250. (16) Benson, H. A. E. Curr. Drug DeliVery 2005, 2, 23–33. (17) Efrat, R.; Aserin, A.; Garti, N. In Food Colloids: Self-Assembly and Material Science; Dickinson, E., Leser, M. E., Eds.; Royal Society of Chemistry: Cambridge, 2007; pp 87-102.

10.1021/la800603f CCC: $40.75  2008 American Chemical Society Published on Web 06/12/2008

Effect of Na-DFC Loads on Mesophase Components

Charged drugs have been shown to interact with amphiphilic molecules, affecting the rate of drug release. The rates of release of charged drugs in LLC systems depend on both the mesophase structure and the electrostatic interactions.13–15,18–20 We have demonstrated that charged drugs such as Na-diclofenac (Na-DFC) can be solubilized in a QL (cubic micellar discontinuous)21,22 type of lyotropic liquid crystal phase, causing a series of consecutive drug concentration-dependent transitions:10

QL f Q f LR f LR/ f disordered phase + crystals The proximity of the Na-DFC to the tail groups or the headgroups was dependent on the type of mesophase. This effect on the Na-DFC partition coefficient varied with pH, ion-pair formation, and hydrophobicity of the solvents. In the present study, 2H and 23Na NMR were used to independently study the interactions of water, ethanol, and NaDFC, and their impact on the lipid GMO and on the structure of the lamellar mesophase. The degree of anisotropic motion seen by quadrupolar coupling of deuterium oxide, deuterated ethanol, and sodium under increased loading capacities of NaDFC indicates the degree of binding and anisotropic orientation of these compounds. The results elucidate the nature of the interfacial interactions that charged drugs may impart on the GMO/ethanol/water mesostructure in particular, or on any other aqueous-based delivery system in general. Studying the different components of the formulation gave a detailed understanding of the changes in the mesophase with increased loads of Na-DFC. The changes in mesophase under increased drug loads may explain the delivery and release patterns of certain ionic drugs and other bioactive compounds.

Experimental Section Materials. Distilled monoolein (distilled glycerol monooleate, GMO) consisting of 97.1 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%) was obtained from Riken Vitamin Co. Ltd., Japan. Ethanol was analytical reagent grade (>99%) purchased from Frutarom Ltd., Israel. Water was double distilled. Diclofenac sodium salt (Na-DFC), deuterated water, and ethanol-d6 were purchased from Sigma (St. Louis, MO). QL Mesophase Preparation. The QL samples were prepared by weighing appropriate amounts of GMO/ethanol/water at a ratio of 41:11:48 wt % into culture tubes sealed with Viton-lined screw caps. The samples were heated to ∼60 °C in a water bath for 2 min, vortexed until transparent, and cooled to 25 ( 0.5 °C in a water bath. The samples were allowed to equilibrate at 25 ( 0.5 °C for 24 h before they were examined. NMR Experiments. Samples for NMR experiments were prepared using 5-6 total wt % D2O or ethanol-d6 as part of the water fraction of the sample. The NMR experiments were performed on a Bruker DRX 400 MHz spectrometer operating at a proton frequency of 400.13 MHz, deuterium frequency of 61.42 MHz, and 23Na frequency of 105.84 MHz, or a Bruker Avance 600 MHz DMX spectrometer operating at a proton frequency of 600.13 MHz and a deuterium frequency of 92.12 MHz, both using 5 mm broadband probes. The temperature (25 °C) was precise to within (0.1 °C on both spectrometers. (18) Clogston, J.; Craciun, G.; Hart, D. J.; Caffrey, M. J. Controlled Release 2005, 102, 441–461. (19) Bordi, F.; Cametti, C.; Sennato, S.; Viscomi, D. J. Colloid Interface Sci. 2006, 304, 512–517. (20) Svensson, O.; Thuresson, K.; Arnebrant, T. Langmuir 2008, 24, 2573– 2579. (21) Efrat, R.; Aserin, A.; Kesselman, E.; Danino, D.; Wachtel, E. J.; Garti, N. Aust. J. Chem. 2005, 58, 762–766. (22) Efrat, R.; Aserin, A.; Kesselman, E.; Danino, D.; Wachtel, E. J.; Garti, N. Colloids Surf., A 2007, 299, 133–145.

