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Lecithin based lamellar liquid crystals as a physiologically acceptable dermal delivery system for ascorbyl palmitate
European Journal of Pharmaceutical Sciences 50 (2013) 114–122
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Lecithin based lamellar liquid crystals as a physiologically acceptable dermal delivery system for ascorbyl palmitate Mirjam Gosenca a, Marija Bešter-Rogacˇ b, Mirjana Gašperlin a,⇑ a b
University of Ljubljana, Faculty of Pharmacy, Aškercˇeva 7, Ljubljana, Slovenia University of Ljubljana, Faculty of Chemistry and Chemical Technology, Aškercˇeva 5, Ljubljana, Slovenia
a r t i c l e
i n f o
Article history: Received 16 December 2012 Received in revised form 8 April 2013 Accepted 25 April 2013 Available online 3 May 2013 Keywords: Pseudoternary phase diagram Lamellar liquid crystalline phase Ascorbyl palmitate Keratinocytes Cytotoxicity
a b s t r a c t Liquid crystalline systems with a lamellar structure have been extensively studied as dermal delivery systems. Ascorbyl palmitate (AP) is one of the most studied and used ascorbic acid derivatives and is employed as an antioxidant to prevent skin aging. The aim of this study was to develop and characterize skin-compliant dermal delivery systems with a liquid crystalline structure for AP. First, a pseudoternary phase diagram was constructed using Tween 80/lecithin/isopropyl myristate/water at a Tween 80/lecithin mass ratio of 1/1, and the region of lamellar liquid crystals was identified. Second, selected unloaded and AP-loaded lamellar liquid crystal systems were physicochemically characterized with polarizing optical microscopy, small-angle X-ray scattering, differential scanning calorimetry, and rheology techniques. The interlayer spacing and rheological parameters differ regarding quantitative composition, whereas the microstructure of the lamellar phase was affected by the AP incorporation, resulting either in additional micellar structures (at 25 and 32 °C) or being completely destroyed at higher temperature (37 °C). After this, the study was oriented towards in vitro cytotoxicity evaluation of lamellar liquid crystal systems on a keratinocyte cell line. The results suggest that the lamellar liquid crystals that were developed could be used as a physiologically acceptable dermal delivery system. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Liquid crystals (LCs) are self-assembled organized mesophases with properties of both liquids (fluidity) and solids (ordered crystalline structure, optical anisotropy). Lyotropic LCs are formed by amphiphilic molecules (i.e., surfactants), precisely by their hydrates or solvates, as well as associates of hydrated or solvated molecules. Hydration or solvation results in various self-assembled liquid crystalline structures, and thereby a wide range of LC structures from lamellar and hexagonal to cubic (Burducea, 2004; Müller-Goymann, 2007; Stevenson et al., 2005). Lyotropic LCs have attracted considerable interest as drug carriers in pharmaceutical technology with major focus put towards hexagonal and cubic phases (Guo et al., 2010; Fong et al., 2009; Sallam et al., 2002; Shah et al., 2001; Shah and Paradkar, 2005). On the other hand, lamellar LCs are particularly suitable for dermal application due to great similarity with the intercellular lipid membrane, hydrating properties, and ideal consistency. Lamellar LCs are formed by hydrated amphiphilic molecules in the shape of cylinders. They arrange themselves in layers, yielding a lamellar phase with alternating polar and nonpolar layers. Various aspects ⇑ Corresponding author. Address: University of Ljubljana, Faculty of Pharmacy, Aškercˇeva 7, 1000 Ljubljana, Slovenia. Tel.: +386 1 476 9634; fax: +386 1 425 8031. E-mail address: [email protected] (M. Gašperlin). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.04.029
of lamellar LC systems for dermal delivery were addressed in the literature concerning stability (Vicentini et al., 2008), improved drug activity (Chorilli et al., 2011 and Nesseem, 2001), or modified release. Prolonged release of very water-soluble as well as waterinsoluble drug was observed by Makai et al. (2003), while inhibited drug release from swollen liquid crystalline systems compared to hexagonal phase was explained with rapid water uptake of less ordered lamellar liquid crystalline structure by Farkas et al. (2000). Moreover, extensive research of topical vehicles with lamellar liquid crystalline structure based on alkylpolyglukoside natural surfactants was performed and their physicochemical properties were evaluated towards in vitro/in vivo skin performance (Savic´ et al., 2006, 2009; Savic et al., 2011). To develop a novel delivery system with LC structure phase behavior of a particular amphiphilic molecule/oil/water system is investigated by the construction of a ternary or a pseudo ternary phase diagram, and adequate methods must be involved to determine the structural features of formed systems. Polarized light microscopy (PLM) (Zhang et al., 2008), transmission electron microscopy (Mondain-Monval, 2005), small-angle X-ray scattering (SAXS) (Zhuang et al., 2008), differential scanning calorimetry (DSC) (Fehér et al., 2005), and rheology measurements (Berni et al., 2002; Németh et al., 1998; Youssry et al., 2008) are applied to achieve this goal as reviewed by Müller-Goymann (2004). The aspect of LC’s biological acceptability is also very important;
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therefore the skin irritation potential of individual components should already be considered in the development stage. Amphiphilic molecules must meet the following requirements: biodegradability, biocompatibility, and non-toxicity. Studies in this area have confirmed the adequately low toxicity of non-ionic surfactants such as polyoxyethylene sorbitan fatty acid derivatives (Tweens) as well as zwitterionic surfactants of natural origin such as lecithin (Fiume, 2001 and Malmsten, 2002). Moreover, it should be pointed out that lecithin has the unique property to form lamellar phases due to its molecular structure. Lecithin is an amphiphilic molecule with two non-polar hydrocarbon chains and zwitterionic polar headgroup, which have dipole moments and are strongly hydrated. The combination of two large moieties of opposite polarities strongly defines lecithin properties. LC structures are just one of the various self-organizing structures of lecithin in water along with micelles, swollen micelles, microemulsions, emulsions, organogels and liposomes. It was reported that the lamellar phase is predominant in lecithin/water binary systems and ternary systems (Shchipunov, 1997). Among oils, fatty acid esters such as isopropyl myristate (IPM) are of particular interest due to their biocompatible and biodegradable nature (Kumar and Katare, 2005). Phase behavior of lecithin/IPM/water (Harms et al., 2005 and Mackeben et al., 2001) and Tween 80/IPM/water system (Bonacucina et al., 2012) is well established in the literature. Phase diagrams comprising Tween 80 were also investigated for solubilization of various oils (Alam et al., 2009; Garti et al., 2001; Sharma and Warr, 2012; Yaghmur et al., 2002), while Brij 97 was used for solubilizing IPM into liquid crystalline phase (Wang et al., 2006). Ascorbyl palmitate (AP), an amphiphilic derivative of ascorbic acid, is widely used as an antioxidant active substance in pharmaceutical and cosmetic formulations to enhance skin protection from oxidative stress and consequently combat skin (photo)ageing as reported in recent review (Gašperlin and Gosenca, 2011a). Its stability and effectiveness have been studied in various dermal delivery systems, i.e. microemulsions, solid lipid nanoparticles and liposomes (Jurkovicˇ et al., 2003 and Kristl et al., 2003). Moreover, AP itself forms self-assembled liquid crystalline structures in water. Namely, a cubic and two lamellar mesophases were reported for AP/water binary system depending on concentration and temperature (Benedini et al., 2011). Semisolid gel with lamellar structure that could be used as dermal delivery system due to improved solubilization and stabilization of drugs combined with antioxidant properties of ascorbic acid is formed on cooling. In order to obtain stable mesophases at room temperatures, polyethyleneglycol 400 was added to AP/water system. However, the reduction in transition temperatures was not low enough to employ the systems as pharmaceutical formulations (Benedini et al., 2012). The aim of this study was to develop novel skin compliant delivery system for AP. Phase behavior of Tween 80/lecithin/IPM/ water system, comprising of biocompatible components, was evaluated. This is the first time that selected components have been studied in order to obtain dermal delivery system, therefore constant surfactant mass ratio Tween 80/lecithin = 1/1 was applied. The microstructure of systems, especially in view of structural alterations following incorporation of AP with amphiphilic charac-
Table 1 The composition of LC systems tested (w/w%). Sample
Lecithin
Tween 80
IPM
Bidistilled water
AP
LC1 LC2 LC1-AP LC2-AP
22.50 22.50 22.50 22.50
