European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 121–132

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Research paper

Effect of nanostructured lipid vehicles on percutaneous absorption of curcumin Elisabetta Esposito a,⇑, Laura Ravani a, Paolo Mariani b, Nicolas Huang c,d, Paola Boldrini e, Markus Drechsler f, Giuseppe Valacchi a,g, Rita Cortesi a, Carmelo Puglia h a

Department of Life Sciences and Biotechnologies, University of Ferrara, Ferrara, Italy Department of Life and Environmental Sciences and CNISM, Università Politecnica delle Marche, Ancona, Italy Univ Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry Cedex, France d CNRS UMR 8612, Institut Galien Paris-Sud, Châtenay-Malabry Cedex, France e Electron Microscopy Center, University of Ferrara, Ferrara, Italy f Macromolecular Chemistry II, University of Bayreuth, Germany g Kyung Hee University, Dept. of Food and Nutrition, Seoul, South Korea h Department of Drug Science, University of Catania, Catania, Italy b c

a r t i c l e

i n f o

Article history: Received 25 October 2013 Accepted in revised form 15 December 2013 Available online 20 December 2013 Keywords: Cubosomes Organogel Curcumin Reflectance spectroscopy Tape-stripping Controlled release

a b s t r a c t The present study describes the production and characterization of monoolein aqueous dispersions (MAD) and lecithin organogels (ORG) as percutaneous delivery systems for curcumin (CUR). In particular, MAD stabilized by sodium cholate/poloxamer and w0 3 ORG lipid carriers, both in the presence and absence of CUR, have been considered: MAD morphology and dimensional distribution have been investigated by Cryogenic Transmission Electron Microscopy (cryo-TEM) and Photon Correlation Spectroscopy (PCS), while the inner structure of MAD and ORG has been studied by X-ray scattering techniques. As a general result, CUR chemical stability has been found to be better controlled by MAD, probably because CUR is more protected in the case of CUR-MAD with respect to CUR-ORG. To investigate the performance of differently composed lipid formulations as CUR delivery system, in vitro studies, based on Franz cell and stratum corneum–epidermis (SCE) membranes, and in vivo studies, based on skin reflectance spectrophotometry and tape stripping, were then performed. The results indicated that ORG induces a rapid and intense initial penetration of CUR probably due to a strong interaction between the peculiar supramolecular aggregation structure of phospholipids in the vehicle and the lipids present in the stratum corneum. Conversely, CUR incorporated into MAD can be released in a controlled fashion possibly because of the formation of a CUR depot in the stratum corneum. In this respect ORG could be employed in pathologies requiring rapid CUR action, while MAD could be proposed for assuring a prolonged CUR activity. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction A wide variety of disorders and diseases affect the skin, e.g. acne, warts, multiple inflammatory dermatoses, skin cancers, autoimmune diseases, occupational dermatoses and contact dermatitis, requiring different investigations and therapies. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide [1]. Treatment protocols include disfiguring surgery, platinum-based chemotherapy and radiation, all of which may

Abbreviations: MAD, monoolein aqueous dispersions; ORG, organogel; CUR, curcumin; x-gum, xanthan gum. ⇑ Corresponding author. Dipartimento SVEB, Via Fossato di Mortara, 19, I-44121 Ferrara, Italy. Tel.: +39 0532 455259; fax: +39 0532 455953. E-mail address: [email protected] (E. Esposito). 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.12.011

result in tremendous patient morbidity [2–4]. As a result, there is significant interest in developing adjuvant chemotherapies to augment currently available treatment protocols, which may allow decreased side effects and toxicity without compromising therapeutic efficacy. Curcumin (CUR) is one such potential candidate, in fact it has been recently demonstrated that CUR has a beneficial role in skin diseases, in particular it can be employed as a single agent in the treatment of HNSCC and used as an adjuvant agent in combination with standard platinum-based chemotherapy [5,6]. Despite the efficacy of CUR, its scarce water solubility may limit its administration. On the matter a number of research work has been done in order to develop delivery strategies for CUR, e.g. liposomes, solid lipid nanoparticles, and cyclodextrins have been investigated [7].

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Recently we have proposed the solubilization of CUR in monoolein aqueous dispersions (MAD) stabilized by different emulsifiers, resulting in the formation of complex lyotropic liquid crystalline phases, depending on the emulsifier used [8]. In order to treat cutaneous pathologies such as HNSCC, a semisolid formulation constituted of biocompatible materials able to assure targeted delivery of actives, minimizing at the same time toxic systemic effects appears the best strategy. A non-aqueous microemulsion system based on lecithin could be a good solution at this aim [9,10]. It is well known that lecithin, thanks to its chemico-physical properties, is one of the most promising and useful agents able to increase the skin permeation. In fact lecithin is a non-toxic, naturally occurring biocompatible surfactant able to form poly-molecular structures such as direct and inverted micelles or hexagonal, cubic and lamellar phases, offering the chance to produce innovative topical forms [10–13]. When a precise amount of water is added to an oil lecithin solution, initially the lecithin molecules form spherical reversed micelles, then the micellar aggregates entangle, forming a three-dimensional network in the bulk phase [14,15]. This peculiar w/o microemulsion is called lecithin organogel (ORG) since it consists of a gel-like reverse micellar system in which the external phase is an organic solvent [14]. By adjusting the amount of added water it is possible to modulate the system viscosity [11,14,16]. ORG are particularly suitable as cutaneous delivery systems for many reasons. Indeed, they are thermodynamically stable, they are able to solubilize hydrophilic and lipophilic molecules and eventually they can be produced with biocompatible solvents [17–21]. In the present study, we investigate the performances of different lipid based formulations for CUR cutaneous administration (i.e. MAD and ORG). In particular, the first part of the present study describes the production and characterization of liquid MAD stabilized by poloxamer in mixture with sodium cholate and ORG. The MAD viscosity was then adjusted by the use of xanthan gum (x-gum). The second part concerns an in vitro and in vivo investigation aimed to compare the release modalities of CUR after the administration of the semisolid lipid formulations on the skin. CUR applied on the skin was studied determining its in vivo topical anti-inflammatory activity after cutaneous application of MAD and ORG. The ultraviolet B (UVB)-induced erythema was chosen as inflammatory model on healthy human volunteers and was monitored by reflectance visible spectrophotometry. Moreover, tape-stripping experiments have been performed on skin after topical administration of MAD and ORG to quantify CUR presence in the stratum corneum.

2. Experimental methods 2.1. Materials Glyceryl monooleate RYLO MG 19 (monoolein) was a gift from Danisco Cultor (Grindsted, Denmark). Pluronic F127 (Poloxamer 407, poloxamer) (PEO98–POP67–PEO98) was obtained from BASF (Ludwigshafen, Germany). Curcumin (CUR), (1E,6E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, sodium cholate (Na cholate) (3a,7a,12a-Trihydroxy-5b-cholan-24-oic acid sodium salt), Xanthan gum (x-gum) and isopropylpalmitate (IPP) were purchased from Sigma Chemical Company (St. Louis, MO, USA). The soybean lecithin (90% phosphatidyl choline) used for ORG preparation was Epikuron 200 from Lucas Meyer, Hamburg, Germany. Solvents were of HPLC grade and all other chemicals were of analytical grade.

