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Langmuir 1999, 15, 3710-3717
Articles Solubilization of Sparingly Soluble Active Compounds in Lecithin-Based Microemulsions: Influence on Phase Behavior and Microstructure Christian von Corswant* and Per E. G. Thore´n† Astra Ha¨ ssle AB, S-431 83 Mo¨ lndal, Sweden Received June 16, 1998. In Final Form: March 8, 1999 Starting from the pharmaceutically interesting Winsor III system of water, 1-propanol, soybean phosphatidylcholine, and medium-chain triglycerides (MCT), the influence of two active drug compounds, felodipine and (R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]argininamide (BIBP3226), on the phase behavior and microstructure was studied by means of phase studies, NMR self-diffusion measurements, and measurements of drug solubility in the aqueous phase and the oil phase. Felodipine, being practically insoluble in water and slightly soluble in MCT, was found to act as a nonpenetrating oil. With increasing concentration of felodipine in the oil phase, the polarity of the oil phase increases, which in turn curves the surfactant film toward water. Thus, water is expelled from the microemulsion phase, and oil is incorporated as felodipine is added. With the composition used here, the microstructure remains bicontinuous, however, even at high felodipine concentrations. The increase in the polarity of the oil phase also has the effect of increasing the partitioning of 1-propanol in the oil phase, which increases the solubility of felodipine. The maximum solubility of felodipine in the system was 9 wt %, defined as the weight percent of felodipine/ (felodipine + MCT). This value should be compared to 3 wt %, which is the solubility in pure MCT. BIBP3226, on the other hand, is a charged molecule and practically insoluble in MCT but slightly soluble in water. Furthermore, it has an affinity for the lecithin monolayer and is therefore partitioned between the water phase and the surfactant film. Mainly because of solubilization of BIBP3226 in the surfactant film and the entropy of the accompanying counterions, the excess water is incorporated in the microemulsion at a very low concentration of BIBP3226. At the drug concentration at which the water phase as well as the surfactant film is saturated with drug, the microstructure has changed from a bicontinuous structure to oil-swollen micelles (oil-in-water microemulsion). At this point, approximately 60% of the drug molecules are located in the surfactant film.
Introduction The growing interest in microemulsions as drug-delivery vehicles arises mainly from their physicochemical properties, such as transparency, low viscosity, thermodynamic stability over a large temperature interval, and high solubilization capacity.1,2 Furthermore, microemulsions are easy to prepare in contrast to emulsions that require the input of a substantial amount of energy. The possibility of increased solubility of sparingly soluble drugs in microemulsions is of great interest, as this may improve the therapeutic efficacy of the drug and allow a reduction in the volume of the vehicle, minimizing toxic side effects.2 In some cases, the capability of a microemulsion to solubilize large amounts of lipophilic and hydrophilic drugs at the same time can be advantageous. Some factors limit the use of microemulsions in pharmaceutical applications. The need for pharmaceutically acceptable components limits the choice of components, leading to difficulties in formulation. Furthermore, the amount of surfactant and cosurfactant used must be kept * To whom correspondence should be addressed. E-mail:
[email protected]. † Present address: Department of Physical Chemistry, Chalmers University of Technology, S-412 96 Gothenburg, Sweden. (1) Lawrence, M. J. Curr. Opin. Colloid Interface Sci. 1996, 1, 826832. (2) Garcia-Celma, M. J. Surf. Sci. Ser. 1997, 66, 123-145.
low, for toxicological reasons.3 For intravenous use, the demands on the nontoxicity of the formulations are rigorous, and very few studies have been reported so far.4 A microemulsion is, however, not an inert vehicle. Adding new components such as drugs to the system may affect its phase behavior. It is of vital importance to examine the influence of solubilized drugs on the microstructure and stability of microemulsions, because this in turn influences the drug delivery from these systems. Solubilized drug molecules may affect the spontaneous curvature, H0, of the surfactant film in different ways, either by being incorporated in the surfactant film or by changing the polarities of the polar and/or apolar phases. The partitioning of the drug between the oil phase, the water phase, and the surfactant film is thus of great interest. So far, very little work has focused on the influence of solubilized compounds on the phase behavior and microstructure of the microemulsions. Recently, Testard et al.5 systematically examined the solubilization of a lipophilic molecule, lindane, in a microemulsion system with a nonionic surfactant. An increased solubility of lindane in the microemulsion regime, compared to the bulk oil, was (3) Gasco, M. R. Surf. Sci. Ser. 1997, 66, 97-122. (4) von Corswant, C.; Thore´n, P.; Engstro¨m, S. J. Pharm. Sci. 1998, 87, 200-208. (5) Testard, F.; Zemb, T.; Strey, R. Prog. Colloid Polym. Sci. 1997, 105, 3332-339.