22.50 22.50 22.50 22.50
30.00 17.50 30.00 17.50
25.00 37.50 25.00 37.50
/ / 1 1
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ter, was studied by various techniques such as PLM, SAXS, DCS, and rheological analysis. The potential cytotoxic effects of LC systems were investigated using a human keratinocyte cell line (NCTC2544) as an in vitro model. 2. Materials and methods 2.1. Materials Lipoid S-100Ò, soybean phospholipid with not less than 94% w/w phosphatidylcholine content was provided by Lipoid GmbH Germany. According to the manufacturer’s specification the fatty acids of the two acyl groups of phosphatidylcholine are palmitic (15%), stearic (3%), oleic and isomers (12%), linoleic (62%) and linolenic (5%). Tween 80Ò, polyoxyethylene (20) sorbitan monooleate, with typical fatty acid composition of approximately 70% oleic acid and other fatty acids (i.e. palmitic acid) and isopropyl myristate (IPM) of declared purity P90% were obtained from Fluka, Sigma– Aldrich GmbH, Germany. Bidistilled water was used throughout the experiments. AP was provided by Fluka, Sigma-Aldrich GmbH, Germany. 2.2. Methods 2.2.1. Construction of pseudoternary phase diagram In order to determine the concentration range of components (water/IPM/Tween 80/lecithin) that form lyotropic LCs, a pseudoternary phase diagram with a 1/1 mass ratio of Tween 80 to lecithin was constructed using a water titration technique. For the titration process, a homogeneous mixture of IPM and surfactant mixture at weight ratios ranging from 95/5 to 5/95 was slowly titrated with aliquots of bidistilled water and stirred at 25 °C for a sufficiently long time to obtained equilibrium. After equilibrium was reached, the mixtures were checked visually for transparency. Visually clear samples of high viscosity were additionally checked through the cross polarizer for the presence of a liquid crystalline phase. Because only lamellar LCs were of our interest, no attempts were made to completely identify the other regions of the pseudoternary phase diagram in detail, and these have been described in terms of their visual and external appearance. 2.2.2. Sample preparation Representative samples of lyotropic LCs, whose composition is presented in Table 1, were further characterized. Samples were prepared by mixing appropriate amounts of IPM, Tween 80, and lecithin to form a homogeneous mixture; in the case of AP-loaded samples, AP was dissolved in a homogeneous oil–surfactant mixture. Water was added afterwards during continuous stirring to form lyotropic LCs. 2.2.3. Polarizing light microscopy The structure of the unloaded and AP-loaded LC samples was examined with a microscope with polarization using a Physica MCR 301 rheometer (Anton Paar, Graz, Austria) at 25 °C. The magnification was 20. 2.2.4. Small-angle X-ray scattering (SAXS) The structure of the unloaded and AP-loaded LC samples was further evaluated with SAXS measurements that were performed with an evacuated Kratky compact camera system (Anton Paar, Graz, Austria) with a block collimating unit, attached to a conventional X-ray generator (Bruker AXS, Karlsruhe, Germany) equipped with a sealed X-ray tube (Cu-anode target type) producing Ni-filtered Cu Ka radiation with a wavelength of k = 0.154 nm. The
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voltage was set to U = 35 kV with an anode current of I = 35 mA. The unloaded and AP-loaded LC samples were transferred to a standard quartz capillary placed in a thermally controlled sample holder centered in the X-ray beam. The scattered intensity was detected by a linear position sensitive detector (PSD 50 m, M. Braun, Garching, Germany). Measurements were performed at 25, 32, and 37 °C with measurement time setting t = 3600 s. The interlayer spacing d was calculated as d = 2p/q1 where q1 is the value of the scattering vector at the first peak maximum in the scattering curve. 2.2.5. Differential scanning calorimetry (DSC) DSC measurements were performed with a differential scanning calorimeter DSC 1 equipped with FRS5 sensor (Mettler Toledo, Switzerland) in order to highlight interactions between surfactant and water molecules. Nitrogen with a flow of 20 ml/min was used as a purge gas. Approximately 10 mg of sample, that is, individual components (Tween 80, lecithin, IPM, and water) and LC systems (unloaded and AP-loaded LC samples), was weighed precisely into a small aluminum pan and quickly sealed hermetically to prevent water evaporation. The empty sealed pan was used as a reference. Samples were cooled from 20 °C to 60 °C (cooling rate: 5 K/min), kept at 60 °C for 15 min, and then heated back to 20 °C (heating rate: 5 K/min). 2.2.6. Rheological measurements Rotational and oscillatory rheological tests were performed for unloaded and AP-loaded LC samples using a Physica MCR 301 rheometer (Anton Paar, Graz, Austria) with a cone-plate measuring system CP50-2 (cone diameter 49.961 mm, cone angle 2.001°, sample thickness 0.209 mm). Rotational tests were performed at 25.0 ± 0.1 °C, 32.0 ± 0.1 °C, and 37.0 ± 0.1 °C, whereas oscillatory tests were performed at a constant temperature of 25.0 ± 0.1 °C. Rotational tests were used to determine the viscosity, which for a cone-plate measuring system is calculated as g = s/c_ where s is the shear stress and c_ is the shear rate. Oscillatory tests were performed to define the elastic and loss moduli, which are calculated as G0 = (s/c) cos d and G00 = (s/c) sin d where s is the shear stress, c is the deformation, and d is the phase shift angle, together with complex viscosity calculated as g = s/(c x) where x is angular frequency. The shear rate during the rotational tests ranged from 2 to 100 s1. For oscillatory analysis, first the stress sweep measurements were performed at a constant frequency of 10.0 s1 in order to determine the linear viscoelastic region. Afterwards, the oscillatory shear measurements were carried out as a function of frequency (0.1–100 s1) at a constant amplitude (10%) chosen within the linear region.
sulfophenyl)-2H-tetrazolium, an inner salt, into the soluble colored formazan product by mitochondrial dehydrogenase enzymes in metabolically active cells. Keratinocytes were seeded at a density of 0.5 104 cells per well in 96-well plates. After one day (the attachment phase) the cells were treated with the test formulations. Test formulations were prepared by diluting unloaded LC1 and LC2 as well as sodium dodecyl sulfate (SDS) (positive control) in cell culture medium to final concentrations of 0.45, 0.90, and 4.50 mg/ml. Keratinocyte proliferation was assessed 4 h after the addition of test formulations. The absorbance of formazan was measured at 492 nm using a Safire2 microplate reader (Tecan, Switzerland). The results were expressed as the absorbance ratio of treated to control cells, and cell proliferation was calculated as cell proliferation = (AS–AS0)/(AC–AC0) where AS is the absorbance of the treated cells (sample), AC the absorbance of untreated cells (control), AS0 the absorbance of test formulation in cell-free medium, and AC0 the absorbance of the medium alone. 2.2.9. Statistical analysis Statistical analysis of MTS results was carried out using the independent samples Student’s t-test. Significance was tested at the 0.05 level of probability. 3. Results and discussion This study describes the preparation and characterization of LC systems as innovative and biocompatible carriers for dermal delivery of AP. Fig. 1 represents the pseudoternary phase diagram of the investigated system Tween 80/lecithin/IPM/water for the applied mass ratio of Tween 80 to lecithin = 1/1. An anisotropic lamellar LC phase appeared in the wide region of the pseudoternary phase diagram. More precisely, adding water at a level of 15–55% and 12.5– 35% of IPM resulted in the formation of lamellar LC systems. In other parts of the diagram, the formation of coarse emulsion and opaque gel-like disperse systems, or nonhomogeneous systems, was detected, which was beyond the interest of our study. Visually, the lamellar LC systems appeared as yellowish, transparent, and
2.2.7. Cell culture and treatment Human keratinocyte cells (cell line NCTC 2544, ICLC, University of Genoa) were cultured as adherent monolayers at 37 °C in a humidified atmosphere of 5% CO2. They were grown in Eagle’s Minimum Essential Medium with Earle’s balanced salt solution supplemented with 10% (v/v) fetal bovine serum (Gibco, Invitrogen, USA), 1% penicillin/streptomycin mixture, 1% 2 mM L-glutamine, and 1% (v/v) non-essential amino acids. Cells were subcultured with trypsin/EDTA when they reached 80 to 90% confluence. Cell culture reagents were from Sigma, Germany unless otherwise indicated. 2.2.8. MTS assay The effect of unloaded LC samples on cell proliferation was assessed using the MTS assay (Cell titer 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI) according to manufacturer’s procedure. The assay is based on conversion of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
Fig. 1. Pseudoternary phase diagram of the system Tween 80/lecithin/IPM/water with empty circles showing areas of existence of lamellar LCs at the Tween 80/ lecithin mass ratio indicated. LCI and LC2 system investigated are marked with full circles.
