European Journal of Pharmaceutical Sciences 50 (2013) 114–122

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European Journal of Pharmaceutical Sciences

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Lecithin based lamellar liquid crystals as a physiologically acceptabledermal delivery system for ascorbyl palmitate

0928-0987/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.ejps.2013.04.029

⇑ Corresponding author. Address: University of Ljubljana, Faculty of Pharmacy,Aškerceva 7, 1000 Ljubljana, Slovenia. Tel.: +386 1 476 9634; fax: +386 1 425 8031.

E-mail address: mirjana.gasperlin@ffa.uni-lj.si (M. Gašperlin).

Mirjam Gosenca a, Marija Bešter-Rogac b, Mirjana Gašperlin a,⇑a University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, Ljubljana, Sloveniab University of Ljubljana, Faculty of Chemistry and Chemical Technology, Aškerceva 5, Ljubljana, Slovenia

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 December 2012Received in revised form 8 April 2013Accepted 25 April 2013Available online 3 May 2013

Keywords:Pseudoternary phase diagramLamellar liquid crystalline phaseAscorbyl palmitateKeratinocytesCytotoxicity

Liquid crystalline systems with a lamellar structure have been extensively studied as dermal delivery sys-tems. Ascorbyl palmitate (AP) is one of the most studied and used ascorbic acid derivatives and isemployed as an antioxidant to prevent skin aging. The aim of this study was to develop and characterizeskin-compliant dermal delivery systems with a liquid crystalline structure for AP. First, a pseudoternaryphase diagram was constructed using Tween 80/lecithin/isopropyl myristate/water at a Tween 80/leci-thin mass ratio of 1/1, and the region of lamellar liquid crystals was identified. Second, selected unloadedand AP-loaded lamellar liquid crystal systems were physicochemically characterized with polarizingoptical microscopy, small-angle X-ray scattering, differential scanning calorimetry, and rheology tech-niques. The interlayer spacing and rheological parameters differ regarding quantitative composition,whereas the microstructure of the lamellar phase was affected by the AP incorporation, resulting eitherin 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 crys-tal systems on a keratinocyte cell line. The results suggest that the lamellar liquid crystals that weredeveloped could be used as a physiologically acceptable dermal delivery system.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Liquid crystals (LCs) are self-assembled organized mesophaseswith properties of both liquids (fluidity) and solids (ordered crys-talline structure, optical anisotropy). Lyotropic LCs are formed byamphiphilic molecules (i.e., surfactants), precisely by their hy-drates or solvates, as well as associates of hydrated or solvatedmolecules. Hydration or solvation results in various self-assembledliquid crystalline structures, and thereby a wide range of LC struc-tures from lamellar and hexagonal to cubic (Burducea, 2004;Müller-Goymann, 2007; Stevenson et al., 2005).

Lyotropic LCs have attracted considerable interest as drug carri-ers in pharmaceutical technology with major focus put towardshexagonal 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 dermalapplication due to great similarity with the intercellular lipidmembrane, hydrating properties, and ideal consistency. LamellarLCs are formed by hydrated amphiphilic molecules in the shapeof cylinders. They arrange themselves in layers, yielding a lamellarphase with alternating polar and nonpolar layers. Various aspects

of lamellar LC systems for dermal delivery were addressed in theliterature concerning stability (Vicentini et al., 2008), improveddrug activity (Chorilli et al., 2011 and Nesseem, 2001), or modifiedrelease. Prolonged release of very water-soluble as well as water-insoluble drug was observed by Makai et al. (2003), while inhibiteddrug release from swollen liquid crystalline systems compared tohexagonal phase was explained with rapid water uptake of less or-dered lamellar liquid crystalline structure by Farkas et al. (2000).Moreover, extensive research of topical vehicles with lamellarliquid crystalline structure based on alkylpolyglukoside naturalsurfactants was performed and their physicochemical propertieswere evaluated towards in vitro/in vivo skin performance (Savicet al., 2006, 2009; Savic et al., 2011).

To develop a novel delivery system with LC structure phasebehavior of a particular amphiphilic molecule/oil/water system isinvestigated by the construction of a ternary or a pseudo ternaryphase diagram, and adequate methods must be involved to deter-mine the structural features of formed systems. Polarized lightmicroscopy (PLM) (Zhang et al., 2008), transmission electronmicroscopy (Mondain-Monval, 2005), small-angle X-ray scattering(SAXS) (Zhuang et al., 2008), differential scanning calorimetry(DSC) (Fehér et al., 2005), and rheology measurements (Berniet al., 2002; Németh et al., 1998; Youssry et al., 2008) are appliedto achieve this goal as reviewed by Müller-Goymann (2004). Theaspect of LC’s biological acceptability is also very important;

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therefore the skin irritation potential of individual componentsshould already be considered in the development stage. Amphi-philic molecules must meet the following requirements: biode-gradability, biocompatibility, and non-toxicity. Studies in thisarea have confirmed the adequately low toxicity of non-ionic sur-factants such as polyoxyethylene sorbitan fatty acid derivatives(Tweens) as well as zwitterionic surfactants of natural origin suchas lecithin (Fiume, 2001 and Malmsten, 2002). Moreover, it shouldbe pointed out that lecithin has the unique property to form lamel-lar phases due to its molecular structure. Lecithin is an amphiphilicmolecule with two non-polar hydrocarbon chains and zwitterionicpolar headgroup, which have dipole moments and are strongly hy-drated. The combination of two large moieties of opposite polari-ties strongly defines lecithin properties. LC structures are justone of the various self-organizing structures of lecithin in wateralong with micelles, swollen micelles, microemulsions, emulsions,organogels and liposomes. It was reported that the lamellar phaseis predominant in lecithin/water binary systems and ternary sys-tems (Shchipunov, 1997). Among oils, fatty acid esters such as iso-propyl myristate (IPM) are of particular interest due to theirbiocompatible and biodegradable nature (Kumar and Katare,2005). Phase behavior of lecithin/IPM/water (Harms et al., 2005and Mackeben et al., 2001) and Tween 80/IPM/water system(Bonacucina et al., 2012) is well established in the literature. Phasediagrams comprising Tween 80 were also investigated for solubili-zation of various oils (Alam et al., 2009; Garti et al., 2001; Sharmaand Warr, 2012; Yaghmur et al., 2002), while Brij 97 was used forsolubilizing IPM into liquid crystalline phase (Wang et al., 2006).