10.1021/la980706v CCC: $18.00 © 1999 American Chemical Society Published on Web 05/06/1999
Active Compounds in Lecithin-Based Microemulsions
observed and explained by the incorporation of lindane in the surfactant layer. This work studied the solubilization of two active compounds in lecithin-based microemulsions6-8 using NMR self-diffusion measurements and UV absorption measurements, combined with phase studies. Because lecithin is slightly too lipophilic in water-oil systems to spontaneously form zero mean curvature surfactant layers and has a strong tendency to form lamellar phases, the presence of a cosurfactant is necessary in the formation of lecithin-based microemulsions.9 Addition of a shortchain alcohol, such as 1-propanol, has two important features: it decreases the polarity of the water phase, which increases H0, and it increases the flexibility of the surfactant film, destabilizing the lamellar phase that is formed without 1-propanol. Using 1-propanol as the cosurfactant makes the microemulsion pharmaceutically unacceptable for parenteral use. For detailed phase studies the system described here is preferred, however, because it contains relatively few components compared to, e.g., the pharmaceutically acceptable system described by von Corswant et al.4 The influence of the drugs on the phase behavior and microstructure of the system will be discussed in terms of the spontaneous curvature of the surfactant film. Experimental Section Materials. Soybean phosphatidylcholine (Epicuron 200) was obtained from Lucas Meyer Co., Hamburg, Germany. The water content was 1.3-1.9% as determined by the Karl-Fisher coulometric method.6 The chain length distribution of the fatty acids in Epicuron 200 has been reported in ref 9: C16:0 ) 13.3%, C18:0 ) 3%, C18:1 ) 10.2%, C18:2 ) 66.9%, and C18:3 ) 6.6%. The medium-chain triglyceride, Miglyol 810N, was purchased from Hu¨ls, Germany. According to the manufacturer, the chain length distribution of the fatty acids was as follows: C6:0 e 2%, C8:0 ) 70-80%, C10:0 ) 18-28%, and C12:0 e 2%. 1-Propanol (HPLC grade) was supplied by Aldrich. The water used was first purified by reverse osmosis, then further treated in a primary and secondary ion-exchange pack with a photooxidation step between, and finally passed through an ultramicrofiltration unit (Elgastat maxima-HPLC, ELGA Ltd., Bucks, England). The quality of the water was checked by measuring the conductance and surface tension. Felodipine and BIBP3226 (HCl salt) were synthesized at Astra. All reagents were used as received. Phase Diagrams and Notations. The phase diagrams in Figures 2 and 4 were constructed by weighing appropriate amounts of the components in glass vials which were sealed with rubber stoppers and aluminum seals. The samples were shaken by a vortex shaker (MS 1 Minishaker, IKA-Works Inc., Wilmington) and placed in water baths at 25 ( 0.05 °C. The samples were allowed to equilibrate for at least 4 days before they were examined. The nature of the different phases was established using ocular and optical (crossed polarized filters) methods. The volume fraction of the different phases was measured with a height measuring instrument (Feinmesszeugfabrik Suhl GmbH, Suhl, Germany). The phase diagram in Figure 3b was constructed in the following way. Stock solutions of SbPC and 1-propanol at various weight ratios were made. Mixtures of SbPC, 1-propanol, MCT, water, and felodipine at appropriate weight ratios were prepared in glass vials, which were sealed with rubber stoppers and aluminum seals and placed in a water bath at 25 ( 0.05 °C. The mixtures were then titrated with the corresponding stock solutions using a syringe equipped with an injection needle, and (6) von Corswant, C.; Engstro¨m, S.; So¨derman, O. Langmuir 1997, 13, 5061-5070. (7) von Corswant, C.; So¨derman, O. Langmuir 1997, accepted. (8) von Corswant, C.; Olsson, C.; So¨derman, O. Langmuir 1998, Submitted. (9) Shinoda, K.; Araki, M.; Sadaghiani, A.; Khan, A.; Lindman, B. J. Phys. Chem. 1991, 95, 989-993.
Langmuir, Vol. 15, No. 11, 1999 3711 the nature of the different phases was examined between each addition of stock solution. The different phases observed in all of the diagrams presented were noted as follows: Wc, clear aqueous phase, consisting mainly of water, 1-propanol, and in some cases BIBP3226; Lc, a generic notation for several different liquid-crystalline phases; O, clear oil phase, consisting mainly of MCT, 1-propanol, and in some cases felodipine; Me, microemulsion phase, characterized by a low viscosity, optical isotropic behavior, and a translucent appearance. No distinction between oil-in-water (o/w), waterin-oil (w/o), or bicontinuous type microemulsions was made. Pulsed Field Gradient Nuclear Magnetic Resonance Measurements. The PFG-NMR studies were performed as described in ref 8. The 1-propanol self-diffusion coefficient was measured through the peak heights of the 1-methylene group, the SbPC self-diffusion coefficient was measured through the peak heights of the choline group, and the MCT self-diffusion coefficient was measured through the peak heights of the protons from the R-methylene group in the fatty acid in the triglyceride. The felodipine and BIBP3226 self-diffusion coefficients were measured through the peak heights of the benzene rings. Because there is a fast exchange of protons between water and the