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highly viscous mixtures and their LC structure was confirmed using PLM. Birefringence was visualized and lamellar mesophases were identified as Maltese crosses, either in a separate arrangement or in chains as oily streaks. Such phase behavior is closely related to lecithin’s structural characteristics as described by the critical packing parameter, the values of which may, being a fundamental geometric quantity, represent some critical conditions for possible aggregate shapes. The critical packaging parameter value for lecithin, namely for phosphatidylcholine as its principal ingredient is ½ 1, which meets the criteria for bilayer formation (Choi et al., 1999). On the other hand, Tween 80, forms micellar solution, a hexagonal phase and inverted micellar solution with increasing concentration in water (Sharma and Warr, 2012). Namely, its critical packing parameter was estimated to be 0.07 (Amani et al., 2011) indicating the ability to form spherical structures. Addition of Tween 80 to lecithin would therefore result in reduced packing parameter and alteration of lamellar phase to hexagonal or micellar one. Regardless of its tendency to form other structures, only lamellar liquid crystalline phase was identified for Tween 80/lecithin/IPM/water mixture investigated in our experiments. This is consistent with literature data (Rong et al., 1996) where the incorporation of Tween 80 into fatty acid/lecithin lamellar LC (i.e. Stratum corneum lipid model) resulted in strong increase in interlayer spacing for the initial addition of Tween 80 followed by formation of two lamellar structures. Separation into two lamellar structures was attributed to space requirement of bulk polyoxyethylene headgroup. In study of parenteral dosage forms performed by Moreno et al. (2003), phase behavior of systems containing water/lecithin/Tween 80/IPM at different lecithin/Tween 80 ratios was evaluated. To the best of our knowledge, this is the only study comprising the same components as used in our investigation. In contrast to our results, only microemulsion region of clear and isotrope low–viscosity samples was established, while gel-like structures were reported, but not characterized. Tween 80 was able to reduce packing parameter of lecithin that resulted in formation of microemulsion, however it must be considered that phosphatidylcholine content of soybean lecithin used was significantly lower (20% w/w) compared to one used in our study (not less than 94% w/w). Further on, structural evaluation of unloaded and AP-loaded LC samples was performed in our study using SAXS together with DSC and rheological measurements. Principally LC structural characteristics following incorporation of antioxidant with amphiphilic properties were of most interest. Lamellar LC1 and LC2 samples (the composition is reported in Table 1 and highlighted in Fig. 1) were selected from the central part of the established LC region avoiding phase boundaries and comprising the same percentage (w/w %) of surfactant mixture. The photomicrographs of unloaded and AP-loaded formulations, analyzed by PLM, are shown in Fig. 2. The presence of Maltese crosses was observed in the case of unloaded LC samples. It was found to be dependent on water content, with a decreasing number of structures in Maltese crosses observed by enhanced water amount (Fig. 2A and B). Incorporation of AP into the system caused a reduction in the well-defined Maltese crosses as seen for AP-loaded LC1 (Fig. 2C), however for AP-loaded LC2 with the highest water content birefringence was not observed (data not shown). A reasonable explanation is that liquid crystalline structures were too small to be observed with used magnifications since ordered structure was clearly confirmed by SAXS measurements. SAXS analysis was performed and diffractograms were taken at various temperatures, including room (25 °C), skin surface (32 °C), and body (37 °C) temperature. SAXS patterns of unloaded and APloaded LC1 and LC2 at different temperatures tested are shown in Fig. 3. It is well known that LCs can be oriented to form one, two, or three-dimensional structures and SAXS curves show Bragg peak
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Fig. 2. Polarized light microscopy photomicrographs of unloaded samples: (A) LC1, (B) LC2, and (C) AP-loaded sample LC1.
intensities for specific values of the scattering vector q. Results obtained from SAXS are in good agreement with those from PLM because the q values for unloaded LC samples show the ratio q1:q2 = 1:2, which is indicative for the presence of lamellar structure. The correlation distances 1:2 of two Bragg peaks remained invariable of temperature. However, at 37 °C additional peak at low q value was observed for both unloaded systems tested. It was attributed to transition to micellar solution due to high concentration of Tween 80 accompanied by dissolution of lamellar liquid crystalline phase at higher temperatures as reported for ternary system lecithin/IPM/water (Harms et al., 2005). Results of interlayer spacing d support temperature effect on inner structure (Table 2). By rising temperature from 25 to 37 °C interlayer spacing increases for unloaded LC1, whereas in case of unloaded LC2 firstly decreases at 32 °C and then increases at 37 °C. The latter is in line with results on phospholipid membranes, which indicated decrease in interlayer spacing due to strengthening of attractive van der Waals interactions with temperature. However, at some point the repulsion prevails due to stronger thermal fluctuations, resulting in increased interlayer spacing (Szekely et al., 2012).
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Fig. 3. Scattering curves of unloaded and AP-loaded (inset) (A) LC1 and (B) LC2 at three different temperatures.
For LC2 minimum of interlayer spacing was not observed as it is most likely beyond 32 °C. Additionally, water content has a great effect on the repeat distance within the lamellar structure. An increasing interlayer spacing ranging from 7.12 nm (LC1) to 7.90 nm (LC2) can be observed with increasing water content and coincides with swelling of surfactants (Mackeben et al., 2001 and Zhuang et al., 2008). It is also expected that addition of Tween 80 with different
Table 2 Interlayer spacing d of unloaded and AP-loaded LC1-2 samples at various temperatures. d (nm)
25 °C 32 °C 37 °C
LC1
LC2
LC1-AP
LC2-AP
7.12 7.24 7.42
7.90 7.57 7.78
9.28 8.73 /
8.56 8.18 /
structural properties compared to lecithin resulted in increased interlayer spacing followed by formation of additional micellar solution at 37 °C. This interpretation is supported by results of AP-loaded LC samples. Namely, the characteristic Braggs reflections corresponding to lamellar phase were observed at 25 and 32 °C, yet less defined. Further, the existence of micellar solution was confirmed due to protuberant scattering peak that was also the only peak observed at 37 °C. Though increasing temperature should result in scattering peak, which moves to lower, not higher q, adequate explanation for this behavior cannot be given at this time. To continue, AP with amphiphilic structure similar to Tween 80 (i.e. large polar moiety and single lipophilic chain) most likely reduces packing parameter of Tween 80/lecithin mixture and thereby exhibits similar effect as Tween 80. Interlayer spacing was evidently increased for APloaded LC samples when compared to unloaded LC samples (Table 2). Widening effect is in agreement to the increase of the interlayer spacing observed with timolol maleat-loaded lameller mesophase of lecithin/IPM/water (Mackeben et al., 2001). In this case formation
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of hydrogen bonds between lecithin and polar headgroup of drug molecules was postulated; therefore the former hydratation water becomes free and could be located between the polar heads of the lecithin molecules within the lamellae. The same mechanism can be proposed for Tween 80 and AP due to multiple polar functional groups on polar moiety followed by structural rearrangement to micellar aggregates with increasing temperature. In addition, this structural rearrangement is also relevant for dermal delivery. AP skin permeation from LC1 was previously evaluated and proven to be superior to other colloidal systems tested (Gosenca and Gašperlin, 2011b). However, not due to lamellar structure as proposed at that time but rather coexistence of lamellar and micellar phase at 32 °C as confirmed by SAXS results presented in this work. DSC analysis was employed in order to highlight the state of water in LC samples to consequently evaluate the strength of interactions between surfactant molecules and water. Firstly, preliminary tests have been carried on individual components (Fig. 4A). In the cooling curve of the bidistilled water, a large and sharp exothermic peak was observed at approximately 19 °C, which indicates freezing of supercooled water. For IPM, three exothermic peaks were detected. Namely, the largest peak appears at approximately 7 °C and represents solidification of the IPM. Furthermore, two exothermic peaks at 14 °C and 20 °C most probably correspond to solidification of the small amount of IPM impurity (the declared purity of IPM used is P90%), which is in agreement with the previous findings (Podlogar et al., 2004). A wide endothermic peak at 1 °C and 7 °C in DSC heating curves corresponds to melting of pure water and IPM (Bonacucina et al., 2012) (Fig. 4A; dotted line). No thermal events were observed in DSC curves of Tween 80, lecithin, or AP (data not shown).
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Cooling curves for unloaded LC samples are presented in Fig. 4B (solid lines). A triple exothermic peak ranging from 7 °C to 20 °C was observed and is attributed to IPM crystallization. The peak shape was similar to that of IPM alone, which thus indicates freezing of IPM without any interactions with other molecules. An additional broad exothermic peak appears at lower temperatures (i.e., between approximately 20 °C and 45 °C), which corresponds to freezing of freezable interlamellar water. Namely, it is possible to distinguish between freezable and nonfreezable water because water molecules form a hydration shell around the polar groups of surfactants and an aqueous interlayer between lamellae in lamellar LC systems. As a general rule, water molecules that interact with polar heads of surfactants are bound so tightly that they cannot form hydrogen bonds with their neighboring water molecules and can be understood as nonfreezable water, whereas the water that exists in the interbilayer region keeps the degree of freedom necessary to form hydrogen bonds and is designated freezable interlamellar water (Bonacucina et al., 2012 and Kodama and Aoki, 2001). The freezing temperature of water can therefore be dependent on water and surfactant content, and consequently the strength of interactions. The lowest water crystallization temperature was observed for unloaded LC1 with the lowest water-tosurfactant ratio that means that the water is strongly bound by headgroup of the surfactants as the freezing temperature (approximately 40 °C) is very low compared to freezing of pure water (19 °C). As for the unloaded LC2 sample, the water crystallization peak is shifted towards higher temperatures. This allows us to presume that water is bound to polar heads of surfactants because surfactants have to be supersaturated with water before a free water freezing peak can be observed (Garti et al., 2000); neverthe-
Fig. 4. (A) DSC curves of individual components (i.e., water and IPM) and (B) unloaded and AP-loaded LC samples. DSC cooling curves are indicated as a solid line, and DSC heating curves as a dotted line.
