Lecithin Organogels Used as Bioactive Compounds Carriers. A

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Lecithin Organogels Used as Bioactive Compounds Carriers. A Microdomain Properties Investigation Spyridon Avramiotis,† Vassiliki Papadimitriou,† Elina Hatzara,† Vlasoula Bekiari,‡ Panagiotis Lianos,‡ and Aristotelis Xenakis*,† Institute of Biological Research & Biotechnology, The National Hellenic Research Foundation, 48, Vassileos Constantinou AVenue, 11635 Athens, Greece, and Engineering Science Department, UniVersity of Patras, 26500 Patras, Greece ReceiVed December 4, 2006. In Final Form: January 23, 2007 Organogels were obtained by adding small amounts of water to a solution of lecithin in organic solvents. Either isooctane or isopropyl palmitate and isopropyl myristate were used as the continuous organic phase of the gels. EPR spectroscopy using both DSA membrane-sensitive and lipophilic spin probes was applied to define the dynamic structure of the surfactant monolayer and the continuous oil phase of lecithin organogels. It was found that by increasing the water quantity, an increase of the polar head area per lecithin molecule was induced, and as a consequence the total interface expanded. It was found that the use of esters as organic solvents induced a decrease of the size of the dispersed structures. The interconnection of the aqueous microdomains and their dynamics were monitored by both static and time-resolved fluorescence quenching spectroscopy using Ru(bipy)32+ as fluorophore and Fe(CN)63as quencher. It was found that the rates of inter- and/or intra-micellar exchange of water molecules were very slow because they appeared quite immobilized close to the lecithin polar heads. According to the results of the dynamic studies, appropriate organogels were formulated and used to incorporate model bioactive compounds with medicinal or cosmetic interest such as caffeine and theophylline. When these systems were tested for trans-membrane diffusion, they showed a 24 h permeation of 20% and 35%, respectively.

Introduction The growing interest in lecithin organogels as functional materials with various technological and biomedical applications arises mainly from the advantages of their physicochemical properties. Lecithin organogels are easily obtained when small but critical water amounts are added into lecithin/organic solvent solutions.1 Macroscopically, lecithin organogel is a viscous gel, isotropic, thermostable, and thermoreversible.2,3 The viscosity depends on lecithin concentration, on water to surfactant molar ratio, and on the nature of the organic solvent. It has been established that the addition of water induces a transition from initial spherical micelles to cylindrical, flexible rodlike micelles via a one-dimensional growth process.4,5 These hypermolecular structures are also called polymer-like or living polymers due to the dynamic equilibrium between the separateness and the recombination of the aggregates. Lecithin organogels have been studied worldwide over the past years with regard to their potential use in novel pharmaceutical and cosmetic formulations especially as topical drug carrier systems.6-9 Topical administration of therapeutic agents * Corresponding author. Tel.: +302107273762. Fax: +302107273758. E-mail: [email protected]. † The National Hellenic Research Foundation. ‡ University of Patras. (1) Scartazzini, R.; Luisi, P. L. J. Phys. Chem. 1988, 92, 829-833. (2) Schurtenberger, P.; Scartazzini, R.; Magid, L. J.; Leser, M. E.; Luisi, P. L. J. Phys. Chem. 1990, 94, 3695-3701. (3) Schurtenberger, P.; Magid, L. J.; Penfold, J.; Heenan, R. Langmuir 1990, 6, 1800. (4) Schurtenberger, P.; Cavaco, C. J. Phys. Chem. 1994, 98, 5481-5486. (5) Schurtenberger, P.; Cavaco, C. Langmuir 1994, 10, 100-108. (6) Von Corswant, C.; Thoren, P. E. G. Langmuir 1999, 15, 3710-3717. (7) Paolino, D.; Ventura, C. A.; Nistico`, S.; Puglisi, G.; Fresta, M. Int. J. Pharm. 2002, 244, 21-31. (8) Willimann, H.; Walde, P.; Luisi, P. L.; Gazzaniga, A.; Stroppolo, F. J. Pharm. Sci. 1992, 81, 871-874. (9) Dreher, F.; Walde, P.; Luisi, P. L.; Elsner, P. Skin Pharmacol. 1996, 9, 124-129.

is currently regarded as an important alternative to the classical oral and intravenous administration.10 The improved topical drug delivery of lecithin organogel-based systems has been attributed mainly to the coexistence of aqueous and organic phases separated by a large interface and the possibility to entrap bioactive compounds within the gel matrix. In addition, the interaction of biolipids with skin tissues induces an enhanced skin penetration and delivery of bioactive molecules into the deeper skin.11 Structural characterization of organogels made by lecithin in biocompatible oils is very important while investigating the potential applications of these systems as model carriers of bioactive compounds. Multiple instrumental techniques based on microscopy, spectroscopy, and scattering were undertaken to elucidate the structural details of the lecithin-based organogels. Phase behavior studies were conducted by employing nuclear magnetic resonance spectroscopy (NMR).2,12 Fourier transformed infrared spectroscopy (FTIR) was used to investigate the nature of binding forces responsible for the self-assembly of monomers in the organic solvents.13,14 Molecular packing within the organogel network has been elucidated by employing dynamic and static light scattering, small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and atomic force microscopy (AFM).3,15-17 Nevertheless, detailed studies concerning (10) Guy, R. R.; Hadgraft, J. J. Pharm. Sci. 1985, 74, 1016. (11) Kumar, R.; Katare, O. P. AAPS PharmSciTech. 2005, 6, E298-E310. (12) Sadagiani, A. S.; Noori, A.; Khan, A. J. Surf. Sci. Technol. 1991, 7, 163-175. (13) Mezzasalma, S. A.; Koper, G. J. M.; Shchipunov, Y. A. Langmuir 2000, 16, 10564-10565. (14) Suzuki, M.; Nakajima, Y.; Yumoto, M.; Kimura, M.; Shirai, H.; Hanabusa, K. Org. Biomol. Chem. 2004, 2, 1155-1159. (15) Aboofazeli, R.; Barlow, D. J.; Lawrence, M. J. AAPS PharmSciTech. 2000, 2, E13. (16) Zemb, T. N.; Barnes, I. S.; Derian, P. J.; Ninham, B. W. Prog. Colloid Polym. Sci. 1990, 81, 20-29. (17) Simmons, B. A.; Taylor, C. E.; Landis, F. A.; McPherson, G. L.; Schwartz, D. K.; Moore, R. J. Am. Chem. Soc. 2001, 123, 2414-2421.