hydroxyl group of 1-propanol, both in the aqueous medium and in the oil medium, and the exchange of water and 1-propanol between the aqueous medium and the oil medium is also fast, as inferred from the appearance of the NMR spectra, all of these species contribute to the observed self-diffusion coefficient obtained from the OH peak. Dw was calculated according to a two-site model as described in ref 9, however, neglecting the contribution of the water and 1-propanol dissolved in the oil medium. This simplification is valid for the water-rich and bicontinuous microemulsions. For the oil-rich microemulsions, on the other hand, the water and 1-propanol dissolved in the oil medium may contribute significantly to the observed diffusion coefficient, as will be further discussed below. To account for random errors in the NMR signal, DMCT/Df and DSbPC/Df were determined directly from the intensities as described in ref 10. Water Content. Karl-Fisher titration was used to determine the water content. Determination of Alcohol Concentration. The concentration of 1-propanol in the MCT phases was measured by 1H NMR on a Varian 400 MHz spectrometer. The concentration was measured through the integrals of the peaks of the 1-methylene group of 1-propanol and the protons from the R-methylene group in the fatty acid in the triglyceride, using a standard curve based on samples with known 1-propanol concentrations. The concentration of 1-propanol in H2O was measured by 1H NMR on a Varian 400 MHz spectrometer. The concentration was measured through the integrals of the peak of the 1-methylene group of 1-propanol and the water peak, using a standard curve based on samples with known 1-propanol concentrations. Oil-Water Partitioning of 1-Propanol. The partitioning of 1-propanol between the oil medium and the aqueous medium of the microemulsion containing felodipine, expressed as the partition coefficient, Ko/w, where
Ko/w ) g of 1-propanol in the oil phase/g of MCT in the oil phase g of 1-propanol in the water phase/g of water was calculated from the 1-propanol concentrations of the excess oil and water phases. UV Absorption Measurements. The concentration of felodipine in the excess oil phase of various samples was measured on a UV-visible spectrophotometer (HP 8451A, Hewlett-Packard). 1-Propanol was used as the solvent. For each sample, an average of at least three measurements of the absorbance at λ ) 362 nm was used (standard deviation < 0.3%). The concentration of felodipine was calculated from Lambert-Beer’s law, using a standard curve. After the concentration of 1-propanol in the oil had been determined, the felodipine concentration was (10) Olsson, U.; Nagai, K.; Wennerstro¨m, H. J. Phys. Chem. 1988, 92, 6675-6679.
3712 Langmuir, Vol. 15, No. 11, 1999
von Corswant and Thore´ n
Figure 2. Volume fractions of the different phases of the water/ 1-propanol/SbPC/MCT/felodipine system at 25 °C and R ) 0.5, as a function of the felodipine concentration. The felodipine concentration is expressed as β, which is the weight percent of felodipine/(felodipine + MCT).
Figure 1. Chemical structures of (a) felodipine and (b) BIBP3226. recalculated and expressed as the weight fraction of felodipine/ (felodipine + MCT). The concentration of BIBP3226 in a water/1-propanol mixture was measured on a UV-visible spectrophotometer (4050 ULTROSPEC II, LKB Biochrom Ltd., Cambridge, England), with 1-propanol as the solvent. An average of at least 10 measurements of the absorbance at λ ) 278 nm was used with a standard curve of mixtures of water/1-propanol at the weight ratio of 84/16 with known BIBP3226 concentrations (standard deviation < 0.3%). Dynamic Viscosity. The dynamic viscosity of water/1propanol mixtures was calculated from the density measured at 25 ( 0.1 °C with a DMA 48 density meter manufactured by Anton Paar KG., Gratz, Austria, and the kinematic viscosity at 25 ( 0.1 °C measured with an Ubbelohde viscometer from Schott Gera¨te GmbH, Hofheim, Germany.
Results and Discussion Two sparingly soluble drugs were selected for the studies, felodipine and (R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]argininamide (BIBP3226). Felodipine is the active compound in the Astra Ha¨ssle product Plendil, a vasoselective calcium antagonist for the treatment of high blood pressure. It is practically insoluble in water and has a low solubility in triglycerides. Its molecular structure is shown in Figure 1a. BIBP3226 is an NPY (Neuropeptide Y) receptor antagonist.11 BIBP3226 has two large polar groups, an arginine group and a tyrosine group, and a large apolar group consisting of two benzene rings; see Figure 1b. Microemulsions Containing Felodipine. (a) Phase Behavior. The phase behavior of the system water/1propanol/SbPC/MCT/felodipine was studied both as a function of the felodipine concentration and as a function of SbPC and 1-propanol concentrations in a so-called “fishcut” representation.12 Figure 2 shows the volume fractions of the different phases in the water/1-propanol/SbPC/MCT/ felodipine system at constant SbPC and 1-propanol concentrations. Here, and throughout this work, the (11) Rudolf, K.; Eberlein, W.; Wolfhard, E.; Wieland, H. A.; Willim, K. D.; Entzeroth, M.; Wienen, W.; Beck-Sickinger, A. G.; Doods, H. N. Eur. J. Pharmacol. 1994, 271, R11-R13. (12) Kahlweit, M.; Busse, G.; Faulhaber, B. Langmuir 1995, 11, 15761583.