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less, the interactions are weaker compared to unloaded LC1 system. Incorporation of AP resulted in shifting water crystallization peak towards higher temperatures, which was especially evident for AP-loaded LC1. This supports our previous assumption that water molecules are less firmly bound due to interactions between AP and surfactant molecules. From heating part of the curve (Fig. 4B; dotted lines) a broad endothermic peak, even though not very pronounced, that begins at 30 °C and corresponds to melting of ice from freezable interlamellar water was observed. Again, the more remote the water molecules are from the bilayer surfaces, the more the peak moves towards 0 °C and the more similar the shape is to that of pure water (Kodama and Aoki, 2001) (Fig. 4A; dotted line). The effect is more evident in the case of unloaded LC2 system due to higher amount of water between the lamellae as confirmed by higher interlayer spacing. For AP-loaded LC systems, the peak became more distinguishable for AP-loaded LC1 system indicating more pronounced structural changes within this system. Rheological analysis is one of the most frequently used techniques for structure characterization at the macroscopic level. The shape of the viscosity curves clearly shows decreasing viscosity with increasing shear rate at all temperatures tested, which is characteristic for non-Newtonian systems exhibiting shear-thinning behavior (Chorilli et al., 2011). Fig. 5 shows changes in viscosity as a function of shear rate for unloaded and AP-loaded LC samples obtained from rotational tests. Further on, when looking the viscosity at the lowest measured shear rate (2 s1) at 25 °C (Fig. 6), it is notably higher for unloaded LC2 compared to unloaded LC1. Evidently surfactant molecules in system with higher waterto-surfactant ratio (LC2) form denser layer structure. With increasing temperature the surfactant molecule aggregate more loosely in the lamellae as seen from decrease in viscosity values, which is in accordance with the published data (Zhao et al., 2011). In our study the decrease was more pronounced for unloaded LC2 at 32 °C, while further decrease at 37 °C was less prominent. A possible explanation is that cohesion of liquid crystalline structure was reduced at 32 °C, most likely due to weaker hydrophilic binding mechanism (Harms et al., 2005), even though micellar aggregates were not observed. The viscosity was also inversely proportional with temperature for AP-loaded LC samples, which also exhibited evidently lower viscosities as unloaded samples, again pointing to transition from lamellar liquid crystalline structure to isotropic solution after incorporation of amphiphilic AP. It is important to emphasize that observed viscosity curves for AP-loaded LC sample, in particular at 37 °C, are in fact viscosity curves for micellar solution. More information about the lamellar structure can be obtained from oscillatory rheology; shear frequency sweep measurements were performed with typical curves of the frequency dependence of the storage modulus G0 , loss modulus G00 , and complex viscosity for unloaded and AP-loaded LC samples presented in Fig. 7. Unloaded LC samples are more elastic than viscous, which is in accordance with literature data (Farkas et al., 2000; Németh et al., 1998; Zhang et al., 2008): G0 is about one order of magnitude higher than G00 throughout the entire frequency range. G0 was nearly frequencyindependent and G00 shows a local minimum, whereas the complex viscosity drops linearly as a function of frequency. The ratio between G0 and G00 (tan d) at various frequencies (100 Hz, 10 Hz) and minimum of G00 (Table 3) clearly demonstrate that the sample becomes more elastic with an increasing water to surfactant ratio, as seen from the decrease in tan d. Higher values of G0 for unloaded LC2 system indicates stronger interactions between the bilayer that is supported also by viscosity measurements, however is not consistent with SAXS and DSC results. DSC results clearly revealed weaker interactions between surfactant mixture and water for unloaded LC2 system while SAXS analysis confirmed increased inter-
Fig. 5. Viscosity curves showing shear-thinning behavior for unloaded (solid line) and AP-loaded (dotted line) (A) LC1 and (B) LC2 at 25 °C (h), 32 °C (e), and 37 °C (s). Data are expressed as mean ± S.D. (n = 3).
Fig. 6. Viscosity of unloaded and AP-loaded LC samples measured at the lowest shear rate 2 s1 and various temperatures. Data are expressed as mean ± S.D. (n = 3).
layer distance, which is connected with weaker, not stronger, interactions (Youssry et al., 2008). The same tendency of rheological parameters was observed for AP-loaded system; both moduli decrease when compared to unloaded LC systems. This point to structural changes and is consistent with SAXS results since lamellar phase coexists with micellar. A decrease was very pronounced for AP-loaded LC1, indicating weaker interactions between the bilayer after incorporation of amphiphilic molecule. Results of SAXS and DSC measurements strengthen this assumption. Namely, the increase of interlayer spacing and shift of water crystallization peak towards higher temperature was more prominent for AP-loaded LC1. And even though explanation for inconsistency of rheological towards SAXS and DSC results for unloaded LC2 samples cannot be given at the moment, it is probably due to this phenomena disturbance of lamellae in APloaded LC2 less distinct as seen from only slight decrease in rheological parameters. What is more, this is also confirmed by less prominent increase in interlayer spacing and only minor changes
M. Gosenca et al. / European Journal of Pharmaceutical Sciences 50 (2013) 114–122
Fig. 7. Elastic modulus (G0 , h), loss modulus (G00 , s), and complex viscosity (g, e) of unloaded (solid line) and AP-loaded (dotted line) (A) LC1 and (B) LC2 sample as a function of frequency (x) at a stress of 10% at 25 °C. Data are expressed as mean ± S.D. (n = 3).
Table 3 Loss tangent (tan d) at various frequencies (100 Hz, 10 Hz, and minimum of G00 ) of unloaded LC samples with corresponding water/surfactant (W/S) ratio. Sample
W/S ratio
tan d (100 Hz)
tan d (10 Hz)
tan d (G00 min)
LC1 LC2
0.55 0.87
0.233 0.202
0.181 0.099
0.185 0.078
Fig. 8. Proliferation of keratinocytes 4 h after addition of unloaded LC1-2 samples and SDS solution to the cells (T = 37 °C). The results are presented relative to the proliferation of untreated control cells. Data are expressed as mean ± S.D. (n = 6). a P < 0.05 compared to SDS solution at the same concentration.
121
for its practical use. In keeping with this, a cytotoxicity assessment of LC systems on a keratinocyte cell line was performed. Unloaded LC samples were tested because AP can directly reduce the MTS reagent and in a concentration range (0.45, 0.90 and 4.50 mg of LC sample/ ml) that can reveal differences in cell proliferation regarding LC composition. Keratinocyte proliferation was measured with an MTS assay following short-term exposure (4 h post-treatment). Exposing keratinocytes to lamellar LC systems at a concentration of 0.45 mg/ml resulted in no considerable impact on cell proliferation, being around 100%, and it also remained at a high level at a concentration of 0.90 mg/ml. The pronounced differences regarding LC composition became significant at the highest concentration tested (4.50 mg/ml); the cell proliferation was highest for LC2 (83.45%), with the highest water content compared to LC1 (71.38%). The cell proliferation measured by the MTS reduction test is summarized in Fig. 8. After exposure to SDS, which is used as a positive control in skin irritation testing (Effendy and Maibach, 1996), the cell proliferation dramatically declined. The LC systems tested performed significantly better than SDS solution, proving lamellar LC to be a potential non-toxic dermal delivery system. These results are in accordance to very low acute and low cumulative irritancy potential that was established with patch test for lecithin microemulsion gel composed of soybean lecithin, IPM and water (Dreher et al., 1996), while IPM was confirmed as safe lipophilic excipient, even though trend of decreased viability was observed (Savic´ et al., 2009). Nevertheless, being aware of the fact that biological assays are not able to evaluate structural damage that may occur at a non-cytotoxic dose, but can promote pathological effects (Lastella et al., 2007), extensive study oriented towards cells’ morphological evaluation using atomic force microscopy is in progress. We strongly believe that the data obtained together with the MTS results presented here will unequivocally answer the question regarding the dermal acceptability of LC formulations investigated.
4. Conclusions This study revealed that (Tween 80/lecithin = 1/1)/IPM/water mixtures were able to form lamellar LC, as confirmed by the PLM and SAXS results. The structure was additionally evaluated regarding its quantitative composition. The interlayer spacing was affected by water content, which can be attributed to differences in the strength of interactions between water and surfactant molecules, as observed by DSC measurements. A rheological study showed that the formulation behaves like a non-Newtonian system, with characteristics of shear-thinning material. The lamellar phase structure also remained organized at skin and physiological temperatures. Structural changes in the system were observed after incorporation of AP, which was used as an antioxidant, and were attributed to micelle formation due to its amphiphilic character and interactions with surfactant molecules. The cell proliferation remained high at 0.45 and 0.90 mg/ml, decreased at 4.50 mg/ml, but remained significantly higher when compared to the standard irritant SDS. Therefore, setting strict selection criteria for individual components together with qualitative structural evaluation were proved as a prosing approach in developing a skin compliant carrier system; namely, promising results obtained on living keratinocytes indicate that the lecithin based LC system shows great potential as a physiologically acceptable dermal delivery system.
Acknowledgements in peaks positions (DSC cooling curves) or shape (DSC heating curves) for AP-loaded LC2 compared to AP-loaded LC1. Biological acceptability is one of crucial aspects that should be considered when developing a novel delivery system and is decisive
The authors are grateful to the ICLC-Interlab Cell Line Collection, University of Genova (Italy), for providing the immortalized human keratinocyte cell line NCTC2544. We thank Tanja Tavcˇar
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and Lucija Kralj for carrying out a lot of the experimental work and Mr. Anton Kokalj for preforming SAXS experiments.
