Characterization of the Nonionic Microemulsions by EPR. I. Effect of

Dec 30, 2008 - Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, NSF IUC...
2 downloads 14 Views 1MB Size
Download PDF
J. Phys. Chem. B 2009, 113, 691–699

691

Characterization of the Nonionic Microemulsions by EPR. I. Effect of Solubilized Drug on Nanostructure Anna Kogan,†,| Shoshana Rozner,† Somil Mehta,‡ Ponisseril Somasundaran,‡ Abraham Aserin,† Nissim Garti,*,† and Maria Francesca Ottaviani*,§ Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel, NSF IUCR Center for AdVanced Studies in NoVel Surfactants, Columbia UniVersity, New York, New York 10027, and Institute of Chemical Sciences, UniVersity of Urbino, Piazza Rinascimento 6, 61029 Urbino, Italy ReceiVed: August 11, 2008; ReVised Manuscript ReceiVed: October 26, 2008

The effect of the solubilized model drug, carbamazepine, on the internal structure of fully dilutable nonionic microemulsions was examined for the first time using electron paramagnetic resonance (EPR). Systems containing different surfactant to oil ratios, at two different pH values (4.6 and 8.5), with continuous dilution implementing structural transformations (micellar solution-W/O-bicontinuous-O/W) were investigated. The internal order, micropolarity, and microviscosity were scrutinized utilizing pH-dependent amphiphilic probe 5-doxylstearic acid (5-DSA). In the basic environment, the probe explored the vicinity of the surfactant head region; the deeper hydrophobic region of the surfactant tails was investigated in the acidic milieu. The study demonstrated that the EPR technique enables efficient monitoring of structural changes and examination of drug influence on structure in surfactant-poor systems. Lower order and microviscosity values were obtained in surfactant-poor systems in comparison to surfactant-rich systems. The drug functioned as a spacer of the surfactant molecules or as a cosurfactant depending on the formed microemulsion structure and the surfactant to oil ratio. The structural changes, pH variation, and presence of the drug did not alter the polarity parameter, indicating that the probe most likely does not sense a water environment in any of the examined systems. Under the basic conditions, higher microviscosity and order values were obtained in comparison to those at low pH, suggesting a higher order packing of the surfactant chains near the surfactant heads. The structural changes initiated in the vicinity of the surfactant heads, therefore, are more apparent in the basic environment. The ability to control and monitor the intramicellar interactions within drug carrier systems may be of significant interest for understanding the kinetics of drug release. Introduction Many drugs are traditionally given via oral administration in their solid form, but due to their poor water solubility, they are characterized by slow and irregular gastrointestinal absorption. One such example is carbamazepine (CBZ)1 (Chart 1A), which is widely used as an antiepileptic agent2 in the therapy of psychomotor seizures and trigeminal neuralgia,3 with a solubility of only about 170 mg/L at 24 °C.4 There is also considerable variability in CBZ plasma concentration.1,3 Given the clinical importance of CBZ, there is heightened interest in improving its dissolution and bioavailability. This will reduce the necessary dosage and frequency of dosing, which will probably reduce its side effects. Previous attempts to increase dissolution and bioavailability have focused on solid dispersions,5-7 physical mixtures with surfactants,4,6 trapping the drug in an amorphous state,8,9 and conversion to less stable crystal forms.10 * To whom correspondence should be addressed. Tel: +972 2 6586574/ 5; Fax: +972 2 6520262; E-mail: [email protected] (N.G.). Tel: +39 0722 303319; Fax: +39 0722 303311; E-mail: [email protected] (M.F.O.). † The Hebrew University of Jerusalem. ‡ Columbia University. § University of Urbino. | The results presented in this paper are part of Anna Kogan’s Ph.D. dissertation in Applied Chemistry, The Hebrew University of Jerusalem, Israel.