2.2. MAD preparation Production of dispersions was based on the emulsification of monoolein and emulsifier in water, as previously described [22]. MAD composition was monolein 4.5% w/w, Na cholate 0.15% w/ w, poloxamer 0.5% w/w and water. After emulsification, the dispersions were subjected to homogenization (15,000 rev min1, Ultra Turrax, Janke & Kunkel, IkaWerk, Sardo, Italy) at 60 °C for 1 min, then cooled and maintained at room temperature in glass vials. To produce CUR containing MAD (CUR-MAD), 7.5 mg of CUR (0.33% w/w with respect to the monoolein, 0.015% w/w with respect to the dispersion) was added to the molten monoolein/emulsifier mixture and dissolved before addition to the aqueous solution. During production the vial was protected from light with an aluminum foil to prevent photo-degradation of CUR. 2.3. Characterization of MAD 2.3.1. Cryo-Transmission Electron Microscopy (cryo-TEM) Samples were vitrified as described in a previous study [22]. The vitrified specimen was transferred to a Zeiss EM922Omega (Carl Zeiss Microscopy, Oberkochen, Germany) transmission electron microscope using a cryoholder (CT3500, Gatan, Munich, Germany). Sample temperature was kept below 100 K throughout the examination. Specimens were examined with reduced doses of about 1000–2000 e/nm2 at 200 kV. Images were recorded by a CCD digital camera (Ultrascan 1000, Gatan, Munich, Germany) and analyzed using a GMS 1.8 software (Gatan, Munich, Germany). 2.3.2. Photon Correlation Spectroscopy (PCS) Submicron particle size analysis was performed using a Zetasizer 3000 PCS (Malvern Instr., Malvern, England) equipped with a 5 mW helium neon laser with a wavelength output of 633 nm. Glassware was cleaned of dust by washing with detergent and rinsing twice with sterile water. Measurements were made at 25 °C at an angle of 90° with a run time of at least 180 s. Samples were diluted with bidistilled water in a 1:10 v:v ratio. Data were analyzed using the ‘‘CONTIN’’ method [23]. Measurements were performed on MAD after production and after 6 months from production. 2.3.3. CUR content of MAD The encapsulation efficiency (EE) of CUR in the MAD was determined as described by Nayak and colleagues [24]. 100 ll aliquot of MAD was loaded in a centrifugal filter (Microcon centrifugal filter unit YM-10 membrane, NMWCO 10 kDa, Sigma Aldrich, St. Louis, MO, USA) and centrifuged (Spectrafuge™ 24D Digital Microcentrifuge, Woodbridge NJ, USA) at 8000 rpm for 20 min. The amount of free and entrapped CUR was determined by dissolving the supernatant with a known amount of ethanol (1:10, v/v). The amount of CUR in the supernatant was determined by high performance liquid chromatography (HPLC) method, as below reported. The EE was determined as follows:

EE ¼ T CUR  SCUR =T CUR  100

ð1Þ

where TCUR stands for the total amount of CUR added to the formulation and SCUR for the amount of drug measured in the supernatant. 2.4. ORG preparation ORG were prepared by dissolving lecithin (200 mM) in IPP. From these reverse micellar solutions, microemulsion gels were prepared by adding water, under magnetic stirring, to obtain the

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gel. The amount of added water has been expressed as w0 that is the molar water to lecithin ratio (w0 = [H2O]/[lecithin]) [11]. w0max was determined, i.e. the highest amount of water that can be incorporated into the lecithin solution with no phase separation. To produce drug containing ORG (CUR-ORG), after 3 h of stirring, CUR was added (0.015% w/v) to the obtained formulations. ORG were then maintained under stirring for 12 h. Afterward, the samples were maintained at 25 °C for 30 min and later examined under a microscope equipped with a polarization device. Solubility of CUR in ORG was determined by saturating the ORG with an excess of CUR (7 mg/ml). The obtained mixture was maintained under stirring at room temperature protected from exposure to light for 72 h. CUR content in the produced formulation was evaluated by extraction of un-dissolved drug with centrifugation cycles. In particular 1 ml of CUR-ORG was subjected to two cycles of centrifugation for 15 min at 6000 rpm (Spectrafuge™ 24D Digital Microcentrifuge, Woodbridge NJ, USA). 100 ll of supernatant was then dissolved with ethanol (1:10, v/v), afterwards the amount of solubilized CUR was determined by HPLC, as below reported.

123

Log (CUR residual content, %) was plotted against time and the slopes (m) were calculated by linear regression. The slopes (m) were then substituted into the following equation for the determination of k values:

k ¼ m  2:303

ð2Þ

Shelf life values (the time for 10% loss, t90) and half-life (the time for 50% loss, t1/2) were then calculated by the following equations:

t90 ¼ 0:105=k

ð3Þ

t1=2 ¼ 0:693=k

ð4Þ

as reported by Wells [29]. 2.7. Gel production The MAD have been viscosized by adding x-gum (1% w/w) directly into the dispersion and by slowly stirring for 1 h until complete dispersion of the gum. The obtained viscous MAD was named CUR-MAD x-gum. 2.8. Rheological measurements

2.5. X-ray scattering measurements Low-angle and wide-angle X-ray diffraction experiments were performed using a laboratory 3.5 kW Philips PW 1830 X-ray generator equipped with a Guinier-type focusing camera operating with a bent quartz crystal monochromator (k = 1.54 Å). Diffraction patterns were recorded on GNR Analytical Instruments Imaging Plate system. Samples, held in a tight vacuum cylindrical cell provided with thin Mylar windows, were analyzed at two different temperatures, 37 and 45 °C. In each experiment, a number of Bragg peaks were eventually detected in the low-angle X-ray diffraction region, and the peak indexing was performed considering the different symmetries commonly observed in lipid phases [25]. From the averaged spacing of the observed peaks, the unit cell dimension, a, was calculated using the Bragg law. The nature of the short-range sample conformation was derived analyzing the high-angle X-ray diffraction profiles [26]. ORG were also investigated by Small-Angle X-ray Scattering (SAXS) experiments, which were performed at the SAXS beamline at the Elettra Synchrotron (Trieste, Italy). The wavelength of the incident beam was k = 1.54 Å and the explored Q-range extended from 0.1 to 0.9 Å1 (Q is the modulus of the scattering vector, defined as 4p sin h/k, where 2h is the scattering angle). At the SAXS beamline, organogels were measured using 1 mm thick quartz capillaries at the 2 different temperatures of 37 and 45 °C. Particular attention was paid to checking for equilibrium conditions and monitoring radiation damage. In a few tests, measurements were repeated several times (up to 10) at the same temperature to account for a constant scattering signal. Experimental intensities were corrected for background, solvent contributions, detector inhomogeneities, and sample transmission, as usual [27]. Unfortunately, no absolute scale calibration of the experimental data was available. 2.6. Prediction of long-term stability The stability of CUR in MAD and in ORG was assessed in the absence of light. CUR-MAD and CUR-ORG were stored in well-closed amber glass containers and kept at room temperature for 6 months. Chemical stability was evaluated on drug loaded formulations, determining CUR content by HPLC analyses. Shelf life values were calculated [28].