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Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Lecithin based lamellar liquid crystals as a physiologically acceptable dermal delivery system for ascorbyl palmitate Mirjam Gosenca a, Marija Bešter-Rogacˇ b, Mirjana Gašperlin a,⇑ a b
University of Ljubljana, Faculty of Pharmacy, Aškercˇeva 7, Ljubljana, Slovenia University of Ljubljana, Faculty of Chemistry and Chemical Technology, Aškercˇeva 5, Ljubljana, Slovenia
a r t i c l e
i n f o
Article history: Received 16 December 2012 Received in revised form 8 April 2013 Accepted 25 April 2013 Available online 3 May 2013 Keywords: Pseudoternary phase diagram Lamellar liquid crystalline phase Ascorbyl palmitate Keratinocytes Cytotoxicity
a b s t r a c t Liquid crystalline systems with a lamellar structure have been extensively studied as dermal delivery systems. Ascorbyl palmitate (AP) is one of the most studied and used ascorbic acid derivatives and is employed as an antioxidant to prevent skin aging. The aim of this study was to develop and characterize skin-compliant dermal delivery systems with a liquid crystalline structure for AP. First, a pseudoternary phase diagram was constructed using Tween 80/lecithin/isopropyl myristate/water at a Tween 80/lecithin mass ratio of 1/1, and the region of lamellar liquid crystals was identified. Second, selected unloaded and AP-loaded lamellar liquid crystal systems were physicochemically characterized with polarizing optical microscopy, small-angle X-ray scattering, differential scanning calorimetry, and rheology techniques. The interlayer spacing and rheological parameters differ regarding quantitative composition, whereas the microstructure of the lamellar phase was affected by the AP incorporation, resulting either in additional micellar structures (at 25 and 32 °C) or being completely destroyed at higher temperature (37 °C). After this, the study was oriented towards in vitro cytotoxicity evaluation of lamellar liquid crystal systems on a keratinocyte cell line. The results suggest that the lamellar liquid crystals that were developed could be used as a physiologically acceptable dermal delivery system. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Liquid crystals (LCs) are self-assembled organized mesophases with properties of both liquids (fluidity) and solids (ordered crystalline structure, optical anisotropy). Lyotropic LCs are formed by amphiphilic molecules (i.e., surfactants), precisely by their hydrates or solvates, as well as associates of hydrated or solvated molecules. Hydration or solvation results in various self-assembled liquid crystalline structures, and thereby a wide range of LC structures from lamellar and hexagonal to cubic (Burducea, 2004; Müller-Goymann, 2007; Stevenson et al., 2005). Lyotropic LCs have attracted considerable interest as drug carriers in pharmaceutical technology with major focus put towards hexagonal and cubic phases (Guo et al., 2010; Fong et al., 2009; Sallam et al., 2002; Shah et al., 2001; Shah and Paradkar, 2005). On the other hand, lamellar LCs are particularly suitable for dermal application due to great similarity with the intercellular lipid membrane, hydrating properties, and ideal consistency. Lamellar LCs are formed by hydrated amphiphilic molecules in the shape of cylinders. They arrange themselves in layers, yielding a lamellar phase with alternating polar and nonpolar layers. Various aspects ⇑ Corresponding author. Address: University of Ljubljana, Faculty of Pharmacy, Aškercˇeva 7, 1000 Ljubljana, Slovenia. Tel.: +386 1 476 9634; fax: +386 1 425 8031. E-mail address: [email protected] (M. Gašperlin). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.04.029
of lamellar LC systems for dermal delivery were addressed in the literature concerning stability (Vicentini et al., 2008), improved drug activity (Chorilli et al., 2011 and Nesseem, 2001), or modified release. Prolonged release of very water-soluble as well as waterinsoluble drug was observed by Makai et al. (2003), while inhibited drug release from swollen liquid crystalline systems compared to hexagonal phase was explained with rapid water uptake of less ordered lamellar liquid crystalline structure by Farkas et al. (2000). Moreover, extensive research of topical vehicles with lamellar liquid crystalline structure based on alkylpolyglukoside natural surfactants was performed and their physicochemical properties were evaluated towards in vitro/in vivo skin performance (Savic´ et al., 2006, 2009; Savic et al., 2011). To develop a novel delivery system with LC structure phase behavior of a particular amphiphilic molecule/oil/water system is investigated by the construction of a ternary or a pseudo ternary phase diagram, and adequate methods must be involved to determine the structural features of formed systems. Polarized light microscopy (PLM) (Zhang et al., 2008), transmission electron microscopy (Mondain-Monval, 2005), small-angle X-ray scattering (SAXS) (Zhuang et al., 2008), differential scanning calorimetry (DSC) (Fehér et al., 2005), and rheology measurements (Berni et al., 2002; Németh et al., 1998; Youssry et al., 2008) are applied to achieve this goal as reviewed by Müller-Goymann (2004). The aspect of LC’s biological acceptability is also very important;
M. Gosenca et al. / European Journal of Pharmaceutical Sciences 50 (2013) 114–122
therefore the skin irritation potential of individual components should already be considered in the development stage. Amphiphilic molecules must meet the following requirements: biodegradability, biocompatibility, and non-toxicity. Studies in this area have confirmed the adequately low toxicity of non-ionic surfactants such as polyoxyethylene sorbitan fatty acid derivatives (Tweens) as well as zwitterionic surfactants of natural origin such as lecithin (Fiume, 2001 and Malmsten, 2002). Moreover, it should be pointed out that lecithin has the unique property to form lamellar phases due to its molecular structure. Lecithin is an amphiphilic molecule with two non-polar hydrocarbon chains and zwitterionic polar headgroup, which have dipole moments and are strongly hydrated. The combination of two large moieties of opposite polarities strongly defines lecithin properties. LC structures are just one of the various self-organizing structures of lecithin in water along with micelles, swollen micelles, microemulsions, emulsions, organogels and liposomes. It was reported that the lamellar phase is predominant in lecithin/water binary systems and ternary systems (Shchipunov, 1997). Among oils, fatty acid esters such as isopropyl myristate (IPM) are of particular interest due to their biocompatible and biodegradable nature (Kumar and Katare, 2005). Phase behavior of lecithin/IPM/water (Harms et al., 2005 and Mackeben et al., 2001) and Tween 80/IPM/water system (Bonacucina et al., 2012) is well established in the literature. Phase diagrams comprising Tween 80 were also investigated for solubilization of various oils (Alam et al., 2009; Garti et al., 2001; Sharma and Warr, 2012; Yaghmur et al., 2002), while Brij 97 was used for solubilizing IPM into liquid crystalline phase (Wang et al., 2006). Ascorbyl palmitate (AP), an amphiphilic derivative of ascorbic acid, is widely used as an antioxidant active substance in pharmaceutical and cosmetic formulations to enhance skin protection from oxidative stress and consequently combat skin (photo)ageing as reported in recent review (Gašperlin and Gosenca, 2011a). Its stability and effectiveness have been studied in various dermal delivery systems, i.e. microemulsions, solid lipid nanoparticles and liposomes (Jurkovicˇ et al., 2003 and Kristl et al., 2003). Moreover, AP itself forms self-assembled liquid crystalline structures in water. Namely, a cubic and two lamellar mesophases were reported for AP/water binary system depending on concentration and temperature (Benedini et al., 2011). Semisolid gel with lamellar structure that could be used as dermal delivery system due to improved solubilization and stabilization of drugs combined with antioxidant properties of ascorbic acid is formed on cooling. In order to obtain stable mesophases at room temperatures, polyethyleneglycol 400 was added to AP/water system. However, the reduction in transition temperatures was not low enough to employ the systems as pharmaceutical formulations (Benedini et al., 2012). The aim of this study was to develop novel skin compliant delivery system for AP. Phase behavior of Tween 80/lecithin/IPM/ water system, comprising of biocompatible components, was evaluated. This is the first time that selected components have been studied in order to obtain dermal delivery system, therefore constant surfactant mass ratio Tween 80/lecithin = 1/1 was applied. The microstructure of systems, especially in view of structural alterations following incorporation of AP with amphiphilic charac-
Table 1 The composition of LC systems tested (w/w%). Sample
Lecithin
Tween 80
IPM
Bidistilled water
AP
LC1 LC2 LC1-AP LC2-AP
22.50 22.50 22.50 22.50
22.50 22.50 22.50 22.50
30.00 17.50 30.00 17.50
25.00 37.50 25.00 37.50
/ / 1 1