Ascorbyl palmitate (AP), an amphiphilic derivative of ascorbicacid, is widely used as an antioxidant active substance in pharma-ceutical and cosmetic formulations to enhance skin protectionfrom oxidative stress and consequently combat skin (photo)ageingas reported in recent review (Gašperlin and Gosenca, 2011a). Itsstability and effectiveness have been studied in various dermaldelivery systems, i.e. microemulsions, solid lipid nanoparticlesand liposomes (Jurkovic et al., 2003 and Kristl et al., 2003). More-over, AP itself forms self-assembled liquid crystalline structuresin water. Namely, a cubic and two lamellar mesophases were re-ported for AP/water binary system depending on concentrationand temperature (Benedini et al., 2011). Semisolid gel with lamel-lar structure that could be used as dermal delivery system due toimproved solubilization and stabilization of drugs combined withantioxidant properties of ascorbic acid is formed on cooling. In or-der to obtain stable mesophases at room temperatures, polyethyl-eneglycol 400 was added to AP/water system. However, thereduction in transition temperatures was not low enough to em-ploy the systems as pharmaceutical formulations (Benedini et al.,2012).

The aim of this study was to develop novel skin compliantdelivery system for AP. Phase behavior of Tween 80/lecithin/IPM/water system, comprising of biocompatible components, was eval-uated. This is the first time that selected components have beenstudied in order to obtain dermal delivery system, therefore con-stant surfactant mass ratio Tween 80/lecithin = 1/1 was applied.The microstructure of systems, especially in view of structuralalterations following incorporation of AP with amphiphilic charac-

Table 1The composition of LC systems tested (w/w%).

Sample Lecithin Tween 80 IPM Bidistilled water AP

LC1 22.50 22.50 30.00 25.00 /LC2 22.50 22.50 17.50 37.50 /LC1-AP 22.50 22.50 30.00 25.00 1LC2-AP 22.50 22.50 17.50 37.50 1

ter, was studied by various techniques such as PLM, SAXS, DCS, andrheological analysis. The potential cytotoxic effects of LC systemswere investigated using a human keratinocyte cell line (NCTC2544)as an in vitro model.

2. Materials and methods

2.1. Materials

Lipoid S-100�, soybean phospholipid with not less than 94%w/w phosphatidylcholine content was provided by Lipoid GmbHGermany. According to the manufacturer’s specification the fattyacids of the two acyl groups of phosphatidylcholine are palmitic(15%), stearic (3%), oleic and isomers (12%), linoleic (62%) and lin-olenic (5%).

Tween 80�, polyoxyethylene (20) sorbitan monooleate, withtypical fatty acid composition of approximately 70% oleic acidand 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 wasprovided by Fluka, Sigma-Aldrich GmbH, Germany.

2.2. Methods

2.2.1. Construction of pseudoternary phase diagramIn order to determine the concentration range of components

(water/IPM/Tween 80/lecithin) that form lyotropic LCs, a pseudo-ternary phase diagram with a 1/1 mass ratio of Tween 80 to leci-thin was constructed using a water titration technique. For thetitration process, a homogeneous mixture of IPM and surfactantmixture at weight ratios ranging from 95/5 to 5/95 was slowlytitrated with aliquots of bidistilled water and stirred at 25 �C fora sufficiently long time to obtained equilibrium. After equilibriumwas reached, the mixtures were checked visually for transparency.Visually clear samples of high viscosity were additionally checkedthrough the cross polarizer for the presence of a liquid crystallinephase. Because only lamellar LCs were of our interest, no attemptswere made to completely identify the other regions of the pseudo-ternary phase diagram in detail, and these have been described interms of their visual and external appearance.

2.2.2. Sample preparationRepresentative samples of lyotropic LCs, whose composition is

presented in Table 1, were further characterized. Samples wereprepared by mixing appropriate amounts of IPM, Tween 80, andlecithin to form a homogeneous mixture; in the case of AP-loadedsamples, AP was dissolved in a homogeneous oil–surfactant mix-ture. Water was added afterwards during continuous stirring toform lyotropic LCs.

2.2.3. Polarizing light microscopyThe structure of the unloaded and AP-loaded LC samples was

examined with a microscope with polarization using a PhysicaMCR 301 rheometer (Anton Paar, Graz, Austria) at 25 �C. The mag-nification 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 performedwith an evacuated Kratky compact camera system (Anton Paar,Graz, Austria) with a block collimating unit, attached to a conven-tional X-ray generator (Bruker AXS, Karlsruhe, Germany) equippedwith a sealed X-ray tube (Cu-anode target type) producing Ni-fil-tered Cu Ka radiation with a wavelength of k = 0.154 nm. The

Fig. 1. Pseudoternary phase diagram of the system Tween 80/lecithin/IPM/waterwith 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 fullcircles.

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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 astandard quartz capillary placed in a thermally controlled sampleholder centered in the X-ray beam. The scattered intensity was de-tected by a linear position sensitive detector (PSD 50 m, M. Braun,Garching, Germany). Measurements were performed at 25, 32, and37 �C with measurement time setting t = 3600 s. The interlayerspacing d was calculated as d = 2p/q1 where q1 is the value of thescattering 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 surfactantand water molecules. Nitrogen with a flow of 20 ml/min was usedas a purge gas. Approximately 10 mg of sample, that is, individualcomponents (Tween 80, lecithin, IPM, and water) and LC systems(unloaded and AP-loaded LC samples), was weighed precisely intoa small aluminum pan and quickly sealed hermetically to preventwater 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 (heatingrate: 5 K/min).