CHART 1: Chemical Structures of (A) Carbamazepine (CBZ) and (B) 5-Doxylstearic Acid (5-DSA)

For several years, microemulsions have attracted much interest in terms of their drug delivery potential. Microemulsions exhibit some superior physical characteristics over other colloidal systems, such as emulsions. The physicochemical properties include nanometric size, transparency, low viscosity, thermal and thermodynamic stability over a wide range of pHs and ionic environments, ease of preparation, and high molecular solubilization capacity.11-15 Microemulsions were also explored as systems mimicking bile salt micelles that play an important role in absorbing poorly soluble drugs.16 In this study, we used a complex mixture of pharmaceutically permitted components: Tween 60 as a surfactant, R-(+)-

10.1021/jp807161g CCC: $40.75  2009 American Chemical Society Published on Web 12/30/2008

692 J. Phys. Chem. B, Vol. 113, No. 3, 2009 limonene as an oil, propylene glycol as a cosolvent, and ethanol as a cosurfactant. These ingredients facilitate formation of fully dilutable nonionic microemulsions, which undergo continuous structural inversion without phase separation all the way from a water-in-oil (W/O) microemulsion via a bicontinuous phase to an oil-in-water (O/W) microemulsion upon dilution with an aqueous phase.17,18 These structured liquids exhibit high surface-to-volume ratios allowing high solubilization capacities for CBZ. Most studies on microemulsions as drug delivery vehicles neglected the effects of dilution on the stability and structure of the microemulsions.16,19 However, it is important to consider the effect of dilution since the vehicle is diluted during consumption and during transport in the digestive tract. The entrapped drug may itself affect the physicochemical properties of the vehicle, consequently influencing its release.13,20 In our previous studies, we investigated the behavior of the dilutable microemulsions in terms of intermicellar interactions of the structures and explored the influence of the solubilized active molecule on the “macro” properties of the system using various techniques (dynamic light scattering, viscosity, conductivity, self-diffusion-NMR, and small-angle X-ray scattering).18,21,22 In this study, we explored the effects of solubilized CBZ molecules in terms of intramicellar interactions using electron paramagnetic resonance (EPR) spectroscopy. EPR is a powerful tool in pharmaceutical analysis since it is nondestructive and sensitive and requires only a small sample size, which can be applied to a wide range of sample forms (solids, liquids, suspensions, solutions, whole tablets, etc.23). Furthermore, the technique is noninvasive, enabling obtaining significant information concerning the accessibility of the drug molecules in the pharmaceutical formulation.24 The EPR technique has been applied by several authors to investigate the microstructure of adsorbed layers of surfactants and polymers at the liquid/liquid and solid/liquid interfaces, in lipid bilayers 25-28 and microemulsion systems.29-31 Most published data on characterization of microemulsions using the EPR technique relate to ionic surfactants, such as AOT (sodium di-2(ethylhexylsulfosuccinate)), forming W/O microemulsions,29-31 and only limited information exists on nonionic microemulsion structures.32,33 Moreover, most investigations on AOT W/O microemulsions have been performed mainly as a function of temperature in a restricted range of composition of the L2 region. These studies did not allow obtaining a complete view of the microstructural evolutions occurring within the whole range of the dilution line.29-31 In this study, we investigated, for the first time, nonionic fully dilutable microemulsions and compared unloaded and CBZloaded formulations utilizing the EPR technique. The spectroscopic behavior of the paramagnetic species used as probes is highly dependent on the environment around them. Hence, they were exploited to characterize the environment in which they reside. Here we used an amphiphilic probe, 5-doxylstearic acid (5-DSA) (Chart 1B), which was embedded into the surfactant layer.34-36 The microenvironment of nonionic systems was monitored as a function of pH,29 probing the surfactant shell closer to the headgroups at high pH (8.4) and closer to the surfactant tails at low pH (4.6). These two pH values also mimic biological conditions along the intestinal tract and can enable a better understanding of the effect of pH on drug behavior.37 Additionally, it provided significant information on packing, local mobility, and environmental micropolarity in different domains of the surfactant layer. It revealed the surfactant shell