temperature is 25 °C, and R, defined as the weight fraction of oil/(oil + water), is equal to 0.5. To make the phase study as illustrative as possible, the concentrations of SbPC and 1-propanol were chosen such that a Winsor III (a microemulsion phase in equilibrium with an excess water phase and an excess oil phase) system was formed in the absence of felodipine. Furthermore, the 1-propanol concentration was kept as low as possible. With 5 wt % SbPC and 12 wt % 1-propanol, the microemulsion phase swells approximately equal amounts of water and oil. The initial amount of felodipine in the oil, β, is defined as the weight percent of felodipine/(felodipine + MCT). As β is increased, water is expelled from the microemulsion phase and oil is incorporated until, at β ) 9 wt %, a Winsor II system (a microemulsion phase in equilibrium with an excess water phase) is formed. This shows that the surfactant film becomes more curved toward water; i.e., the spontaneous curvature, H0, decreases with increasing β. β ) 9 wt % was found to be the solubility of felodipine in the system. Figure 3a, adapted from ref 6, shows the fish-cut diagram of the system water/1-propanol/SbPC/MCT as a function of 1-propanol and SbPC concentrations. The three-phase body of the system is asymmetrical, because a certain amount of 1-propanol is needed to destabilize the lamellar phase that is otherwise formed. When the 1-propanol concentration is high enough to destabilize the lamellar phase, the lower boundary of the three-phase body has already been passed. The corresponding diagram at β ) 8 wt %, with felodipine and MCT as pseudocomponents, is shown in Figure 3b. Here, the boundaries of the three-phase body are moved to higher 1-propanol concentrations compared to the diagram at β ) 0 wt %, and the full three-phase body is observed. This is in accordance with the observation in Figure 2 that the addition of felodipine decreases H0. Furthermore, the location of the fish tail, i.e., the minimum surfactant concentration for a balanced one-phase system, is moved to a higher SbPC concentration. This is most probably a consequence of the higher 1-propanol concentrations needed to reach the balanced state, which increases the flexibility of the film and thereby reduces its ability to swell large volumes of water and oil.13 The observed H0 dependence of the felodipine concentration can be a consequence of either a change in the (13) Daicic, J.; Olsson, U.; Wennerstro¨m, H. Langmuir 1995, 11, 2451-2458.
Active Compounds in Lecithin-Based Microemulsions
Langmuir, Vol. 15, No. 11, 1999 3713
Figure 4. (a) Self-diffusion coefficients for water (b), MCT (O), SbPC (∆), and felodipine (2) in the microemulsion phase of the samples from Figure 2. (b) Relative self-diffusion coefficients, D/D0, for water (b), MCT (O), and felodipine (2). D0 in the oil phase could not be measured at β ) 8 and 9 wt %.
Figure 3. Phase behavior of the water/1-propanol/SbPC/MCT/ felodipine system at 25 °C and R ) 0.5. (a) The “fish-cut” diagram in the absence of felodipine (β ) 0 wt %). The figure is modified from ref 6. (b) The “fish-cut” diagram at β ) 8 wt %. The dotted lines represent one or more phase boundaries which were not observed but must be present according to the phase rule.
polarity of the oil phase, caused by solubilized felodipine in the bulk oil phase, or a change in the curvature of the surfactant film because of an incorporation of felodipine in the surfactant layer, or a combination of these two effects. These different types of interactions cannot be distinguished from the phase behavior alone, however. The ability of an oil to be incorporated in the hydrophobic part of the surfactant film is commonly denoted the penetration power of the oil. Oils with a high penetration
power (such as hexane) are called penetrating oils while oils that are not incorporated in the surfactant film are named nonpenetrating oils.14-16 (b) NMR Self-Diffusion Measurements. In microemulsion structures in which one of the solvents forms a continuous medium and the other is confined in discrete droplets, the observed self-diffusion coefficients, D, can be interpreted in the following way. The self-diffusion coefficient of the continuous solvent is close to that of the neat liquid, D° (although D is somewhat lower as a result of obstruction by the dispersed droplets).17 With the observation times commonly used in self-diffusion measurements by PFG-NMR, the length scale is on the order of micrometers. Because the droplets are typically as small as 3-20 nm,18 the diffusion of the dispersed solvent within the droplets becomes unimportant and the observed diffusion of the dispersed solvent is the thermal translation of the droplets themselves. Consequently, as long as the solubility of the surfactant in the continuous solvent is negligible, the diffusion coefficients of the dispersed solvent and the surfactant are equal and relatively low. As for bicontinuous microemulsions, studies have shown that the diffusion process of the oil is a molecular diffusion in (14) Chen, S. J.; Evans, D. F.; Ninham, B. W.; Mitchell, D. J.; Blum, F. D.; Pickup, S. J. Phys. Chem. 1986, 90, 842-847. (15) Chen, S. J.; Evans, D. F.; Ninham, B. W. J. Phys. Chem. 1984, 88, 1621. (16) Ninham, B. W.; Chen, S. J.; Evans, D. F. J. Phys. Chem. 1984, 88, 5855. (17) Lindman, B.; Shinoda, K.; Olsson, U.; Anderson, D.; Karlstro¨m, G.; Wennerstro¨m, H. Colloids Surf. 1989, 38, 205-224. (18) Paul, B. K.; Moulik, S. P. J. Dispers. Sci. Technol. 1997, 18, 301-367.
