In vitro permeation of diclofenac salts from lyotropic liquid crystalline systems

Colloids and Surfaces B: Biointerfaces 78 (2010) 185–192

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In vitro permeation of diclofenac salts from lyotropic liquid crystalline systems Doron Yariv 1 , Rivka Efrat, Dima Libster, Abraham Aserin, Nissim Garti ∗ Casali Institute of Applied Chemistry, The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel

a r t i c l e

i n f o

Article history: Received 16 February 2010 Received in revised form 24 February 2010 Accepted 26 February 2010 Available online 4 March 2010 Keywords: Diclofenac Lamellar mesophase Cubic mesophase Monoolein Transdermal delivery Drug delivery

a b s t r a c t In this paper we examined feasible correlations between the structure of different lyotropic mesophases and transdermal administration of three diclofenac derivatives with varying degrees of kosmotropic or chaotropic properties, solubilized within the mesophases. It was found that the most chaotropic derivative of diclofenac diethyl amine (DEA-DFC) interacted with the polar heads of glycerol monooleate (GMO), thus expanding the water–lipid interface of the lamellar and cubic mesophases. This effect was detected by an increase in the lattice parameter of both mesophases, enhanced elastic properties, and increased solid-like response of the systems in the presence of DEA. Potassium diclofenac (K-DFC), a less chaotropic salt, had less pronounced effect on the structural features of the mesophases. Kosmotropic Na+ salt (Na-DFC) had only minor influence on both lamellar and cubic structures. The locus of solubilization of the molecules with the host mesophases was correlated with their delivery. It was suggested that transdermal delivery of kosmotropic Na-DFC was accelerated by the aqueous phase and less constrained by the interaction with monoglyceride. On the other hand, the chaotropic cations (K+ and DEA+ ), presumably entrapped in the water–lipid interface, interacted with monoglyceride headgroups, which is likely to be the key cause for their sustained administration. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The major benefits of transdermal delivery are its relatively easier accessibility to the skin, assisting in patient compliance, avoidance of the gastrointestinal tract, and the ability to achieve sustained release. These advantages have led to considerable advancement in transdermal administration development over the last decade [1]. The transdermal route is considered a promising approach to advance the delivery of drugs and minimize side effects and first-pass metabolism [2]. However, the skin has evolved to be a highly effective barrier around the human body. The properties of the skin barrier are derived from the outermost layer of the skin, the stratum corneum (SC). The SC consists of 10–15 layers of corneocytes and varies in thickness from approximately 10–15 ␮m in the dry state to about 40 ␮m when hydrated. The limitations and the difficulty in crossing the barrier of the SC by various molecules can be addressed and partially overcome via the skin application of penetration enhancers and various colloidal carriers that increase the diffusion coefficient of the drug through the SC. Colloidal drug carriers, and in particular lyotropic liquid crystals (LLC), seem to be promising candidates as transdermal delivery

∗ Corresponding author. Tel.: +972 2 658 6574/5; fax: +972 2 652 0262. E-mail address: [email protected] (N. Garti). 1 The results presented in this manuscript will appear in the thesis of D.Y. in partial fulfillment of the requirements for the M.Sc. degree in Applied Chemistry. 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.02.029

means for various pharmaceuticals. Such vehicles can provide enhanced drug solubility, relative protection of the solubilized drugs, and controlled release of drugs, thus avoiding substantial side effects [3–6]. In addition, it was recently demonstrated that better bio-distribution of drug pharmaceutical molecules can be achieved with such LC vehicles [7–10]. The potential of utilizing LLC as efficient transdermal delivery vehicles was elucidated in our lab. Using reverse hexagonal mesophases (HII ), we have demonstrated that small peptide drugs, represented by the lipophilic peptide cyclosporin A (CSA) [11,12] and the hydrophilic peptide desmopressin [13,14], could be solubilized within the HII mesophase. Moreover, it was demonstrated by Franz diffusion cell measurements that the hexagonal mesophases had a great potential for a sustained transdermal delivery of desmopressin [13,14] and high transdermal permeation of CSA [15]. Also studied in our lab were the effects of a model electrolytic drug diclofenac on intermolecular interactions, conformational changes, and phase transitions in structured discontinuous cubic QL lyotropic liquid crystals [16,17]. In our previous reports, transdermal permeation of sodium diclofenac was studied via various lyotropic mesophase, revealing the critical role of the carriers’ nanostructure on the diffusion profiles [16,18]. In the current work, we examined the impact of three salt derivatives of diclofenac (Na+ , K+ , and DEA+ ) with varying kosmotropic/chaotropic properties on structural features of different lyotropic mesophases and transdermal release profiles from these carriers. Our main objective was to find out whether the kos-

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motropic or chaotropic nature of these molecules can be correlated with their transdermal releases profiles.

