Preparation and In Vivo Evaluation of Rosmarinic Acid-Loaded Transethosomes After Percutaneous Application on a Psoriasis Animal Model

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1208/s12249-021-01966-3” 1 Preparation and In vivo Evaluation of Rosmarinic Acid-Loaded Transethosomes After Percutaneous Application on a Psoriasis Animal Model Azahara Rodríguez-Luna1, Elena Talero1, Javier Ávila-Román2, Ana María Fernández Romero3, Antonio M. Rabasco3, Virginia Motilva1, and María Luisa González- Rodríguez3,4 1 Department of Pharmacology, Faculty of Pharmacy, Universidad de Sevilla, Seville, Spain 2 Department of Biochemistry and Biotechnology, Faculty of Chemistry, Universitat Rovira i Virgili, Tarragona, Spain 3 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, Universidad de Sevilla, C/ Prof. García González, 2, 41012 Seville, Spain 4 To whom correspondence should be addressed. (e–mail: [email protected]) 2 ABSTRACT The topical use of rosmarinic acid (RA) in skin inflammatory pathologies is restricted due to its poor water solubility, poor permeability and chemical instability. In this study, RA- loaded transethosomes-in-Carbopol® formulations have been developed to evaluate its anti-inflammatory activity on imiquimod-induced psoriasis-like skin inflammation in mice. In vitro release profiles demonstrated sustained behaviour due to the retentive action of gel and the entrapment of RA into the vesicles. However, the low viscosity of the combined formulation increased the drug release rate. Animal evaluation of antiinflammatory activity demonstrated that transethosomes-in-gel containing dexamethasone (Dex-TE-Gel), as positive control, showed effect in all the proinflammatory parameters evaluated, evidencing that these drug-loaded nanocarriers have been effectively reached the site of action. In addition, transethosomes-in-gel containing RA (RA-TE-Gel) formulations produced a great reduction in the punch edema (P < 0.001) and in TNF-α and IL-6 (P < 0.05). However, non-significant differences were obtained for IL-1β, IL17 and MPO. Despite the protecting effect of Carbopol® and transethosomes on oxidation index and antioxidant activity of RA over the seven days of treatment, however, a degradation process of this antioxidant to caffeic acid may be the cause of these in vivo results. We have also checked that the pH existing into the intercellular space of damaged cells (pH 6.8) may be affecting. Therefore, our results suggest that RA-TE-Gel could act as an effective RA formulation for skin delivery; further studies will help to understand the loss of activity at the cellular level. KEY WORDS: transethosomes; rosmarinic acid; psoriasis; stability; caffeic acid 3 Abbreviations: Ch, Cholesterol; Dex, Dexamethasone; EPC, L-α Phosphatidylcholine from egg-yolk; IL, Interleukin; IMQ, Imiquimod; MPO, Myeloperoxidase; PdI, Polydispersity index; pKa, Ionization constant; RA, Rosmarinic acid; SDC, Sodium deoxycholate; TNF-α, Tumor necrosis factor alpha; RA-TE, RA- loaded transethosomes; Dex-TE, Dex-loaded transethosomes; RA- Gel, RA in gel; Dex-Gel, Dex in gel; Empty- TE-Gel, Transethosomesin-gel; RA-TE-Gel, Transethosomes-in-gel containing RA; Dex-TE- Gel, Transethosomes-in-gel containing dexamethasone; TE, Transethosomes. 4 INTRODUCTION Psoriasis is a common immune-mediated inflammatory skin disease that affects at 2– 4% of the population (1). Nowadays, treatment of this pathology comprises a wide range of possibilities, including topical agents (corticoids, vitamin D derivatives and retinoids) and phototherapy for mild cases, as well as systemic treatment (immunosuppressants and biological drugs) for severe cases. However, some of these treatments can cause important side effects. Moreover, the majority of patients remain untreated or undertreated, which leads to poor adherence and dissatisfaction with therapies (2). For this reason, it is necessary to search for new alternatives for psoriasis management. In this line, an interesting option is the use of natural products such as polyphenols. These compounds are the most popular antioxidants in our diet and exhibit antioxidant, antiinflammatory and antineoplastic properties. Hence, they have been considered an important alternative for the management of skin protection (3). Nowadays, the development of new delivery systems to administer certain compounds is crucial to improve psoriasis treatment (4, 5). Rosmarinic acid (RA) is a phenolic ester widely studied with remarkable biological and pharmacological activities as antimicrobial, antioxidant and anti-inflammatory (6). Nevertheless, its percutaneous use after incorporation in conventional drug delivery systems is limited due to its poor water solubility, discoloration, chemical instability and poor permeability properties (7–10). To overcome these limitations, nanocarrier-based systems are being explored as alternative and interesting