3714 Langmuir, Vol. 15, No. 11, 1999
an oil medium. The same is true for the water in the water domains.19 Thus, any deviation of D from D° is mainly due to a geometrical obstruction. In a bicontinuous structure, both the oil and water domains extend over macroscopic distances and, consequently, D is relatively close to D° for both solvents. The theoretical value of the relative self-diffusion coefficient, D/D°, for equal volume fractions of water and oil is 2/3 for both water and oil if the film is infinitely thin.17 The self-diffusion coefficients of the components in the microemulsion phase, at values of β corresponding to the phase diagram in Figure 2, are shown in Figure 4a. In Figure 4b, the relative self-diffusion coefficients, D/D°, for MCT, water, and felodipine are plotted as a function of β. D° is the self-diffusion coefficient in the excess oil and water phases. The system at β ) 0 wt % forms a nearly balanced microemulsion, with D/D° for both water and MCT being relatively close to 2/3. As β is increased, DMCT/DMCT0 increases to 0.74, while Dw/Dw0 decreases to 0.14 at β ) 9 wt %. In terms of H0, there is a transition from a state in which H0 is close to zero to a system with negative values of H0, leading to an increased obstruction of the water molecules, with the necks between the bulges in the aqueous medium becoming narrower as H0 decreases.20 The observed value of Dw at β ) 9 wt % is more than an order of magnitude larger than that of DSbPC (1.85 × 10-10 and 0.70 × 10-11 m2/s, respectively), suggesting that the microemulsion in the Winsor II system remains bicontinuous rather than changing to a structure of waterswollen reverse micelles. Dw is calculated without considering the contribution from dissolved “free” water and 1-propanol in the oil medium, however, which may have a significant effect on the observed self-diffusion coefficient in the Winsor II system, where the total water concentration has decreased considerably. To discriminate between the two possible microstructures, it is convenient to assume a reverse micellar structure, calculate the resulting diffusion coefficient Dobs for the OH peak, and then compare it with the measured value.8 Dobs is calculated as
Dobs ) xw,dDw,d + xw,oDw,o + xa,dDa,d + xa,oDa,o where xw,d, xw,o, xa,d, and xa,o are the proton molar fractions (w ) water, a ) 1-propanol, d ) droplet, o ) oil medium) and Dw,d, Dw,o, Da,d, and Da,o are the corresponding diffusion coefficients. When the oil medium in the microemulsion is approximated with the excess oil phase from the sample where β ) 4 wt % (there is no excess oil phase at β ) 9 wt %), the “free” water content was determined to be 0.9 wt %, Dw,o ) 2.3 × 10-10 m2/s, and Da,o ) 1.6 × 10-10 m2/s. The total amount of water in the microemulsion phase at β ) 9 wt % was measured to be 17.7 wt %. Using a Ko/w of 0.75 (see Table 1) and Dw,d ) Da,d ) DSbPC ) 7 × 10-12 m2/s, Dobs was calculated to be 2.5 × 10-10 m2/s. This should be compared with the observed value of 18.5 × 10-10 m2/s. Thus, the contribution from dissolved water and 1-propanol in the oil medium cannot explain the high value of the observed self-diffusion coefficient of water, and it can be concluded that the microstructure of the microemulsion containing 9 wt % felodipine is still of a bicontinuous nature. To distinguish between the two situations for felodipine discussed in the previous section, one can study the ratio (19) So¨derman, O.; Stilbs, P. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 445-482. (20) Anderson, D. M.; Wennerstro¨m, H. J. Phys. Chem. 1990, 94, 8683-8694.
von Corswant and Thore´ n Table 1. Measured 1-Propanol and Felodipine Concentrations in the Oil Phase βa (wt %)
b (wt %)
Ko/w
βobsc (wt %)
0 2 4 6 9
7.1 8.0 8.7 9.3 10.5
0.39 0.47 0.54 0.60 0.75
0 1.7 3.4 5.1
βsd (wt %)
7.2 7.4 7.9
a Initial felodipine concentration. b 1-Propanol concentration in the excess oil phase; the value for β ) 9 wt % was extrapolated from the 1-propanol concentrations at β ) 0, 2, 4, and 6 wt %. c The observed felodipine concentration in the excess oil phase. d The solubility of felodipine in MCT/1-propanol at the actual 1-propanol concentration.
Figure 5. DMCT/Df (O) for the excess oil phase and DMCT/Df (∆) and DSbPC/Df (0) for the microemulsion phase. The error within each experiment expressed as the standard deviation is smaller than the symbols representing each data point in the figure. For β ) 6 and 8 wt %, three and four replicates, respectively, were performed to show the variation, including both sample preparation and NMR measurement.