Calculations of the in vitro data were performed as detailed in [18].

2. Experimental

2.2.2. Rheological experiments Oscillatory shear experiments were performed on a Rheoscope 1 rheometer (Thermo-Haake, Karlsruhe, Germany). The rheometer was a thermostatically controlled (Thermo-Haake C25P) at 25 ± 0.5 ◦ C. A cone-plate sensor with a diameter of 35 mm, cone angle of 1◦ , and a gap of 0.024 mm was used. The sample quantity was ca. 0.2 g, a sinusoidal stress was applied to the sample and the induced response was measured (strain). After identification of the linear viscoelastic region (data not shown), samples were investigated over a frequency of 0.01–100 Hz. All measurements were performed in triplicate. The viscoelasticity of the samples was characterized in terms of the elastic modulus G , and complex viscosity (*).

2.1. Materials and methods 2.1.1. Materials Sodium diclofenac (sodium salt of 2-(2,6-dichloroanilino) phenylacetic acid, Na-DFC) was purchased from Sigma (St. Louis, MO, USA), diethylamine diclofenac (diethylamine salt of 2(2,6-dichloroanilino) phenylacetic acid, DEA-DFC) and potassium diclofenac (potassium salt of 2-(2,6-dichloroanilino) phenylacetic acid, K-DFC) were purchased from Amplachem (Carmel, IN, USA). High-performance liquid chromatography (HPLC) grade solvents (water, acetonitrile) were obtained from J.T. Baker (Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA) and Merck (Darmstadt, Germany). Monoolein (distilled glycerol monooleate, GMO) that consists of 97.1 wt% monoglyceride, 2.5 wt% diglyceride, and 0.4 wt% free glycerol (acid value 1.2, iodine value 68.0, melting point 37 ◦ C) was obtained from Riken Vitamin Co. Ltd. (Tokyo, Japan). Ethanol (EtOH; analytical reagent >99%) was purchased from Frutarom Ltd. (Haifa, Israel). Triflouroacetic acid (TFA) was purchased from Fluka (Buchs, Switzerland). Water was double distilled. Phosphatebuffered saline (PBS) was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). All components were used without any further purification. 2.1.2. Preparation of liquid crystals Mixtures of GMO/EtOH/water were prepared in culture tubes sealed with Viton-lined screw caps in predetermined weight ratios, stirred by vortex, heated to 60 ◦ C for 2 min, and allowed to equilibrate at 25 ± 0.5 ◦ C for at least 24 h before they were examined. The diclofenac salts were dissolved in the water/EtOH mixture for the preparation of the DFC-loaded samples. 2.1.3. In vitro skin permeation study The permeability of DFC through porcine skin was determined in vitro with a Franz diffusion cell system (PermeGear, Inc., Hellertown, PA, USA). The porcine skin was excised from ears of slaughtered white pigs (locally grown in the Institute of Animal Research, Kibbutz Lahav, Israel) carefully dissected and dermatomized, stored at −20 ◦ C, and used within a month. Before the experiments, the skin was thawed and mounted on Franz cells (diffusion area of 0.635 cm2 ) with the stratum corneum facing the donor compartment. The receptor compartment was filled with PBS (pH 7.2). The receptor phase was kept under constant stirring at 37 ± 0.5 ◦ C. 500 mg of the liquid crystalline formulations containing 1 wt% DFC salt were applied to the surface of the stratum corneum. 2.2. Analytical method 2.2.1. High performance liquid chromatography (HPLC) DFC content in the samples was determined by highperformance liquid chromatography (Waters 600 series, Milford, MA, USA) and autosampler (Waters 717plus) equipped with photodiode array detector (Waters 996). Isocratic elution was carried out with 35% acetonitrile and 65% trifluoroacetic acid aqueous solution (0.1%, w/v). The wavelength for UV detection was 275 nm. The column used was Luna 5 ␮m, C18, 250 mm × 4.6 mm (Phenomenex, Torrance, CA, USA). The experiments were performed at ambient temperature at a flow rate of 1 mL/min. The injection volume was 20 ␮L. Retention time of the drug was 10 min.