Langmuir, Vol. 24, No. 14, 2008 7591 The deuterium spectra were acquired with 16K points and a spectral width of 80 ppm, giving a repetition rate of approximately 2 s. They were acquired without field-frequency lock, using 80 transients, and exponential apodization with line broadening of 5 Hz. The chemical shifts were calibrated to an external signal of deuterium oxide that was set to 0 ppm. Anisotropic 2H signals with small coupling values from the deuterium oxide were separated from the strong isotropic signal that masked them by using an in-phase double quantum filtered pulse sequence with refocusing.23 23Na spectra were acquired with 8k points, 1024 transients, and a repetition time of 30 ms and were processed using exponential apodization and line broadening of 5 Hz. The observed quadrupolar splitting (∆) for an anisotropic liquid crystalline phase is given24–27 by eq 1:

∆)

∑ |Pi · χi · Si|

(1)

where Pi is the fraction of nuclei at site i having the quadrupolar coupling constant χi (ca. 220 and 167 kHz for O–D and C–D couplings, respectively), and the order parameter Si where S ) ((3 cos2 θ – 1)/2) and θ is the angle between the external magnetic field and the director (axis of symmetry) of the corresponding phase. The “two-site” model is applied to the 2H of water in the lyotropic mesophases in which two types of water were considered: water bound to the aggregates and free bulk water. Therefore, eq 1 can be rewritten (eq 2).

∆ ) Pb · χb · Sb + Pf · χf · Sf

(2)

Here, b denotes the bound and f the free water molecules; Sb is the order parameter for water bound to the amphiphile headgroups and orientated toward their hydroxyl groups. Sf is the order parameter of the free water molecules; often, Sf approaches zero because the water molecules have no order and freely tumble without any restriction. In this case, the term including the order parameter of the free molecules vanishes. This assumption cannot be made if Pb is very small or if the second term cannot be neglected. pH Measurements. Apparent pH measurements were carried out using a Mettler-Toledo pH meter (model SevenEasy) at room temperature, using a silver-silver chloride electrode.

Results and Discussion Na-DFC can be solubilized in the GMO/ethanol/water system in the discontinuous cubic phase called QL (Figure 1) in quantities up to 12.5 wt %.10 We have shown that drug-loaded mesophases undergo significant curvature modulation with increasing solubilization loads, resulting in several consecutive phase transitions and ultimately phase separation at very high loads. The effect of the ionic drug Na-DFC on individual components of the QL structure was studied using 2H and 23Na NMR spectroscopy. The water, ethanol, and Na-DFC components of the mesophase itself were observed by directly measuring NMR spectra of the specific component. This was done using samples in which the water or ethanol was exchanged by deuterated components or by following the naturally abundant Na-DFC by 23Na-NMR. The structure and properties of the phases were essentially unchanged by substitution with deuterated components as seen by all other methods and by changing the relative concentrations of D2O/H2O. (23) Eliav, U.; Navon, G. J. Magn. Reson. 1999, 137, 295–310. (24) Zhang, K.; Khan, A. Macromolecules 1995, 28, 3807–3812. (25) Fairhurst, C. E.; Holmes, M. C.; Leaver, M. S. Langmuir 1997, 13, 4964– 4975. (26) Coppola, L.; Gianferri, R.; Nicotera, I.; Oliviero, C. Mol. Cryst. Liq. Cryst. 2003, 398, 157–167. (27) Fournial, A.-G.; Zhu, Y.; Molinier, V.; Vermeersch, G.; Aubry, J.-M.; Azaroual, N. Langmuir 2007, 23, 11443–11450.

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Figure 1. Phase diagram of the GMO/ethanol/water ternary system at 25 °C. The phase boundaries of the single-phase regions are indicated with solid lines. The phases are lamellar (LR), disordered lamellar (LR*), bicontinuous reverse cubic (Q), and three isotropic phases: micellar isotropic (L), sponge (L3), and the QL phase.