10.1021/la0634995 CCC: $37.00 © 2007 American Chemical Society Published on Web 03/06/2007

Microdomain Properties of Lecithin Organogels

the interfacial properties of the lecithin monolayer as well as the nature of the entrapped water in lecithin organogels are still lacking. In the present study, electron paramagnetic resonance spectroscopy (EPR) was undertaken as a valuable method to define the dynamic structure of the surfactant monolayer and the continuous oil phase in lecithin organogels.18 The influence of the various constituents on the fluidity and structure of the gels membrane and also the restrictions imposed by them in the intramicellar organic space can be estimated. We investigated and compared the EPR spectra of five different spin probes, the membrane spin probes 5-doxyl stearic acid (5-DSA), 12-doxyl stearic acid (12-DSA), 16-doxyl stearic acid (16-DSA), and the lipophilic spin probes 12-doxyl methyl stearate (12-DMS) and 10-doxyl nonadecane (10-DN) in three systems: organic solvents, binary systems, and gels. The doxyl stearic acid derivatives (nDSA’s) are labeled at different positions of the fatty acid aliphatic chain (n ) 5, 12, 16), thus reflecting the dynamics of the membrane at different depths.19 The ester and the hydrocarbon spin probes, 12-DMS and 10-DN, respectively, are expected to monitor the dynamics of the continuous organic phase due to their specific location in the system. Isooctane, isopropyl palmitate, and isopropyl myristate were used as organic solvents. Lecithin solutions in the above-mentioned solvents were considered as binary systems. Gels were prepared by adding tiny amounts of water in the binary systems. To extend the EPR findings, both static and time-resolved fluorescence quenching spectroscopy were undertaken. When the fluorophore and the quencher are hydrophilic molecules, the quenching process occurs exclusively in the aqueous domains, providing information about the nature and the dynamics of the entrapped water. By applying the Stern-Volmer relationship, the interconnection of the aqueous droplets of the lecithin/ isopropyl palmitate/water organogels was tested.20,21 To investigate the dynamics of the water entrapped in the above-mentioned lecithin-based organogels, the fluorescence quenching reaction of Ru(bipy)32+ from Fe(CN)63- was followed, and the corresponding decay profiles were analyzed with a model of stretched exponentials.22-25 In this paper, we also investigated the ability of lecithin-based gels to incorporate bioactive compounds, and their transfer yield through a membrane was considered by employing an in vitro permeation study. The compounds, which have been used, were caffeine and theophylline. These xanthine derivatives have extended medicinal and cosmetic applications due to their effect on the central nervous system, the myocardium, the kidney, and the regulation of lipid metabolism.26,27 Caffeine and its salts are the most active in stimulating the central nervous system and are principally used for this purpose. Caffeine is sometimes administered in combination with medicines to increase their effectiveness. Theophylline preparations are used to relax bronchial smooth muscle for asthma and chronic obstructive pulmonary disease. The diuretic effects of caffeine are less than (18) Griffith, O. H.; Jost, P. C. In Lipid Spin Labels in Biological Membrane. In: Spin Labeling, Theory and Applications; Berliner, L. J., Ed.; Academic Press: New York, 1976; pp 454-484. (19) Avramiotis, S.; Cazianis, C. T.; Xenakis, A. Langmuir 1999, 15, 23752379. (20) Malliaris, A. AdV. Colloid Interface Sci. 1987, 27, 153-168. (21) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951. (22) Lianos, P.; Modes, S.; Staikos, G.; Brown, W. Langmuir 1992, 8, 1054. (23) Lianos, P.; Argyrakis, P. J. Phys. Chem. 1994, 98, 7278. (24) Duportail, G.; Merola, F.; Lianos, P. J. Photochem. Photobiol., A: Chem. 1995, 89, 135. (25) Lianos, P. Heterog. Chem. ReV. 1996, 3, 53. (26) MARTINDALE. The Extra Pharmacopoeia, 27th ed.; 1977. (27) Kobayashi-Hattori, K.; Mogi, A.; Matsumoto, Y.; Takita, T. Biosci. Biotechnol. Biochem. 2005, 69, 2219-2223.

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those of theophylline, but the usefulness of the latter is limited by its poor solubility. Tolerance to some of the effects of the xanthines, particularly the diuretic and smooth muscle effects, may develop by applying specific formulation of the compounds for topical administration. Experimental Procedures Materials. Soybean lecithin (Epikuron 200) containing 96% phosphatidylcholine was purchased from Lucas Meyer. Isooctane was from Merck; isopropyl palmitate [IPP] and isopropyl myristate [IPM] were from Fluka. Caffeine and theophylline were from Sigma. The spin-labeled doxylated derivatives 5-doxyl stearic acid [5-(1oxyl-2,2-dimethyl-oxazolidin) stearic acid; 5-DSA], 12-doxyl stearic acid [12-DSA], 16-doxyl stearic acid [16-DSA], 12-doxyl methyl stearate [12- DMS], and 10-doxyl nonadecane [10-DN] were obtained from Sigma. The fluorescent probe tris (2,2-bipyridine) ruthenium dichloride hexahydrate, Ru(bpy)3Cl2, was from GFS Chemicals, and the quencher potassium ferricyanide, K3Fe(CN)6, was from Merck. High-purity water was obtained by a Millipore Milli Q Plus water purification system. Preparation of the Samples. EPR Measurements. Solutions of the spin-labeled doxylated derivatives, 5-DSA, 12-DSA, 16-DSA, 12-DMS, and 10-DN, in three solvents, isooctane, isopropyl palmitate, or isopropyl myristate, were prepared as follows: each spin probe was added in each solvent to give a final concentration of 10-2 mM. The binary systems were prepared as follows: each spin probe was added in either 5% w/w lecithin/isooctane or 10% w/w lecithin/ isopropyl palmitate or 10% w/w lecithin/isopropyl myristate to give a final concentration of 10-2 mM. The gel systems were prepared like the binary systems with the addition of small amounts of water to give a desired final wo value. In the case of organogels, to examine the effect of the pH of the aqueous phase on the EPR spectra of the spin probes, the following solutions were used instead of water: for pH 2, 0.01 M HCl solution; for pH 5.5, 5 mM phosphate buffer solution; for pH 8.5, 5 mM Tris/HCl buffer solution; and for pH 12, 0.01 M NaOH solution. Fluorescence Quenching Measurements. In the case of fluorescence spectroscopy studies, lecithin organogels were prepared by adding appropriate amounts of aqueous stock solutions of fluorophore Ru(bpy)3Cl2 and quencher K3Fe(CN)6 to a 10% w/w lecithin/ isopropyl palmitate solution. The final concentrations of the fluorophore and the quencher were 10-5 M and 2 × 10-5 to 10-4 M, respectively. Three different wo values were considered, 2.1, 3.0, and 3.7. In Vitro Permeation Studies. Lecithin organogels containing bioactive compounds were prepared at room temperature by adding a few milliliters of a concentrated aqueous solution of the bioactive compound (caffeine, theophylline) to a 10% w/w lecithin/isopropyl palmitate or isopropyl myristate solution. The final overall concentrations of caffeine and theophylline in the organogel were 0.76 and 0.34 mM, respectively. The final wo of the gel was 3.5 throughout the experiments (wo ) [H2O]/[lecithin]). The pH of the aqueous phase was 5.5 to simulate the real pH of the skin. Methods. EPR Measurements. EPR spectra were recorded at room temperature using a Bruker ER 200 D spectrometer operating at the X-band. The samples were contained in a flat E-248 cell. Typical settings were: center field, 3471 G; scan width, 100 G; time constant, 0.5 s; scan time, 100 ms; microwave power, 7.5 mW; microwave frequency, 9.76 GHz; modulation amplitude, 1 G. Data collection was performed using the computerized program DAT200 (Data Acquisition Program, University Lubeck, Germany) and analyzed with the GEP (Graphic Evaluation Program version 1.2) program for personal computer. Simulations of the experimental spectra were conducted with a simulation program, Winsim, developed at the National Institute of Environment and Health Sciences. Interpretation of the EPR Data. The results reported in this work were analyzed in terms of both order parameter, S, and rotational correlation time, τR. The above-mentioned parameters can monitor the dynamics of a spin probe in membranes or in viscous media. The

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AVramiotis et al.