2.2.3. Synchrotron small angle X-ray scattering (SAXS) Synchrotron small angle X-ray scattering (SAXS) experiments were performed using two bending magnet sources, ESRF beamline BM26B (Grenoble), and EMBL beamline X33 (Hamburg), and an undulator source, Soleil beamline SWING (Paris), in six different runs, using 2D multi-wire gas-filled detectors (133 mm × 133 mm)—a 2D MAR345 image plate (345 mm2 ) with online readout, and a 2D PCCD (170 mm × 170 mm) detector (Aviex). The beam sizes were 600 ␮m × 600 ␮m, 500 ␮m × 500 ␮m, and 450 ␮m × 80 ␮m FWHM in the experimental hutch. X-ray scattering patterns were reproducible for different samples from different preparations on different runs. For all sources, the beam was monochromatic using a Ge (1,1,1) crystal and the X-ray photon energy was kept at 9 keV. Samples were scanned for 10 min at BM26B and X33 beamlines and for about 1–5 s at SWING beamline, during which no sample damage was detected. The sample-todetector distance was determined in each setup using silver behenate as a standard sample. Samples were not oriented, thus SAXS scans collected on a 2D detector exhibited a powder pattern and were radially averaged. Intensity as a function of momentum transfer vector, q, was plotted. The components of q are (qx , qy , qz ) in Cartesian coordinates or (q sin  cos , q sin  sin , q cos ) in polar coordinates, as we will use later. 2.2.4. Cryogenic-transmission electron microscopy (cryo-TEM) Samples were equilibrated at 25 ◦ C either in the Controlled Environment Vitrification System (CEVS) or in the Vitrobot (FEI) for 20 min, in the presence of water and EtOH to avoid evaporation of volatile components during specimen preparation. Vitrified specimens were prepared on 400 mesh copper grid coated with a perforated formvar film (Ted Pella). A small drop (5–8 ␮L) was applied to the grid and blotted with filter paper to form a thin liquid film of solution. The blotted sample was immediately plunged into liquid ethane at its freezing point (−196 ◦ C). The procedure was performed manually in the CEVS, and automatically in the Vitrobot. The vitrified specimens were transferred into liquid nitrogen for storage. Some samples were examined in a Philips CM120 transmission electron microscope, operated at 120 kV, using an Oxford 3500 cryo-holder maintained below −178 ◦ C. Images were recorded on a Gatan 791 MultiScan cooled charge-coupled device (CCD) camera. Other samples were studied using a Philips Tecnai 12 G2 TEM, at 120 kV with a Gatan cryo-holder maintained below −173 ◦ C, and images were recorded on an Ultrascan 1000 2k × 2k CCD camera. In both microscopes, images were recorded with the Digital Micrograph software package, at low dose conditions, to minimize electron beam radiation damage. Brightness and contrast enhancement were done using the Adobe Photoshop 7.0 ME package.

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3. Results and discussion Three different salts of diclofenac (Na+ , K+ , and DEA+ ) were chosen to examine the possibility of controlling the transdermal administration of this drug. It was supposed that the different physicochemical performances of the salts can potentially influence the permeability through the skin. The main difference in the expected impact of these derivatives on the LLC is their structural behavior according to the kosmotropic or chaotropic characteristics. Kosmotropic (water-structure makers) and chaotropic (waterstructure breakers) solutes have a very significant impact on the properties of liquid crystalline mesophases by indirect (Hofmeister) interactions with these structures. It was shown that kosmotropic solutes stabilize the structure of bulk water [19–21]. These ions are normally incorporated within the bulk water and excluded from the interfacial areas. Water structures (kosmotropes) interfere with the tetrahedral network of water and, as a result, dehydration of the surfactant polar heads takes place (“salting-out” effect) and the amount of interfacial water is decreased. While the sodium ion of diclofenac is a kosmotropic solute, the behavior of K+ and DEA ions in water solutions is generally accepted as chaotropic [22,23]. However, it should be noted that the kosmotropic/chaotropic properties of the diclofenac salts also strongly depend on the diclofenac molecule itself (the anion). Therefore, the overall impact of the diclofenac salts is a function of a delicate balance between the anion (diclofenac molecule in our case) and the explored cations. In the current study three lyotropic mesophases with different structures, composed of glycerol monooleate (GMO), ethanol (EtOH), and water were chosen for solubilization of the diclofenac derivatives. The phase diagram of the ternary GMO/EtOH/water system (Fig. 1) was earlier presented and extensively investigated by Efrat et al. [16]. Three compositions (water/GMO ratios 2/7, 5/4, 7/2) with a constant concentration of EtOH (10 wt%) and increasing water/GMO ratio were chosen for incorporation of the drugs (Fig. 1). Concerning the unloaded systems, it should be noted that while the composition denoted 2/7 appeared to be a homogeneous formulation, the formulations denoted by 5/4 and 7/2 were biphasic solutions. However, all three systems loaded with diclofenac salts developed as homogeneous systems and were characterized by SAXS and rheological techniques to explore the embedment effects of the guest molecules on microscopic and macroscopic scales, respectively.