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263, 230, 191, and 167 Hz, respectively, characteristic of an anisotropic phase. The coupling values, measured between the coupled peaks, are relatively small for a lamellar phase as compared to 2154 Hz for a lamellar phase of the same system with different GMO/water ratios, such as the GMO-rich lamellar phase of GMO/ethanol/water/Na-DFC (69/10/20/1 wt %) (unpublished results) and lamellar phases of GMO–water.31 The lower values of QL loaded Na-DFC may be due to the low GMO/ water ratio of 0.9, while in the GMO-rich mixture it is 3.5. The results correlate the Scherrer parameters32 as calculated from SAXS data, which have also shown that the GMO-enriched empty phases have more lamellar order than do the Na-DFC-loaded QL (346 versus 697, respectively), which also correlates with the NMR results. We conclude that at these load levels QL has inverted into a slightly disordered lamellar structure. With increasing Na-DFC loads, the lamellar phase becomes increasingly disordered and the D2O coupling is reduced to below the line width of the broad central signal (Figure 2C and D). The line broadening of the D2O signal is due to a distribution of orientations in the system and may be due to enhanced undulations that occur in the disordered lamella layers of the lamellar mesophase, as previously shown.33–36 A plot of the observed quadrupolar splitting (∆) for an anisotropic liquid crystalline phase versus the molar ratio of amphiphile-to-water should give a straight line24,37–39 with a slope of the average number of water molecules bound per amphiphile molecule as shown in eq 3. Neglecting the free water gives the observed quadrupolar splitting as shown in eq 3:

∆ ) χ · S · nSW

Figure 2. 2H spectrum of isotropic and anisotropic phases containing deuterium oxide and Na-DFC at: (A) 0, (B) 1, (C) 3, and (D) 10 wt % at different magnifications.

Deuterated Water. We followed the orientation of D2O in the QL mesophases loaded with Na-DFC by 2H NMR. Changes in the anisotropic orientation of water molecules affect the molecular ordering tensor that can be detected by the degree of quadrupolar splitting.28,29 The D2O spectrum of the empty QL phase comprising GMO/ ethanol/water (42:11:47 wt %, respectively) gave an isotropic deuterium oxide signal, as expected, for discontinuous micelles organized in a cubic array.19,22 The time-averaged isotropic distribution of the phase and the random tumbling of the D2O molecules caused the quadrupolar coupling to average out (Figure 2A). At low solubilization loads of Na-DFC (0-1.2 wt %) in the QL phase, the same isotropic D2O signal was observed.28,30 At these loading concentrations, the drug content does not induce any change visible by these NMR methods (Figure 2B). At medium solubilization loads of Na-DFC (1.5, 2, 2.4, and 3 wt %), the D2O signal showed decreasing coupling values of (28) Lawson, K. D.; Flautt, T. J. J. Phys. Chem. 1968, 72, 2066–2074. (29) Hoff, B.; Strandberg, E.; Ulrich, A. S.; Tieleman, D. P.; Posten, C. Biophys. J. 2005, 88, 1818–1827. (30) Chiccoli, C.; Pasini, P.; Skacˇej, G.; Zannoni, C.; Zˇumer, S. Phys. ReV. E 1999, 60, 4219–4225.

XS XW

(3)