strategies for skin drug delivery. Among them, many researchers have worked on lipid-based nanocarriers like solid lipid nanoparticles, nanostructured lipid carriers, lipid nanocrystals, liposomes and derivatives, niosomes, and so on. These nanosystems topically administered have 5 provided an enhancement in the drug permeability and have achieved an extended release. The use of lipid vesicles has been widely exploited in this research field because of their similarity with cell membranes. Liposomes are lipid vesicles composed of one or more phospholipid bilayers surrounding inner aqueous compartments. These biodegradable and biocompatible nanocarriers present the advantage of having a lipid composition like the intercellular lamellae and keratinocytes, which increases their affinity by the skin surface. In addition, they act as penetration enhancers, altering the intercellular lipid matrix. The versatility of these vesicles to entrap hydrophilic molecules in the aqueous phase, and also lipophilic and amphipathic substances into the lipid bilayer, make liposomes very interesting systems for skin drug delivery (11). However, conventional liposomes have poor permeation capability because they can act as a reservoir (4) or passing through the skin by using the intercellular lipid matrix (12, 13). Based on these previous premises, modified vesicles have been developed to enhance and optimize the skin permeation of drugs (12, 14), such as transfersomes, ethosomes, transethosomes, cubosomes, vesosomes, and novasomes (15). Transethosomes are produced by combining the advantages of both transfersomes and ethosomes. Structurally, they comprise the basic components of ethosomes (ethanol, 20– 40%) and a single chain surfactant as edge activator. Both of them act destabilizing the phospholipid bilayers of vesicles by increasing their deformability (elasticity) and squeezing through pores in the stratum corneum (13). The advantages of transethosomes have been reported in several papers that demonstrated successful stabilizing and topical delivery of antioxidants (16, 17). Also, the lipid composition makes these vesicles sensitive to peroxidative phenomena. It has been reported that transethosomes may improve the stability of 6 encapsulated antioxidants. Indeed, the presence of edge activators (mainly ionic surfactants) becomes in higher zeta potential values, which contribute to possess high electrostatic repulsion to surround vesicles and to decrease the aggregation of vesicles, improving the stability of transethosomes. This fact gives rise to an improvement in the durability of vesicles as a reduction of antioxidant leakage from them. Taking into account these findings, the present study was focused on evaluating the anti-inflammatory activity of (RATE-Gel) in an imiquimod (IMQ)-based animal psoriatic model, using dexamethasone (Dex)-loaded transethosomes as a positive control. MATERIALS AND METHODS Materials RA (98%) from Rosemarinus officinalis L. and Dex were obtained from Sigma Chemical Co. (St. Louis, MO, USA). L-α phosphatidylcholine from egg-yolk (EPC), cholesterol ( Ch), sodium deoxycholate ( SDC) and dicethylphosphate (DCP) were purchased from Sigma- BioChemika (Steinheim, Germany). Carbopol® 940 was ob tain ed from E s c u der ( Barcelo n a, S p a in ). Trichloromethane, methanol, o-phosphoric acid, ethanol, and 2-[4 (2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (Hepes) were obtained from Panreac Química (Barcelona, Spain). Polycarbonate membranes with 400 and 200 nm pore size were provided by Millipore (County Cork, Ireland). Solvents used for chromatographic analysis were HPLC quality. All other chemicals were of analytical degree. Preparation of Transethosomes Transethosomes (TE) were prepared by using the thin film hydration method, widely 7 employed for entrapping drugs (18, 19). The composition of these modified liposomes was given based on previous formulations proposed by our research group. The ratio of EPC and Ch was 62.81 and 20.90 mol-percent, respectively. DCP was added as negative charge donor, at 10.41 mol-percent and SDC was selected from previous studies, as edge activator to produce ultradeformable liposomes, at 5.87 mol-percent. Briefly, RA-loaded transethosomes were prepared as follows: EPC, Ch and DCP were dissolved in chloroform (4 mL) and SDC was dissolved in methanol (2 mL); both solutions were mixed and the solvent was evaporated under vacuum using a rotary evaporator (Büchi R-210 with Heating Bath Buchi B-491, Switzerland) at 58°C for 30 min. Once removed the residual solvent, the lipid film was hydrated and vortexed (Velp Scientifica Zx3) with 6 mL of ethanol:Hepes buffer pH 7.4 mixture (20:80 v/v) containing 5 mg/mL of RA (13.9 mM). The formulation was quickly sealed in glass containers and stored in the dark at 4°C. Finally, unilamellar transethosomes (RATE) were obtained after extruding the