2.2.6. Rheological measurementsRotational and oscillatory rheological tests were performed for

unloaded and AP-loaded LC samples using a Physica MCR 301 rhe-ometer (Anton Paar, Graz, Austria) with a cone-plate measuringsystem CP50-2 (cone diameter 49.961 mm, cone angle 2.001�, sam-ple thickness 0.209 mm). Rotational tests were performed at25.0 ± 0.1 �C, 32.0 ± 0.1 �C, and 37.0 ± 0.1 �C, whereas oscillatorytests were performed at a constant temperature of 25.0 ± 0.1 �C.Rotational tests were used to determine the viscosity, which fora cone-plate measuring system is calculated as g = s/ _c where s isthe shear stress and _c is the shear rate.

Oscillatory tests were performed to define the elastic and lossmoduli, which are calculated as G0 = (s/c) � cosd and G00 =(s/c) � sind where s is the shear stress, c is the deformation, andd is the phase shift angle, together with complex viscosity calcu-lated as g� = s/(c �x) where x is angular frequency.

The shear rate during the rotational tests ranged from 2 to100 s�1. For oscillatory analysis, first the stress sweep measure-ments were performed at a constant frequency of 10.0 s�1 in orderto determine the linear viscoelastic region. Afterwards, theoscillatory shear measurements were carried out as a function offrequency (0.1–100 s�1) at a constant amplitude (10%) chosenwithin the linear region.

2.2.7. Cell culture and treatmentHuman keratinocyte cells (cell line NCTC 2544, ICLC, University

of Genoa) were cultured as adherent monolayers at 37 �C in ahumidified atmosphere of 5% CO2. They were grown in Eagle’s Min-imum Essential Medium with Earle’s balanced salt solution supple-mented 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 tryp-sin/EDTA when they reached 80 to 90% confluence.

Cell culture reagents were from Sigma, Germany unless other-wise indicated.

2.2.8. MTS assayThe effect of unloaded LC samples on cell proliferation was

assessed using the MTS assay (Cell titer 96 Aqueous One SolutionCell Proliferation Assay; Promega, Madison, WI) according tomanufacturer’s procedure. The assay is based on conversion of3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium, an inner salt, into the soluble coloredformazan product by mitochondrial dehydrogenase enzymes inmetabolically active cells. Keratinocytes were seeded at a densityof 0.5 � 104 cells per well in 96-well plates. After one day (theattachment phase) the cells were treated with the test formula-tions. Test formulations were prepared by diluting unloaded LC1and LC2 as well as sodium dodecyl sulfate (SDS) (positive control)in cell culture medium to final concentrations of 0.45, 0.90, and4.50 mg/ml.

Keratinocyte proliferation was assessed 4 h after the addition oftest formulations. The absorbance of formazan was measured at492 nm using a Safire2 microplate reader (Tecan, Switzerland).The results were expressed as the absorbance ratio of treated tocontrol cells, and cell proliferation was calculated as cell prolifera-tion = (AS–AS0)/(AC–AC0) where AS is the absorbance of the treatedcells (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 theindependent samples Student’s t-test. Significance was tested atthe 0.05 level of probability.

3. Results and discussion

This study describes the preparation and characterization of LCsystems as innovative and biocompatible carriers for dermal deliv-ery of AP.

Fig. 1 represents the pseudoternary phase diagram of the inves-tigated system Tween 80/lecithin/IPM/water for the applied massratio of Tween 80 to lecithin = 1/1. An anisotropic lamellar LCphase appeared in the wide region of the pseudoternary phase dia-gram. More precisely, adding water at a level of 15–55% and 12.5–35% of IPM resulted in the formation of lamellar LC systems. Inother parts of the diagram, the formation of coarse emulsion andopaque 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

Fig. 2. Polarized light microscopy photomicrographs of unloaded samples: (A) LC1,(B) LC2, and (C) AP-loaded sample LC1.

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highly viscous mixtures and their LC structure was confirmedusing PLM. Birefringence was visualized and lamellar mesophaseswere identified as Maltese crosses, either in a separate arrange-ment or in chains as oily streaks. Such phase behavior is closely re-lated to lecithin’s structural characteristics as described by thecritical packing parameter, the values of which may, being a funda-mental geometric quantity, represent some critical conditions forpossible aggregate shapes. The critical packaging parameter valuefor lecithin, namely for phosphatidylcholine as its principal ingre-dient is ½ � 1, which meets the criteria for bilayer formation (Choiet al., 1999). On the other hand, Tween 80, forms micellar solution,a hexagonal phase and inverted micellar solution with increasingconcentration in water (Sharma and Warr, 2012). Namely, its crit-ical packing parameter was estimated to be 0.07 (Amani et al.,2011) indicating the ability to form spherical structures. Additionof Tween 80 to lecithin would therefore result in reduced packingparameter and alteration of lamellar phase to hexagonal or micel-lar one. Regardless of its tendency to form other structures, onlylamellar liquid crystalline phase was identified for Tween 80/leci-thin/IPM/water mixture investigated in our experiments. This isconsistent with literature data (Rong et al., 1996) where the incor-poration of Tween 80 into fatty acid/lecithin lamellar LC (i.e. Stra-tum corneum lipid model) resulted in strong increase in interlayerspacing for the initial addition of Tween 80 followed by formationof two lamellar structures. Separation into two lamellar structureswas attributed to space requirement of bulk polyoxyethyleneheadgroup. In study of parenteral dosage forms performed byMoreno et al. (2003), phase behavior of systems containingwater/lecithin/Tween 80/IPM at different lecithin/Tween 80 ratioswas evaluated. To the best of our knowledge, this is the only studycomprising the same components as used in our investigation. Incontrast to our results, only microemulsion region of clear and iso-trope low–viscosity samples was established, while gel-like struc-tures were reported, but not characterized. Tween 80 was able toreduce packing parameter of lecithin that resulted in formationof microemulsion, however it must be considered that phosphati-dylcholine 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 LCsamples was performed in our study using SAXS together with DSCand rheological measurements. Principally LC structural character-istics following incorporation of antioxidant with amphiphilicproperties 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 regionavoiding phase boundaries and comprising the same percentage(w/w %) of surfactant mixture.