Kogan et al. structure under the influence of a solubilized drug and upon dilution. The dependence of microemulsion microstructure on different surfactant to oil ratios was also investigated in order to develop a complete understanding of the system. Materials and Methods Materials. Commercial pharma-grade carbamazepine, 5Hdibenz(b,f)-azepine-5-carboxamide (Chart 1A), was obtained from Teva Pharmaceutical Industries Ltd. (Kfar Saba, Israel). Tween 60 [polyoxyethylene-(20)-sorbitan monostearate], commercial grade, R-(+)-limonene (98%), and spin probe 5-doxylstearic acid [2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3oxazolidinyloxy] (Chart 1B), free radical, were purchased from Sigma-Aldrich (St. Louis, MO). The spin probe was used as received. Ethanol (EtOH) (99.8%) and hydrochloric acid (32%) were obtained from Frutarom (Haifa, Israel). Propylene glycol (PG) (99.5%) was purchased from Merck KGaA, (Darmstadt, Germany). Disodium borate tetradecahydrate and acetic acid were purchased from Merck-BDH, (Lutterworth, UK). Sodium acetate trihydrate was purchased from J. T. Baker (Deventer, The Netherlands). Water was tridistilled. All components were used without further purification. Buffer solutions. For basic buffer (pH ) 8.5), 50 mL of 0.025 M disodium borate tetradecahydrate was added to 15.2 mL of 0.1 M hydrochloric acid. This solution was diluted to 100 mL with TDW and for acidic buffer (pH ) 4.6), 49 mL of 0.2 M sodium acetate trihydrate was added to 51 mL of 0.2 M acetic acid. CBZ Solubilization in the Microemulsion System. CBZ was solubilized in the five-component system based on the pseudoternary phase diagram that was previously constructed in our laboratory.17,18 Transparent structured mixtures of Tween 60/[R-(+)-limonene/EtOH (1:1)] in ratios of 6:4, 7:3, 8:2, and 9:1 were prepared. The compositions were termed 64ME, 73ME, 82ME, and 91ME, respectively. The microemulsion capacity for CBZ solubilization is dependent on surfactant to oil ratio (S/O).18 Thus, in order to study the effect of the formulation on the drug, all the systems were loaded with 3 wt % CBZ that allows complete solubilization and progressive dilution with no drug precipitation. Appropriate quantities (5, 10, 20, 40, and 70 wt %) of aqueous phase ((acidic or basic buffer)/PG (1:1) wt %) were added to form various microstructures along the dilution lines.18,21 Insertion of the Probe. The probe was first dissolved in chloroform at a concentration of 2.5 × 10-3 M; the solvent was then evaporated before adding the prepared microemulsion. The inserted microemulsions were then incubated for 12 h, at 37 °C, 45 rpm to permit insertion of the probe into the surfactant assemblies. A probe concentration of 4 × 10-5 M in the tested microemulsion samples was used for all EPR studies. Such a low concentration is not expected to have any effect on the microemulsion droplet structure.25 EPR Instrumentation and Method. EPR spectra were recorded at room temperature using a Bruker EMX spectrometer operating at X band (9.5 GHz) using Pyrex capillary tubes (∼1 mm inner diameter) as sample containers. The spectra were recorded 24 h after sample preparation. The EPR experimental setup is as follows: modulation amplitude 0.1 mT; microwave frequency 9.87 GHz; center magnetic field 352 mT; sweep width 10 mT; resolution 1024; time constant 40.96 ms; and conversion time 81.92 ms. Computation of the EPR Spectra. Analysis of the EPR spectra was carried out using the simulation program by Schneider and Freed38 and Budil et al.,39 which takes into

Characterization of the Nonionic Microemulsions

J. Phys. Chem. B, Vol. 113, No. 3, 2009 693

Figure 1. Schematic illustration of 5-DSA probe and the CBZ molecule’s location within the microemulsion system at different pH. (A) pH 8.5 where the probe is ionized and the drug is un-ionized, (B) pH 4.6 where the probe is un-ionized and the drug is ionized.