of the self-diffusion coefficients DMCT/Df in the microemulsion and compare it with the values obtained from the excess oil phase. If all felodipine is solubilized in the oil medium and there is no interaction with the surfactant layer for either felodipine or MCT, DMCT/Df in the excess oil phase and in the microemulsion should be equal and should not vary with H0. If, on the other hand, every felodipine molecule would be incorporated in the surfactant film, the observed Df in the microemulsion would be equal to the lateral self-diffusion of felodipine in the mixed felodipine/SbPC layer. The explicit values of this lateral self-diffusion of felodipine are not known, however, but it is reasonable to assume that the ratio DSbPC/Df would be constant for 0 < β < 9 wt % because the bicontinuous microstructure is maintained and, thus, variations in D would mainly originate from geometric obstruction effects, which would be the same for both felodipine and SbPC. Figure 5 shows DMCT/Df in both the excess oil phase and the microemulsion phase and DSbPC/Df in the microemulsion phase. DMCT/Df in both the excess oil phase and the microemulsion phase appears, within experimental errors, to be constant, while DSbPC/Df decreases with increasing β. This indicates that felodipine is not located in the surfactant layer but acts as a nonpenetrating oil, increasing the polarity of the oil phase and thereby decreasing H 0. (c) Solubility Measurements. The solubility of felodipine in pure MCT is 29.8 mg/g of MCT, corresponding to a β value of 2.9 wt %. Because the PFG-NMR study revealed that felodipine is not incorporated in the surfactant layer, the increased solubility in the microemulsion (β ) 9 wt %) remains to be explained.
Active Compounds in Lecithin-Based Microemulsions
It is customary to assume that the composition of the oil domains in the microemulsion phase is the same as the composition of the excess oil phase.10 When the concentration of felodipine and 1-propanol in the excess oil phase and water phase is studied, the concentration in the microemulsion phase can therefore be determined. There were no traces of felodipine found in the excess water phases. The measured concentrations of felodipine in the excess oil phases of the Winsor III systems, βobs, are relatively close to the initial concentrations of β (Table 1), indicating that essentially all of the felodipine is solubilized in the bulk oil phase. The 1-propanol concentration in the excess oil phase, expressed as the weight percent of 1-propanol/(1-propanol + MCT) and denoted , was found to increase with increasing β; see Table 1. That is, the addition of felodipine increases the partitioning coefficient, Ko/w, of 1-propanol, thereby increasing the solubility of felodipine in the oil phase until an equilibrium is reached. It is known that Ko/w is dependent on the polarity of the oil phase and increases with increasing polarity.21 Thus, felodipine seems to be more polar than MCT. To verify the conclusion above, the solubility, βs, of felodipine in MCT/1-propanol mixtures of the same ratios as in the bulk oil phase of the samples from the Winsor III systems was determined; see Table 1. It is evident that the solubility of felodipine increases with increasing 1-propanol concentration in the oil, and it was found that the solubility of felodipine in the system (β ) 9 wt %) is close to the observed solubility in the corresponding MCT/ 1-propanol mixture (βs ∼ 8 wt %). From the phase diagrams, the NMR self-diffusion measurements, and the measurements of the solubility of felodipine in the oil phase, it can now be concluded that felodipine seems to act as a nonpenetrating oil, curving the surfactant film toward water by increasing the polarity of the oil phase. The increased solubility of felodipine in the system compared to the solubility in pure MCT is explained by the presence of 1-propanol in the MCT phase and an increase in the partitioning coefficient of 1-propanol as felodipine is added to the oil phase, reaching an equilibrium at β ) 9 wt %. Microemulsions Containing BIBP3226. (a) Phase Behavior. The Winsor representation diagram of the system water/1-propanol/SbPC/MCT/BIBP3226 at constant surfactant concentration and 1-propanol concentration is shown in Figure 6. Note that the concentration of BIBP3226 is expressed as the weight percent of BIBP3226/ (BIBP3226 + SbPC), denoted δ. The 1-propanol concentration and total surfactant concentration (BIBP3226 + SbPC) are 12 and 5 wt %, respectively. The first point in the diagram is identical with the first point in Figure 2, a Winsor III system. The system exhibits a dramatic change in phase behavior as BIBP3226 is added. At δ ) 0.8 wt %, all of the water is incorporated in the microemulsion phase. As δ is increased further, more oil is expelled from the microemulsion. The solubility of BIBP3226 in the system corresponds to δ ) 20 wt %. BIBP3226 has a very low solubility in MCT but was found to be somewhat soluble in water (5 mg/g), lowering the pH to approximately 4. To examine the influence of pH on the phase behavior of the system, the water phase of the sample at δ ) 0 wt % was substituted with a phosphate buffer of pH ) 3 (ionic strength ) 50 mM). It was found that lowering the pH to 3 had no effect on the phase behavior. The strong influence of BIBP3226 on the phase behavior is attributed to the fact that BIBP3226, (21) Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525-616.
Langmuir, Vol. 15, No. 11, 1999 3715
Figure 6. Volume fractions of the different phases of the water/ 1-propanol/SbPC/MCT/BIBP3226 system at 25 °C and R ) 0.5, as a function of BIBP3226 concentration. The BIBP3226 concentration is expressed as δ, which is the weight percent of BIBP3226/(BIBP3226 + SbPC). The inset shows the phase behavior at low δ.