Rheological measurements were performed with an AR-G2 rotational rheometer (TA Instruments). The geometry used was an aluminum cone-plate with a diameter of 40 mm and an angle of 1°. Flow curve was obtained in increasing the shear rate from 0.01 s1 to 5000 s1 with 5 points per decade, and each point was maintained for a duration of 180 s in order to perform measurements in the permanent regime. The temperature was set at 25 °C or 35 °C and controlled with a Peltier plate. A solvent trap was used to prevent water evaporation. Measurements were performed in triplicate for each sample, to ensure reproducibility. 2.9. In vitro skin permeation experiments Samples of adult human skin (mean age 36 ± 8 years) were obtained from breast reduction operations. Subcutaneous fat was carefully trimmed and the skin was immersed in distilled water at 60 ± 1 °C for 2 min, after which SCE (stratum corneum/epidermis) was removed from the dermis using a dull scalpel blade. SCE membranes were dried in a desiccator at 25% relative humidity. The dried samples were wrapped in aluminum foil and stored at 4 ± 1 °C until use. SCE were then rehydrated by immersion in distilled water at room temperature for 1 h before being mounted in Franz-type diffusion cells supplied by LGA (Berkeley, CA) as reported by Puglia and colleagues [8]. The exposed skin surface area was 0.78 cm2 area (1 cm diameter orifice). The receptor compartment contained 5 ml of a mixture of phosphate buffer 60 mM pH 7.4 (PBS) and ethanol (50:50, v/v) to allow the establishment of the sink conditions and to sustain permeant solubilization [30]. This solution was stirred with the help of a magnetic bar at 500 rpm and thermostated at 32 ± 1 °C during all the experiments [31]. Approximately 500 ll of each formulation (CUR-MAD, CUR MAD x-gum and CUR-ORG) was placed on the skin surface in the donor compartment and the latter was sealed to avoid evaporation. At predetermined time intervals comprised between 1 and 24 h, samples (0.15 ml) of receptor phase solution were withdrawn and the CUR concentration in the receptor phase was measured using HPLC. Each removed sample was replaced with an equal volume of simple receptor phase. The CUR concentrations were determined six times in independent experiments and the mean values ± standard deviations were calculated. The mean values

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were then plotted as a function of time. The diffusion coefficients were computed from the linear portion of the accumulation curve and expressed both as experimentally observed fluxes (Fo) and as normalized fluxes Fn

F n ¼ F o =C

ð5Þ

where C is the CUR concentration in the analyzed form, expressed in mg/ml. After Franz cell experiments, skin samples were treated with reducing agents and fixed with glutaraldehyde 2.5% (w/v) in phosphate buffer 0.1 M pH 7.4, post-osmicated with osmium tetroxide 2% in the same buffer, dehydrated with increasing quantities of acetone and included in Araldite Durcupan ACM (Fluka). Semifine sections were made on ultramicrotome Reichert Ultracut S, stained with an aqueous solution of Toluidin blue 1% (w/v) and studied by light microscope Nikon Eclipse E800. 2.10. In vivo studies 2.10.1. Volunteers recruitment In vivo experiments were performed on two groups of ten volunteers: group A enrolled for the first in vivo experimentation (evaluation of anti-inflammatory activity) and group B enrolled for the second one (tape-stripping). Experiments were conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The volunteers were of both sexes in the age range 25–55 years and they were recruited after medical screening including the filling of a health questionnaire followed by physical examination of the application sites. After they were fully informed on the nature of the study and on the procedures involved, they gave their written consent. The participants did not suffer from any ailment and were not on any medication at the time of the study. They were rested for 15 min prior to the experiments and room conditions were set at 22 ± 2 °C and 40–50% relative humidity. 2.10.2. In vivo anti-inflammatory activity UVB-induced skin erythema was monitored by using a reflectance visible spectrophotometer X-Rite model 968 (XRite Inc., Grandville, MI, USA), calibrated and controlled as previously reported [32,33]. Reflectance spectra were obtained over the wavelength range 400–700 nm using illuminant C and 2° standard observer. From the spectral data obtained, the erythema index (EI) was calculated using Eq. (6) [34]:      1 1 1 1 1 EI ¼ 100 log þ 1:5 log þ log þ log  2 log R560 R540 R580 R510 R610 ð6Þ where 1/R is the inverse reflectance at a specific wavelength (560, 540, 580, 510 and 610). The skin erythema was induced by UVB irradiation using a UVM-57 ultraviolet lamp (UVP, San Gabriel, CA, USA) whose specific parameters are reported elsewhere [33]. The MED was preliminarily determined, and an irradiation dose corresponding to twice the value of MED was used throughout the study. For each subject (group A), seven sites on the ventral surface of each forearm were defined using a circular template (1 cm2) and demarcated with permanent ink. One of the seven sites of each forearm was used as control, three sites were treated with 300 mg of CUR-MAD and the remaining three with 300 mg of CUR-ORG. The preparations were spread uniformly by means of a solid glass rod and then the sites were occluded for 6 h using Hill Top Chambers (Hill Top Research, Cincinnati, OH). After the occlusion period, the chambers were removed and the skin surfaces were gently washed to remove the gel and allowed to dry for 15 min. Each pretreated

site was exposed to UV-B irradiation 1, 3 and 6 h (t = 1, t = 3 and t = 6, respectively) after CUR-MAD and CUR-ORG removal and the induced erythema was monitored for 52 h. EI baseline values were taken at each designated site before application of gel formulation and they were subtracted from the EI values obtained after UV-B irradiation at each time point to obtain DEI values. For each site, the AUC was computed using the trapezoidal rule. To better outline the results obtained, the PIE was calculated from the AUC values using following equation:

Inhibition ð%Þ ¼

AUCðCÞ  AUCðTÞ  100 AUCðCÞ

ð7Þ

where AUC(C) is the area under the response/time curve of the vehicle-treated site (control) and AUC(T) is the area under the response/ time curve of the drug-treated site. Statistical differences of in vivo data were determined using repeated measure analysis of variance (ANOVA) followed by the Bonferroni-Dunn post hoc pair-wise comparison procedure. A probability, P, of less than 0.05 was considered significant in this study. 2.10.3. Tape stripping The first steps of the experimental protocol previously described were also employed in this second in vivo study; in fact, for each subject of group B, seven sites (2 cm2) were defined on the ventral surface of each forearm, and 200 mg of each formulations (CUR-MAD and CUR-ORG) was applied on these cutaneous sites (three sites of application for each formulation in duplicate). The preparations were spread uniformly on the site by means of a solid glass rod and were then occluded for 6 h. After the occlusion period, the residual formulations were removed by gently wiping with cotton balls (different for each pretreated site). Ten individual 2 cm2 squares of adhesive tape (Scotch Book Tape 845, 3M) were utilized to sequentially tape-strip the stratum corneum on the application sites. To obtain a realistic comparison between the results of this experimentation and the ones obtained in the previous in vivo study, the removal of stratum corneum in each pretreated site was effected at 1 h (t = 1), 3 h (t = 3) and 6 h (t = 6) after gel removal [32]. Each adhesive square, before and after skin tape stripping, was weighed on a Sartorius balance (model ME415S, sensitivity 1 mg) to quantify the weight of stratum corneum removed. After each stripping, the tapes were put in the same vial containing 2 ml of the HPLC mobile phase (methanol, 2% acetic acid and acetonitrile, 5:30:65 v/v) and subjected to vortical stirring over 30 s. The extracted CUR was then quantified by HPLC. The recovery of CUR was validated by spiking tape-stripped samples of untreated stratum corneum with 2 ml of a mobile phase containing CUR 10 mg/ ml. The extraction efficiency of CUR was 96.8 ± 0.9% (n = 3). 2.11. Statistical analysis Statistical differences of in vivo data were determined using repeated-measures analysis of variance (ANOVA) followed by the Bonferroni–Dunn post hoc pairwise comparison procedure. The employed software was Prism 5.0, Graph Pad Software Inc. (La Jolla, CA, USA). A probability of less than 0.05 is considered significant in this study. 2.12. HPLC procedure HPLC determinations were performed using a two-plungers alternative pump (Jasco, Japan), an UV-detector operating at 425 nm, and a 7125 Rheodyne injection valve with a 50 ll loop. Samples were loaded on a stainless steel C-18 reverse-phase

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column (15  0.46 cm) packed with 5 lm particles (GraceÒ – Alltima, Alltech, USA). Elution was performed with a mobile phase containing methanol, 2% acetic acid and acetonitrile 5:30:65 v/v at a flow rate of 0.5 ml/min. Retention time of CUR was 7.0 min. 3. Results 3.1. Preparation of MAD It is well known that monoolein is able to form cubosome dispersions when it is emulsified in water in the presence of a surfactant [35–37]. In particular poloxamer 407 is extensively employed [38–40]. Recently we found that stable monoolein dispersions can be obtained by the use of Na cholate in mixture of poloxamer as emulsifiers [41]. In this study MAD and CUR-MAD were produced by the mixture of Na cholate and poloxamer, resulting in translucent dispersions, yellowish in the case of CUR-MAD. Cryo-electron transmission microscope analyses were conducted in order to investigate the internal structures of MAD and to compare the influence of the emulsifier on the nanostructure of the dispersed phase. Fig. 1 reports cryo-TEM images of MAD (panel A) and CUR-MAD (panel B). One can observe the presence of unilamellar vesicles in both panels. The black points are ascribed to ice crystals contamination due to sample preparation. The presence of CUR does not seem to affect MAD aspect. 3.1.1. MAD dimensional analyses PCS studies were conducted to determine the dimensional distribution of MAD dispersions, in the absence and in the presence of CUR. Table 1 summarizes the obtained results. One can observed that the mean diameter of the MAD disperse phase after production is around 150 nm. The presence of CUR leads to a slight decrease in mean diameters and polydispersity index. After 6 months from production, mean diameters are almost unvaried in the absence of MAD (2% increase with respect to the initial mean diameters), while they undergo a 20% increase in the presence of CUR.

Fig. 1. Cryo-transmission electron microscopy images (cryo-TEM) of MAD in the absence (panel A) and in the presence (panel B) of CUR.

3.2. Preparation of ORG Solutions of 50–250 mM phosphatidylcholine in organic solvent can be transformed into transparent gels by addition of critical and well defined amounts of water [11]. The addition of water leads to a strong change in viscosity until a transparent gel is obtained [13]. It should be stressed that the transparency of the system has considerable advantages with respect to topical and transdermal applications [21]. In lecithin gels in fact, complete solubilization of solid compounds can be easily evaluated [14,15]. It is well known that the viscosity of the gel is a function of the added water, expressed as w0 [11]. ORG and CUR-ORG with w0 values 1, 2 and 3 were studied, the determined w0max was 3. For in vitro and in vivo studies the w0 3 CUR-ORG was chosen, thus providing suitable viscosity for topical application. The produced organogels were transparent, yellow, macroscopically monophasic and isotropic under polarized light. In the case of CUR-ORG the yellow color was more intense. 3.3. X-ray diffraction analyses X-ray diffraction was used to investigate the inner structural organization of MAD and ORG. Experiments were performed as a function of temperature, both in the presence and in the absence

of CUR. A first example of diffraction results is shown in Fig. 2. Considering the expected Bragg peak sequence, the X-ray diffraction profiles indicate the presence of a Pn3m inverted bicontinuous cubic phase both in the absence and in the presence of CUR [42]. However, two results should be underlined: first, as no changes are detected in diffraction profiles, the inner structure of MAD and CUR-MAD samples is the same, but a larger unit cell dimension is observed in the presence of CUR: at 37 °C, the unit cell parameter changes from 95.5 to 102.4 Å. Second, no phase transitions are observed on heating. In both cases, heating only determines a small reduction of the unit cell size (when temperature increases from 37 to 45 °C, the unit cell reduces from 95.5 to 83.8 Å and from 102.4 to 98.7 Å for MAD and CUR-MAD samples, respectively), as expected considering the temperature-induced decrease in the hydrocarbon chain order parameter. Finally, no changes were detected in wide-angle (WAXS) profiles (data not shown), indicating that the fluid nature of the short-range lipid conformation is not modified by the presence of CUR. In the case of ORG samples, synchrotron SAXS experiments were performed in addition (Fig. 3A), as no low-angle scattering was observed using a standard X-ray diffractometer. While in the absence of CUR a typical SAXS profile for a disordered micellar system is detected, in the CUR-ORG sample two very low and large

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Table 1 Dimensional parameters of MAD and CUR-MAD after production (0 d) and after 6 months (180 d) from production. Formulation

Mean diameter (nm) ± SD Average

MAD CUR-MAD

Polydispersity index Analysis by intensity

Analysis by volume

Analysis by number

0 (d)

180 (d)

0 (d)

180 (d)

0 (d)

180 (d)

0 (d)

180 (d)

0 (d)

180 (d)