account the relaxation process and therefore enables correctly computing the EPR line shape: the main parameters extracted from the spectral analysis are as follows: (a) The coupling tensor between the electron spin and the nuclear spin, A, mainly the Azz component, whose increase is related to an increase in environmental polarity of the radicals. (b) The correlation time for the rotational motion of the probe, τ: the Brownian diffusion (Di ) 1/(6τi)) or the jump diffusion (Di ) 1/τi) models are tried in the computation. In both cases the main component of the correlation time for motion is the perpendicular one, τperp. (c) The order parameter, D20, or S, which measures the wobbling mobility of the surfactants in an ordered structure. In some cases, the spectra were very noisy, due to probe degradation or self-aggregation, and the computation became less accurate; in these and other cases, different parameter sets might reproduce the experimental line shape. We selected, among several attempts of computations, the ones which well reproduced the experimental spectra in a series of EPR measurements on similar systems. Mainly, we selected for the graphs of τ and S as a function of the water content, the values obtained by assuming a variation of one (τ or S) parameter, taking the other as constant: if we analyze the variation of τ, the S parameter was assumed constant (equal to the one calculated at the lowest τ value); conversely, if we analyze the variation of S, the τ value was assumed constant (equal to the one calculated at the lowest S value). This is of course an approximation, which allows us to get more accurate information on the microviscosity and order variations. The accuracy of the simulations (5%) was determined by computation: a variation of a parameter greater than 5% led to a perceptible variation of the computed line shape and, consequently, to worse fitting between the computed and the experimental spectra. Results and Discussion Microemulsion structure depends on the physicochemical factors, including interfacial molecular conformation, surfactant structure, surfactant and cosurfactant concentrations, oil and water concentrations, and temperature.15 The temperature remained constant during the study; the nonionic microemulsions investigated in this study were not influenced by the pH, but rather by the surfactant to oil ratio used for their formation,

dilution with aqueous phase and the presence of the drug,18 the structure of which depends on pH.21,40,41 It is important to investigate the effect of pH on the structure of drug carrier, since selective permeation through the intestinal mucosa and absorption is favored in the un-ionized form, which can be manipulated by a change in pH.37 Microemulsion systems used in this study were investigated using a 5-DSA probe (Chart 1B), EPR spectral parameters of which are also found to be pHdependent.29 In the basic environment, the probe is ionized (carboxylate anion),25 and consequently it is pulled toward the water phase due to ion-dipole interactions between the water and the probe,42 increasing the hydration shell of the latter (Figure 1A). This polar structure causes the doxyl group to sense the environment just below the surfactant heads, which are located closer to the aqueous phase (Figure 1A). However, at an acidic pH of 4.6, the probe exists in the un-ionized form and interactions between the carboxylic acid and water molecules are relatively weak (dipole-dipole interactions);42 therefore, the hydration shell is smaller (Figure 1B). Accordingly, the structure is less polar and the doxyl group is located deeper within the structure, closer to the fluid oily phase (pKa (stearic acid) ) 7.45) (Figure 1B).25,31,42 The EPR spectra for 5-DSA were recorded for CBZ-loaded and -unloaded systems in different microstructures along several dilution lines under acidic and basic conditions. The effect of the CBZ-loaded and -unloaded microemulsions in different pH environments on hyperfine coupling constant, correlation time, and the order parameters along dilution line 64 are shown in Figure 2. The spectra in the bicontinuous region (Figure 3) reveal the resolution of the parallel and perpendicular components of the hyperfine A tensor for the coupling between the electron and the nuclear spin (indicated in the figure as Apar and Aperp). This kind of resolution is characteristic of a partially ordered system and determines the introduction of an order parameter (S) in the spectral calculation. The visual evidence of the increase in Apar in Figure 3 nicely reflects the increase in the S parameter. As noted in Azz measurements (results not shown), the probe shows a very slight increase in environment polarity upon dilution with aqueous phase (Azz ) 3.13mT at W/O to Azz ) 3.20 mT at O/W structures) along different dilution lines, in

694 J. Phys. Chem. B, Vol. 113, No. 3, 2009

Kogan et al.

Figure 2. EPR spectra (black lines) and their simulations in unloaded (red lines) and CBZ-loaded microemulsions (blue lines) in (A) basic environment and (B) acidic environment along 64 dilution line at 25 °C.

Figure 3. EPR spectra of unloaded (dash dot) and CBZ-loaded microemulsion (solid line) at basic conditions: unloaded (dot) and CBZ loaded microemulsion (dash) at acidic conditions. The microemulsion is based on a 64ME system containing 40 wt % aqueous phase.