Scheme 1. Schematic Presentation of the Spin Probe 12-Doxyl Stearic Acid (12-DSA) and Its Tumbling around the x-, y-, and z-Axesa

a The z-axis is the long molecular axis that is parallel to the direction of the nitrogen 2p π orbital of the doxyl ring. The axes of the other doxylstearic acids are analogous.31

order parameter S provides a measure of the spin probe’s arrangement in a supramolecular assembly and varies from 0 to 1, with S ) 1 for the completely ordered state and S ) 0 for the completely random state.18 The rotational correlation time, τR, is relevant to the spin probe’s rotational motion. We must say that for the fast motion region (τR < 3 × 10-9 s) the τR values are more accurate because they depict precisely the spin probe’s molecular motion. For the slow motion region (τR > 3 × 10-9 s), the determination of τR values is difficult and inaccurate. In the last case, the order parameter S is a much better approach to the immobilization degree. Calculation of the Rotational Correlation Time, τR. The rotational correlation time, τR, was calculated by the following relationship:28 τR ) 6 × 10-10[(h0/h+1)1/2 + (h0/h-1)1/2 - 2]∆H0

(1) S ) 1/2(cos γ + cos2 γ)

where ∆H0 is the width (in Gauss) of the central peak and h+1, h0, and h-1 are the intensities of the low, center, and high field peaks, respectively. The relationship of eq 1 is applicable in the fast motion region, that is, τR < 3 × 10-9 s. For the slow motion region, that is, τR > 3 × 10-9 s, the rotational correlation time, τR, is calculated according to Kuznetsov’s method.29 Calculation of the Order Parameter, S. From the spectral characteristics, we have calculated two more parameters: the order parameter, S, and the isotropic hyperfine splitting constant, RN. The order parameter, S, is defined as:18,30 S ) (A| - A⊥)/[Azz - 1/2(Axx + Ayy)](RN0/RN)

When doxyl stearic acid spin probes are anchored in phospholipidic membranes, their molecular motion becomes anisotropic. In that case, DSA’s are not free to tumble in all directions but mainly rotate around the long molecular axis zz′ (Scheme 1).31 This anisotropic motion has been described as a restricted random walk of the long molecular axis on the surface of a sphere, as a rapid motion within a cone, and as a rapid fluctuation of the angle θ between the long molecular axis and the sample axis zz′. The angle θ fluctuates randomly between the limits θ ) 0° and a maximum half amplitude, γ, so-called wobbling angle, which gives a more geometrical sense of the membrane rigidity. The wobbling angle γ was calculated from the order parameter S by the following relationship:18

(2)

where Axx, Ayy, and Azz are the single-crystal values of the spin probe equal to 6.3, 5.8, and 33.6 G, respectively, and are indicative for doxyl derivatives.18 A| and A⊥ are the hyperfine splitting constants, where A| is the half distance of the outermost EPR lines while A⊥ is the half distance of the inner EPR lines. The A| and A⊥ values were also calculated with computer simulations in all spectra and in particular in those where the spectral characteristics were not well resolved. The ratio RN0/RN is the polarity correction factor (hyperfine splitting constants) where RN0 ) 1/3(Azz + Axx + Ayy) and RN ) 1/3(A| + 2A⊥). RN0 is the isotropic hyperfine splitting constant for the group molecule in the crystal state, and RN is the isotropic hyperfine splitting constant for the spin probe in the membrane. RN values are sensitive to the polarity of the environment of the spin probe and are increased when the polarity of the medium is increased. (28) Kommaredi, N. S.; O’Connor, K. C.; John, V. T. Biotechnol. Bioeng. 1994, 43, 215. (29) Kuznetsov, A. N.; Wasserman, A. M.; Volkov, A. U.; Korst, N. N. Chem. Phys. Lett. 1971, 12, 103-106. (30) Hubbel, W. L.; McConnell, H. M. J. Am. Chem. Soc. 1971, 93, 314-326.

(3)

Fluorescence Quenching Measurements. Steady-State Fluorescence Quenching. Fluorescence quenching measurements were recorded at room temperature using a Perkin-Elmer 650-40 fluorescence spectrophotometer. Fluorescence intensities were measured in the absence and presence of various concentrations of quencher. All solutions were measured without previous degassing, in equilibrium with atmospheric oxygen. The method of steadystate fluorescence quenching allows distinction between quenching processes taking place in homogeneous and microheterogeneous media.20 Thus, in homogeneous solutions, the usual Stern-Volmer relationship applies; that is, plots of Fo/Fi versus [Q] produce straight lines. Fi and Fo are the fluorescence intensities in the presence and in the absence of quencher, respectively, whereas [Q] is the concentration of the added quencher. In small confinements, on the other hand, Stern-Volmer curves show an upward curvature. This modification of the Stern-Volmer relationship in microheterogeneous media is the result of the Poisson distribution of fluorophores and quenchers in small confinements.21 The excitation wavelength used was 450 nm, at which no absorption was detected due to any of the constituents of the solution, whereas the emission wavelength was fixed at 620 nm. Fluorescence Quenching Decay Measurements. Nanosecond decay profiles were recorded with the photon counting technique using a homemade hydrogen flash and ORTEC electronics. A MellesGriot interference filter was used for excitation (450 nm), and a cutoff filter (600 nm) was used for emission. All samples were deoxygenated by the freeze-pump-thaw method. The decay profiles were recorded in 1000 channels at 2.6 ns per channel and were analyzed by least-square fits using the distribution of the residuals (31) Smith, I .C. P.; Butler, K. W. In Oriented Lipid Systems as Model Membranes; Berliner, L. J., Ed.; Academic Press: New York, 1976; pp 411-451.

Microdomain Properties of Lecithin Organogels

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Table 1. Rotational Correlation Times, τR, and Hyperfine Coupling Constants, rN, of the Spin Probes in Pure Solvents As Calculated from the Corresponding EPR Spectra 5-DSA

12-DSA RN (G)

τR (s)

10-12

7.0 × 7.3 × 10-10 5.3 × 10-10

solvent

τR (s)

isooctane isopropyl palmitate isopropyl myristate

16-DSA RN (G)

τR (s)

13.09 14.09

10-10

1.4 × 8.5 × 10-10

14.29

6.0 × 10-10

12-DMS RN (G)

τR (s)

12.90 13.90

10-12

2.1 × 1.4 × 10-11

14.15

1.5 × 10-11

10-DN RN (G)

τR (s)

RN (G)

13.89 13.93

10-12

3.4 × 4.5 × 10-10

14.09 14.12

10-12

6.3 × 4.9 × 10-10

14.10 14.10

14.30

3.3 × 10-10

14.29

3.6 × 10-10

15.10

Table 2. Rotational Correlation Times, τR, Order Parameters, S, and Wobbling Angle, γ, Values for the Spin Probes 5-DSA, 12-DSA, 16-DSA, 12-DMS, and 10-DN Calculated in Binary Systems and Organogelsa 5-DSA

12-DSA

16-DSA

12-DMS

10-DN

system

τR (ns)

S

γo

τR (ns)

S

γo

τR (ns)

τR (ns)

τR (ns)

lecithin/isooctane lecithin/isooctane/water pH 2 pH 5.5 pH 8.5 pH 12 lecithin/IPM 10% w/w lecithin/IPM/water pH 2 pH 5.5 pH 8.5 pH 12 lecithin/IPP 10% w/w lecithin/IPP/water pH 2 pH 5.5 pH 8.5 pH 12