158.9 ± 2.0 147.6 ± 3.1

161.9 ± 4.2 178.7 ± 2.8

164.3 ± 5.1 154.5 ± 6.2

166.7 ± 5.3 157.0 ± 3.2

137.3 ± 3.3 131.5 ± 2.9

141.2 ± 6.3 132.4 ± 6.3

98.2 ± 4.1 95.7 ± 3.3

99.1 ± 3.8 96.5 ± 2.5

0.43 ± 0.07 0.34 ± 0.01

0.30 ± 0.05 0.15 ± 0.04

Data are the mean of 5 independent determinations of different MAD batches.

on the use of a stirrer and a homogenizer, so the drug can be lost partly on the paddle employed for MAD production. For these reason, to determine CUR recovery in MAD, the Ultracentrifugation method (UC) was employed [8,24]. Because the method allows to separate CUR associated with the disperse phase from CUR free in the dispersion, it enables to obtain both recovery and encapsulation data. The latter are given indirectly by difference and refer to free CUR found in the supernatant after UC of CUR-MAD. CUR EE was almost quantitative (99.19% with respect to the CUR amount used for the preparation). CUR recovery data determined by MAD dilution in ethanol and stirring were superposable (data not shown). The data indicate that the whole CUR employed for MAD production was encapsulated into the MAD disperse phase and that no drug has been lost during production. It should be emphasized the solubility power of ORG with respect to CUR. It was found that ORG is able to solubilize 5.5 mg/ ml of CUR. This value is 5.5-fold higher than the CUR solubility in ethanol (1 mg/ml). In the case of MAD, the solubility power for CUR was 350 lg/ml that is 15-fold lower than its solubility in ORG, but dramatically higher than its solubility in water (11 ng/ ml). 3.5. CUR stability in MAD and in ORG Fig. 2. X-ray scattering results from MAD samples, both in the presence and absence of CUR. Scattering profiles have been measured at 37 °C (continuous line) and 45 °C (dotted lines) and curves have been displaced along the intensity axis for clarity. The vertical lines indicate the expected positions of the Bragg peaks in the case of a cubic Pn3m structure [42].

Bragg peaks were observed. As the position of the two peaks scales p as 1: 3, a local, short-range 2D hexagonal order between the lecithin cylindrical reverse micelles can be suggested [14]. Noticeable is the fact that the unit cell parameter of the Hexagonal (H) phase, a, (which corresponds to the lateral distance between two rodshaped micelles) is practically independent on temperature and equal to about 170 Å. Wide-angle diffraction profiles (WAXS) shown in Fig. 3B evidence a broad band at Q = 1.35 Å1, which corresponds to a repeat distance of 4.65 Å and which suggests a fluid conformation of the hydrocarbon chains. In order to derive structural information, the SAXS curves have been analyzed by a very simple and phenomenological model already used to describe the clustering effects in macromolecular systems [43–46] (Supplementary Data, SD). From such a model, the clustering strength and the correlation length for the micellar cylinders, e.g. the so-called ‘‘blob’’ size, can be derived.

CUR content in the different formulations was calculated as a function of time and expressed as percentage of the total amount used for the preparation. Fig. 4 reports the first order kinetics obtained plotting Log (CUR residual content, % with respect to drug content at time 0) against time. From the slopes (m), determined by linear regression, shelf life (t90) and half life (t1/2) values were calculated and reported in Table 2. All data were statistically significant (p < 0.0001). It was found that CUR-MAD was decidedly more efficacious than CUR-ORG in controlling CUR stability. In fact MAD could maintain 90% of CUR stability for almost 23 years, while in the case of ORG, t90 is around one month. The t1/2 values reach 154 years for CUR-MAD and 7 months for CUR-ORG. Anyway the efficacy in controlling CUR stability is startling for both formulations, in fact it should be underlined that CUR in phosphate buffer 0.1 M is rapidly decomposed (t1/2 9.4 min) [47]. The macroscopic aspect of both CUR-ORG and CUR-MAD did not change by time, in fact they did not show phase separation phenomena, maintaining the almost absence of aggregates also after six months from production. 3.6. Production of viscous MAD

3.4. Efficiency of CUR encapsulation In the case of CUR-ORG, the preparation method prevents drug losses, allowing to obtain 100% recovery of drug into the formulation. Conversely, in the case of CUR-MAD the production is based

In order to obtain vehicles suitable for possible administration on the skin and to compare ORG with MAD for in vitro and in vivo studies, the low viscosity of the MAD was adequately improved by the use of xanthan gum x-gum. The plain x-gum (1% w/w) in water has a viscosity of 0.4 Pa s.

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A

2

Log (% CUR residual)

1.96

1.92

1.88

1.84

1.8

0

20

40

60

80

100

time (days) Fig. 4. Variation of CUR residual content in CUR-MAD (s) and CUR-ORG (h) as a function of time. Data are the means of 5 analyses on different batches of the same type of dispersions.

Table 2 Curcumin recovery in MAD and ORG by time.

B

Formulation

CUR-MAD CUR-ORG

Shelf life values K

t90 (d)a

t1/2 (d)b

0.000012 0.00325

254,010 968.4

56,341 213.23

The reported results represent the average of four independent experiment, SD were within 5%. a Time at which the drug concentration has lost 10% with respect to drug recovery at 0 day. b Time at which the drug concentration has lost 50% with respect to drug recovery at 0 day.

The ORG and CUR-ORG viscosity at a shear rate value of 10 s1 were 8.082 and 8.216 Pa s respectively. Thus the presence of CUR induces a light increase in MAD x-gum and ORG viscosity.

3.7. Rheological analyses

Fig. 3. X-ray scattering results from ORG samples, both in the presence and absence of CUR. SAXS profiles (panel A) have been measured at 37 °C (continuous line) and 45 °C (dotted lines), while WAXS results (panel B) refer to 37 °C. Curves have been displaced along the intensity axis for clarity. Note the different Q-ranges. The vertical lines indicate the expected positions of the Bragg peaks in the case of a 2D H phase.

The viscosity value of MAD x-gum and CUR-MAD x-gum measured at 25 °C were 1.290 and 1.649 Pa s respectively, at a shear rate value of 10 s1.