3.1. Formulation 2/7 (water/GMO ratio: 2/7) The structural modifications caused by solubilization of the diclofenac derivatives into the lamellar mesophase were examined by different techniques. SAXS diffraction pattern of lamellar mesophases (Fig. 2) is characterized by scattering peaks, in positional ratio of q, 2q, 3q, etc. (q – scattering vector) and the position of the first order Bragg peaks corresponds to q = 2п/d [24]. The interlamellar spacing (lattice parameter) between adjacent bilayers of the lamellar structures was calculated and summarized in Table 1. A significant increase in the lattice parameter took place in the

Fig. 1. Phase diagram of the GMO/EtOH/water ternary system at 25 ◦ C. The phase boundaries of the one-phase regions are drawn with solid lines. The phases indicated are lamellar phase (L␣ ), bicontinuous reverse cubic phase (V), and three isotropic phases: micellar isotropic phase (L), sponge phase (L3 ), and the QL phase. The marked formulations contain constant 10 wt% ethanol and they lay in a parallel line starting at 9:1 water/GMO and are diluted with water.

Fig. 2. X-ray diffraction profiles of samples from point 2/7 with Na-DFC, K-DFC, DEA-DFC, and without DFC.

presence of DEA-DFC, from 42 Å in the empty system to 48 Å in the loaded one. It depicts the chaotropic behavior of DEA+ , which probably increased the concentration of free water molecules that can form hydrogen bonds with GMO, promoting the hydration of the surfactant layer. Hence, DEA+ tends to expand the water lipid interphase of the lamellar structure as compared to the DEA-free system. Two other cations, K+ and Na+ , did not seem to affect the dimensions of the unit cells of the mesophase (lattice parameter). K+ is less chaotropic than DEA+ and probably its relatively low ion concentration was insufficient to induce significant structural alterations. We assume that kosmotropic influence of Na+ on the lamellar system was also not reflected on the lattice parameter because of its relatively low ion content. We did not use higher concentra-

Table 1 Results from SAXS and rheological experiments performed on samples of formulation 2/7 (lamellar mesophase) with Na-DFC, K-DFC, DEA-DFC, and empty system (without DFC). Lamellar mesophases

Empty system (without DFC)

Na-DFC

K-DFC

DEA-DFC

Lattice parameter (Å) S (Pa s2 /rad) m

42.2 1875 −0.93

42.4 733 −0.86

43.6 512 −0.85

48.5 36,022 −1.01

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its morphology possesses fractal characteristics. Indeed, lyotropic lamellar mesophases were shown to exhibit fractal structure when fractal analysis of the birefringence patterns observed in optical polarized microscopy structure was applied [28]. In principle, such properties can potentially enhance drug delivery through the large surface area of these carriers, owing to the fractal nature of the surface. The incorporation of DEA into the lamellar system caused a significant increase in both storage and loss moduli (Fig. 3B). Moreover, contrary to other lamellar systems examined, a sharp decrease in the loss modulus values was detected, while no changes were observed in the behavior of the elastic one. These modifications may be assigned to strong interactions between the flowing units, which in the case of lamellar phase are large planar lamellar domains. The complex viscosity (*) of the samples showed no plateau and it decreased sharply with increasing frequency. The dynamic rheology of the weak gel-like behavior described above is characteristic of the planar lamellar phase [29]. Such frequency dependence of the storage and loss moduli was explained by Jones and McLeish [30], who introduced a slip-plane theory describing relaxation in weak solids. The applicability of this theory was investigated and proved in the case of the lamellar linear viscoelastic region [31]. The frequency dependence of complex viscosity was analyzed in terms of a power law model (Eq. (1)) ∗ = Sωm