in contrast to felodipine, is a charged molecule which introduces electrostatic interactions in the system. The effect of adding small amounts of an ionic amphiphile has been suggested to originate from the entropy associated with the counterions of the ionic amphiphile.22 In this case it is the presence of the chloride counterions of BIBP3226 which makes a phase separation with excess water, corresponding to a compression of the counterion volume, less favorable. (b) NMR Self-Diffusion Measurements. Figure 7a plots the self-diffusion coefficients of the components in the microemulsion phase corresponding to the phase diagram in Figure 6. The self-diffusion coefficient of BIBP3226 at δ values below 8 wt % was not measured, because the drug concentration was too low. The starting point is a bicontinuous structure, as stated in the discussion of the felodipine system. As δ increases, Dw increases rapidly to a constant value while DSbPC and DMCT decrease gradually and become equal first at δ ) 20 wt % (Figure 7b). This implies that although the volume fraction of oil in the microemulsion phase is markedly decreased at low δ, the oil domains seem to remain connected, transforming gradually to an oil-in-water droplet structure as δ is increased to 20 wt %. The self-diffusion coefficient of BIBP3226 at δ ) 20 wt % is much higher, as compared with DSbPC and DMCT, which indicates that a significant amount of the solubilized drug is not associated with the surfactant layer of the swollen micelles but is dissolved in the continuous aqueous phase. The fraction of drug molecules located in the surfactant film can be determined quantitatively with a two-site model, which is frequently used to study solubilization in micelles.19 For a drug that is partially solubilized in or otherwise associated with micelles, the observed selfdiffusion coefficient of the drug (Dobs) is at a good approximation, equal to
Dobs ) pmicDmic + (1 - pmic)Dfree where pmic is the fraction of drug molecules associated with the micelles, Dmic is the self-diffusion coefficient of the drug molecules associated with the micelles, and Dfree (22) Fukuda, K.; Olsson, U. Langmuir 1994, 10, 3222-3229.
3716 Langmuir, Vol. 15, No. 11, 1999
von Corswant and Thore´ n
as well as SbPC, the fraction of bound water, pb, can be calculated as
pb ) (nw,bound,BIBP3226nBIBP3226 + nw,bound,SbPCnSbPC)/nw,tot
Figure 7. (a) Self-diffusion coefficients for water (b), MCT (O), SbPC (∆), and BIBP3226 (2) in the microemulsion phase of samples from Figure 6. (b) Enlargement showing only the self-diffusion coefficients of MCT and SbPC.
is the self-diffusion coefficient of the drug in the continuous medium. The equation can be rearranged to
Pmic )
Dfree - Dobs Dfree - Dmic
Applying this equation to our system of oil-swollen micelles, we know from the discussion of the felodipine system that solubilizate diffusion within the micelle is unimportant, and Dmic can be taken to be equal to the micellar diffusion coefficient, which in turn is approximately equal to Dsurfactant as long as the concentration of free surfactant molecules in solution is negligible.23 Dfree can be obtained from the self-diffusion coefficient of BIBP3226, Dfree,0, in a water/1-propanol mixture saturated with BIBP3226, corresponding to the continuous phase in the microemulsion at δ ) 20 wt %. The presence of swollen micelles in the solution lowers Dfree compared with the measured value in the water/1propanol mixture. To correct for this obstruction effect, the ratio Dw/Dw0 for water can be used, assuming the obstruction effect created by the micelles to be equal for both BIBP3226 molecules and water molecules. Dw/Dw0 was determined to be 0.82, where Dw0 was measured in the same saturated solution as Dfree,0 for the drug. The 1-propanol concentration, 16.4 wt %, was chosen according to the measurements described in the next section. However, to obtain the obstruction factor, Dw/Dw0 must be corrected for the hydration effect (some water molecules are bound to the surface of the micelles, lowering the value of Dw). Because the surfactant film includes drug molecules (23) Stilbs, P. J. Colloid Interface Sci. 1981, 87, 385-394.
Here, nw,bound,BIBP3226 is the number of water molecules bound to one drug molecule, nw,bound,SbPC is the number of bound water molecules per SbPC molecule, and nBIBP3226 and nSbPC are the total number of BIBP3226 molecules and SbPC molecules in the interfacial film, respectively. The total number of water molecules, nw, tot, is known from the preparation of the sample, because all of the water is in the microemulsion phase. nSbPC is also known, neglecting the small concentration of SbPC in the oil phase. Assuming nw,bound,SbPC and nw,bound,BIBP3226 to be 15,6 pb is calculated at 0.05. Correcting the measured value of Dw/Dw0 for the hydration effect, we arrive at an aggregate obstruction factor of 0.86, which gives pmic ) 0.58 (Dobs ) 7.54 ×10-11 m2/s, Dmic ) DSbPC ) 4.8 ×10-12 m2/s, and Dfree,0 ) 2.01 ×10-10 m2/s). The assumed values of nw,bound,SbPC and nw,bound,BIBP3226 are not critical for the result obtained for pmic because, if the hydration effect is totally neglected, pmic ) 0.56. The aggregate obstruction factor for the water molecules, Dw/Dw0, calculated above as 0.86 also contains information on the shape of the aggregates.24 This value should be compared with the value for hard spheres given by 1/(1 + Φagg/2),20 where Φagg is the volume fraction of aggregates. Φagg can be estimated by assuming the densities of water, 1-propanol, MCT, and SbPC all to be equal to 1 g/cm3 and by disregarding the drug molecules in the aggregates. By adding the volume fractions of SbPC, MCT, and some of the 1-propanol, corresponding to the alcohol concentration in the oil phase, and by excluding the volume fraction of the excess oil phase, we obtain Φagg ) 0.16 at δ ) 20 wt %. Allowing an uncertainty in Φagg of (0.05, we obtain an obstruction factor of 0.93 ( 0.02. Thus, the difference between the observed obstruction effect (0.86) and the corresponding value for hard spheres is small and, consequently, the shape of the swollen micelles appears to be close to spherical. The hydrodynamic radius of the oil-swollen micelles can now be calculated using the Stokes-Einstein relation