Fig. 5 shows flow curves for CUR-MAD x-gum (panels A and B) and CUR-ORG (panels C and D), at 25 °C and 35 °C. In particular the viscosity vs. shear rate (panels A and C) and vs. shear stress (panels B and D) has been plotted. The points on the curves are the means of three experiments, and error bars represent standard deviation. In Fig. 5A, CUR-MAD x-gum shows a marked non-Newtonian shear thinning behavior: the steady shear viscosity sharply decreased as an increase in shear rate while the first and second plateaux (Newtonian viscosity regions) are not observed at low and high shear rates respectively [48]. The temperature does not influence the behavior, in fact the curves at 25 °C or 35 °C are superposable. A similar trend is reported in the flow curve of Fig. 5B, referring to CUR-MAD x-gum vs. shear stress. In Fig. 5C, CUR-ORG, at 25 °C or 35 °C, exhibits a Newtonian plateau at low shear rate, and is shear thinning at higher shear rate [49]. More precisely, at 25 °C, the Newtonian plateau extends from 0.01 s1 to 1 s1, and the viscosity decreases significantly above 10 s1. For this temperature, the viscosity curve as a function of stress (Fig. 5D) also shows a sharp decrease in the viscosity above

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A

B

C

D

Fig. 5. Flow curves for CUR-MAD x-gum (panels A and B) and CUR-ORG (panels C and D) determined at 25 and 35 °C. Viscosity vs. shear rate (panels A and C) and vs. shear stress (panels B and D) has been plotted.

a stress of about 100 Pa (yield stress). At 35 °C, the same shear thinning behavior is observed one decade higher in shear rate (but no yield stress is observed): the Newtonian plateau extends from 0.01 s1 to 10 s1, and the viscosity decreases significantly above 100 s1. For shear rates corresponding to the Newtonian plateau, viscosity at 35 °C ( 0.8 Pa s) is about one decade lower that viscosity at 25 °C ( 11 Pa s). At high shear rate (>400 s1), viscosities at 25 °C and 35 °C are identical: the shear is high enough to break significantly the structure of the gel (shear thinning behavior) and to conceal the effect of temperature. It should be noted that the second Newtonian plateau, normally observed at very high shear rate, was not reached at 10,000 s1. However, for the CURORG (Fig. 5C) at 25 °C, the slight decrease in the viscosity slope at 10,000 s1 may be the sign of the very beginning of this plateau. Flow curves of MAD x-gum and ORG are not shown since they are superposable to the reported curves, thus viscosity is not affected by the presence of CUR.

Statistical analysis revealed significant differences (p < 0.01) between the Fn values obtained for all formulations. An important question preliminary to pharmaceutical applications is whether, and to what extent, CUR-MAD and CUR-ORG are harmful to human skin [21]. To try to clarify this point, we carried out a light microscopic investigation of human skin after in vitro diffusion experiments. In particular, samples of skin treated for 8 h with either PBS (taken as control), CUR-MAD or CUR-ORG were analyzed (Fig. 7). No significant alterations of the skin were apparent for all the treated samples. In particular, the stratum corneum was still intact after treatment. There was no difference

y = 0.020028 + 0.12301x R= 0.99417 y = 0.0034273 + 0.013116x R= 0.99135

3.8. In vitro CUR diffusion kinetics

0.8

2

CUR (ug/cm )

The in vitro CUR diffusion was studied by Franz cell in order to investigate the efficiency of CUR-MAD and CUR-ORG designed as topical vehicles. In particular CUR diffusion from liquid CUR-MAD, viscous MAD (CUR-MAD x-gum) and CUR-ORG were compared. A non-physiological receptor phase with 50% v/v of ethanol was used due to CUR scarce solubility in aqueous media, in fact it was found previously that the use of physiological media as receptor phases led to negligible diffusion kinetics [31]. In Fig. 6 the cumulative plots of the amount of CUR permeated through SCE membranes as a function of time are reported. From the obtained equation, the CUR steady state flux values (Fn) for the different vehicles were calculated. The Fn of CUR formulated in ORG resulted 0.82 lg/cm2, while the Fn was 0.129 lg/cm2 and 0.087 lg/cm2 for CUR formulated in CUR-MAD and in CUR-MAD x-gum respectively.

y = 0.011447 + 0.019436x R= 0.99148

1

0.6

0.4

0.2

0 0

2

4

6

8

10

time (hours) Fig. 6. In vitro permeation profiles of CUR from CUR-MAD (s), CUR MAD x-gum (d) and CUR-ORG (j). Data represent the mean of six independent experiments ± SD.

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between the control samples treated with PBS (Fig. 7A) and skin samples treated with CUR-MAD and CUR-ORG. 3.9. In vivo anti-inflammatory activity CUR is able to inhibit skin erythema by cutaneous application due to its anti-inflammatory activity [50,51]. Formulation CUR-ORG and CUR-MAD x-gum were further studied in vivo to determine their ability to inhibit the UVB-induced skin erythema on healthy human volunteers. Skin reflectance spectrophotometry was used to determine the extent of the erythema and to assess the inhibition capacity of the formulations after their preventive application onto the skin [32]. The AUC was determined for each subject plotting DEI values vs. time. Table 3 reports the obtained AUC0–52 values. An inverse relationship was found between the AUC and the inhibition of UVB-induced erythema. Fig. 8A reports the PIE values.

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CUR-ORG showed to be more effective than CUR-MAD x-gum in inhibiting the induced erythema 1 h after its removal (P < 0.05), while at 6 h, the formulation CUR-MAD x-gum showed the best inhibitory ability (P < 0.05; Table 3, Fig. 8A). No difference between the formulations was found, in terms of anti-inflammatory activity, after 3 h after their removal (p > 0.05). 3.10. Tape-stripping technique The technique was useful for quantifying drug depletion in the viable epidermis and the amount of CUR responsible for the antiinflammatory effect [32]. From the analysis of the data, a decrease in the amount of CUR in the stratum corneum was detected for both CUR-MAD x-gum and CUR-ORG (Fig. 8B). Nonetheless the amount of CUR recovered in the stratum corneum after removal of CUR-ORG was significantly different with respect to the one from the CUR-MAD x-gum. In fact in the case of CUR-ORG the amount decreased from 37 ng/cm2 after 1 h to 20 ng/cm2 after 6 h, while in the case of CUR-MAD x-gum the CUR amount passed from 192 ng/cm2 after 1 h to 69 ng/cm2 after 6 h (Fig. 8B). 4. Discussion