Fig. 3. Dynamic frequency sweep test for samples from point 2/7: (A) Na-DFC and K-DFC, (B) DEA-DFC, and empty system.

tions of these drugs in order to stay within accepted therapeutic range. Frequency-dependent rheological measurements were performed to characterize the viscoelasticity of the lamellar phases. Such analysis is particularly useful to establish the correlation between the microscopic and macroscopic organizations of these systems. Furthermore, in transdermal delivery, viscoelastic properties of liquid crystals are important for proper adhesion to the skin and the ability to penetrate into the micro-fissures [25]. Rheological characterization was performed by determination of the storage modulus (G (ω)), loss modulus (G (ω)), and the complex viscosity *, as a function of frequency. The storage moduli G (ω) and the loss moduli G (ω) were plotted against the frequency of the applied oscillations (ω) (Fig. 3). The behavior of the lamellar phase loaded with K-DFC and Na-DFC is depicted in Fig. 3A. Elastic modulus (G ) is higher by about one order of magnitude over the viscous modulus (G ) in the whole examined frequency range. Both moduli are moderately dependable on the angular frequency, starting from 1 rad/s. While the storage modulus has a weak dependence on the applied frequency, the loss modulus shows a minimum. It should be noted that a lamellar mesoscopic structure consists of polycrystalline, randomly aligned domains composed of flexible bilayers. Hence, from a macroscopic point of view it looks disoriented, with structural defects [26]. The occurrence of such a minimum in lost modulus indicates formation of an elastic network as a consequence of strong interactions developing within and among the monodomains. For frequencies higher than 7 s−1 the elastic modulus scales as: G (ω) ≈ ωn with n values of 0.28 for empty system, 0.19 for K+ , and 0.25 for Na+ . This power law behavior with the relaxation exponent n suggests that the mesophase structure is self-similar over a wide range of length scales [27] and

(1)

where S is the gel strength parameter, which depends on the strength of intermolecular interactions and m is the complex viscosity relaxation exponent. Values of m close to zero indicate a liquid-like behavior, while values of m approaching −1 suggest a solid-like response of the system (Table 1). It was observed that incorporation of sodium and potassium salts induced more liquid-like behavior of the mesophases, according to the lower S and m absolute values (Table 1, Fig. 3). Bearing in mind that the lattice parameters of these mesophases stayed almost intact following the incorporation of the salts, it can be inferred that the major impact of both derivatives is mostly reflected by a weakening of intermolecular interactions in the water–lipid interface. Presumably this effect was achieved by the diclofenac molecule itself (the anion) rather than the cations. It seems that kosmotropic (in the case of sodium) or weak chaotropic (in the case of potassium) influence was negligible, since opposite structural effects could be expected otherwise. On the other hand, in the presence of DEA the obtained increase in the S values (Table 1) and increased m values suggested enhancement of the intermolecular interactions and more pronounced solid-like response of the systems. Thus, the major effects of the entrapment of DEA on the macroscopic rheological properties of the systems are greater elasticity and increased solid-like response. These findings are well correlated with microscopic picture of the lamellar mesophase, as reflected by SAXS measurements. Probably, the increase in the lattice parameter in the presence of DEA is the main reason for enhancement of elastic characteristics. It is well recognized that the major rheological properties of LLC depend primarily on the topology of the water–lipid interface [32]. As a consequence, it can be suggested that enlarged GMO–water interface induced by the strong chaotropic influence of DEA-DFC is responsible for the transformation into more solid-like behavior. 3.2. Formulation 5/4 (water/GMO ratio: 5/4) Polarized optical microscopy observations of three compositions loaded with diclofenac derivatives did not reveal any image, suggesting 3D isotropic organization of the mesophases. The SAXS patterns of these samples displaying peaks at q = 0.151, 0.170, 0.287, √ √ √ √ 0.312 Å−1 (Fig. 4). The ratios of the d-spacing are 2: 3: 8: 9,

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Fig. 4. X-ray diffraction profiles of samples from point 5/4 with Na-DFC, K-DFC, and DEA-DFC.