RH )
kBT 6πηD
where T is the temperature, η is the viscosity of the continuous medium, kB is the Boltzmann constant, and D is the self-diffusion constant of the micelle. Because the Stokes-Einstein relation is valid only for infinitely dilute spherical aggregates, the observed value of Dmic must be corrected for the aggregate obstruction effects according to Dmic ) Dmic,0(1 - kΦagg), where Dmic,0 is the self-diffusion coefficient of the aggregate in an infinitely dilute system and k is a constant, which, for hard spheres with no hydrodynamic interaction, is equal to 2. The viscosity of the water/1-propanol solution at 25 °C was measured at 1.7 mPa s. Using the values of k ) 2 and Φagg ) 0.16 ( 0.05, the hydrodynamic radius of the aggregates at δ ) 20 wt % was calculated to be 18 ( 3 nm. (c) Solubility Measurements. For comparison, the solubility of BIBP3226 in the continuous water phase was measured. It was first necessary to determine the 1-propanol concentration. Because no excess water phase was (24) Jo¨nsson, B.; Wennerstro¨m, H.; Nilsson, P. G.; Linse, P. Colloid Polym. Sci. 1986, 264, 77-88.
Active Compounds in Lecithin-Based Microemulsions
observed with BIBP3226 present in the system, the excess oil phase in each sample was examined instead. By 1H NMR measurements, it was found that the 1-propanol concentration in the oil phase was constant for all δ values and equal to 7.1 wt %, corresponding to Ko/w ) 0.39. This means that the concentration of 1-propanol in the water phase must also be constant. With Ko/w equal to 0.39, the 1-propanol concentration in the water phase should be 16.4 wt %, calculated as the weight fraction of 1-propanol/ (1-propanol + water). The accuracy of this prediction is confirmed by an experiment in which equal amounts of water and MCT were mixed with 1-propanol at a concentration of 12 wt %. The 1-propanol concentration in the water phase was measured by 1H NMR and was found to be 16.4 wt %. This mixture was saturated with BIBP3226, and the drug concentration in this solution, now known to correspond to the water phase of the sample at δ ) 20 wt %, was measured by UV absorption spectroscopy. The measured value of the drug concentration was 8.3 mg/g. This is the maximum solubility of BIBP3226 in the continuous aqueous phase, and the value should be compared to the total amount of BIBP3226 at δ ) 20 wt %, divided by the weight of the water phase, including 1-propanol, which gives 20.1 mg/g. The difference is the increased solubility in the microemulsion, due to solubilization in the lecithin film. Calculated with this method, the fraction of drug molecules bound to the surfactant layers of the oil droplets, pmic, is thus 0.59 and is in excellent agreement with the value obtained in the PFG-NMR study. Conclusions Evaluation of phase diagrams, combined with NMR measurements of the self-diffusion coefficients of the components and studies of the solubility of the drug in the aqueous phase and the nonaqueous phase, has proven to be a useful method when characterizing the influence of solubilized active compounds on the phase behavior and microstructure of microemulsions. Starting from a Winsor
Langmuir, Vol. 15, No. 11, 1999 3717
III system of water, 1-propanol, SbPC, and MCT, the two drugs studied here, felodipine and BIBP3226, were found to affect the phase behavior and microstructure in different ways. Felodipine, which is practically insoluble in water and slightly soluble in MCT, acts as a nonpenetrating oil. With an increasing concentration of felodipine in the oil phase, the polarity of the oil phase increases, which in turn curves the surfactant film toward water. Thus, water is expelled from the microemulsion phase, and oil is incorporated as felodipine is added. With the composition used here, the microstructure remains bicontinuous, however, even at high felodipine concentrations. The increase in the polarity of the oil phase also has the effect of increasing the partitioning of 1-propanol in the oil phase, which increases the solubility of felodipine. BIBP3226, on the other hand, is a charged molecule and practically insoluble in MCT but slightly soluble in water. Furthermore, it has an affinity for the lecithin monolayer and is therefore partitioned between the water phase and the surfactant film. Mainly because of solubilization of BIBP3226 in the surfactant film and the entropy of the accompanying counterions, the excess water is incorporated in the microemulsion at a very low concentration of BIBP3226. The drug does not appear to influence the partitioning coefficient of 1-propanol. At the drug concentration at which the water phase and the surfactant film are saturated with the drug, the microstructure has changed from a bicontinuous structure to oil-swollen micelles (o/w microemulsion). At this point, approximately 60% of the drug molecules are located in the surfactant film, according to both the NMR selfdiffusion measurements and the UV absorption measurements. Acknowledgment. Dr. Olle So¨derman is acknowledged for his critical reading of the manuscript. LA980706V