Fig. 7. Light micrographs of SCE membranes treated for 8 h with PBS (panel A) taken as control or with CUR-MAD (panel B) and CUR-ORG (panel C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Lipid based formulations have a potential as matrixes able to dissolve and deliver active molecules in a controlled fashion, thereby improving their bioavailability and reducing side-effects [37,52,53]. Their topical application allows the treatment of cutaneous pathologies improving local delivery of the incorporated drugs or modulating drug diffusion, as a function of the lipid components. In the present study we have produced lipid based topical forms with the aim to find new vehicles for cutaneous administration of the lipophilic CUR molecule. For this purpose we focused our efforts toward two different colloidal systems: the first based on a o/w monoolein nanodispersion (MAD) mainly constituted of vesicles and cubosomes and the second based on a w/o lecithin microemulsion (ORG) constituted of an entanglement of elongated reverse micelles. With regard to MAD, from cryo-TEM images it can be asserted that the use of Na cholate as emulsifier in mixture with poloxamer, does not allow the formation of cubic or sponge-like structures but only unilamellar vesicles (Fig. 1). Indeed by the presence of Pn3m inverted bicontinuous cubic phases was detected both in the absence and in the presence of CUR (Fig. 2). Cryo-TEM and X-ray data apparently disagree but the latter are in accordance with the observations by Lindstrom and colleagues which found that monoolein in dilute micellar bile salt solutions forms vesicles and different liquid crystalline phases such as the cubic one [54]. In this regard it should be underlined that in few cases inner structured cubosomes or hexasomes could not be trapped during freezing processes, mostly it should be observed that only a very small volume of the sample is examined by TEM. By contrast, a very large sample volume is analyzed by X-ray diffraction, and even small amounts of ordered phases can be easily detected. Therefore, the whole results for plain CUR-MAD suggest the coexistence of vesicles and inner structured particles. Concerning X-ray diffraction results, noticeable is the relatively large unit cell increase induced by CUR (Fig. 2): this fact clearly suggests that CUR, according to its amphiphilic nature, promotes an increase in the negative curvature of the polar/apolar interface with a subsequent increased hydration. Rheology studies indicated for MAD x-gum, in the presence and in the absence of CUR, a steady shear flow behavior typical of xanthan gum dispersions [48]. The large molecules of xanthan

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Table 3 AUC0–52 values obtained pre-treating skin sites with formulation CUR-ORG and CUR-MAD x-gum and applying UVB radiations after 1 h (t = 1), 3 h (t = 3) or 6 h (t = 6) from their removal. Subjects

AUC0–52 t=1

t=3

t=6

Control

CUR-ORG

CUR-MAD x-gum

CUR-ORG

CUR-MAD x-gum

CUR-ORG

CUR-MAD x-gum

A B C D E F

590.2 621.4 670.5 700.2 632.5 690.4

1321.4 1286.3 1279.1 1325.2 1295.5 1288.6

921.8 1000.2 896.4 912.6 1058.3 954.2

980.2 975.1 880.2 926.4 958.1 944.3

1164.3 1120.2 1100.3 1200.8 1190.6 1160.4

831.2 823.6 770.2 790.4 754.3 780.2

1625.9 1568.3 1489.4 1410.3 1184.9 1320.1

Mean

650.9

1299.4

957.3

944.1

1156.1

791.7

1433.2

A

60

50

P.I.E.

40

30

20

10

0

CUR-ORG

CUR-MAD x-gum

B 0.25

Curcumin ug/cm 2

0.2

0.15

0.1

0.05

0

CUR-ORG

CUR-MAD x-gum

Fig. 8. In vivo comparative study of CUR anti-inflammatory effect (panel A) and CUR amount in the stratum corneum (panel B) after topical application and removal of formulation CUR-ORG and CUR-MAD x-gum. PIE: percentage of erythema index; t = 1, 3, and 6: hours from the removal of formulations. Data represent the mean for ten subjects.

gum form aggregates through hydrogen bonding and polymer entanglement, resulting in a high shear viscosity at low shear rates or at rest. As shear rate increases, the steady shear viscosity decreases due to the disentanglement of the polymer network and to the alignment of individual macromolecules in the direction of the shear flow, this results in a low shear viscosity at high shear rate region. The network structure reforms rapidly upon removing

of shear, hence the viscosity is recovered almost instantaneously [55]. The presence of monooleine–poloxamer–sodium cholate based nanosystems does not seem to affect the rheology behavior of MAD x-gum. The lecithin organogel is characterized by a typical supramolecular organization described by other authors as a three-dimensional network of entangled interpenetrating polymer-like or worm-like micelles [14,56]. In the micellar evolution there are few stages upon successive addition of water, resulting in a branching of aggregates from the initial linear growth [13]. Then the micelles disintegrate into a mixture consisting of shorter and longer ones that persist in constituting a three-dimensional network stabilized by hydrogen bonds between lecithin and polar solvent molecules. The ORG were prepared using of a lecithin solution in IPP, a biocompatible oil which has been demonstrated to be particularly suitable for transdermal delivery [52,57]. Lecithin concentration was fixed at 200 mM, since this concentration was found to be effective for penetration enhancement [12]. In the case of ORG, the SAXS profile suggests that the presence of CUR induces the formation of a short-range, local 2D hexagonal organization of the lecithin cylindrical reverse micelles, while the broad band in WAXS profile confirms the fluid conformation of the hydrocarbon chains [26], independently on the presence of CUR. It is also interesting to observe that the results obtained fitting the SAXS curves by equation (S) in the SD confirms the increased ordering of the organogel after the addition of CUR (e.g., the entanglement length, which is a measure of the distance along the micelle separating neighboring entanglements, increases from about 420 to 620 Å). Therefore, the presence of CUR induces an increased ordering of the system, possibly due to an increase in the intermolecular hydrogen bonding in the CUR-ORG [58,59]. From a rheological point of view, CUR-ORG exhibited the typical behavior of lecithin organogels both in the absence and in the presence of CUR. As found by other authors, a Newtonian flow was found for over the low shear rate range, as indicated by a near constant viscosity over shear rate [49,60]. This suggests that the network structure formed by the worm-like micelles does not break in this range. At higher shear rates, CUR-ORG displayed non-Newtonian flow, giving a decrease in viscosity with increasing shear rate. This phenomenon may be a result of the collapse of the network structure due to disentanglement and/or breakage of the worm-like micelles as the shear rate increases [49,60]. It was found that CUR solubility in ORG is 5.5-fold higher than the CUR solubility in ethanol (1 mg/ml). Within the organogel, CUR is probably localized partially between the acyl chains of the lecithin molecules by hydrophobic interactions [61], as previously found for other lipophilic molecules [21,62] and partially dissolved in the external oil phase. Conversely in the MAD, that is constituted of an external aqueous phase and an inner nanostructured lipophilic phase, CUR