where nSW is the average number of hydration of each surfactant molecule, and XS and XW are molar fractions of the surfactant and the D2O in the lamellar mesophase, respectively. In our experiments, the ratio of GMO surfactant to water was kept constant (XGMO/Xwater ) 0.045) while increasing the content of solubilized Na-DFC. In this case, the ordered lamellar phase should give a plateau in quadrupolar coupling (∆) as a function of the Na-DFC concentration as seen in eq 3. The sharp decrease in splitting for Na-DFC loads of up to 4 wt % in an ordered lamellar domain is followed by a plateau (O in Figure 3). This indicates that Na-DFC loading levels directly affect the coupling values. The decrease in coupling values with increasing NaDFC load showed that the water-surfactant interface became less hydrated and/or less oriented. There may also be an increase in the undulation of the lamellar structure that is induced by the rigidity of the surfactant layer. Defects in the lamellae have been found to increase the curvature of the surfactant-water interface in a hexaethylene glycol n-hexadecyl ether/water system, decreasing the order parameter of bound water, and thus reducing coupling values.25 (31) Feiweier, T.; Geil, B.; Pospiech, E.-M.; Fujara, F.; Winter, R. Phys. ReV. E 2000, 62, 8182–8194. (32) Landh, T. J. Phys. Chem. 1994, 98, 8453–8467. (33) Stubenrauch, C.; Burauer, S.; Strey, R.; Schmidt, C. Liq. Cryst. 2004, 31, 39–53. (34) Wachowicz, M.; Jurga, S.; Vilfan, M. Phys. ReV. E 2004, 70, 0317011–031701-9. (35) Lutti, A.; Callaghan, P. T. Phys. ReV. E 2006, 73, 011710-1–011710-9. (36) Sparrman, T.; Westlund, P.-O. J. Phys. Chem. B 2001, 105, 12524– 12528. (37) Persson, N.-O.; Lindman, B. J. Phys. Chem. 1975, 79, 1410–1418. (38) Morley, W. G.; Tiddy, G. J. T. J. Chem. Soc., Faraday Trans. 1993, 89, 2823–2831. (39) Pacios, E.; Renamayor, C. S.; Horta, A.; Lindman, B.; Thuresson, K. J. Colloid Interface Sci. 2006, 299, 378–387.

Effect of Na-DFC Loads on Mesophase Components

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Figure 3. Observed quadrupolar coupling ((50 Hz) of D2O as a function of Na-DFC load in GMO/ethanol/water systems: (×) smaller and (O) larger quadrupolar coupling. 23Na NMR quadrupolar splitting (+) measured upon increasing Na-DFC concentration at a constant ratio of GMO/ethanol/water.

We found that at higher loads of Na-DFC (>4 wt %), the splitting reaches a plateau (103 ( 5 Hz), showing no further measurable effect on the hydration of water molecules and/or the internal order of the structure of water bound to the surfactant. These results show that at Na-DFC levels above 4 wt %, the water-surfactant–drug interactions are no longer involved in the competitive hydration process over the surfactant. 2H NMR measurements of D2O were not sufficiently sensitive to identify the change in the phase transition that occurred at higher loads of Na-DFC. The broad water signal masked anisotropic signals with small coupling values. We used an in-phase double quantum filtered pulse sequence, refocusing on the deuterium signal23 to filter out the isotropic deuterium signal from water with unrestricted motion, and revealed another population of anisotropic water with small coupling values. Systems loaded with less than 4 wt % Na-DFC showed two coupling values at 103 ( 5 Hz (× symbols in Figure 3) and the values seen by 1D 2H NMR at 100-260 Hz. The smaller coupling remained constant at all of the loading ranges of Na-DFC. The smaller couplings are probably due to fast exchange with ethanol and/or exchange of D2O itself between bound and unbound states. The larger coupling is probably due to slow exchange with the GMO headgroups, whose motion is restricted. The decrease in this coupling is caused by the NaDFC, which has a salting-out effect on the hydration water of the lipid headgroups (consistent with our previous SAXS results10). This behavior has been shown in lamellar samples composed of the ionic surfactant octanoic acid/D2O,37 and in systems composed of C6-18:0 monoglyceride/D2O.38 The effect of Na-DFC at higher loads was further determined by following sodium ion and deuterated ethanol components. Na-DFC. 23Na NMR was used to study the binding effect of Na-DFC over the entire range of concentrations. The isotropic singlet and splitting that occur in the lamellar phase due to quadrupolar interactions are shown in Figure 4.40–42 23Na coupling showed the four main regions with different couplings as a function of Na-DFC content (Figure 3). In the first region (up to 1.2 wt % Na-DFC), there was no splitting. In the second region (1.5–3.5 wt % Na-DFC), there was significant

Figure 4. 23Na NMR spectra of (A) isotropic phase (QL) and (B) anisotropic phase (lamellar).