The photomicrographs of unloaded and AP-loaded formula-tions, analyzed by PLM, are shown in Fig. 2. The presence of Mal-tese crosses was observed in the case of unloaded LC samples. Itwas found to be dependent on water content, with a decreasingnumber of structures in Maltese crosses observed by enhancedwater amount (Fig. 2A and B). Incorporation of AP into the systemcaused a reduction in the well-defined Maltese crosses as seen forAP-loaded LC1 (Fig. 2C), however for AP-loaded LC2 with the high-est water content birefringence was not observed (data notshown). A reasonable explanation is that liquid crystalline struc-tures were too small to be observed with used magnifications sinceordered structure was clearly confirmed by SAXS measurements.

SAXS analysis was performed and diffractograms were taken atvarious temperatures, including room (25 �C), skin surface (32 �C),and body (37 �C) temperature. SAXS patterns of unloaded and AP-loaded LC1 and LC2 at different temperatures tested are shown inFig. 3. It is well known that LCs can be oriented to form one, two, orthree-dimensional structures and SAXS curves show Bragg peak

intensities for specific values of the scattering vector q. Resultsobtained from SAXS are in good agreement with those from PLMbecause the q values for unloaded LC samples show the ratioq1:q2 = 1:2, which is indicative for the presence of lamellar struc-ture. The correlation distances 1:2 of two Bragg peaks remainedinvariable of temperature. However, at 37 �C additional peak atlow q value was observed for both unloaded systems tested. Itwas attributed to transition to micellar solution due to high con-centration of Tween 80 accompanied by dissolution of lamellar li-quid crystalline phase at higher temperatures as reported forternary system lecithin/IPM/water (Harms et al., 2005). Results ofinterlayer spacing d support temperature effect on inner structure(Table 2). By rising temperature from 25 to 37 �C interlayer spacingincreases for unloaded LC1, whereas in case of unloaded LC2 firstlydecreases at 32 �C and then increases at 37 �C. The latter is in linewith results on phospholipid membranes, which indicated de-crease in interlayer spacing due to strengthening of attractivevan der Waals interactions with temperature. However, at somepoint the repulsion prevails due to stronger thermal fluctuations,resulting in increased interlayer spacing (Szekely et al., 2012).

Fig. 3. Scattering curves of unloaded and AP-loaded (inset) (A) LC1 and (B) LC2 at three different temperatures.

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For LC2 minimum of interlayer spacing was not observed as it ismost likely beyond 32 �C. Additionally, water content has a great ef-fect on the repeat distance within the lamellar structure. An increas-ing interlayer spacing ranging from 7.12 nm (LC1) to 7.90 nm (LC2)can be observed with increasing water content and coincides withswelling of surfactants (Mackeben et al., 2001 and Zhuang et al.,2008). It is also expected that addition of Tween 80 with different

Table 2Interlayer spacing d of unloaded and AP-loaded LC1-2 samples at varioustemperatures.

d (nm)

LC1 LC2 LC1-AP LC2-AP

25 �C 7.12 7.90 9.28 8.5632 �C 7.24 7.57 8.73 8.1837 �C 7.42 7.78 / /

structural properties compared to lecithin resulted in increasedinterlayer spacing followed by formation of additional micellar solu-tion at 37 �C. This interpretation is supported by results of AP-loadedLC samples. Namely, the characteristic Braggs reflections corre-sponding to lamellar phase were observed at 25 and 32 �C, yet lessdefined. Further, the existence of micellar solution was confirmeddue to protuberant scattering peak that was also the only peak ob-served at 37 �C. Though increasing temperature should result inscattering peak, which moves to lower, not higher q, adequate expla-nation for this behavior cannot be given at this time. To continue, APwith amphiphilic structure similar to Tween 80 (i.e. large polar moi-ety and single lipophilic chain) most likely reduces packing param-eter of Tween 80/lecithin mixture and thereby exhibits similar effectas Tween 80. Interlayer spacing was evidently increased for AP-loaded LC samples when compared to unloaded LC samples (Table 2).Widening effect is in agreement to the increase of the interlayerspacing observed with timolol maleat-loaded lameller mesophaseof lecithin/IPM/water (Mackeben et al., 2001). In this case formation

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of hydrogen bonds between lecithin and polar headgroup of drugmolecules was postulated; therefore the former hydratation waterbecomes free and could be located between the polar heads of thelecithin molecules within the lamellae. The same mechanism canbe proposed for Tween 80 and AP due to multiple polar functionalgroups on polar moiety followed by structural rearrangement tomicellar aggregates with increasing temperature. In addition, thisstructural rearrangement is also relevant for dermal delivery. APskin permeation from LC1 was previously evaluated and proven tobe superior to other colloidal systems tested (Gosenca and Gašper-lin, 2011b). However, not due to lamellar structure as proposed atthat time but rather coexistence of lamellar and micellar phase at32 �C as confirmed by SAXS results presented in this work.

DSC analysis was employed in order to highlight the state ofwater in LC samples to consequently evaluate the strength of inter-actions between surfactant molecules and water. Firstly, prelimin-ary tests have been carried on individual components (Fig. 4A). Inthe cooling curve of the bidistilled water, a large and sharp exo-thermic peak was observed at approximately �19 �C, which indi-cates freezing of supercooled water. For IPM, three exothermicpeaks were detected. Namely, the largest peak appears at approx-imately �7 �C and represents solidification of the IPM. Further-more, two exothermic peaks at �14 �C and �20 �C most probablycorrespond to solidification of the small amount of IPM impurity(the declared purity of IPM used is P90%), which is in agreementwith the previous findings (Podlogar et al., 2004). A wide endother-mic peak at �1 �C and 7 �C in DSC heating curves corresponds tomelting of pure water and IPM (Bonacucina et al., 2012) (Fig. 4A;dotted line). No thermal events were observed in DSC curves ofTween 80, lecithin, or AP (data not shown).