8.8

0.76

34.2

2.4

0.20

71.9

0.41

0.06

0.05

6.3 6.5 8.1 8.3 8.5

0.49 0.50 0.59 0.66 0.69

52.1 51.8 45.9 43.9 38.8

1.6 1.8 2.5 2.7 2.6

0.18 0.19 0.21 0.22 0.18

73.4 72.6 71.3 70.9 73.5

0.36

0.004

0.04

0.38

0.38

0.40

6.5 6.8 7.8 8.3 7.7

0.49 0.54 0.60 0.64 0.60

52.2 49.3 45.1 42.4 45.1

1.6 1.7 2.1 2.8 2.7

0.14 0.14 0.15 0.15 0.18

76.7 76.7 76.0 76.0 73.5

0.06

0.31

0.35

0.49

0.50

0.49

5.9 6.0 6.5 7.5

0.51 0.52 0.54 0.65

51.2 50.5 49.2 41.2

1.8 1.9 1.9 2.6

0.14 0.15 0.15 0.21

76.7 76.0 76.0 71.3

0.32

0.36

0.44

a

IPM, isopropyl myristate; IPP, isopropyl palmitate.

and the autocorrelation function of the residuals as fitting criterion.32 All measurements were performed in thermostatic cells to obtain the desired temperature. The fluorophore was Ru(bpy)3 2+. Its concentration was maintained at 10-5 M. Two series of experiments were performed at two different concentrations of the quencher, Fe(CN)63-, 0.5 × 10-4 and 10-4 M. The analysis of the fluorescence decay profiles was carried out with a model of stretched exponentials given by the following equation:22-25 I(t) ) Io exp(-t/τ0) exp[-C1(t/τ0)f + C2(t/τ0)2f]

(4)

where 0 < f < 1, whereas the first-order quenching rate was calculated by the following equation: K(t) ) 1/τo[fC1(t/τ0)f-1 - 2fC2(t/τ0)2f-1]

(5)

Fitting eq 4 to the experimental decay profile gives the values of the constant parameters C1, C2, and f, which can be used to find K(t) through eq 5. Equation 4 was previously shown to apply to profiles recorded by the photon counting technique (i.e., to noisy data), as it is the present case and it models all quenching reactions, where the excited fluorophore can in principle be quenched by any quencher present.25 Such a reaction is a diffusion-controlled quenching, as in the present case. Diffusion-controlled reactions in restricted geometries are distance dependent, which, in terms of rates, creates time dependence. Thus, K is time dependent. Because it is not practical to tabulate all K values for all time values, we usually choose to tabulate K1, that is, the value of K(t) at the first recorded time channel and the value at the last channel KL. Thus, K1 is the reaction probability at short times and KL at long times. In the case of compartmentalized reactants, K1 is the reaction probability between close-lying reactants and KL is the diffusion-limited rate, which is equivalent to the rate of intercompartment migration of reactants. Finally, the value of the non-integer exponent f can be understood in the following manner. (32) Grinvald, A.; Steinberg, I. Z. Anal. Biochem. 1974, 59, 583.

Diffusion in organized molecular assemblies is successfully modeled by random walk in a percolation cluster, either above or below the percolation threshold. Low f values correspond to non-communicating clusters, for example, isolated, non-interacting reverse micelles. Low f values also correspond to localized (or aggregated) reactants. For example, if the number of reactants is relatively large as compared to the number of micelles, then the probability of coexistence of a fluorescent molecule and a quencher molecule in the same micelle is high. As a consequence, quenching is rapid. This situation is represented by low f values and relatively high K1 values. In Vitro Permeation Studies. The in vitro permeation studies were performed by using a Franz-type diffusion cell.33 The cell had a diffusion surface area of 10 mm and a 5.2 mL cell volume. 1 mL of the lecithin gel containing the dissolved compound (caffeine, theophylline) was placed in the donor compartment of the cell. The acceptor compartment was filled with PBS isotonic buffer solution, pH 7.4, and was constantly stirred with a small magnetic stirring bar. A Visking dialysis tubing 36/32 membrane separated the upper donor compartment and the lower acceptor compartment. Prior to use, the membrane was incubated for 24 h in the PBS isotonic buffer solution. The cells were thermostated at 34 °C. At 2 h time intervals, 0.2 mL of the receptor phase was withdrawn and analyzed by UV spectroscopy to determine the amount of permeated compounds. The concentration of the bioactive compounds was determined spectroscopically by using standard curves. Each sample removed was replaced by equal volume of fresh PBS buffer, and correction for dilution was carried out. Each experiment was carried out in duplicate.

Results and Discussion Microdomain Properties Studies. EPR Probes in Organic SolVents. To determine the behavior of each spin probe used in this work, at first we have monitored their EPR spectra in pure (33) Franz, T. J. Curr. Probl. Dermatol. 1978, 7, 58-68.

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solvents. These solvents were used for the preparation of the gels, and their properties reflect the behavior of the continuous organic phase. Table 1 shows the calculated rotational correlation time values for 5-DSA, 12-DSA, 16-DSA, 12-DMS, and 10-DN in the three solvents examined: isooctane, isopropyl palmitate, and isopropyl myristate. All probes have rotational correlation time values in the fast motion region, for example, τR < 3 × 10-9 s. This indicates that all probes tumble free at all directions (x-, y-, and z-axes), while the z-axis is the long molecular axis that is parallel to the direction of the nitrogen 2p π orbital of the doxyl ring (Scheme 1). Nevertheless, the rotation correlation time values calculated in isooctane are smaller as compared to those obtained in both esters. This can be explained from the higher viscosity of the esters. As it can be seen from Table 1, the highest τR value was observed in the case of 12-DSA and the lowest τR in the case of 16-DSA. This observation indicates that the position of the paramagnetic ring on the fatty acid aliphatic chain, the chemical nature of the probe (fatty acid, ester, hydrocarbon), and also the viscosity of the medium affect the tumbling of the spin probe. Table 1 also shows the hyperfine coupling constant values, RN, of the spin probes measured in pure solvents. The hyperfine coupling constant reflects the polarity of the medium. As it can be observed, the lowest values of RN are obtained in isooctane, while the highest ones are obtained in isopropyl myristate. This finding indicates that the polarity of the solvents examined was in the order: isooctane < isopropyl palmitate < isopropyl myristate. EPR Probes in Binary Systems. The rotation correlation times, τR, of the spin probes obtained in binary lecithin/organic solvent systems are presented in Table 2. These τR values are much higher than the rotation correlation time values obtained in pure organic solvents (Table 1) due to the existence of lecithin layers. The reduction of the mobility as expressed from the τR is more pronounced in the case of DSA’s. The rotation correlation time of 5-DSA in the lecithin/isooctane binary system is about 1260-fold higher than the respective value in the same organic solvent, and the corresponding values are in the slow motion region. The τR of 16-DSA is about 195-fold higher in the lecithin/isooctane system than the respective value in the pure organic solvent. The τR value of 12-DSA in the isooctane/lecithin system is about 17-fold higher, respectively. In the lecithin/isopropyl palmitate and lecithin/isopropyl myristate binary systems, the reduction of the 5-DSA and 16DSA mobility is remarkable concerning their mobility in pure solvents. The τR values of 5-DSA and 16-DSA in binary systems are more than 10-fold higher from the corresponding τR values in isopropyl palmitate and isopropyl myristate. The τR values of 5-DSA in both ester binary systems are in the slow motion region. The reduction of 12-DSA molecule mobility in the binary systems is smaller, as compared to 5-DSA and 16-DSA, but is still remarkable. It is known that lecithin polar heads are very strongly bound with 0.6-1.5 water molecules per lecithin molecule.34,35 As a consequence, lecithin solutions in organic solvents can be considered as microemulsions with wo ≈ 1. When lecithin is dissolved in organic solvents, self-organized aggregates are formed. The lecithin threshold concentration for the formation of aggregates in isooctane was determined in a previous work between 0.2 and 0.9 mM.19 The DSA’s are well-known membrane probes.18 Because of their amphiphilic character, these stearic (34) Giomini, M.; Giulliani, A. M.; Trotta, E.; Boicelli, C. A. Chem. Phys. Lett. 1989, 158, 334-340. (35) Avramiotis, S.; Cazianis, C. T.; Xenakis, A. Prog. Colloid Polym. Sci. 2000, 115, 196-200.