the presence of CBZ or in different pH environments. These values are very similar to these obtained for the 5-DSA probe in the oil phase (R-(+)-limonene/EtOH (1:1)) of 3.16 mT. It can be concluded that in all the examined systems the environment polarity of the doxyl group increases only slightly at high aqueous phase content upon formation of direct micelles. It depicts that the probe most likely does not sense the water environment in any of the examined systems. It may be concluded that neither the drug introduction nor surfactant to oil ratio variations or continuous dilution perturbs the structure sufficiently to modify the polarity of the environment near the probe. Yaghmur et al.43 have also indicated that surfactant/ alcohol/PG can strongly bind water in the inner phase (shown as the hydration shell in Figure 1). Changes of Correlation Time and Order Parameters. The correlation time (τ) parameter gives information on the microviscosity among the surfactant molecules within the structure.35,44 S parameter characterizes the degree of space ordering of the surfactant molecules.35,25

I. Basic Environment. It is known that internal organization of microemulsion constituents is altered upon dilution due to the structural changes occurring in the system,11,12 thus varying the microviscosity of the environment around the probe. As mentioned above, in the basic environment the probe is ionized and the doxyl group is located within the ordered surfactant chain aggregates, just below the polar head groups (Figure 1A).25,31 The variation of τ and S as a function of the surfactant to oil ratio and dilution with the aqueous phase is shown in Figure 4. W/O Region (5-20 wt %). At low aqueous phase concentrations ( 64ME, and this is due to the surfactant quantities within the structures as previously discussed. Comparison between the CBZ-loaded and -unloaded systems at 40 wt % aqueous phase shows that insertion of the drug did not affect the microviscosity in surfactant-rich systems, but decreased it by 34% and 10% in the 73ME and 64ME, respectively. Therefore, it may be assumed that the drug perturbs the structural modification of 73ME and 64ME, causing the surfactant aggregates to become more spaced. Yet, the impact of the CBZ on microviscosity is less pronounced under acidic conditions compared to the basic environment since, in the latter case, the probe was located in the vicinity of the drug, which was accommodated near the surfactant headgroups (Figures 4B, C, 6B, C). Examination of the order demonstrates that surfactant-poor systems in the presence of CBZ achieve a maximum order in the W/O region that remains constant upon dilution (Figure 6D). In surfactant-rich systems the order increases upon formation of bicontinuous structures. This is most probably due to greater quantities of aqueous phase needed to hydrate more surfactant heads in the surfactant-rich systems in order to invert it to bicontinuous structures. Similarly to unloaded systems, the order increases in the 20-40 wt % region (7%, 4%, 23%, 26% at 64ME, 73ME, 82ME, and 91ME, respectively, while in unloaded systems the system exhibited an increase of 30%, 50%, 22%, and 23%, respectively). Once more the surfactant-poor system shows considerably more change. Furthermore, the order values are still S/O-dependent. In the 73ME CBZ-loaded system

698 J. Phys. Chem. B, Vol. 113, No. 3, 2009 the order is greater than in 64ME (by 35%), and in 91ME is higher than 82ME (by 10%) (Figure 6D). Additionally, the microviscosity (Figure 6C) and order values in 73ME reach values similar to those in the 82ME system (Figure 6D). One can note that, even though the 73ME CBZloaded system exhibits an increase of order upon dilution, the spacing effect of the drug is still considerably significant (i.e., at 40 wt % aqueous phase the unloaded system exhibits an order that is 39% greater than in a CBZ-loaded one). O/W Region (40-70 wt %). Upon further dilution, the structures invert to O/W droplets where the tails strongly interact.18,21 The unloaded surfactant-rich systems do not exhibit a microviscosity change, while surfactant-poor systems show a decrease in microviscosity due to the lower packing of the structure (Figure 6A). The order decreases upon inversion to direct micelles in all the unloaded systems since the tails are less ordered in the micellar core (Figure 6B). The CBZ-loaded systems reach maximum microviscosity and order in the bicontinuous region and are not affected by the transition to direct droplets except for the 64ME (Figure 6C). In 64ME the order decreases in the presence of the drug as in the unloaded system, meaning that CBZ cosurfactant activity is not sufficient to compensate for the disorder caused by the structural inversion. Comparing the order values at 70 wt % aqueous phase, one can note that CBZ significantly increases the order of the surfactant-rich systems (by 24% in 91ME and 57% in 82ME (Figure 6D)) relative to the unloaded system. This is assumed to be due to the CBZ that spaces the dense packed surfactant tails allowing the probe to rotate. In general, comparison between the acidic and basic environments within unloaded and loaded systems shows that under basic conditions the values of order and microviscosity are greater, since the doxyl group is located in the well-packed surfactant chains region below the polar heads, while in acidic region the doxyl group is located deeper within the surfactant tails where the tail packing is low (Figures 1, 4, and 6). Consequently, the structural changes in the basic environment are more evident. The trends observed in both environments are similar. The influence of the CBZ is more significant in the surfactantpoor systems (64ME and 73ME). Conclusions The EPR technique gives an insight into the influence of aqueous dilution on the nonionic microemulsion structures in unloaded and CBZ-loaded microemulsions in various structural regions (O/W, bicontinuous, and W/O) by changing the pH environment. The effects of pH-dependent CBZ structures and different surfactant/oil ratios on the microemulsion structures formed upon dilution with aqueous phase can be explored using the EPR technique. The amphiphilic probe (5-DSA) that was used in this study enabled monitoring different regions of the surfactant aggregates. Due to probe’s deprotonated form, in the basic environment structural changes occurring below the surfactants heads were examined, while in the acidic environment the probe was protonated; therefore, the deeper hydrophobic region near the surfactant tails was studied. The order parameters were in good agreement with microviscosity results. In the basic environment, where the probe was located closer to the ordered vicinities of surfactant heads, the values of the order and microviscosity are greater than in the acidic environment. Consequently, the structure transformations