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ter, was studied by various techniques such as PLM, SAXS, DCS, and rheological analysis. The potential cytotoxic effects of LC systems were investigated using a human keratinocyte cell line (NCTC2544) as an in vitro model. 2. Materials and methods 2.1. Materials Lipoid S-100Ò, soybean phospholipid with not less than 94% w/w phosphatidylcholine content was provided by Lipoid GmbH Germany. According to the manufacturer’s specification the fatty acids of the two acyl groups of phosphatidylcholine are palmitic (15%), stearic (3%), oleic and isomers (12%), linoleic (62%) and linolenic (5%). Tween 80Ò, polyoxyethylene (20) sorbitan monooleate, with typical fatty acid composition of approximately 70% oleic acid and other fatty acids (i.e. palmitic acid) and isopropyl myristate (IPM) of declared purity P90% were obtained from Fluka, Sigma– Aldrich GmbH, Germany. Bidistilled water was used throughout the experiments. AP was provided by Fluka, Sigma-Aldrich GmbH, Germany. 2.2. Methods 2.2.1. Construction of pseudoternary phase diagram In order to determine the concentration range of components (water/IPM/Tween 80/lecithin) that form lyotropic LCs, a pseudoternary phase diagram with a 1/1 mass ratio of Tween 80 to lecithin was constructed using a water titration technique. For the titration process, a homogeneous mixture of IPM and surfactant mixture at weight ratios ranging from 95/5 to 5/95 was slowly titrated with aliquots of bidistilled water and stirred at 25 °C for a sufficiently long time to obtained equilibrium. After equilibrium was reached, the mixtures were checked visually for transparency. Visually clear samples of high viscosity were additionally checked through the cross polarizer for the presence of a liquid crystalline phase. Because only lamellar LCs were of our interest, no attempts were made to completely identify the other regions of the pseudoternary phase diagram in detail, and these have been described in terms of their visual and external appearance. 2.2.2. Sample preparation Representative samples of lyotropic LCs, whose composition is presented in Table 1, were further characterized. Samples were prepared by mixing appropriate amounts of IPM, Tween 80, and lecithin to form a homogeneous mixture; in the case of AP-loaded samples, AP was dissolved in a homogeneous oil–surfactant mixture. Water was added afterwards during continuous stirring to form lyotropic LCs. 2.2.3. Polarizing light microscopy The structure of the unloaded and AP-loaded LC samples was examined with a microscope with polarization using a Physica MCR 301 rheometer (Anton Paar, Graz, Austria) at 25 °C. The magnification was 20. 2.2.4. Small-angle X-ray scattering (SAXS) The structure of the unloaded and AP-loaded LC samples was further evaluated with SAXS measurements that were performed with an evacuated Kratky compact camera system (Anton Paar, Graz, Austria) with a block collimating unit, attached to a conventional X-ray generator (Bruker AXS, Karlsruhe, Germany) equipped with a sealed X-ray tube (Cu-anode target type) producing Ni-filtered Cu Ka radiation with a wavelength of k = 0.154 nm. The
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voltage was set to U = 35 kV with an anode current of I = 35 mA. The unloaded and AP-loaded LC samples were transferred to a standard quartz capillary placed in a thermally controlled sample holder centered in the X-ray beam. The scattered intensity was detected by a linear position sensitive detector (PSD 50 m, M. Braun, Garching, Germany). Measurements were performed at 25, 32, and 37 °C with measurement time setting t = 3600 s. The interlayer spacing d was calculated as d = 2p/q1 where q1 is the value of the scattering vector at the first peak maximum in the scattering curve. 2.2.5. Differential scanning calorimetry (DSC) DSC measurements were performed with a differential scanning calorimeter DSC 1 equipped with FRS5 sensor (Mettler Toledo, Switzerland) in order to highlight interactions between surfactant and water molecules. Nitrogen with a flow of 20 ml/min was used as a purge gas. Approximately 10 mg of sample, that is, individual components (Tween 80, lecithin, IPM, and water) and LC systems (unloaded and AP-loaded LC samples), was weighed precisely into a small aluminum pan and quickly sealed hermetically to prevent water evaporation. The empty sealed pan was used as a reference. Samples were cooled from 20 °C to 60 °C (cooling rate: 5 K/min), kept at 60 °C for 15 min, and then heated back to 20 °C (heating rate: 5 K/min). 2.2.6. Rheological measurements Rotational and oscillatory rheological tests were performed for unloaded and AP-loaded LC samples using a Physica MCR 301 rheometer (Anton Paar, Graz, Austria) with a cone-plate measuring system CP50-2 (cone diameter 49.961 mm, cone angle 2.001°, sample thickness 0.209 mm). Rotational tests were performed at 25.0 ± 0.1 °C, 32.0 ± 0.1 °C, and 37.0 ± 0.1 °C, whereas oscillatory tests were performed at a constant temperature of 25.0 ± 0.1 °C. Rotational tests were used to determine the viscosity, which for a cone-plate measuring system is calculated as g = s/c_ where s is the shear stress and c_ is the shear rate. Oscillatory tests were performed to define the elastic and loss moduli, which are calculated as G0 = (s/c) cos d and G00 = (s/c) sin d where s is the shear stress, c is the deformation, and d is the phase shift angle, together with complex viscosity calculated as g = s/(c x) where x is angular frequency. The shear rate during the rotational tests ranged from 2 to 100 s1. For oscillatory analysis, first the stress sweep measurements were performed at a constant frequency of 10.0 s1 in order to determine the linear viscoelastic region. Afterwards, the oscillatory shear measurements were carried out as a function of frequency (0.1–100 s1) at a constant amplitude (10%) chosen within the linear region.
sulfophenyl)-2H-tetrazolium, an inner salt, into the soluble colored formazan product by mitochondrial dehydrogenase enzymes in metabolically active cells. Keratinocytes were seeded at a density of 0.5 104 cells per well in 96-well plates. After one day (the attachment phase) the cells were treated with the test formulations. Test formulations were prepared by diluting unloaded LC1 and LC2 as well as sodium dodecyl sulfate (SDS) (positive control) in cell culture medium to final concentrations of 0.45, 0.90, and 4.50 mg/ml. Keratinocyte proliferation was assessed 4 h after the addition of test formulations. The absorbance of formazan was measured at 492 nm using a Safire2 microplate reader (Tecan, Switzerland). The results were expressed as the absorbance ratio of treated to control cells, and cell proliferation was calculated as cell proliferation = (AS–AS0)/(AC–AC0) where AS is the absorbance of the treated cells (sample), AC the absorbance of untreated cells (control), AS0 the absorbance of test formulation in cell-free medium, and AC0 the absorbance of the medium alone. 2.2.9. Statistical analysis Statistical analysis of MTS results was carried out using the independent samples Student’s t-test. Significance was tested at the 0.05 level of probability. 3. Results and discussion This study describes the preparation and characterization of LC systems as innovative and biocompatible carriers for dermal delivery of AP. Fig. 1 represents the pseudoternary phase diagram of the investigated system Tween 80/lecithin/IPM/water for the applied mass ratio of Tween 80 to lecithin = 1/1. An anisotropic lamellar LC phase appeared in the wide region of the pseudoternary phase diagram. More precisely, adding water at a level of 15–55% and 12.5– 35% of IPM resulted in the formation of lamellar LC systems. In other parts of the diagram, the formation of coarse emulsion and opaque gel-like disperse systems, or nonhomogeneous systems, was detected, which was beyond the interest of our study. Visually, the lamellar LC systems appeared as yellowish, transparent, and
2.2.7. Cell culture and treatment Human keratinocyte cells (cell line NCTC 2544, ICLC, University of Genoa) were cultured as adherent monolayers at 37 °C in a humidified atmosphere of 5% CO2. They were grown in Eagle’s Minimum Essential Medium with Earle’s balanced salt solution supplemented with 10% (v/v) fetal bovine serum (Gibco, Invitrogen, USA), 1% penicillin/streptomycin mixture, 1% 2 mM L-glutamine, and 1% (v/v) non-essential amino acids. Cells were subcultured with trypsin/EDTA when they reached 80 to 90% confluence. Cell culture reagents were from Sigma, Germany unless otherwise indicated. 2.2.8. MTS assay The effect of unloaded LC samples on cell proliferation was assessed using the MTS assay (Cell titer 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI) according to manufacturer’s procedure. The assay is based on conversion of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
Fig. 1. Pseudoternary phase diagram of the system Tween 80/lecithin/IPM/water with empty circles showing areas of existence of lamellar LCs at the Tween 80/ lecithin mass ratio indicated. LCI and LC2 system investigated are marked with full circles.