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Figure 1. EPR spectra of 5-DSA in: (a) isopropyl palmitate; (b) lecithin/isopropyl palmitate; (c) lecithin/isopropyl palmitate/water gels at pH 2; (d) lecithin/isopropyl palmitate/water gels at pH 8.5; and (e) lecithin/isopropyl palmitate/water gels at pH 12. wo ) 3.0. Straight line, experiment; dotted line, simulation.

acid probes are preferably located in the lecithin interface, between the oriented lecithin molecules. The spin probe’s mobility is hindered from the presence of the neighbor lecithin hydrophobic tails. The degree of 5-DSA, 12-DSA, and 16-DSA immobilization reflects the rigidity of the interface at different depths. The τR values of 5-DSA, 12-DSA, and 16-DSA obtained in the isooctane/ lecithin binary system were 8.8, 2.4, and 0.4 ns, respectively. In the lecithin/isopropyl palmitate and lecithin/isopropyl myristate binary systems, the τR values of 5-DSA, 12-DSA, and 16-DSA follow the same profile as in the lecithin/isooctane binary system. The observed differences of the spin probes mobility can be attributed to the different position of the nitroxide ring on the fatty acid aliphatic chain, reflecting the rigidity of lecithin monolayer at different depths. Figure 1a,b shows the EPR spectra of 5-DSA in isopropyl palmitate and 10% w/w lecithin/isopropyl palmitate solution. The observed differences in the spectral characteristics between spectra a and b are attributed to the immobilization of the spin probe in the lipid layer. The higher mobility of 12-DMS and 10-DN in all binary systems as compared to the DSA spin probe’s mobility can be attributed to their specific location in these systems. These two nonpolar spin probes are most probably located in the continuous organic phase, which is the major component of the systems. The mobilities of the ester (12-DMS) and the alkane (10-DN) both in lecithin/isopropyl myristate and in lecithin/isopropyl

Microdomain Properties of Lecithin Organogels

palmitate binary systems are not affected by the presence of lecithin. When the same spin probes were added in the lecithin/ isooctane binary system, the τR values increased about 1 order of magnitude. This may be attributed to the increased viscosity of the isooctane after the dilution of lecithin, which indicates stronger intermolecular forces in this system. Table 2 also shows the order parameter S values for the spin probes 5-DSA and 12-DSA in lecithin/organic solvent binary systems. The high S values in the case of 5-DSA confirm the strong immobilization of the probe and thus the tight packing of lecithin molecules in the interface. The corresponding S values in binary systems are the following: S ) 0.76 in lecithin/isooctane, S ) 0.69 in lecithin/isopropyl myristate, and S ) 0.60 in lecithin/ isopropyl palmitate solution. The immobilization of 5-DSA in lecithin binary systems became more perceptible from the evaluation of the half-maximum wobbling amplitude expressed as angle γ. When dissolved in pure organic solvents, 5-DSA molecules tumble free at all three directions. Nevertheless, in the presence of lecithin, 5-DSA molecules are mainly tumbling around their long molecular axis. It was estimated that 5-DSA wobbles at an angle of 34.2° in the lecithin/isooctane system, 38.8° in the lecithin/isopropyl myristate system, and 45.1° in the lecithin/ isopropyl palmitate system. These γ values reflect the rigidity of the membrane from the depth of the membrane where the doxyl-ring of the 5-DSA is located, for example, at the fifth carbon atom. From the order parameter S and the wobbling angle γ, it is apparent that lecithin packing in isooctane is tighter than lecithin packing in the more polar esters considered in this study, isopropyl palmitate and isopropyl myristate. From computer simulations, we found that the 5-DSA EPR spectra recorded in binary systems are the result of superimposed spectra of two marginal species: the immobilized and the nonimmobilized and somehow all of the intermediate situations. In the lecithin/isooctane system, the immobilized fraction is about 53% with A| ) 22.9 and the non-immobilized fraction about 47% with A⊥) 12.0. In the lecithin/isopropyl palmitate system, the immobilized fraction is about 55% with A| ) 22.0 and the non-immobilized about 45% with A⊥ ) 13.4, and in the lecithin/ isopropyl myristate system the immobilized fraction is about 55% with A| ) 22.1 and the non-immobilized about 45% with A⊥ ) 13.1. These results can have the following interpretation. As the aggregates are dynamic self-assembling structures, the above fractions express somehow the equilibrium between immobilized and non-immobilized spin probes, and within experimental error we can say that the degree of immobilization is the same for all systems examined. The calculated S values in the case of 12-DSA presented in Table 2 are much smaller than the respective S values of 5-DSA in the three binary systems studied. This indicates less obstructed spin probe mobility at this specific depth of the membrane, for example, at the twelfth carbon atom. Nevertheless, the calculated value of the order parameter in lecithin/isooctane, S ) 0.20, is slightly higher than the same value calculated in both lecithin/ ester systems, S ) 0.18. Consequently, the resulting wobbling angle γ is 71.9° for the isooctane system and 73.5° for both the isopropyl myristate and isopropyl palmitate systems. This result confirms the assumption that lecithin is more rigidly packed in isooctane. EPR Probes in Gel Systems. We have monitored the EPR spectra of all of the spin probes in three different lecithin-based gel systems. To evaluate the effect of the pH of the aqueous phase on the interfacial properties of lecithin gels, four different pH values were examined: 2, 5.5, 8.5, and 12. Figure 1c-e shows the EPR spectra of 5-DSA in lecithin/isopropyl palmitate/

Langmuir, Vol. 23, No. 8, 2007 4443

Figure 2. EPR spectra of (a) 10-DN; (b) 12-DMS; (c) 16-DSA; (d) 12-DSA; and (e) 5-DSA in lecithin/isopropyl palmitate/water organogels at wo ) 3.0. The pH of the aqueous phase was 5.5. The final concentration of the spin probes in the gels was 10-2 mM. Straight line, experiment; dotted line, simulation.

water gel systems (wo ) 3.0) at three different pH values, pH 2, 8.5, and 12. Table 2 shows the rotational correlation times, τR, the order parameter, S, and the wobbling angle, γ, values calculated in three different gel systems. When the spin probes 16-DSA, 12DMS, and 10-DN were considered, the values obtained at four different pH’s did not alter, so we present only one value in each gel system. Figure 2a-e shows the EPR spectra of the spin probes 10-DN, 12-DMS, 16-DSA, 12-DSA, and 5-DSA obtained in lecithin/ isopropyl palmitate/water (pH 5.5) gels at wo ) 3.0. Let us first focus on the comparison between the τR values obtained in gel systems and the τR values obtained in binary systems. It is surprising that, despite the increased viscosity of the gels, the τR values of the spin probes in gels are lower than the corresponding τR values in binary systems. This behavior clearly shows an increase of the spin probe’s mobility by increasing the water content of the organogels. Particularly, in the case of 5-DSA, the calculated order parameter S was decreased as the water content was increased from wo ≈ 1 (binary system) to wo > 1 (gel systems). When isooctane was used as the organic solvent, the corresponding order parameters decreased from S ) 0.76 in binary systems to S ) 0.51-0.65 in gel systems. In the case of isopropyl myristate, the order parameters decreased from S ) 0.69 in binary systems to S ) 0.49-0.64 in gel systems. Finally, in the case of isopropyl palmitate, the order parameters decreased from S ) 0.60 in binary systems to S ) 0.51-0.54 in gel systems. This must be the result of less tight packing of the lecithin molecules in the gel systems. IR experiments have shown the existence of a hydrogen-bonding network as a result