Kogan et al. in the basic environment were detected at lower aqueous phase content than in the acidic environment. Intramicellar structural changes occurring upon addition of aqueous phase were revealed by the EPR analysis. No significant changes in polarity values were observed; i.e., the probe was not exposed to the water molecules. In the W/O droplets the microviscosity and the order were low, but upon adding aqueous phase they increased due to the swelling effect followed by formation of a more ordered bicontinuous structure. Another decrease in the order values could be registered as a result of inversion into an O/W structure formation with further dilution. Good correlation between the surfactant/oil ratio comprising the vehicle and its internal structure was found. Formulations with large surfactant/oil ratios exhibited high values of microviscosity and order parameters even in very diluted systems. It is important to note that structural changes were better detected in surfactant-poor systems (64ME and 73ME) than in surfactant-rich systems (82ME and 91ME). This is due to greater surfactant packing in the later systems, which restricts the probe’s mobility. Within the bicontinuous structures (40 wt %), at basic conditions the internal order and microviscosity of the 64ME microemulsion was as high as in the one consisting of greater quantities of surfactant (73ME). It may be speculated that the system containing smaller quantities of surfactant will show a drug release profile in transdermal applications similar to that of the surfactant-rich system. The presence of the CBZ perturbs the surfactant molecules’ organization in the W/O region. Therefore, greater aqueous phase contents are required for structural transformations to occur in surfactant-poor systems. The impact of the CBZ solubilization within the surfactant-rich systems is less pronounced due to the packed organization of the surfactant molecules. In the direct micelles, the CBZ serves as a cosurfactant and causes the surfactant-poor system (73ME) to exhibit behavior similar to that of the surfactant-rich systems (82ME and 91ME). This means that incorporation of the drug has an effect on the structure similar to that of an increase in surfactant to oil ratio. The decrease in pH influences the ionization state of the CBZ resulting in an increase of its effective area. As a result the CBZ is accommodated closer to the aqueous phase and the surfactant heads are more distanced, resulting in lower microviscosity sensed by the probe. We can conclude from these results that the CBZ is solubilized in the microemulsion closer to the aqueous phase and, therefore, its release from direct droplets can be controlled by the appropriate choice of surfactant to oil ratio within the delivery vehicle.52 Additionally, the EPR technique is more sensitive and provides more detailed information on systems containing low surfactant to oil ratios. Revealing the difference or similarity of internal structure within the formulation may lead to understanding and controlling the intermicellar or the intramicellar interactions within the vehicle that influence the drug release. Acknowledgment. We thank Mrs. Zehava Cohen for graphical support and the NSF I/UCRC for the facilities. References and Notes (1) El-Zein, H.; Riad, L.; Abd El-Bary, A. Int. J. Pharm. 1998, 168, 209–220. (2) Voudris, K. A.; Attilakos, A.; Katsarou, E.; Drakatos, A.; Dimou, S.; Mastroyianni, S.; Skardoutsou, A.; Prassouli, A.; Garoufi, A. Epilepsy Res. 2006, 70, 211–217.