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would be encapsulated into the inner phase of the dispersion [8], its solubility in this case is only 350 lg/ml. The difference in CUR location within the two formulations should be responsible of the different CUR stability. It can be hypothesized that CUR is more exposed to degradation in the case of CUR-ORG and more protected in the case of CUR-MAD. In vitro CUR diffusion studies demonstrated that CUR Fn values from CUR-ORG were 6-fold higher with respect to CUR-MAD xgum and 9-fold higher than CUR-MAD. The differences in Fn of CUR-MAD and CUR-MAD x-gum may be ascribed to the vehicle viscosity, indeed the presence of x-gum represents an obstacle to CUR diffusion through the vehicle. Conversely the difference in CUR fluxes from CUR-ORG and CUR-MAD has to be related to the technological aspect of the vehicle. In fact, as above reported, in CUR-ORG the molecule is partially solubilized in the external phase constituted of IPP, so that it is promptly available for diffusion through the SCE membrane [21]. Whereas in CUR-MAD the drug is associated with the nanostructure present in the disperse phase while the external phase is constituted of water. Thus the lower CUR fluxes from CUR-MAD and CUR-MAD x-gum could be justified by the presence of different structural systems (i.e. cubosomes and vesicles) which could slow CUR diffusion. In vivo experiments gave precious information about CUR cutaneous biodistribution after topical application with MAD or ORG. The low anti-inflammatory effect exerted by CUR-MAD x-gum after 1 h from the removal of the formulation followed by a progressive increase after 3 and 6 h, could be tentatively justified by the formation of a CUR depot in the stratum corneum induced by CUR-MAD presence in this layer, as indicated by experimental evidences reported by other authors [32,52]. Since Algrem and colleagues have found a similarity between the cubic phase structure and the structure of the stratum corneum [63], it is reasonable to suppose the formation of a mix of cubosomal monoolein with stratum corneum lipids. According to this hypothesis, from the stratum corneum depot a sustained release of CUR toward the deeper skin layers could take place. Thus the stratum corneum lipids might play a role in modulating CUR pharmacodynamic response [32]. On the other hand, the high initial anti-inflammatory effect induced by CUR-ORG followed by a rapid decrease could suggest a rapid and intense initial penetration of CUR. This phenomenon could be justified by a strong interaction with the stratum corneum lipids and results in a high concentration of CUR in the vascularized section of the skin, from which CUR is rapidly removed by the blood stream [52]. This strong interaction could be due to the peculiar supramolecular aggregation structure of phospholipids in the CUR-ORG promoting a CUR penetration enhancer effect [52]. Tape stripping experiments helped to better elucidate the ‘‘in vivo’’ behavior of CUR applied on the skin [64]. In the case of CUR-MAD x-gum, 1 h after the occlusion, the CUR amount in the stratum corneum was 5-fold higher than that one found for CURORG. Afterward CUR contents decreased time dependently in both forms with similar trend and CUR-ORG constantly lower in CUR concentration. This profile justifies the above reported hypothesis of the formation of a cubosome depot in the stratum corneum from which CUR can be released in a controlled fashion [32]. Therefore, the decrease in CUR has to be ascribed to a drug slow release from this reservoir. The same amounts were probably responsible for the CUR-MAD x-gum anti-inflammatory profile recorded. The lower CUR amount found in the stratum corneum in the case of CUR-ORG could be related to a different kind of interaction between the formulation and the skin. Considering both lecithin and CUR-ORG abilities to promote CUR absorption through the skin [65–67], it appears reasonable to suppose that already after 1 h of occlusion, CUR has penetrated

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through the upper compartments of the epidermis and then can reach the dermal zone, where it displays an anti-inflammatory activity. Afterward in the time points corresponding to 3 and 6 h from the occlusion period the amounts of CUR undergo a further decrease. 5. Conclusions This study has highlighted the CUR performances in two different nanostructured lipid vehicles. Both are able to solubilize this lipophilic molecule and to protect it from degradation. In vivo data have demonstrated that CUR-ORG could be a promising strategy to treat skin diseases (e.g. scleroderma, psoriasis and skin cancer) because the vehicle promotes CUR absorption through skin, while CUR-MAD x-gum could be employed to treat the disease by time in a controlled fashion, since CUR delivery can be modulated and its action prolonged. To verify this hypothesis animal studies should be performed. Acknowledgements The authors are grateful to Sarah Mosbah from Institut Galien Paris-Sud, Châtenay-Malabry, France for rheological characterization and Clelia Miracco from Department of Medicine and Surgery, University of Siena, Italy for her expert advice about microscopy data of skin samples. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2013.12.011. References [1] D.L. Miller, M.A. Weinstock, Nonmelanoma skin cancer in the United States: incidence, J. Am. Acad. Dermatol. 30 (1994) 774–778. [2] F. Farshadpour, H. Kranenborg, E.V. Calkoen, Survival analysis of head and neck squamous cell carcinoma: influence of smoking and drinking, Head Neck 33 (2011) 817–823. [3] S. Marur, A.A. Forastiere, Head and neck cancer: changing epidemiology, diagnosis, and treatment, Mayo Clin. Proc. 83 (2008) 489–501. [4] V. Bouvard, R. Baan, K. Straif, Y. Grosse, B. Secretan, F. El Ghissassi, A review of human carcinogens – Part B: Biological agents, Lancet Oncol. 10 (2009) 321– 322. [5] M.M. LoTempio, M.S. Veena, H.L. Steele, B. Ramamurthy, T.S. Ramalingam, A.N. Cohen, R. Chakrabarti, E.S. Srivatsan, M.B. Wang, Curcumin suppresses growth of head and neck squamous cell carcinoma, Clin. Cancer Res. 11 (2005) 6994– 7002. [6] R. Wilken, M.S. Veena, M.B. Wang, E.S. Srivatsan, Curcumin: a review of anticancer properties and therapeutic activity in head and neck squamous cell carcinoma, Mol. Cancer 10 (2011) 1–19. [7] R.K. Bhawana, H.S. Basniwal, V.K. Buttar, N. Jain, Curcumin nanoparticles: preparation, characterization, and antimicrobial study, J. Agric. Food Chem. 59 (2011) 2056–2061. [8] C. Puglia, V. Cardile, A.M. Panico, L. Crascì, A. Offerta, S. Caggia, M. Drechsler, P. Mariani, R. Cortesi, E. Esposito, Evaluation of monooleine aqueous dispersions as tools for topical administration of curcumin: characterization, in vitro and ex-vivo studies, J. Pharm. Sci. 102 (2013) 2349–2361. [9] E. Esposito, E. Menegatti, R. Cortesi, Design and characterization of fenretinide containing organogels, Mater. Sci. Eng. C 33 (2013) 383–389. [10] F. Dreher, P. Walde, P.L. Luisi, P. Elsner, Human skin irritation studies of a lecithin microemulsion gel and of lecithin liposomes, Skin Pharmacol. 9 (1996) 124–129. [11] R. Scartazzini, P.L. Luisi, Organogels from lecithins, J. Phys. Chem. 92 (1988) 829–833. [12] M.B. Fawzi, U.R. Iyer, M. Mahjour, Use of Commercial Lecithin as Skin Penetration Enhancer, US Patent 4, 783, 450, 1988. [13] P.L. Luisi, R. Scartazzini, G. Haering, P. Schurtenberger, Organogels from waterin-oil microemulsions, Colloid Polym. Sci. 268 (1990) 356–374. [14] Y.A. Shchipunov, A micellar system with unique properties, Colloids Surf. A 185 (2001) 541–554. [15] P. Schurtenberger, R. Scartazzini, L.J. Magid, M.E. Leser, P.L. Luisi, Structural and dynamic properties of polymer like reverse micelles, J. Phys. Chem. 94 (1990) 3695–3701.

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Effect of nanostructured lipid vehicles on percutaneous absorption of curcumin.

The present study describes the production and characterization of monoolein aqueous dispersions (MAD) and lecithin organogels (ORG) as percutaneous d...
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