splitting that decreased with concentration. In the third region (4-7.3 wt % Na-DFC), splitting increased with concentration, and the fourth region (8-12 wt % Na-DFC) showed a slighter increase of coupling values with increasing concentration. The pH values of the different phases differed according to the degree of dissociation of the acidic Na-DFC into its cation and weakly basic anion. The hydrophilicity of the environment of the drug, dependent on the degree of hydration and its position in the mesophase, determines its degree of dissociation. The above regions with different 23Na coupling values also correlated with the pH values of the phases, where the three anisotropic regions have pH values of ca. 6, 7, and 7.5 for regions II, III, and IV, respectively, and the isotropic phase is more acidic (pH 5-6). Na-DFC itself is an acidic salt (pKa 3.9) and more hydrophobic than the dissociated form, where the anion is a weak acid and raises the pH with its increasing degree of ionization. The Na-DFC partition coefficient has been shown to vary with pH, ion-pair formation, and hydrophobicity of its environment.43,44 The location of the drug changes with loading, causing a change in the mesophase, which is evident in the pH values. Changing the pH externally did not transform QL to the lamellar phase (results not shown). The singlet 23Na NMR peak in the first region indicated an isotropic liquid crystalline phase (Figure 4A). Here, the drug is at low concentrations and is dissolved in the hydrophobic tails (40) Persson, N.-O.; Lindblom, G.; Lindman, B.; Arvidson, G. Chem. Phys. Lipids 1974, 12, 261–270. (41) Kilpatrick, P. K.; Bogard, M. A. Langmuir 1988, 4, 790–796. (42) Mosca, M.; Murgia, S.; Ceglie, A.; Monduzzi, M.; Ambrosone, L. J. Phys. Chem. B 2006, 110, 25994–26000. (43) Fitzpatrick, D.; Corish, J. Int. J. Pharm. 2005, 301, 226–236. (44) Llinas, A.; Burley, J. C.; Box, K. J.; Glen, R. C.; Goodman, J. M. J. Med. Chem. 2007, 50, 979–983.

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of the GMO. The concentration is not sufficient to influence the curvature and hence the isotropic phase. The environment is not conducive to ion dissociation, giving low pH values and a singlet 23Na NMR peak. In the second region, at 1.5-3.5 wt % Na-DFC, the 23Na coupling values were 2500-5500 Hz, about 36 times larger than those of the deuterium oxide. The splitting decreases, in parallel with the decrease in deuterium splitting, and goes through a minimum. The pH values in region II increased with Na-DFC content: 5.8, 6.2, and 6.3 for 1.5, 2.0, and 3 wt % Na-DFC, respectively, indicating an increase in drug dissociation due to a more hydrophilic environment. Presumably this is because sodium ions extract water from the interfacial region, thereby increasing their isotropic motion and reducing their coupling. In region III, above 4 wt % Na-DFC, the 23Na splitting increased while the splitting values of deuterium remained constant (Figure 3). The change in the slope of the increase in coupling indicates a transition from LR to LR*. This is in good agreement with self-diffusion NMR results.10 At higher Na-DFC concentrations, in region IV, the pH values were higher as the ionic population increased, relative to the previous regions, and there was increased anisotropy in the 23Na signal. There seems to be an equilibrium with free Na+ and ions that are adsorbed on the surface of the headgroups. Apparently, the sodium binds the lipid headgroups, which are more ordered. In the transitions from region II to III and IV, the sodium ion properties change from kosmotropic to chaotropic due to its adsorption on the surfactant headgroups because of a change in the location of the Na-DFC. The Na+ coupling for ionic systems has been explained in terms of diffusion of ions and counterions to the surface across the Stern double layer.45,46 Ions in the diffusion layer are not associated with any specific surfactant group and would be expected to cause an electric field gradient. The diffuse layer is assumed to have a positive order parameter. Ions in the Stern layer may be located between the headgroups or adjacent to a particular charged anion consisting of specifically adsorbed counterions that have negative order parameters. The presence of both types of ions could give rise to the complex composition dependence of the splitting.45 Deuterated Ethanol (EtOH-d6). Ethanol is an essential interfacial component for forming the QL phase. The interfacial location of the ethanol varies because it can interact with both the hydrophilic lipid groups at the interface and the methylene groups of the hydrophobic surfactant tail. Its partitioning depends on the properties of the surfactant including hydrocarbon chain length, the nature of its headgroups, the orientation of the headgroup dipoles, and surfactant-water interactions.47–49 NaDFC is also an interfacial electrolyte and can affect the interactions between ethanol and GMO, and alter the location of the interface components. We followed deuterated ethanol by 2H NMR to investigate this phenomenon. The 2H spectrum of EtOH-d6 in an empty QL system showed three peaks with an integral ratio of 1.8:2.0:3.0, at -0.02, -1.30, and -3.77 ppm (relative to external D2O), which represent the hydroxyl, methylene, and methyl groups, respectively, based on ratio and relative chemical shifts (Figure 5A). The high integral value for the hydroxyl peak is probably due to augmentation by its exchange with water. (45) Lindblomn, G.; Lindman, B.; Tiddy, G. J. T. J. Am. Chem. Soc. 1978, 100, 2299–2303. (46) La Mesa, C.; Khan, A.; Fontell, K.; Lindman, B. J. Colloid Interface Sci. 1985, 103, 373–391. (47) Barry, J. A.; Gawrisch, K. Biochemistry 1994, 33, 8082–8088. (48) Holte, L. L.; Gawrisch, K. Biochemistry 1997, 36, 4669–4674.