indicating on bicontinuous cubic symmetry, but the determination of the exact space group was practically impossible because of the absence of additional required reflections. Apparently, the main reason for the absence of several reflections of cubic symmetry is the relatively low packing order of the systems, which is attributed to relative high concentration of EtOH in these samples. Although EtOH allows the existence of bicontinuous cubic organization, it is known as a hydrotrope, which has been shown to disrupt liquid crystalline structures and/or induce transitions into discontinuous mesophase [16,18]. The lattice parameters for Na+ , K+ , and DEA-DFC were calculated to be 128, 136, and 144 Å, respectively (Table 2). Analogous to the SAXS results obtained with the lamellar mesophase, the chaotropic solutes (K+ and DEA+ ) triggered swelling of the lattice, compared to the kosmotropic Na+ . The more marked effect of DEA+ on the lattice was assigned to its higher chaotropic nature, increasing the hydration of the lipid polar head layer, and hence swelling GMO–aqueous phase interface. In order to elucidate how the mentioned microscopic modifications influenced the cubic structure on a macroscopic scale, rheological characterization of the mesophases was utilized. As depicted in Fig. 5, at low frequencies the cubic systems loaded with the drug were found to be more viscous than elastic (G > G ). The viscous regime, at low frequencies, is a characteristic property of viscoelastic fluids. With an increase in angular frequency, both G and G increased monotonically, and, finally, at the crossover point, G dominated G . Above the crossover point, the elastic properties of the systems dominate (G > G ), indicating that the stored energy in the structure prevails over the energy that was dissipated by the viscous forces. At frequencies close to the crossover point, the cubic

189

Fig. 5. Dynamic frequency sweep test for samples from point 5/4: (A) K-DFC, (B) Na-DFC and DEA-DFC.

phases revealed viscoelastic behavior that can be classified as the “transition to the flow region”. It should be noted that in the case of DEA+ salt, the values of both elastic and loss moduli are a decade higher than those of the K+ and Na+ derivatives. Moreover, greater dependence of the moduli on the frequency was clearly observed in the case of K+ and Na+ salts, compared to DEA+ . These findings are a sign of weaker network structure and less distinct interactions in the mesophases loaded with K+ and Na+ salts. This was confirmed by frequency dependence of complex viscosity (Fig. 6). A significant increase in the S values from 7536 in the case of Na+ and 8900 of K+ to 27,326 in the presence of DEA+ as well as higher m values (from −0.76 and −0.78 in the system with Na+ and K+ to −0.92 in the presence of DEA+ ) demonstrated more distinct solid-like behavior of the system as a result of DEA+ incorporation (Table 2). The experimental data from the viscoelastic measurements were tested for quasi-Maxwellian behavior, which has been demonstrated in several LLC systems. Fluids exhibiting Maxwellian behavior possess the following frequency dependence of storage and dissipative moduli [32]: G (ω) =

ω2 0 1 + (ω)2

(2)

Table 2 Results from SAXS and rheological experiments performed on samples of formulation 5/4 (cubic mesophase) with Na-DFC, K-DFC, and DEA-DFC. Cubic mesophases

Na-DFC

K-DFC

DEA-DFC

Lattice parameter (Å) S (Pa s2 /rad) m Zero-shear viscosity (Pa s)

128 7536 −0.76 8012

136 8899 −0.78 9332

144 27,326 −0.92 89,784

Fig. 6. Complex viscosity * of samples from point 5/4: (A) K-DFC, (B) Na-DFC and DEA-DFC.

190

G (ω) =

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ω0

(3)

1 + (ω)2

In these equations G is the storage modulus, G is the dissipative modulus, ω is the angular frequency,  is the relaxation time, and 0 is a zero-shear-rate viscosity. The Maxwell model can be characterized by a single relaxation time . It implies that at high angular frequencies the elastic modulus (G ) should reach a plateau value while the loss modulus should pass through a maximum. In the present study, the results obtained for all the cubic phases comply with the Maxwell model only at low frequencies, where the plot G /G against ω is a straight line (data not shown). Similar results were found for reverse hexagonal systems [21,31,33]. At higher frequencies, the systems deviated from the Maxwell model. In liquid crystals, longest relaxation time ( max ) is regarded as the time scale for relaxation to the equilibrium configuration of the water–lipid interface, following perturbation by shear deformations. Hence, variation of  max with composition has been shown to be the most reliable rheological method to detect order–disorder transitions in liquid crystalline phase [32].  max can be calculated as the inverse of the frequency at which the crossover takes place ( max = 1/ω) or by the plot G /G against ω at low frequencies. The “liquid-like” behavior that was measured upon the addition of K+ or Na+ , was reflected in the decrease of  max (0.75 and 0.64 s, respectively) compared to the  max obtained with DEA (5.8 s). In order to compare three given systems in terms of viscosity, the zero-shear rate viscosities (0 ) were calculated (Table 2). For quasiMaxwellian systems, the zero-shear viscosity can be estimated from G (ωm ), where G (ωm ) and G (ωm ) cross each other (Eq. (4)) [34]. 0 = 2G (ω) · max

Fig. 7. A typical cryo-TEM image of sample 7/2 with Na-DFC.