Fig. 4. (A) DSC curves of individual components (i.e., water and IPM) and (B) unloaded aheating curves as a dotted line.

Cooling curves for unloaded LC samples are presented in Fig. 4B(solid lines). A triple exothermic peak ranging from �7 �C to�20 �Cwas observed and is attributed to IPM crystallization. The peakshape was similar to that of IPM alone, which thus indicates freez-ing of IPM without any interactions with other molecules. An addi-tional broad exothermic peak appears at lower temperatures (i.e.,between approximately �20 �C and �45 �C), which correspondsto freezing of freezable interlamellar water. Namely, it is possibleto distinguish between freezable and nonfreezable water becausewater molecules form a hydration shell around the polar groupsof surfactants and an aqueous interlayer between lamellae inlamellar LC systems. As a general rule, water molecules that inter-act with polar heads of surfactants are bound so tightly that theycannot form hydrogen bonds with their neighboring water mole-cules and can be understood as nonfreezable water, whereas thewater that exists in the interbilayer region keeps the degree offreedom necessary to form hydrogen bonds and is designatedfreezable interlamellar water (Bonacucina et al., 2012 and Kodamaand Aoki, 2001). The freezing temperature of water can thereforebe dependent on water and surfactant content, and consequentlythe strength of interactions. The lowest water crystallization tem-perature was observed for unloaded LC1 with the lowest water-to-surfactant ratio that means that the water is strongly bound byheadgroup of the surfactants as the freezing temperature (approx-imately �40 �C) is very low compared to freezing of pure water(�19 �C). As for the unloaded LC2 sample, the water crystallizationpeak is shifted towards higher temperatures. This allows us to pre-sume that water is bound to polar heads of surfactants becausesurfactants have to be supersaturated with water before a freewater freezing peak can be observed (Garti et al., 2000); neverthe-

nd AP-loaded LC samples. DSC cooling curves are indicated as a solid line, and DSC

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 lowestshear rate 2 s�1 and various temperatures. Data are expressed as mean ± S.D. (n = 3).

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less, the interactions are weaker compared to unloaded LC1 sys-tem. Incorporation of AP resulted in shifting water crystallizationpeak towards higher temperatures, which was especially evidentfor AP-loaded LC1. This supports our previous assumption thatwater molecules are less firmly bound due to interactions betweenAP and surfactant molecules.

From heating part of the curve (Fig. 4B; dotted lines) a broadendothermic peak, even though not very pronounced, that beginsat �30 �C and corresponds to melting of ice from freezable interla-mellar water was observed. Again, the more remote the water mol-ecules are from the bilayer surfaces, the more the peak movestowards 0 �C and the more similar the shape is to that of purewater (Kodama and Aoki, 2001) (Fig. 4A; dotted line). The effectis more evident in the case of unloaded LC2 system due to higheramount of water between the lamellae as confirmed by higherinterlayer spacing. For AP-loaded LC systems, the peak becamemore distinguishable for AP-loaded LC1 system indicating morepronounced structural changes within this system.

Rheological analysis is one of the most frequently used tech-niques for structure characterization at the macroscopic level.The shape of the viscosity curves clearly shows decreasing viscos-ity with increasing shear rate at all temperatures tested, which ischaracteristic for non-Newtonian systems exhibiting shear-thin-ning behavior (Chorilli et al., 2011). Fig. 5 shows changes in viscos-ity as a function of shear rate for unloaded and AP-loaded LCsamples obtained from rotational tests. Further on, when lookingthe viscosity at the lowest measured shear rate (2 s�1) at 25 �C(Fig. 6), it is notably higher for unloaded LC2 compared to unloadedLC1. Evidently surfactant molecules in system with higher water-to-surfactant ratio (LC2) form denser layer structure. With increas-ing temperature the surfactant molecule aggregate more loosely inthe lamellae as seen from decrease in viscosity values, which is inaccordance with the published data (Zhao et al., 2011). In our studythe decrease was more pronounced for unloaded LC2 at 32 �C,while further decrease at 37 �C was less prominent. A possibleexplanation is that cohesion of liquid crystalline structure was re-duced at 32 �C, most likely due to weaker hydrophilic bindingmechanism (Harms et al., 2005), even though micellar aggregateswere not observed. The viscosity was also inversely proportionalwith temperature for AP-loaded LC samples, which also exhibitedevidently lower viscosities as unloaded samples, again pointingto transition from lamellar liquid crystalline structure to isotropicsolution after incorporation of amphiphilic AP. It is important toemphasize that observed viscosity curves for AP-loaded LC sample,in particular at 37 �C, are in fact viscosity curves for micellarsolution.

More information about the lamellar structure can be obtainedfrom oscillatory rheology; shear frequency sweep measurementswere performed with typical curves of the frequency dependenceof the storage modulus G0, loss modulus G00, and complex viscosityfor unloaded and AP-loaded LC samples presented in Fig. 7. Un-loaded LC samples are more elastic than viscous, which is in accor-dance with literature data (Farkas et al., 2000; Németh et al., 1998;Zhang et al., 2008): G0 is about one order of magnitude higher thanG00 throughout the entire frequency range. G0 was nearly frequency-independent and G00 shows a local minimum, whereas the complexviscosity drops linearly as a function of frequency. The ratio be-tween G0 and G00 (tand) at various frequencies (100 Hz, 10 Hz)and minimum of G00 (Table 3) clearly demonstrate that the samplebecomes more elastic with an increasing water to surfactant ratio,as seen from the decrease in tand. Higher values of G0 for unloadedLC2 system indicates stronger interactions between the bilayerthat is supported also by viscosity measurements, however is notconsistent with SAXS and DSC results. DSC results clearly revealedweaker interactions between surfactant mixture and water for un-loaded LC2 system while SAXS analysis confirmed increased inter-

layer distance, which is connected with weaker, not stronger,interactions (Youssry et al., 2008).