above samples through 400 and 200 nm pore sized polycarbonate membrane filters mounted in the Lipex Thermobarrel extruder (Northern Lipids Inc., Canada) under high pressure of nitrogen (N2). Dex-loaded transethosomes (Dex-TE) were obtained by following the previous protocol, being added the drug into the aqueous phase also. Dex concentration in liposome sample was 2 mg/mL (5.1 mM). Control liposomal formulations without drug (Empty-TE) were also obtained by following the same methodology. Control solutions for RA and Dex were prepared by dissolving 5 and 2 mg, respectively, in 1 mL of ethanol:Hepes buffer pH 7.4 mixture (20:80 v/v). Preparation of Gelled Formulations Hydrophilic gels of Carbopol® 940 were used to enhance the contact and to prolong 8 the exposure time of liposomal formulation after application onto the skin site for the in vivo study. They were prepared as described earlier with slight modifications (20). In brief, they were prepared by blending Carbopol® 940 powder in deionized water (1% w/w), staying for 24 h at room temperature to swell and followed by the addition of triethanolamine up to pH 7.4 for gelation and swelling. The resulting gel was stored in capped glass containers, at 4°C, in the dark. RA and Dex liposomal gels (RA-TE-Gel and Dex-TE- Gel) were prepared after the swelling time. The transethosomal dispersion (33.3% w/w) was added and stirred carefully until a uniform distribution within the hydrogel was achieved. Final concentrations of 0.167 and 0.067% w/w for RA and Dex, respectively, were obtained. Reference gels without liposomes were also formulated (RA-Gel and Dex- Gel) by adding RA solution and Dex solution in the same ratio into the gel. Characterization Studies Size, Polydispersity Index, and Zeta Potential The hydrodynamic diameter and polydispersity index (PdI) were determined by dynamic light scattering technique by using Zetasizer Nano-S equipment (Malvern Instruments, Malvern, UK). Results of size were expressed as the average liposomal hydrodynamic diameter (nm). Values of the dimensionless parameter PdI less than 0.2 indicate a homogeneous and monodisperse population whereas larger PdI (> 0.3) implies heterogeneity. The surface charge of vesicles was determined by correlation spectroscopy from electrophoretic mobility (μ) measurements, by using the same equipment as above. Results were expressed as zeta potential (Z, mV) after conversion of μ to Z by the 15 for Dex samples to clarify the release behavior of Dex from the different formulations (Dex control solution, Dex-TE, Dex- Gel, and Dex-TE-Gel) by using a volume or amount equivalent to 2 mg. Finally, samples were analyzed by HPLC method as described above for RA and spectrophotometrically for Dex. Although pH 7.4 was initially used for release test, however, unexpected in vivo results for RA samples were forced to make these experiments at pH 6.8 to mimic the conditions into the inflammatory tissues. In Vivo Efficacy Studies Animals For this study, 8-week-old female BALB/c mice were supplied by Janvier-Labs (Le Genest St Isle, France). Mice were maintained in our Animal Laboratory under standard conditions (temperature of 24–25°C, humidity of 70–75% and 12 h light–12 h dark cycle). Mice were allowed free access to a standard diet (Panlab, Barcelona, Spain) and water ad libitum. Dorsal region of the mice was shaved by using an electric clipper and skin hair removal cream (Deliplus, Barcelona, Spain) without scratching or damaging the surface to maintain the integrity of the skin (27). The study was performed following the recommendations of the European Union regarding animal experimentation (Directive of the European Council 2010/63/EU). The experiments followed a protocol approved by the Animal Ethics Committee of the University of Seville. Imiquimod-Induced Psoriasis-Like Inflammation in Mice A daily topical dose of 62.5 mg from commercially available IMQ cream (Aldara 5%; Meda AB, Solna, Sweden) was administered on the shaved dorsal selected areas of female BALB/c mice for 6 consecutive days (27). In the same way, a vehicle cream (Vaseline; 16 Acofar, Madrid, Spain) was applied to the IMQ-untreated mice (sham). Repeated topical application of IMQ promotes skin inflammation with structural features typical of psoriasis (28). Mice were stratified randomly into 5 experimental groups: 1. Sham: mice were pre-treated with Empty-TE-Gel (100 mg per site) and vaseline. 2. IMQ: mice were pre-treated with Empty-TE-Gel (100 mg per site) and IMQ cream. 3. Dex-TE-Gel: Dex-loaded transethosomes dispersed in gel (100 mg per site, containing 67 µg of Dex) (0.067 % w/w) and IMQ cream. 4. RA-TE-Gel: RA-loaded transethosomes dispersed in gel (100 mg per site, containing 167 µg of RA) (0.167 % w/w) and IMQ cream. 