4444 Langmuir, Vol. 23, No. 8, 2007

of the interactions between water molecules and phosphate groups of lecithin polar heads.36,37 Further addition of water lowers the packing of the lecithin molecules.37 Increasing the water quantity induces an increase of the polar head area per lecithin molecule and, as a consequence, expansion of the interface. Subsequently, the micellar length and the fusing ability of the micelles are also increased. The calculated γ values (Table 2) provide a more geometrical sense of the changes induced on the interface by increasing the water content. For the lecithin/isooctane/water gel system, the wobbling angle of 5-DSA was varied from 52.1° to 43.9°, for the lecithin/IPM/water gel system it was from 52.2° to 42.4°, and for the lecithin/IPP/water gel system it was from 51.2° to 41.2°, depending on the pH of the aqueous phase. These results emphasize the differences of lecithin packing in the three solvents considered, isooctane, isopropyl myristate, and isopropyl palmitate. Depending on the nature of the solvent used, a wide range of viscosities can be obtained.1 This is probably due to the organic molecules penetration between the lecithin lipophilic tails and the induced curvature changes of the lecithin interface.2,38,39 The viscosity of the lecithin/isooctane/water gels is about 2 orders of magnitude higher than the viscosity of the lecithin/ ester/water gels.1,40 The determination of higher average wobbling angles γ of 5-DSA and 12-DSA in systems based on esters can be probably attributed to a greater average negative curvature of the lecithin layer in both ester systems. The formation of smaller and more curved reverse structures in organogels based on isopropyl myristate and isopropyl palmitate is considered. In a previous work, we have examined the mobility of 5-DSA in AOT/isooctane/water and lecithin/isoctane/1-propanol/water microemulsions.35 The behavior of 5-DSA in lecithin/isooctane/ water microemulsion gels examined in the present work is completely different from the behavior of the same spin probe in the microemulsion systems mentioned above. In AOT/ isooctane/water microemulsions, as the water content increased, the mobility of 5-DSA decreased. This behavior was interpreted concerning properties of bulk water. The wo values (wo ) 5-30) obtained in those microemulsion systems are far larger than the wo values (wo ) 1-5) obtained in the lecithin organogels of the present study. At large wo values, bulk water is present and the carboxylic group of 5-DSA can be partially ionized and sank deeper in the microemulsion interface.35 In lecithin organogels, the water does not behave like bulk water. The 2-4 excess water molecules per lecithin polar head probably induce an expansion of lecithin interface, thus reducing lecithin packing and lowering the membrane rigidity. With simple trigonometric calculations ((sin γgel - sin γbin)/ sin γbin), we have calculated this interface expansion from binary systems to gel systems. For the isooctane organogel system, this interface expansion per lecithin molecule was found to be about 40%. In the case of isopropyl myristate, the expansion was about 21% and for isopropyl palmitate about 9%. For the above calculations, γgel values obtained in lecithin organogels with an aqueous buffer phase pH 5.5 were considered. The reported interface expansions could possibly explain the increased viscosities observed in isooctane organogel systems in comparison to those obtained in ester organogel systems. (36) Shchipunov, Y. A.; Shumilina, E. V. Mater. Sci. Eng., C 1995, 3, 43-50. (37) Aliotta, F.; Fontanella, M. E.; Pieruccini, M.; Salvato, G.; Trusso, S.; Vasi, C.; Lecnher, R. E. Colloid Polym. Sci. 2002, 280, 193-202. (38) Angelico, R.; Palazzo, G.; Colafemmina, G.; Cirkel, P. A.; Giustini, M.; Ceglie, A. J. Phys. Chem. 1998, 102, 2883-2889. (39) Shchipunov, Y. A.; Schmiedel, P. Langmuir 1996, 12, 6443-6445. (40) Angelico, R.; Ceglie, A.; Colafemmina, G.; Lopez, F.; Murgia, S.; Olsson, U.; Palazzo, G. Langmuir 2005, 21, 140-148.

AVramiotis et al.

The τR, S, and γ values obtained in gel systems at different pH values (Table 2) demonstrate substantial modification of the EPR spectral characteristics depending on the acidic-basic character of the aqueous phase. In the case of 5-DSA, for instance, when the pH of the aqueous phase in lecithin/isooctane/water organogels was increased from 2 to 12, the rotation correlation time, τR, was increased from 6.3 to 8.3 ns, indicating a more restricted motion of the spin probe. At the same time, the order parameter S was increased from 0.56 to 0.62, reflecting an increased rigidity of the interface. The same monotonic decrease of the 5-DSA’s mobility, as expressed from τR and S values, when the pH value of the aqueous phase increases, appeared in both ester gel systems. Such a trend was also observed for 12-DSA in all gel systems. Interestingly, the maximum wo value obtained in the examined gel systems does not vary in accordance with the pH of the water used. We interpret this finding taking into consideration the zwitterionic polar head of the phosphatidylcholine polar group, N(CH3)3+, and the phosphate group, PO4-. At low pH values, the choline polar group is favored to be closer to the interface, at intermediate pH values, both N(CH3)3+ and PO4- groups are favored, and finally at high pH values, only the phosphate group is favored to take this position. Most likely, the carboxylic group of the DSA’s follows an analogous position alteration depending on the pH of the dispersed water. Spectra of 5-DSA and 12-DSA in gel systems prepared with water from MilliQ system with pH 5.3 had the same characteristics as the spectra monitored in gels where the water used was buffered in pH 5.5. The interest of this finding is the emphasis on the monotonically pH dependence followed by the substantial spectral differences on the 5-DSA spectra in such systems where the actual properties of the water, in the reverse micelle water core, are considered that differentiated strongly from the bulk water. The EPR spectra of 10-DN and 12-DMS obtained in lecithin gels are characteristic of their location in the system. As these two probes are located in the continuous organic phase, which has been trapped between the micellar network, their spectral characteristics are similar and reflect increased mobility (Figure 2a and b). Rotational correlation times, τR, of 10-DN and 12-DMS in organogels are systematically lower than the τR values obtained in binary systems (Table 2). This behavior denotes that the organic phase is less restricted when jellification takes place and there are less oligomeric aggregates or monomers of lecithin in the intra-micellar organic space. Figure 2c-e shows the EPR spectra of 16-DSA, 12-DSA, and 5-DSA, respectively, in the same lecithin/isopropyl palmitate/ water (pH 5.5) gel system at wo ) 3.0. The spectrum of 16-DSA (c) has characteristics similar to those of the spectra of the more lipophilic spin probes 12-DMS and 10-DN. These spectra consist of three peaks. The low and central field peaks are equal, while the high field peak is smaller. This is the result of the fast motion of the nitroxide ring. On the contrary, the EPR spectrum of 12-DSA, Figure 2d, is characterized from three unequal peaks, indicating a more restricted motion. The high field peak of the spectrum is broadened, while a slight splitting of the low field peak can be observed. In the case of 5-DSA, Figure 2e, the significant splitting of both low and high field peaks underlines the strong immobilization of the spin probe in the membrane. From the EPR measurements presented above, we can conclude that all of the spin probes considered in this study tumble free at all directions when solubilized in organic solvents. Nevertheless, the chemical nature and structure of the spin probe as well as the viscosity of the organic solvent affect the spin probe’s