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highly viscous mixtures and their LC structure was confirmed using PLM. Birefringence was visualized and lamellar mesophases were identified as Maltese crosses, either in a separate arrangement or in chains as oily streaks. Such phase behavior is closely related to lecithin’s structural characteristics as described by the critical packing parameter, the values of which may, being a fundamental geometric quantity, represent some critical conditions for possible aggregate shapes. The critical packaging parameter value for lecithin, namely for phosphatidylcholine as its principal ingredient is ½ 1, which meets the criteria for bilayer formation (Choi et al., 1999). On the other hand, Tween 80, forms micellar solution, a hexagonal phase and inverted micellar solution with increasing concentration in water (Sharma and Warr, 2012). Namely, its critical packing parameter was estimated to be 0.07 (Amani et al., 2011) indicating the ability to form spherical structures. Addition of Tween 80 to lecithin would therefore result in reduced packing parameter and alteration of lamellar phase to hexagonal or micellar one. Regardless of its tendency to form other structures, only lamellar liquid crystalline phase was identified for Tween 80/lecithin/IPM/water mixture investigated in our experiments. This is consistent with literature data (Rong et al., 1996) where the incorporation of Tween 80 into fatty acid/lecithin lamellar LC (i.e. Stratum corneum lipid model) resulted in strong increase in interlayer spacing for the initial addition of Tween 80 followed by formation of two lamellar structures. Separation into two lamellar structures was attributed to space requirement of bulk polyoxyethylene headgroup. In study of parenteral dosage forms performed by Moreno et al. (2003), phase behavior of systems containing water/lecithin/Tween 80/IPM at different lecithin/Tween 80 ratios was evaluated. To the best of our knowledge, this is the only study comprising the same components as used in our investigation. In contrast to our results, only microemulsion region of clear and isotrope low–viscosity samples was established, while gel-like structures were reported, but not characterized. Tween 80 was able to reduce packing parameter of lecithin that resulted in formation of microemulsion, however it must be considered that phosphatidylcholine content of soybean lecithin used was significantly lower (20% w/w) compared to one used in our study (not less than 94% w/w). Further on, structural evaluation of unloaded and AP-loaded LC samples was performed in our study using SAXS together with DSC and rheological measurements. Principally LC structural characteristics following incorporation of antioxidant with amphiphilic properties were of most interest. Lamellar LC1 and LC2 samples (the composition is reported in Table 1 and highlighted in Fig. 1) were selected from the central part of the established LC region avoiding phase boundaries and comprising the same percentage (w/w %) of surfactant mixture. The photomicrographs of unloaded and AP-loaded formulations, analyzed by PLM, are shown in Fig. 2. The presence of Maltese crosses was observed in the case of unloaded LC samples. It was found to be dependent on water content, with a decreasing number of structures in Maltese crosses observed by enhanced water amount (Fig. 2A and B). Incorporation of AP into the system caused a reduction in the well-defined Maltese crosses as seen for AP-loaded LC1 (Fig. 2C), however for AP-loaded LC2 with the highest water content birefringence was not observed (data not shown). A reasonable explanation is that liquid crystalline structures were too small to be observed with used magnifications since ordered structure was clearly confirmed by SAXS measurements. SAXS analysis was performed and diffractograms were taken at various temperatures, including room (25 °C), skin surface (32 °C), and body (37 °C) temperature. SAXS patterns of unloaded and APloaded LC1 and LC2 at different temperatures tested are shown in Fig. 3. It is well known that LCs can be oriented to form one, two, or three-dimensional structures and SAXS curves show Bragg peak
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Fig. 2. Polarized light microscopy photomicrographs of unloaded samples: (A) LC1, (B) LC2, and (C) AP-loaded sample LC1.
intensities for specific values of the scattering vector q. Results obtained from SAXS are in good agreement with those from PLM because the q values for unloaded LC samples show the ratio q1:q2 = 1:2, which is indicative for the presence of lamellar structure. The correlation distances 1:2 of two Bragg peaks remained invariable of temperature. However, at 37 °C additional peak at low q value was observed for both unloaded systems tested. It was attributed to transition to micellar solution due to high concentration of Tween 80 accompanied by dissolution of lamellar liquid crystalline phase at higher temperatures as reported for ternary system lecithin/IPM/water (Harms et al., 2005). Results of interlayer spacing d support temperature effect on inner structure (Table 2). By rising temperature from 25 to 37 °C interlayer spacing increases for unloaded LC1, whereas in case of unloaded LC2 firstly decreases at 32 °C and then increases at 37 °C. The latter is in line with results on phospholipid membranes, which indicated decrease in interlayer spacing due to strengthening of attractive van der Waals interactions with temperature. However, at some point the repulsion prevails due to stronger thermal fluctuations, resulting in increased interlayer spacing (Szekely et al., 2012).
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Fig. 3. Scattering curves of unloaded and AP-loaded (inset) (A) LC1 and (B) LC2 at three different temperatures.
For LC2 minimum of interlayer spacing was not observed as it is most likely beyond 32 °C. Additionally, water content has a great effect on the repeat distance within the lamellar structure. An increasing interlayer spacing ranging from 7.12 nm (LC1) to 7.90 nm (LC2) can be observed with increasing water content and coincides with swelling of surfactants (Mackeben et al., 2001 and Zhuang et al., 2008). It is also expected that addition of Tween 80 with different
Table 2 Interlayer spacing d of unloaded and AP-loaded LC1-2 samples at various temperatures. d (nm)
25 °C 32 °C 37 °C
LC1
LC2
LC1-AP
LC2-AP
7.12 7.24 7.42
7.90 7.57 7.78
9.28 8.73 /
8.56 8.18 /
structural properties compared to lecithin resulted in increased interlayer spacing followed by formation of additional micellar solution at 37 °C. This interpretation is supported by results of AP-loaded LC samples. Namely, the characteristic Braggs reflections corresponding to lamellar phase were observed at 25 and 32 °C, yet less defined. Further, the existence of micellar solution was confirmed due to protuberant scattering peak that was also the only peak observed at 37 °C. Though increasing temperature should result in scattering peak, which moves to lower, not higher q, adequate explanation for this behavior cannot be given at this time. To continue, AP with amphiphilic structure similar to Tween 80 (i.e. large polar moiety and single lipophilic chain) most likely reduces packing parameter of Tween 80/lecithin mixture and thereby exhibits similar effect as Tween 80. Interlayer spacing was evidently increased for APloaded LC samples when compared to unloaded LC samples (Table 2). Widening effect is in agreement to the increase of the interlayer spacing observed with timolol maleat-loaded lameller mesophase of lecithin/IPM/water (Mackeben et al., 2001). In this case formation
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of hydrogen bonds between lecithin and polar headgroup of drug molecules was postulated; therefore the former hydratation water becomes free and could be located between the polar heads of the lecithin molecules within the lamellae. The same mechanism can be proposed for Tween 80 and AP due to multiple polar functional groups on polar moiety followed by structural rearrangement to micellar aggregates with increasing temperature. In addition, this structural rearrangement is also relevant for dermal delivery. AP skin permeation from LC1 was previously evaluated and proven to be superior to other colloidal systems tested (Gosenca and Gašperlin, 2011b). However, not due to lamellar structure as proposed at that time but rather coexistence of lamellar and micellar phase at 32 °C as confirmed by SAXS results presented in this work. DSC analysis was employed in order to highlight the state of water in LC samples to consequently evaluate the strength of interactions between surfactant molecules and water. Firstly, preliminary tests have been carried on individual components (Fig. 4A). In the cooling curve of the bidistilled water, a large and sharp exothermic peak was observed at approximately 19 °C, which indicates freezing of supercooled water. For IPM, three exothermic peaks were detected. Namely, the largest peak appears at approximately 7 °C and represents solidification of the IPM. Furthermore, two exothermic peaks at 14 °C and 20 °C most probably correspond to solidification of the small amount of IPM impurity (the declared purity of IPM used is P90%), which is in agreement with the previous findings (Podlogar et al., 2004). A wide endothermic peak at 1 °C and 7 °C in DSC heating curves corresponds to melting of pure water and IPM (Bonacucina et al., 2012) (Fig. 4A; dotted line). No thermal events were observed in DSC curves of Tween 80, lecithin, or AP (data not shown).
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Cooling curves for unloaded LC samples are presented in Fig. 4B (solid lines). A triple exothermic peak ranging from 7 °C to 20 °C was observed and is attributed to IPM crystallization. The peak shape was similar to that of IPM alone, which thus indicates freezing of IPM without any interactions with other molecules. An additional broad exothermic peak appears at lower temperatures (i.e., between approximately 20 °C and 45 °C), which corresponds to freezing of freezable interlamellar water. Namely, it is possible to distinguish between freezable and nonfreezable water because water molecules form a hydration shell around the polar groups of surfactants and an aqueous interlayer between lamellae in lamellar LC systems. As a general rule, water molecules that interact with polar heads of surfactants are bound so tightly that they cannot form hydrogen bonds with their neighboring water molecules and can be understood as nonfreezable water, whereas the water that exists in the interbilayer region keeps the degree of freedom necessary to form hydrogen bonds and is designated freezable interlamellar water (Bonacucina et al., 2012 and Kodama and Aoki, 2001). The freezing temperature of water can therefore be dependent on water and surfactant content, and consequently the strength of interactions. The lowest water crystallization temperature was observed for unloaded LC1 with the lowest water-tosurfactant ratio that means that the water is strongly bound by headgroup of the surfactants as the freezing temperature (approximately 40 °C) is very low compared to freezing of pure water (19 °C). As for the unloaded LC2 sample, the water crystallization peak is shifted towards higher temperatures. This allows us to presume that water is bound to polar heads of surfactants because surfactants have to be supersaturated with water before a free water freezing peak can be observed (Garti et al., 2000); neverthe-
Fig. 4. (A) DSC curves of individual components (i.e., water and IPM) and (B) unloaded and AP-loaded LC samples. DSC cooling curves are indicated as a solid line, and DSC heating curves as a dotted line.