5. RA-Gel: RA-Gel formulation (100 mg per site, containing 167 µg of RA) (0.167 % w/w) and IMQ cream. Mice were anaesthetized with ketamine (100 mg/kg of animal) and diazepam (5 mg/kg of animal) during treatments and IMQ challenge. Dex is a corticosteroid highly employed as anti-inflammatory topical reference agent (27). The pretreatments of Dex- TE-Gel, RA-TE-Gel and RA-Gel were applied daily on the selected back area thirty min before the IMQ application (62.5 mg). A day after last application (day 7), mice were euthanized and punch biopsies from the treated dorsal skin were weighed to evaluate edema, before further processing for biochemical parameters. MPO Activity Myeloperoxidase (MPO) activity was evaluated as a marker of neutrophil infiltration following the method of Grisham et al. (29). The tissue was processed as previously reported (30). One unit of MPO activity was defined as the amount of enzyme present 17 that produced a change in absorbance of 1.0 unit/min at 37°C in the final reaction volume containing the acetate. Results are expressed as MPO units/mg tissue. Measurement of Cytokines in Skin Homogenates Frozen skin biopsies were homogenized, centrifuged and supernatants fluids were stored at −80°C until measurements as detailed in Ávila-Román J. et al. (30). Levels of TNF-α, IL- 1β, IL-6, and IL-17 were measured by quantitative Enzyme- Linked ImmunoSorbent Assay kits (ELISA) (Peprotech, Germany), according to the manufacturer’s instructions. Data are reported as ng/mg tissue. Statistical Analysis All values in the figures and text are indicated as arithmetic means ± SEM. Data were evaluated with GraphPad Prism version 5.00 software (GraphPad Software, Inc., San Diego, CA, USA). In all cases, the Shapiro-Wilk test was used to verify the normality of the data. The Mann- Whitney U-test was chosen for non-parametric values. The parametric values groups were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s Multiple Comparison Test. P values < 0.05 were considered statistically significant. RESULTS Stability Studies Physicochemical and Morphological Properties of Formulations Transethosomes were prepared by thin-layer evaporation technique, wherein they were composed of EPC, Ch, DCP, and SDC. This composition was fixed based on our experience and our previous studies (31, 32). This drug delivery system was selected 18 because recent studies have considered it as effective and promising delivery system for skin disease treatment, such as atopic dermatitis or psoriasis (33). The characterization study was carried out with all formulations analyzed in this work. In terms of size, before extrusion, transethosomes had 900 and 604 nm for RA and Dex liposomes, respectively (data not shown), whereas for empty vesicles, size was 750 nm approx. As expected, an increase in vesicle size caused by the drug loading was observed for RA. However, despite using different molar concentration than RA, interesting results were obtained with Dex formulations, since reduced mean diameters were found compared to empty liposomes. The average size, PdI, and zeta potential of transethosomal samples after extrusion are summarized in Table I. Initially, RA-TE represented 74.5% mean entrapment efficacy of RA and had sizes of about 200 nm approx. with a PdI of 0.18. Higher sizes of RA-TE compared with Empty-TE (P < 0.001) and lower sizes of Dex-TE than Empty- TE (P < 0.001) were obtained. Values next to the extrusion filter size denote certain elasticity in RA vesicles, as demonstrated with the deformability assay. Our results showed higher values in RA-TE and Empty-TE compared with Dex-TE, denoting the addition of rigidity of Dex to the lipid bilayer (Fig. 1). The zeta potential of RA-TE was negative (−21.63 mV) and had higher negative values compared to Empty-TE (P < 0.01). However, no statistical differences were obtained between Dex-TE and Empty-TE values. After 7 days of storing, the physicochemical properties of formulations in size were practically maintained, showing a slightly significant difference in the vesicle size (P<0.05). However, zeta potential values decreased in RA-TE samples (P < 0.01) while acquired similar values in Empty-TE. 19 Encapsulation efficiency for RA was 74.5%, indicating that approximately 25% was free in the Hepes medium. The maintenance of this amount of RA unentrapped was important to create a burst release phase for initiating the pharmacological effect. After 7 days of storing, there were significant differences in RA-TE formulations (P < 0.05), decreasing PDE (%) by 5% over the 7 days. The properties of vesicles inside the hydrogel were also obtained, but results showed confused conclusions because of the difficulty of vesicle extraction from gel. Figure 2 shows TEM images of Empty-TE, RA-TE, Dex- TE, Empty-TE-Gel, and RA-TE-gel. All transethosomes had nano-sized globular shape and their sizes were lower than those measured by DLS, probably due to the loss of water for analysis. TEM images for Empty-TE and RA-TE showed more regular and well-defined