Characterization of the Nonionic Microemulsions (3) Stevens, R. E.; Limsakun, T.; Evans, G.; Mason, D. H. J. Pharm. Sci. 1998, 87, 1531–1534. (4) Gavini, E.; Hegge, A. B.; Rassu, G.; Sanna, V.; Testa, C.; Pirisino, G.; Karlsen, J.; Giunchedi, P. Int. J. Pharm. 2006, 307, 9–15. (5) Londhe, V.; Nagarsenker, M. Indian J. Pharm. Sci 1999, 61, 237– 240. (6) Naima, Z.; Siro, T.; Juan-Manuel, G. D.; Chantal, C.; Rene, C.; Jerome, D. Eur. J. Pharm. Sci 2001, 12, 395–404. (7) Friedrich, H.; Fussnegger, B.; Kolter, K.; Bodmeier, R. Eur. J. Pharm. Biopharm 2006, 62, 171. (8) Li, Y.; Han, J.; Zhang, G.; Grant, D.; Surayanarayanan, R. Pharm. DeV. Technol. 2000, 5, 257–266. (9) Han, J.; Surayanarayanan, R. Pharm. DeV. Technol. 1998, 3, 587– 596. (10) Kobayashi, Y.; Ito, S.; Itai, S.; Yamamaoto, K. Int. J. Pharm. 2000, 193, 137–146. (11) Teo, B. M.; Ashokkumar, M.; Grieser, F. J. Phys. Chem. B. 2008, 112, 5265–5267. (12) Mcllwaine, R. E.; Fenton, H.; Scott, S. K.; Taylor, A. F. J. Phys. Chem. C 2008, 112, 2499–2505. (13) Kogan, A.; Garti, N. AdV. Colloid Interface Sci. 2006, 123-126, 369–385. (14) Walderhaug, H. J. Phys. Chem. B 2007, 111, 9821–9827. (15) Spernath, A.; Aserin, A. AdV. Colloid Interface Sci. 2006, 128130, 47–64. (16) Kawakami, K.; Yoshikawa, T.; Moroto, Y.; Kanaoka, E.; Takahashi, K.; Nishihara, Y.; Masuda, K. J. Controlled Release 2002, 81, 65–74. (17) Garti, N.; Yaghmur, A.; Leser, M. E.; Clement, V.; Watzke, H. J. J. Agric. Food Chem. 2001, 49, 2552–2562. (18) Kogan, A.; Aserin, A.; Garti, N. J. Colloid Interface Sci. 2007, 315, 637–647. (19) Kim, S. K.; Lee, E. H.; Vaishali, B.; Lee, S.; Lee, Y. K.; Kim, C. Y.; Moon, H. T.; Byun, Y. J. Controlled Release 2005, 105, 32–42. (20) Kriwet, K.; Mu¨ller-Goymann, C. C. Int. J. Pharm. 1995, 125, 231– 242. (21) Garti, N.; Avrahami, M.; Aserin, A. J. Colloid Interface Sci. 2006, 299, 352–365. (22) Garti, N.; Spernath, A.; Aserin, A.; Lutz, R. Soft Matter 2005, 1, 206–218. (23) Williams, H.; Claybourn, M. Spec. Eur. 2006, 18, 12–14. (24) Jores, K.; Mehnert, W.; Ma¨der, K. Pharm. Res. 2003, 20, 1274– 1283. (25) Wines, T. H.; Somasundaran, P.; Turro, N. J.; Jockusch, S.; Ottaviani, M. F. J. Colloid Interface Sci. 2005, 285, 318–325. (26) Ottaviani, M. F.; Andechaga, P.; Turro, N. J.; Tomalia, D. A. J. Phys. Chem. 1997, 101, 6057–6065. (27) Deo, P.; Deo, N.; Somasundaran, P.; Moscatelli, A.; Jockusch, S.; Turro, N. J.; Ananthapadmanabhan, K. P.; Ottaviani, M. F. Langmuir 2007, 23, 5906–5913. (28) Deo, P.; Jockusch, S.; Ottaviani, M. F.; Moscatelli, A.; Turro, N. J.; Somasundaran, P. Langmuir 2003, 19, 10747–10752. (29) Haering, G.; Luisi, P. L.; Hauser, H. J. Phys. Chem. 1988, 92, 3574– 3581.