(4) DEA+

0 was found to be ten times higher when was embedded into the system, suggesting enhanced molecular interactions in the water–lipids interface. Following this interpretation, DEA-DFC guest molecules should be involved in a network of interactions with the polar moieties of monoglyceride molecules of the hosting system, in contrast to Na-DFC and K-DFC. Such interactions should take place to enable the effect described above, affecting the overall rheological behavior of the system. 3.3. Water-rich formulation: 7/2 (water/GMO ratio: 7/2) This mesophase was previously characterized in detail [18] and identified as an “intermediate structure”, caught between the cubic to lamellar transition induced by high aqueous phase concentration. This structure is characterized by a low viscosity network of disordered lamellar threads, as was demonstrated by cryo-TEM analysis (Fig. 7). These systems did not show a linear viscoelastic region at room temperature and hence were not examined further. In general, structural characterization of the mesophases loaded with DFC derivatives indicated that the most chaotropic DEA-DFC increased the water–lipid interface of both the lamellar and cubic structures. The measured increase of the lattice parameter of structures, enhanced elasticity, and increased solid-like response can be assigned to the hydrogen bonding enhancement between the lipid hydroxyls and its environment. In contrast, physical properties of both mesophases underwent similar and minor modifications in presence of less chaotropic K-DFC and kosmotropic Na-DFC. Apparently, greater concentrations of these molecules are required for a significant structural impact.

Fig. 8. In vitro permeation of Na-DFC from sample 7/2. A linear regression of the data at steady state is depicted (n = 6).

using the characterized mesophases. Characteristic profiles of the cumulative drug permeation (Q) per unit of skin surface area of diclofenac derivatives from the mesophases after 24 h are shown in Fig. 8. The potential of using these mesophases as sustained delivery vehicles for DFC is evident, avoiding burst release of the drug,

3.4. Delivery studies Franz diffusion cells were employed to test the applicability of three salt derivatives of diclofenac on transdermal release profiles

Fig. 9. In vitro cumulative permeation of DFC salts through porcine skin from three different mesophases.

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Another possible explanation of the observed differences is the detailed mechanism by which hydrophilic drugs penetrate the skin via the lyotropic mesophase and via water solution. In a recent pioneering work by Bender et al. [25], the authors demonstrated that the inter-cluster penetration pathway is preferable for delivery of hydrophilic compounds via the elastic cubic LLC, in contrast to the intercellular pathway when such molecules are delivered via water solution. According to the authors’ interpretation, the elastic lipid cubic phases penetrate into the micro-fissures, which are approximately 5 ␮m wide, with an irregular and entangled structure. It was suggested that the hydrophilic drug diffuses into the surrounding intercellular lipid from the micro-fissures matrix, where the latter acts as a drug source for sustained release. Therefore, while water promotes faster diffusion through the skin, monoglyceride sustains transdermal delivery. Consequently, the differences in delivery of diclofenac between kosmotropic and chaotropic salts were noticeable in water-rich samples (7/2 and 5/4) and were undetectable in lamellar water-poor sample 2/7. Certainly, further research is required to determine the detailed mechanism of delivery of diclofenac derivatives and similar pharmaceuticals via proposed carriers. 4. Conclusions Fig. 10. Schematic presentation of the structural modifications to the lamellar mesophase caused by the solubilization of different DFC salts.