The same tendency of rheological parameters was observed forAP-loaded system; both moduli decrease when compared to un-loaded LC systems. This point to structural changes and is consis-tent with SAXS results since lamellar phase coexists withmicellar. A decrease was very pronounced for AP-loaded LC1, indi-cating weaker interactions between the bilayer after incorporationof amphiphilic molecule. Results of SAXS and DSC measurementsstrengthen this assumption. Namely, the increase of interlayerspacing and shift of water crystallization peak towards higher tem-perature was more prominent for AP-loaded LC1. And even thoughexplanation for inconsistency of rheological towards SAXS and DSCresults for unloaded LC2 samples cannot be given at the moment, itis probably due to this phenomena disturbance of lamellae in AP-loaded LC2 less distinct as seen from only slight decrease inrheological parameters. What is more, this is also confirmed by lessprominent increase in interlayer spacing and only minor changes

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 afunction of frequency (x) at a stress of 10% at 25 �C. Data are expressed asmean ± S.D. (n = 3).

Table 3Loss tangent (tand) at various frequencies (100 Hz, 10 Hz, and minimum of G00) ofunloaded LC samples with corresponding water/surfactant (W/S) ratio.

Sample W/S ratio tand (100 Hz) tand (10 Hz) tand (G00 min)

LC1 0.55 0.233 0.181 0.185LC2 0.87 0.202 0.099 0.078

Fig. 8. Proliferation of keratinocytes 4 h after addition of unloaded LC1-2 samplesand SDS solution to the cells (T = 37 �C). The results are presented relative to theproliferation of untreated control cells. Data are expressed as mean ± S.D. (n = 6).aP < 0.05 compared to SDS solution at the same concentration.

M. Gosenca et al. / European Journal of Pharmaceutical Sciences 50 (2013) 114–122 121

in peaks positions (DSC cooling curves) or shape (DSC heatingcurves) for AP-loaded LC2 compared to AP-loaded LC1.

Biological acceptability is one of crucial aspects that should beconsidered when developing a novel delivery system and is decisive

for its practical use. In keeping with this, a cytotoxicity assessment ofLC systems on a keratinocyte cell line was performed. Unloaded LCsamples were tested because AP can directly reduce the MTS reagentand 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 com-position. Keratinocyte proliferation was measured with an MTSassay following short-term exposure (4 h post-treatment). Exposingkeratinocytes to lamellar LC systems at a concentration of 0.45mg/ml resulted in no considerable impact on cell proliferation,being around 100%, and it also remained at a high level at a concen-tration of 0.90 mg/ml. The pronounced differences regarding LCcomposition 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 cellproliferation measured by the MTS reduction test is summarizedin Fig. 8. After exposure to SDS, which is used as a positive controlin skin irritation testing (Effendy and Maibach, 1996), the cell prolif-eration dramatically declined. The LC systems tested performed sig-nificantly better than SDS solution, proving lamellar LC to be apotential non-toxic dermal delivery system. These results are inaccordance to very low acute and low cumulative irritancy potentialthat was established with patch test for lecithin microemulsion gelcomposed of soybean lecithin, IPM and water (Dreher et al., 1996),while IPM was confirmed as safe lipophilic excipient, even thoughtrend of decreased viability was observed (Savic et al., 2009). Never-theless, being aware of the fact that biological assays are not able toevaluate structural damage that may occur at a non-cytotoxic dose,but can promote pathological effects (Lastella et al., 2007), extensivestudy oriented towards cells’ morphological evaluation using atom-ic force microscopy is in progress. We strongly believe that the dataobtained together with the MTS results presented here will unequiv-ocally answer the question regarding the dermal acceptability of LCformulations investigated.

4. Conclusions

This study revealed that (Tween 80/lecithin = 1/1)/IPM/watermixtures were able to form lamellar LC, as confirmed by the PLMand SAXS results. The structure was additionally evaluated regard-ing its quantitative composition. The interlayer spacing was affectedby water content, which can be attributed to differences in thestrength of interactions between water and surfactant molecules,as observed by DSC measurements. A rheological study showed thatthe formulation behaves like a non-Newtonian system, with charac-teristics of shear-thinning material. The lamellar phase structurealso remained organized at skin and physiological temperatures.Structural changes in the system were observed after incorporationof AP, which was used as an antioxidant, and were attributed to mi-celle formation due to its amphiphilic character and interactionswith surfactant molecules. The cell proliferation remained high at0.45 and 0.90 mg/ml, decreased at 4.50 mg/ml, but remained signif-icantly higher when compared to the standard irritant SDS. There-fore, setting strict selection criteria for individual componentstogether with qualitative structural evaluation were proved as aprosing approach in developing a skin compliant carrier system;namely, promising results obtained on living keratinocytes indicatethat the lecithin based LC system shows great potential as a physio-logically acceptable dermal delivery system.

Acknowledgements

The authors are grateful to the ICLC-Interlab Cell Line Collec-tion, University of Genova (Italy), for providing the immortalizedhuman keratinocyte cell line NCTC2544. We thank Tanja Tavcar

122 M. Gosenca et al. / European Journal of Pharmaceutical Sciences 50 (2013) 114–122

and Lucija Kralj for carrying out a lot of the experimental work andMr. Anton Kokalj for preforming SAXS experiments.

References

Alam, M.M., Ushiyama, K., Aramaki, K., 2009. Phase behavior, formation, andrheology of cubic phase and related gel emulsion in Tween 80/water/oilsystems. J. Oleo Sci. 58 (7), 361–367.

Amani, A., York, P., de Waard, H., Anwar, J., 2011. Molecular dynamics simulation ofa polysorbate 80 micelle in water. Soft Matter 7, 2900–2908.

Benedini, L., Schulz, E.P., Messina, P.V., Palma, S.D., Allemandi, D.A., Schulz, P.C.,2011. The ascorbyl palpitate-water system: phase diagram and state of thewater. Colloids Surf. A.: Physcicochem. Eng. Aspects 375, 178–185.