Microdomain Properties of Lecithin Organogels

Figure 3. (A) Stern-Volmer plots and (B) modified Stern-Volmer plots for fluorescence quenching of Ru(bipy)32+ from Fe(CN)63- in lecithin/isopropyl palmitate/water organogels at different wo values. [Ru(bipy)32+] ) 10-5 M. [Q] ) [Fe(CN)63-] from 10-5 to 10-4 M.

rotational motion. When the spin probes are solubilized in binary systems consisting of lecithin and organic solvent, a considerable decrease of the rotational motion can be observed due to the existence of lecithin layers and the formation of self-organized aggregates. The rigidity of the lecithin interface is altered depending on the depth, showing the maximum rigidity close to the surfactants polar head. When small amounts of water are added in lecithin solutions, viscous organogel systems are formed. Increasing the water quantity induces an increase of the polar head area per lecithin molecule and as a consequence expansion of the interface. Subsequently, the membrane rigidity is lowered, and the fusing ability of the micelles is increased. Smaller and more curved reverse structures are formed when esters are used as the continuous organic phase instead of isooctane. In lecithin organogels, the water molecules are tightly bound on the lecithin’s polar groups. On the other hand, the organic solvent trapped between the micellar networks is not affected by the presence of the reverse structures as reflected by the mobility of the lipophilic spin probes. Steady-State Fluorescence Quenching. The nature of the water entrapped in the interior of the reverse micellar structures of lecithin organogels was examined via the fluorescence quenching technique. The water-soluble compounds Ru(bipy)32+ and Fe(CN)63- were used as fluorophore and quencher, respectively. When the lecithin/isooctane/water gel system was considered, the fluorescence quenching reaction was not possible to be monitored. This finding is in agreement with previous experimental results concerning the fluorescence quenching of Ru(bipy)32+ from Fe(CN)63- in lecithin/alcohol/isooctane/water microemulsions at low wo values.41 In these systems, the reactants were either aggregated and thus no fluorescence occurred or the fluorescence quenching had an anomalous dependence on stirring. It seems that the water molecules, in these specific lecithinbased reverse micellar structures, are very strongly bound on the (41) Avramiotis, S.; Bekiari, V.; Lianos, P.; Xenakis, A. J. Colloid Interface Sci. 1997, 194, 326-331.

Langmuir, Vol. 23, No. 8, 2007 4445

Figure 4. Arrhenius plots of fluorescence quenching reaction rate K1 in lecithin/isopropyl palmitate/water organogels at different wo values. [Ru(bipy)32+] ) 10-5 M, [Fe(CN)63-] ) 10-4 M.

phospholipids polar heads, not allowing the occurrence of the quenching reaction. When the fluorescence quenching reaction was monitored in lecithin/isopropyl palmitate/water gels, analyzable experimental results were obtained. Figure 3A shows the Stern-Volmer plots of Fo/Fi as a function of quencher concentration obtained in lecithin/isopropyl palmitate/water organogels at different water content as expressed by wo. The wo values considered in this study were 2.1, 3.0, and 3.7, respectively. In all of the examined cases, the plots gave straight lines. This behavior is characteristic of a homogeneous environment and not of the restricted environment provided by these structures. Curved lines with an upward curvature characterize the normal behavior for a restricted environment, that is, compartmentalized reaction space.20,21 Because both reactants, fluorophore and quencher, are insoluble in isopropyl palmitate and soluble in water, we interpret the linearity of the Stern-Volmer plots as a result of the reactants aggregation in the microdomain of the bound water of the reverse micelles, presenting the same kinetics as in homogeneous systems. Besides, in isopropyl palmitate organogels, the water molecules seem less immobilized than in the case of isooctane organogels, and the slopes of the fitting lines obtained are monotonically dependent on the wo values (Figure 3A). That means that fluorescence quenching is more efficient as the water content of the gels is increased. To follow the distribution of the reactants in lecithin/isopropyl palmitate/water organogels, we interpret the above steady-state fluorescence quenching data according to a modification of the Stern-Volmer equation.42 Assuming that there are two populations of the fluorophore, one accessible from the quencher molecules Fo,a and the other not accessible Fo,b, Fo can be presented as Fo ) Fo,a + Fo,b. The presence of the quencher molecules causes a decrease of the fluorescence given from the Stern-Volmer equation:

F ) (Fo,a/1 + K[Q]) + Fo,b (42) Lehrer, S. S. Biochemistry 1971, 10, 3254-3263.

4446 Langmuir, Vol. 23, No. 8, 2007

AVramiotis et al.

Table 3. Values of K1, KL, and f for the Lecithin/Isopropyl Palmitate/Water Organogels at wo ) 2.1, 3.0, and 3.7, Obtained by Analyzing Time-Resolved Luminescence Quenching Profiles of 10-5 M Ru(bipy)32+ in the Presence of 5 × 10-5 and 10-4 M Fe(CN)63-, According to Equation 5 at 298, 310, 320, and 330 Ka

wo 2.1 3.0 3.7 a

[quencher] M 5 × 10-5 10-4 5 × 10-5 10-4 5 × 10-5 10-4

298 Kτ ) 332 ns K1 KL f 139 186 70 78 62 81

0 0 0 0 0 0

0.39 0.39 0.37 0.37 0.38 0.33

K1

310 K τ ) 310 ns KL

f

195 207 86 102 73 85

0 0 0 0 0 0

0.36 0.37 0.38 0.37 0.38 0.36

K1

320 K τ ) 292 ns KL

f

199 217 107 121 84 87

0 0 0 0 0 0

0.34 0.34 0.38 0.34 0.46 0.46

K1

330 K τ ) 233 ns KL

f

205 253 116 142 146 155

0 0 0 0 0 0

0.44 0.49 0.48 0.49 0.42 0.46

K1, KL (106 s-1).

From this expression, the following relationship can be extracted:

Fo/∆F ) (1/fK[Q]) + 1/f where ∆F ) Fo - Fi and f ) Fo,a/(Fo,a + Fo,b). The distribution of the reactants in the gel systems was calculated from the plots of Fo/∆F as a function of 1/[Q] (Figure 3B). From the reciprocals, we estimate that for the systems with wo ) 2.1 and 3.0, about 82% of the fluorophore is accessible to the quencher molecules, while for the system with wo ) 3.7, this accessibility rise to 90%. Because the fluorescence quenching reaction occurs in lecithin/ isopropyl palmitate/water organogels showing high mutual accessibilities of the reactant species, the corresponding fluorescence decay profiles could be further analyzed with a model of stretched exponentials to monitor the dynamics of the dispersed water. Fluorescence Quenching Decay Measurements. To investigate the dynamics of the water entrapped in lecithin/isopropyl palmitate/water organogels, we followed the quenching reaction between the fluorophore Ru(bipy)32+ and the quencher Fe(CN)63at different wo values and temperatures. The concentration of Ru(bipy)32+ was kept constant, 10-5 M, throughout the experiment, while two different concentrations of Fe(CN)63- were considered, 5 × 10-5 and 10-4 M. The wo values considered in this study were 2.1, 3.0, and 3.7, respectively. The temperatures studied were 298, 310, 320, and 330 K. Table 3 shows the effect of wo, temperature, and quencher concentration on the reaction kinetics as expressed by K1, KL, and f values. In the sense that f is a measure of the restrictions imposed on the fluorescence quenching reactants by the reaction domain, its values are in all cases low. In addition, f values do not alter when wo increases, within experimental error and in the narrow range of wo obtained in such systems. At high temperature, 330 K, f values increase, indicating a less restricted environment of the quenching reaction but still below the percolation state. The term “percolation” refers to the dimensionality of the reaction space as it is sensed by the reactants in the time scale from 350 to 230 ns (the fluorophore excited state) and not to the actual dimensionality of the physicochemical system of the reverse micelles. An increase of the f values can be observed even at lower temperature, 320 K, in the system with the highest water content (wo ) 3.7), indicating the existence of a less restricted domain. Table 3 also presents the quenching rate at the beginning of the reaction and the quenching rate at long time as expressed by K1 and KL, respectively. In all cases, KL is equal to zero excluding percolation phenomena even at the highest temperature tested during the lifetime of the fluorophore’s excited state. In all cases examined, the fluorophore and the quencher are solubilized in the water pockets, and the quenching reaction appears to be topical with restricted water diffusion.