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Figure 5. 2H NMR spectra of deuterated ethanol (CD3CD2OD) as a function of wt % of Na-DFC in GMO/ethanol/water systems: (A) 0, (B) 1.0, (C) 2.4, (D) 6.4, and (E) 12.5 wt % Na-DFC. Left panel is in scale, and the right panel shows magnifications. Table 1. Splitting Values ((10 Hz) of EtOH-d6 Na-DFC [wt %] 0 0.2 1 2.4 6.4 12.5

∆ν OD [Hz]

120 144 156

∆ν CD2 [Hz]

1500 1770 1910

∆ν CD3 [Hz]

structure

144 117 95

QL QL Q LR transition structure LR*

At loads of Na-DFC below 1.2 wt %, the ethanol within the QL mesophase shows three major peaks with no quadrupolar coupling, as in the empty system, indicating that the system is an isotropic phase, consistent with the cubic phase as shown (Figure 4C). Samples with more than 1.5 wt % Na-DFC were lamellar and exhibited quadrupolar splitting of all three ethanol peaks, indicating that ethanol binds the lipid molecules thereby restricting isotropic motion. There was no unbound (free) isotropic ethanol in the spectrum, indicating that all of the ethanol interacted with the interface where it adopted the same anisotropic motion as the lipid or that any residual isotropic signal was masked by the broad anisotropic peaks (Figure 5C–E). The CD2 coupling was the largest seen among the ethanol coupling values, ranging between 1500 and 1910 Hz, indicating a strong interaction with the GMO (Figure 5C–E and Table 1) presumably via hydrogen bonding between hydroxyls.48,50 This quadrupolar splitting is larger than the D2O splitting values because, in addition to the interaction with the GMO lipid, the CD2 is also tightly packed among the GMO molecules at the interface near the hydroxyl groups of the lipid. These results are in good agreement with findings that have shown that in dimyristyl phosphatidylcholine-based lamellar phases the majority of the ethanol molecules are located near the bilayer interface, in the (49) Chanda, J.; Bandyopadhyay, S. Chem. Phys. Lett. 2004, 392, 249–254. (50) Koenig, B. W.; Gawrisch, K. J. Phys. Chem. B 2005, 109, 7540–7547.