J. Phys. Chem. B, Vol. 113, No. 3, 2009 699 (30) Barelli, A.; Eicke, H. F. Langmuir 1986, 2, 780–786. (31) Kommareddi, N. S.; John, V. T.; Waguespack, Y. Y.; McPherson, G. L. J. Phys. Chem. 1993, 97, 5752–5761. (32) Xenakis, A.; Cazianis, C. T.; Malliaris, A. Colloids Surf. 1992, 62, 315–319. (33) Baglioni, P.; Bongiovanni, R.; Rivara-Minten, E.; Kevan, L. J. Phys. Chem. 1989, 93, 5574–5578. (34) Baglioni, P.; Rivara-Minten, E.; Dei, L.; Ferronis, E. J. Phys. Chem. 1990, 94, 8218–8222. (35) Zuev, Y. F.; Vylegzhanina, N. N.; Zakhartchenko, N. L. Appl. Magn. Reson. 2003, 25, 29–42. (36) Ottaviani, M. F.; Daddi, R.; Brustolon, M.; Turro, N. J.; Tomalia, D. A. Appl. Magn. Reson. 1997, 13, 347–363. (37) Pang, K. S. Drug Metab. Dispos. 2003, 31, 1507–1519. (38) Schneider, D. J.; Freed, J. H. Slow motional magnetic resonance spectra: a user’s guide. In Biological Magnetic Resonance; Berliner, L. J., Reuben J., Eds.; Plenum Press: New York, 1989; Vol. 8, pp 1-76. (39) Budil, D. E.; Lee, S.; Saxena, S.; Freed, J. H. J. Magn. Reson. A 1996, 120, 155–189. (40) Andrade, S. M.; Costa, S. M. B.; Pansu, R. J. Colloid Interface Sci. 2000, 226, 260–268. (41) Kim, Y.; Choi, K.; Jung, J.; Park, S.; Kim, P. G.; Jeongim, P. EnViron. Int. 2007, 33, 370–375. (42) Solomons, T. W. G. In Organic Chemistry, 6th ed.; Rose, N., Magida, S., Malinowski, S., Eds.; John Wiley & Sons, Inc. Press: New York, 1996; pp 77, 81, 111. (43) Yaghmur, A.; Aserin, A.; Tiunova, I.; Garti, N. J. Thermal Anal. Cal. 2002, 69, 163–177. (44) Somasundaran, P.; Huang, L.; Fan, A. In Fluorescence and ESR spectroscopy. Surfactant Science Series; Binks, B. T., Ed.; Marcel Dekker Inc. Press: New York, 1999; Vol. 83, pp 229-254. (45) Bidyut, K. P.; Rajib, K. M. Colloids Surf. A. 2006, 273, 129–140. (46) Caboi, F.; Capuzzi, G.; Baglioni, P.; Monduzzi, M. J. Phys. Chem. B 1997, 101, 10205–10212. (47) Mantegazza, F.; Degiorgio, V.; Giardini, M. E.; Price, A. L.; Steytler, D. C.; Robinson, B. H. Langmuir 1998, 14, 1–7. (48) Avramiotis, S.; Cazianis, C. T.; Xenakis, A. Langmuir 1999, 15, 2375–2379. (49) Podlogar, F.; Besˇter Roga, M.; Gasˇperlin, M. Int. J. Pharm. 2005, 302, 68–77. (50) Rege, B. D.; Kao, J. P. Y.; Pollia, J. E. Eur. J. Pharm. Sci. 2002, 16, 237–246. (51) Anwar, K.; Kayden, H. J.; Hussain, M. M. J. Lipid Res. 2006, 47, 1261–1273. (52) Kogan, A.; Kesselman, E.; Danino, D.; Aserin, A.; Garti, N. J. Colloids Surf. B 2008, 66, 1–12. (53) Gershanik, T.; Haltner, E.; Lehr, C. M.; Benita, S. Int. J. Pharm. 2000, 211, 29–36. (54) Wang, W.; Lu, W.; Jiang, L. J. Phys. Chem. B 2008, 112, 1409–1413.

JP807161G