which was obtained in the case of water solution. As seen in Fig. 8, the diffusion was fitted to Fick’s law and the permeability coefficients through the skin (KP × 103 ) were calculated (Fig. 9). On the whole, in accordance to our earlier report of sodium diclofenac permeation [18], the lyotropic structures possessing more free water induced higher permeation rates of all the diclofenac salts, in the order of 7/2 > 5/4 > 2/7. It was obvious that DFC solubilized in water can penetrate more easily and freely through the skin, compared to any lyotropic mesophase vehicles. Probably, the reason is that water is a remarkable permeation enhancer [35]. Regarding the influence of each diclofenac salt, two trends of permeation were obtained, depending on the chaotropic/kosmotropic nature of the molecules. There was no difference in permeation of any of the diclofenac salts from the lamellar mesophase, resulting in the same relatively low permeability coefficients (KP × 103 ) of 2 cm h−1 . In contrast to the lamellar mesophase (2/7), cubic structure (5/4) and the network of disordered lamellar thread (7/2) induced greater permeation of kosmotropic sodium salt, intermediate permeation of more chaotropic potassium salt, and low diffusivity of highly chaotropic DEA derivative (Fig. 9). 3.5. Discussion on delivery studies It appears that if higher permeation rates can be reached with molecules possessing more kosmotropic qualities. The diffusion of a drug in the aqueous channels of a lyotropic mesophase is hindered by two major factors [13]. The first is the physical restriction of the drug motion due to the geometrical constrain, owing to its diffusion within the water domains. This aspect is derived from the microstructure of the carrier. The second factor is the strength of chemical interactions of the drug with lipid hydrophilic moieties. It could be suggested that delivery through the skin of more kosmotropic molecules such as sodium diclofenac, which are mainly solubilized within bulk water of the mesophase (Fig. 10), is facilitated by water and less restricted by the interaction with the lipid hydrophilic heads. On the other hand, more chaotropic molecules (K+ and DEA) tend to be intercalated in the interfacial area and interact with polar moieties of the lipids (Fig. 10). This may sustain their transdermal delivery.

In the present study we investigated structural alterations in lyotropic mesophases caused by the solubilization of DFC salts with varying degrees of kosmotropic or chaotropic qualities and examined transdermal permeation of these DFC derivatives from the carriers. From the above structural data it could be suggested that the strongly chaotropic DEA derivative of DFC interacted with monoglyceride polar moieties and therefore increased the water–GMO interface of the lamellar and cubic structures. This was reflected by an increase of the lattice parameter of both mesophases. The hydrogen bonding enhancement between the hydroxyls of GMO and its environment is most likely responsible for greater elasticity and increased solid-like response of the systems in the presence of DEA. Kosmotropic Na+ salt and the weak chaotropic salt, K+ had only minor structural effects on both lamellar and cubic mesophases. Their major impact was mostly reflected in a weakening of intermolecular interactions in the water–lipid interface, as shown by rheological measurements. On a more practical aspect, it was shown that transdermal administration of kosmotropic sodium diclofenac, was speeded up by water and less constrained by the interaction with the lipid polar heads. On the other hand, more chaotropic molecules (K+ and DEA) are likely to be entrapped in the interfacial area and are delayed by the polar moieties of the lipids, which is the main reason for their sustained transdermal delivery. References [1] A. Otto, J. du Plessis, J.W. Wiechers, Int. J. Pharm. 257 (2003) 41. [2] A. Pagliara, M. Reist, S. Geinoz, P.A. Carrupt, B. Testa, J. Pharm. Pharmacol. 51 (1999) 1339. [3] A. Kogan, N. Garti, Adv. Colloid Interface Sci. 123–126 (2006) 369. [4] A. Spernath, A. Aserin, Adv. Colloid Interface Sci. 128–130 (2006) 47. [5] T. Mishraki, D. Libster, A. Aserin, N. Garti, Colloids Surf. B 75 (2010) 391. [6] L. Bitan-Cherbakovsky, I. Yuli-Amar, A. Aserin, N. Garti, Langmuir 25 (2009) 13106. [7] F.O. Costa-Balogh, E. Sparr, J.J.S. Sousa, A.A.C.C. Pais, Prog. Colloids Polym. Sci. 135 (2008) 119. [8] J.A. Bouwstra, P.L. Honeywell-Nguyen, G.S. Gooris, M. Ponec, Prog. Lipid Res. 42 (2003) 1. [9] N.H. Gabboun, N.M. Najib, H.G. Ibrahim, S. Assaf, Int. J. Pharm. 212 (2001) 73. [10] C. Cervin, P. Vandoolaeghe, C. Nistor, F. Tiberg, M. Johnsson, Eur. J. Pharm. Sci. 26 (2009) 377. [11] D. Libster, A. Aserin, E. Wachtel, G. Shoham, N. Garti, J. Colloid Interface Sci. 308 (2007) 514.

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