Benedini, L., Messina, P.V., Palma, S.D., Allemandi, D.A., Schulz, P.C., 2012. Theascorbyl palmitate-polyethyleneglycol 400-water system phase behavior.Colloids Surf. B.: Biointerfaces 89, 265–270.

Berni, M.G., Lawrence, C.J., Machin, D., 2002. A review of the rheology of thealmmelar phase in surfactant systems. Adv. Colloid Interface Sci. 98, 217–243.

Bonacucina, G., Cespi, M., Mencarelli, G., Palmieri, G.F., 2012. Characterization ofternary phase diagrams by means of thermal and rheological analyses. DrugDev. Ind. Pharm., 1–8.

Burducea, G., 2004. Lyotropic liquid crystals I. Specific structures. Rom. Rep. Phys.56 (1), 66–86.

Choi, S.-Y., Oh, S.-G., Bae, S.-Y., Moon, S.-K., 1999. Effect of short-chain alcohols asco-surfactants on pseudo-ternary phase diagrams containing lecithin. Korean J.Chem. Eng. 16 (3), 377–381.

Chorilli, M., Prestes, P.S., Rigon, R., Leonardi, G.R., Chiavacci, L.A., Sermento, V.H.V.,Oliveira, A.G., Scarpa, M.V., 2011. Structural characterization and in vivoevaluation of retinyl palmitate in non-ionic lamellar liquid crystalline system.Colloids Surf. B.: Biointerfaces 85, 182–188.

Dreher, F., Walde, P., Luisi, P.L., Elsner, P., 1996. Human skin irritation studies of alecithin microemulsion gel and of lecithin liposomes. Skin Pharmacol. 9, 124–129.

Effendy, I., Maibach, H.I., 1996. Detergent and skin irritation. Clin. Dermatol. 14, 15–21.

Farkas, E., Zelkó, R., Németh, Z., Pálinkás, J., Marton, S., Rácz, I., 2000. The effect ofliquid crystalline structure on chlorhexidine diacetate release. Int. J. Pharm. 193,239–245.

Fehér, A., Csányi, E., Csóka, I., Kovács, A., Er}os, I., 2005. Thermoanalyticalinvestigation of lyotropic liquid crystals and microemulsions forpharmaceutical use. J. Therm. Anal. Calorim. 82, 507–512.

Fiume, Z., 2001. Final report on the safety assessment of lecithin and hydrogenatedlecithin. Int. J. Toxicol. 20, 21–45.

Fong, W.-K., Hanley, T., Boyd, B.J., 2009. Stimuli responsive liquid crystals provide‘on-demand’ drug delivery in vitro and in vivo. J. Control. Release 2009, 218–226.

Garti, N., Aserin, A., Tiunova, I., Fanun, M., 2000. A DSC study of water behavior inwater-in-oil microemulsions stabilized by sucrose esters and butanol. ColloidsSurf. A Physicochem. Eng. Aspects 170, 1–18.

Garti, N., Yaghmur, A., Leser, M.E., Clement, V., Watzke, H.J., 2001. Improved oilsolubilization in oil/water food grade microemulsions in the presence of polyolsand ethanol. J. Agric. Food Chem. 49, 2552–2562.

Gašperlin, M., Gosenca, M., 2011. Main approaches for delivering antioxidantvitamins through the skin to prevent skin ageing. Expert Opin. Drug Deliv. 8 (7),905–919.

Gosenca, M., Gašperlin, M., 2011. Dermal delivery of ascorbyl palmitate: thepotential of colloidal delivery systems. J. Drug Del. Sci. Technol. 21 (6), 535–537.

Guo, C., Wang, J., Cao, F., Lee, R.J., Zhai, G., 2010. Lyotropic liquid crystal systems indrug delivery. Drug Discov. Today 15 (23/24), 1032–1040.

Harms, M., Mackeben, S., Müller-Goymann, C.C., 2005. Thermotropic transitionstructures in the ternary system lecithin/isopropyl myristate/water. ColloidsSurf. A Physicochem. Eng. Aspects 259, 81–87.

Jurkovic, P., Šentjurc, M., Gašperlin, M., Kristl, J., Pecar, S., 2003. Skin protectionagainst ultraviolet induced free radicals with ascorbyl palmitate inmicroemulsions. Eur. J. Pharm. Biopharm. 56 (1), 59–66.

Kodama, M., Aoki, H., 2001. Water behaviour in phospholipid bilayer systems. In:Garti, N. (Ed.), Thermal Behaviour of Dispersed Systems. Marcel Dekker Inc.,New York, pp. 247–293.

Kristl, J., Volk, B., Gašperlin, M., Šentjurc, M., Jurkovic, P., 2003. Effect of colloidalcarriers on ascorbyl palmitate stability. Eur. J. Pharm. Sci. 19, 181–189.

Kumar, R., Katare, O.P., 2005. Lecithin organogels as a potential phospholipid-structured system for topical drug delivery: a review. AAPS Pharm. Sci. Technol.6 (2), 298–310.

Lastella, M., Lasalvia, M., Perna, G., Biagi, P.F., Capozzi, V., 2007. Atomic forcemicroscopy study on human keratinocytes treated with HgCl2. J. Phys.: Conf.Ser. 61, 920–925.

Mackeben, S., Müller, M., Müller-Goymann, C.C., 2001. The influence of water onphase transition of a drug-loaded reverse micellar solution into lamellar liquidcrystal. Colloids Surf. A Physicochem. Eng. Aspects 183–185, 699–713.

Makai, M., Csányi, E., Németh, Z., Pálinkás, J., Er}os, I., 2003. Structure and drugrelease of lamellar liquid crystals containing glycerol. Int. J. Pharm. 256, 95–107.

Malmsten, M., 2002. Surfactants and Polymers in Drug Delivery. Marcel Dekker Inc.,New York.

Mondain-Monval, O., 2005. Freeze fracture TEM investigations in liquid crystals.Curr. Opin. Colloid Interface Sci. 10, 250–255.