Effect of Na-DFC Loads on Mesophase Components

regions occupied by the carbonyl groups, glycerol backbone, and methylene groups of the lipid acyl chains.48 The CD3 groups of the ethanol showed quadrupolar splitting in the range of 95-144 Hz, which is more than an order of magnitude smaller than that of the methylene couplings. The CD3 groups are aligned with the GMO but are less proximate to the headgroups. They are in the vicinity of C1-C3 of the lipid and have more motional freedom, such that their splitting values are significantly smaller than those of the methylene groups.51 It has been shown that ethanol interacts with phospholipids in lipid bilayers via its hydroxyl groups,48,50 in which case the anisotropic splitting of OD of ethanol should be largest. The relatively low coupling in our case is probably due to the combined effect of fast exchange and slow motion, the OD being in fast exchange with the water and its motion being restricted by interaction with the lipid, as described also in phosphatidylcholine and phosphatidylethanolamine bilayers.50 This was evident also in the broad peaks of the split OD signals. The increase in the quadrupolar splitting of the OD may reflect increased binding to the GMO headgroups and increasingly restricted motion of this part of the ethanol molecule due to tighter packing and/or alignment of the lipid headgroups. The methylene in the ethanol still undergoes the same restriction of motion due to its proximity to the GMO headgroups. At the position of the methyl group from the headgroup, the lipid tails have increased conformational freedom with increasing loads of Na-DFC, seen in the decreased in splitting values. In the less ordered LR* phase, the CD3 splitting values were smaller than those of the LR phase, indicating a decrease in the order and alignment orientation of the methyl group as reflected in the increased isotropic motion of the CD3 group. The OD and CD2 splittings were larger in LR* than in LR, indicating a stronger interaction between the ethanol and GMO headgroups, possibly due to hydrogen bonding. The ethanol probably interacts via hydrogen bonds with the hydroxyl group of the hydrophilic GMO headgroup, allowing more freedom of the hydrophobic tails. The anionic DFC probably relocates to the region of the GMO interface and can form intermolecular electron donor-acceptor interactions, thereby competing with ethanol for the same site at the surfactant headgroup.

Conclusions NMR spectroscopy was used to investigate the influence of increasing concentrations of an ionic drug (Na-DFC) on mesophase structures and transitions. Increasing concentrations of solubilized Na-DFC caused structural transitions from the isotropic QL mesophase at low (51) Ward, J. I.; Friberg, S. E. Langmuir 1985, 1, 24–28.

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drug loads, through lamellar and disordered lamellar phases. The lamellar phase showed three regions that could only be differentiated using a combination of methods, which can follow the water and ethanol components of the phase, and the solubilized drug. The isotropic region, I, was clearly delineated by all components, while regions II, III, and IV gave anisotropic signals. Region II was distinguished from the other regions by its two water species and the decreasing coupling values in one of them. Regions III and IV were differentiated according to the different slopes of increasing 23Na-DFC couplings and the ethanol coupling values that together characterized the dissimilar regions of the phase. Upon increased drug load, Na-DFC began to interact with the GMO headgroups, effectively increasing their area and changing the curvature of the surfactant from cubic to lamellar, giving region II. In this region, the ionic drug resides in the more hydrophilic region of the GMO headgroups as also seen by the slight increase in pH. The sodium from the drug binds to the interfacial water, extracting it from the interface. The ethanol hydroxyl group binds the GMO headgroups, leaving its aliphatic region more mobile. At the transition from region II to region III, the sodium ion properties go from kosmotropic to chaotropic and the ions are absorbed onto the interface GMO headgroups more strongly. This increases the order among the headgroups while the tails undergo increased motion and undulations. At the transition from region III to region IV, the motion of the sodium ions is reduced, the ethanol more strongly interacts at its hydroxyl, and the aliphatic regions of the ethanol within the GMO tails are more mobile. The interaction of the drug and the carrier changed with increasing drug concentration. We characterized the nature of the drug–carrier interactions and their effect on the interactions among the components of the carrier. These methods show the importance of following a number of components of such systems to understand their complex behavior. Acknowledgment. Some of the results presented in this Article are included in the dissertation of R.E. for the Ph.D. degree in Applied Chemistry, The Hebrew University of Jerusalem, Israel. We thank Prof. Aharon Loewenstein, Dr. Uzi Eliav, and Dr. Daniel Waysbort for their valuable and constructive assistance. This research was supported in part by the United States-Israel Binational Science Foundation, No. 2003260, and the Israel Science Foundation, No. 9059/03. LA800603F