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less, the interactions are weaker compared to unloaded LC1 system. Incorporation of AP resulted in shifting water crystallization peak towards higher temperatures, which was especially evident for AP-loaded LC1. This supports our previous assumption that water molecules are less firmly bound due to interactions between AP and surfactant molecules. From heating part of the curve (Fig. 4B; dotted lines) a broad endothermic peak, even though not very pronounced, that begins at 30 °C and corresponds to melting of ice from freezable interlamellar water was observed. Again, the more remote the water molecules are from the bilayer surfaces, the more the peak moves towards 0 °C and the more similar the shape is to that of pure water (Kodama and Aoki, 2001) (Fig. 4A; dotted line). The effect is more evident in the case of unloaded LC2 system due to higher amount of water between the lamellae as confirmed by higher interlayer spacing. For AP-loaded LC systems, the peak became more distinguishable for AP-loaded LC1 system indicating more pronounced structural changes within this system. Rheological analysis is one of the most frequently used techniques for structure characterization at the macroscopic level. The shape of the viscosity curves clearly shows decreasing viscosity with increasing shear rate at all temperatures tested, which is characteristic for non-Newtonian systems exhibiting shear-thinning behavior (Chorilli et al., 2011). Fig. 5 shows changes in viscosity as a function of shear rate for unloaded and AP-loaded LC samples obtained from rotational tests. Further on, when looking the viscosity at the lowest measured shear rate (2 s1) at 25 °C (Fig. 6), it is notably higher for unloaded LC2 compared to unloaded LC1. Evidently surfactant molecules in system with higher waterto-surfactant ratio (LC2) form denser layer structure. With increasing temperature the surfactant molecule aggregate more loosely in the lamellae as seen from decrease in viscosity values, which is in accordance with the published data (Zhao et al., 2011). In our study the decrease was more pronounced for unloaded LC2 at 32 °C, while further decrease at 37 °C was less prominent. A possible explanation is that cohesion of liquid crystalline structure was reduced at 32 °C, most likely due to weaker hydrophilic binding mechanism (Harms et al., 2005), even though micellar aggregates were not observed. The viscosity was also inversely proportional with temperature for AP-loaded LC samples, which also exhibited evidently lower viscosities as unloaded samples, again pointing to transition from lamellar liquid crystalline structure to isotropic solution after incorporation of amphiphilic AP. It is important to emphasize that observed viscosity curves for AP-loaded LC sample, in particular at 37 °C, are in fact viscosity curves for micellar solution. More information about the lamellar structure can be obtained from oscillatory rheology; shear frequency sweep measurements were performed with typical curves of the frequency dependence of the storage modulus G0 , loss modulus G00 , and complex viscosity for unloaded and AP-loaded LC samples presented in Fig. 7. Unloaded LC samples are more elastic than viscous, which is in accordance with literature data (Farkas et al., 2000; Németh et al., 1998; Zhang et al., 2008): G0 is about one order of magnitude higher than G00 throughout the entire frequency range. G0 was nearly frequencyindependent and G00 shows a local minimum, whereas the complex viscosity drops linearly as a function of frequency. The ratio between G0 and G00 (tan d) at various frequencies (100 Hz, 10 Hz) and minimum of G00 (Table 3) clearly demonstrate that the sample becomes more elastic with an increasing water to surfactant ratio, as seen from the decrease in tan d. Higher values of G0 for unloaded LC2 system indicates stronger interactions between the bilayer that is supported also by viscosity measurements, however is not consistent with SAXS and DSC results. DSC results clearly revealed weaker interactions between surfactant mixture and water for unloaded LC2 system while SAXS analysis confirmed increased inter-
Fig. 5. Viscosity curves showing shear-thinning behavior for unloaded (solid line) and AP-loaded (dotted line) (A) LC1 and (B) LC2 at 25 °C (h), 32 °C (e), and 37 °C (s). Data are expressed as mean ± S.D. (n = 3).
Fig. 6. Viscosity of unloaded and AP-loaded LC samples measured at the lowest shear rate 2 s1 and various temperatures. Data are expressed as mean ± S.D. (n = 3).
layer distance, which is connected with weaker, not stronger, interactions (Youssry et al., 2008). The same tendency of rheological parameters was observed for AP-loaded system; both moduli decrease when compared to unloaded LC systems. This point to structural changes and is consistent with SAXS results since lamellar phase coexists with micellar. A decrease was very pronounced for AP-loaded LC1, indicating weaker interactions between the bilayer after incorporation of amphiphilic molecule. Results of SAXS and DSC measurements strengthen this assumption. Namely, the increase of interlayer spacing and shift of water crystallization peak towards higher temperature was more prominent for AP-loaded LC1. And even though explanation for inconsistency of rheological towards SAXS and DSC results for unloaded LC2 samples cannot be given at the moment, it is probably due to this phenomena disturbance of lamellae in APloaded LC2 less distinct as seen from only slight decrease in rheological parameters. What is more, this is also confirmed by less prominent increase in interlayer spacing and only minor changes
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Fig. 7. Elastic modulus (G0 , h), loss modulus (G00 , s), and complex viscosity (g, e) of unloaded (solid line) and AP-loaded (dotted line) (A) LC1 and (B) LC2 sample as a function of frequency (x) at a stress of 10% at 25 °C. Data are expressed as mean ± S.D. (n = 3).
Table 3 Loss tangent (tan d) at various frequencies (100 Hz, 10 Hz, and minimum of G00 ) of unloaded LC samples with corresponding water/surfactant (W/S) ratio. Sample
W/S ratio
tan d (100 Hz)
tan d (10 Hz)
tan d (G00 min)
LC1 LC2
0.55 0.87
0.233 0.202
0.181 0.099
0.185 0.078
Fig. 8. Proliferation of keratinocytes 4 h after addition of unloaded LC1-2 samples and SDS solution to the cells (T = 37 °C). The results are presented relative to the proliferation of untreated control cells. Data are expressed as mean ± S.D. (n = 6). a P < 0.05 compared to SDS solution at the same concentration.
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for its practical use. In keeping with this, a cytotoxicity assessment of LC systems on a keratinocyte cell line was performed. Unloaded LC samples were tested because AP can directly reduce the MTS reagent and in a concentration range (0.45, 0.90 and 4.50 mg of LC sample/ ml) that can reveal differences in cell proliferation regarding LC composition. Keratinocyte proliferation was measured with an MTS assay following short-term exposure (4 h post-treatment). Exposing keratinocytes to lamellar LC systems at a concentration of 0.45 mg/ml resulted in no considerable impact on cell proliferation, being around 100%, and it also remained at a high level at a concentration of 0.90 mg/ml. The pronounced differences regarding LC composition became significant at the highest concentration tested (4.50 mg/ml); the cell proliferation was highest for LC2 (83.45%), with the highest water content compared to LC1 (71.38%). The cell proliferation measured by the MTS reduction test is summarized in Fig. 8. After exposure to SDS, which is used as a positive control in skin irritation testing (Effendy and Maibach, 1996), the cell proliferation dramatically declined. The LC systems tested performed significantly better than SDS solution, proving lamellar LC to be a potential non-toxic dermal delivery system. These results are in accordance to very low acute and low cumulative irritancy potential that was established with patch test for lecithin microemulsion gel composed of soybean lecithin, IPM and water (Dreher et al., 1996), while IPM was confirmed as safe lipophilic excipient, even though trend of decreased viability was observed (Savic´ et al., 2009). Nevertheless, being aware of the fact that biological assays are not able to evaluate structural damage that may occur at a non-cytotoxic dose, but can promote pathological effects (Lastella et al., 2007), extensive study oriented towards cells’ morphological evaluation using atomic force microscopy is in progress. We strongly believe that the data obtained together with the MTS results presented here will unequivocally answer the question regarding the dermal acceptability of LC formulations investigated.
4. Conclusions This study revealed that (Tween 80/lecithin = 1/1)/IPM/water mixtures were able to form lamellar LC, as confirmed by the PLM and SAXS results. The structure was additionally evaluated regarding its quantitative composition. The interlayer spacing was affected by water content, which can be attributed to differences in the strength of interactions between water and surfactant molecules, as observed by DSC measurements. A rheological study showed that the formulation behaves like a non-Newtonian system, with characteristics of shear-thinning material. The lamellar phase structure also remained organized at skin and physiological temperatures. Structural changes in the system were observed after incorporation of AP, which was used as an antioxidant, and were attributed to micelle formation due to its amphiphilic character and interactions with surfactant molecules. The cell proliferation remained high at 0.45 and 0.90 mg/ml, decreased at 4.50 mg/ml, but remained significantly higher when compared to the standard irritant SDS. Therefore, setting strict selection criteria for individual components together with qualitative structural evaluation were proved as a prosing approach in developing a skin compliant carrier system; namely, promising results obtained on living keratinocytes indicate that the lecithin based LC system shows great potential as a physiologically acceptable dermal delivery system.
Acknowledgements in peaks positions (DSC cooling curves) or shape (DSC heating curves) for AP-loaded LC2 compared to AP-loaded LC1. Biological acceptability is one of crucial aspects that should be considered when developing a novel delivery system and is decisive
The authors are grateful to the ICLC-Interlab Cell Line Collection, University of Genova (Italy), for providing the immortalized human keratinocyte cell line NCTC2544. We thank Tanja Tavcˇar
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and Lucija Kralj for carrying out a lot of the experimental work and Mr. Anton Kokalj for preforming SAXS experiments.
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