The high K1 values obtained at all wo values considered in this study indicate increasing reaction probability at short times and consequently a high degree of restricted movement of the reactants. High values of K1 correspond to localized interactions. Nevertheless, the K1 values decrease appreciably as the water content varies from wo ) 2.1 to 3.0 and 3.7, clearly indicating an expansion of the reaction domain. In parallel, as the temperature increases, the reaction rate K1 also increases, showing a considerable temperature sensibility of the organogels considered in this study. In particular, a considerable increase of the K1 values is observed in the system with the highest water content (wo ) 3.7) and for the highest temperature tested (330 K). From the experimental data mentioned above (Table 3), Arrhenius plots were obtained using the K1 values calculated at the higher quencher concentration, 10-4 M, for wo ) 2.1, 3.0, and 3.7, respectively. The slope of the Arrhenius plots in each system gives the corresponding activation energy, which is the energy barrier that the reactants overcome to achieve higher states of motion. Slope changes possibly indicate phase transition or micellar reorganization. The Arrhenius plots of Figure 4 gave straight lines for wo ) 2.1 and wo ) 3.0, whereas a slope change can be observed for the system with the highest wo value (wo ) 3.7). The activation energy values calculated for the systems with wo ) 2.1 and wo ) 3.0 were 7.4 and 15.1 kJ/mol, respectively, indicating a more restricted motion of the reactants. Such a behavior was expected, as the water content of these organogel systems is relatively low. When the water content was increased (wo ) 3.7), the activation energy was 2.6 kJ/mol over the range of 298-320 K followed by a dramatic increase to 48.0 kJ/mol over the range of 320-330 K. This increase indicates a transition of the reactants mobility possibly due to a different state of water molecules present in this system. The same activation energy profiles can be obtained using the K1 values obtained with the lower quencher concentration, 5 × 10-5 M (data not shown). These findings provide useful information concerning the dynamics of the water molecules in lecithin organogels. The rate of inter- and/or intra-micellar exchange of water must be very slow because these molecules appear quite immobilized close to the lecithin polar heads, as was also reported by Angelico et al.43 These results can be extrapolated to the exchange rate of lecithin molecules considering that the strongly bounded water molecules accompany any inter- or intra-micellar migration of phospholipids on the lecithin polar heads. In Vitro Permeation Studies. The main aim of the present work was to use the spectroscopic techniques of EPR and fluorescence quenching to investigate some structural characteristics of lecithin-based organogels. The interfacial properties of the lecithin layer as well as the nature of the entrapped water (43) Angelico, R.; Balinov, B.; Ceglie, A.; Olsson, U.; Palazzo, G.; So¨derman, O. Langmuir 1999, 15, 1679-1684.

Microdomain Properties of Lecithin Organogels

Figure 5. Effect of gel composition on the transfer yield of caffeine. Gel composition: (9) 10% w/w lecithin in isopropyl palmitate, (0) 10% w/w lecithin in isopropyl myristate. wo ) 3.5.

Figure 6. Effect of gel composition on the transfer yield of theophylline. Gel composition: (9) 10% w/w lecithin in isopropyl palmitate, (0) 10% w/w lecithin in isopropyl myristate. wo ) 3.5.

were evaluated. These dynamic studies were then considered for the formulation of appropriate organogels to serve as carriers of bioactive molecules for topical administration. From the EPR studies, it was concluded that increasing the water quantity of the gels induces a decrease of the membrane rigidity, and, as a consequence, the fusing ability of the micelles is increased. Static and time-resolved fluorescence quenching spectroscopy enabled the evaluation of the interconnection of the aqueous droplets and also the dynamics of the entrapped water. From these studies, it was clear that the use of isopropyl esters as the organic solvent was preferable because in the case of isooctane the interconnection of the aqueous droplets was not verified. In addition, it was obvious that the higher was the water content of the system, the more efficient were the interconnection of the aqueous domains and the consequent exchange of the reactants. To conclude, lecithin organogels to be used as carriers of bioactive molecules should be preferably formulated using isopropyl esters as the organic solvent, isopropyl palmitate and isopropyl myristate. In addition, the water content of the system

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must be the highest possible. Although the structural studies clearly showed that the best communication of the droplets was achieved at wo ) 3.7, in practice a slightly lower value, wo ) 3.5, was preferred. This was due to stability reasons because the organogels are affected by the humidity of the atmosphere during the in vitro permeation study and destabilize after a certain period of time. The pH of the dispersed aqueous phase was kept constant at 5.5 throughout the permeation study to simulate the pH of the skin. To evaluate the ability of these specific lecithin organogels to serve as carriers of bioactive molecules for topical administration, in vitro permeation studies were carried out using the Franztype diffusion cells and a synthetic model membrane. Lecithin organogels containing either caffeine or theophylline were prepared. Initial concentrations of caffeine and theophylline were 0.76 and 0.34 mM, respectively. These are the highest concentrations of the respective compounds that can be achieved in lecithin organogels. The permeated amounts of caffeine and theophylline were calculated by using the following standard curves: y ) 10.197x, R2 ) 0.999 for caffeine, and y ) 8.303x, R2 ) 0.999 for theophylline. Figures 5 and 6show the permeation profiles of caffeine and theophylline, respectively, in both lecithin/ isopropyl palmitate- and lecithin/isopropyl myristate-based organogels for a time period of 24 h. Both organogel systems exhibited similar transfer yields for each compound tested. As we can observe (Figure 5), 20% of caffeine was permeated after 24 h regardless of the nature of the oil used for the formulation of the gels. In the case of theophylline, almost 35% of the compound permeated the membrane after 24 h (Figure 6). The higher transfer yield of theophylline can be possibly attributed to the smaller size of the molecule in comparison to the molecule of caffeine. Nevertheless, both compounds gave satisfactory transfer yields in both lecithin-based organogels.

Conclusion Organogels can be obtained by adding small amounts of water to a solution of lecithin in organic solvents such as isooctane and the isopropyl esters of palmitic and myristic acids. By applying EPR spectroscopy, the dynamic structure of the surfactant monolayer and the continuous oil phase of lecithin organogels were monitored, showing that the water added induces an expansion of the total interface. The membrane rigidity was decreased, and the fusing ability of the reverse micelles increased. Although the nature of the organic solvent affects the size and the shape of the reverse structures, the solvent itself is not affected by the presence of the micellar network. Static and time-resolved fluorescence quenching spectroscopy measurements showed that the dispersed structures were interconnected dynamically. Lecithin organogels formulated according to the results of the dynamic studies can be used as novel media for the transdermal transport of compounds with medicinal or cosmetic interest such as caffeine and theophylline. LA0634995