Moreno, M.A., Ballesteros, M.P., Frutos, P., 2003. Lecithin-based oil-in-watermicroemulsions for parenteral use: pseudoternary phase diagrams,characterization and toxicity studies. J. Pharm. Sci. 92 (7), 1428–1437.

Müller-Goymann, C.C., 2004. Physicochemical characterization of colloidal drugdelivery systems such as reverse micelles, vesicles, liquid crystals andnanoparticles for topical administration. Eur. J. Pharm. Biopharm. 58, 343–356.

Müller-Goymann, C.C., 2007. Drug delivery liquid crystals. In: Swarbrick, J. (Ed.),Encyclopedia of Pharmaceutical Technology, vol. 2, third ed. InformaHealthcare, New York, pp. 1115–1131.

Németh, Z., Halász, L., Pálinkás, J., Bóta, A., Horányi, T., 1998. Rheological behaviourof a lamellar liquid crystalline surfactant-water system. Colloids Surf. APhysicochem. Eng. Aspects 145, 107–119.

Nesseem, D.I., 2001. Formulation and evaluation of itraconazole via liquid crystalfor topical delivery system. J. Pharm. Biomed. Anal. 26, 387–399.

Podlogar, F., Gašperlin, M., Tomšic, M., Jamnik, A., Bešter Rogac, M., 2004. Structuralcharacterisation of water-Tween 40�/Imwitor 308�-isopropyl myristatemicroemulsions using different experimental methods. Int. J. Pharm. 276,115–128.

Rong, G., Yang, J., Friberg, S.E., Aikens, P.A., Greenshields, J.N., 1996. Complexlamellar structure of polyoxyethylene 20 sorbitan oleate and a fatty acid/lecithin lamellar liquid crystal. Langmuir 12, 4286–4291.

Sallam, A.-S., Khalil, E., Ibrahim, H., Freij, I., 2002. Formulation of an oral dosage formutilizing the properties of cubic liquid crystalline phases of glycerylmonooleate. Eur. J. Pharm. Biopharm. 53, 343–352.

Savic, S.D., Savic, M.M., Vesic, S.A., Vuleta, G.M., Müller-Goymann, C.C., 2006.Vehicles based on sugar surfactant: colloidal structure and its impact onin vitro/in vivo hydrocortisone permeation. Int. J. Pharm. 320, 86–95.

Savic, S., Weber, C., Savic, M.M., Müller-Goymann, C., 2009. Natural surfactant-based topical vehicles for two model drugs: influence of different lipophilicexcipients on in vitro/in vivo skin performance. Int. J. Pharm. 381, 220–230.

Savic, S., Lukic, M., Jaksic, I., Reichl, S., Tamburic, S., Müller-Goymann, C., 2011. Analkyl polyglucoside-mixed emulsifier as stabilizer of emulsion system: theinfluence of colloidal structure on emulsion skin hydration potential. J. ColloidInterface Sci. 358, 182–191.

Shah, M.H., Paradkar, A., 2005. Cubic liquid crystalline glyceryl monooleate metricesfor oral delivery of enzyme. Int. J. Pharm. 294, 161–171.

Shah, J.C., Sadhale, Y., Chilukuri, D.M., 2001. Cubic phase gels as drug deliverysystems. Adv. Drug Del. Rev. 47, 229–250.

Sharma, S.C., Warr, G.G., 2012. Phase behavior, self-assembly, and emulsification ofTween 80/water mixtures with limonene and perfluoromethyldecalin.Langmuir 28, 11707–11713.

Shchipunov, Y.A., 1997. Self-organising structures of lecithin. Russ. Chem. Rev. 66(4), 301–322.

Stevenson, C.L., Bennett, D.B., Lechuga-Ballesteros, D., 2005. Pharmaceutical liquidcrystals: the relevance of partially ordered systems. J. Pharm. Sci. 9, 1861–1880.

Szekely, P., Asor, R., Dvir, T., Szekely, O., Raviv, U., 2012. Effect of temperature on theinteractions between dipolar membranes. J. Phys. Chem. B 116, 3519–3524.

Vicentini, F.T., Casagrande, R., Verri Jr., W.A., Georgetti, S.R., Bentley, M.V., Fonseca,M.J., 2008. Quercetin in lyotropic liquid crystalline formulations: physical,chemical and functional stability. AAPS Pharm. Sci. Technol. 9 (2), 591–596.

Wang, Z., Diao, Z., Liu, F., Li, G., Zhang, G., 2006. Microstructure and rheologicalproperties of liquid crystallines formed in Brij 97/water/IPM system. J. ColloidInterface Sci. 297, 813–818.

Yaghmur, A., Aserin, A., Garti, N., 2002. Phase behavior of microemulsions based onfood-grade nonionic sufractants: effect of polyols and short-chain alcohols.Colloids Surf. A Physicochem. Eng. Aspects 209, 71–81.

Youssry, M., Coppola, L., Nicotera, I., Morán, C., 2008. Swollen and collapsedlyotropic lamellar rheology. J. Colloid Interface Sci. 321, 459–467.

Zhang, J., Dong, B., Zheng, L., Li, N., Li, X., 2008. Lyotropic liquid crystalline phaseformed in ternary mixtures of 1-cetyl-3-methylimidazolium bromide/p-xylene/water: a SAXS, POM, and rheology study. J. Colloid Interface Sci. 321, 159–165.

Zhao, J., Wang, Z.N., Wei, X.L., Liu, F., Zhou, W., Tang, X.L., Wu, T.H., 2011. Phasebehaviour and rheological properties of the lamellar liquid crystal formed indodecyl polyoxyethylene polyoxypropylene ether/water system. Indian J.Chem. 50A, 641–649.

Zhuang, W., Chen, X., Cai, J., Zhang, G., Qiu, H., 2008. Characterisation of lamellarphases fabricated from Brij-30/water/1-butyl-3-methylimidazolium saltsternary systems by small-angle X-ray scattering. Colloids Surf. APhysicochem. Eng. Aspects 318, 175–183.