spherical liposomes than those of Dex-TE. In addition, it was possible to see the deformability property in the former vesicles. Morphological analysis showed the integrity of vesicles in a week, demonstrating the stability of formulations. With the aim of better visualizing the integrity of vesicles into the gelled formulations, samples were suitably prepared for scanning laser confocal microscopy. Labeled vesicles with Nile Red allow us to discern their morphology. As depicted in Fig. 3, the spherical shape of vesicles was maintained in all the formulations tested (RA-TE, RA-TE-Gel and Empty-TE- Gel) along 7 days of storing, although it was more difficult the identification of vesicles in gel formulations due to the presence of Carbopol®. Unexpected spicule-shaped of nile red in the preparation were appreciated, probably due to the solution (RA-sol) and this activity was decreased when RA was formulated into the gel and/or into liposomes. However, after 7 days, a higher antioxidant loss of RA from RA-sol was evident, whereas the presence of gel and/or transethosomes protected the antioxidant molecule from oxidation and contributed to maintaining its 20 activity. With the aim to evaluate the efficacy of nano-gelled formulation on decreasing the oxidation RA, the differences between EC50 in both times were plotted. This parameter showed that RA-TE-Gel exhibited an activity approximately four times more powerful than RA-sol. From these results, it was revealed that RA can avoid o diminish the lipid peroxidation into the liposome bilayer and staying in a stabilizing medium within the hydrogel. In Vitro Release Studies Results of in vitro release studies (Fig. 6) showed sustained release of the drug from transethosomal, gel, and transethosomal-gel formulations, which was evident by the cumulative percentage of drug released. Concerning RA- loaded samples (Fig. 6a), two main effects can be visualized in the obtained profiles; firstly, the retardant effect of drug release from the gel (RA-Gel) and the improved RA release from transethosomes (RATE and RA-TE-Gel). On the other hand, the protective effect of gel and liposomes on the integrity of RA contributes to having a higher percentage of drug released. Although Dex has been included in this work as positive control for its antiinflammatory activity in vivo, it was interesting to analyze the exhibited release profiles, which showed a rational behavior of sustained drug release in the presence of gel, and no significant differences among the different formulations were detected (Fig. 6b); as an exception, Dex release from transethosomal formulations was improved, remarking the enhancer effect of the nanosystem employed to solubilize the drug. Therefore, the composition of transethosomal-in-gel formulations could be considered suitable for achieving sustained drug release. As pointed in the methodology section, unexpected results were obtained in the 21 in vivo studies due to the reduced anti-inflammatory activity of RA in RA-TE- Gel formulation. In order to delve into this point, it was suggested to make in vitro release studies, at pH 6.8. This pH value mimics the pathological conditions of the affected inflamed area to detect any influence of the pH on drug release an stability. While Dex followed a similar release profile than at pH 7.4 (data not shown), an increase in RA release rate from gelled formulations at pH 6.8 was obtained compared with the previous pH (Fig. 7a). Moreover, during the first 5 h, RA was quickly released and from this point, the drug began to decrease its concentration, achieving less than 30% after 24 h. To clarify the oxidative mechanism of RA released at pH 6.8 from liposomal gel formulations, chromatograms of RATE-Gel 6.8 with time, were collected (Fig. 7b). Two defined peaks were well-defined in each chromatogram, one at about 7 min corresponding to RA and the other one at 3.5 min approx. identified as caffeic acid, a degradant product of RA at pH 6.8. Figure 7c illustrates the chromatogram and the absorption spectrum of standard solution of caffeic acid, identifying the same peak in Fig. 7b. Anti-Inflammatory Effect of RA Formulations on IMQ- Induced Psoriasis Animal Model In an attempt to evaluate the in vivo effectiveness of RA formulations, we evaluated the topical effect of gel formulations on the murine IMQ-induced psoriasis-like inflammation model, which reproduces certain biochemical and morphological parameters typical of human psoriasis. IMQ- cream administration to dorsal skin led to the development of macroscopic lesions as peeling and skin edema (Fig. 8a), confirmed by a higher weight of the 1 cm2 punch biopsies in comparison with the sham group (P < 0.01). As expected, pre-treatment with the reference compound Dex-TE-Gel 22 significantly reduced the skin punch weight (P < 0.001) (Fig. 8a). Similarly, topical treatment with both RA-Gel and RA-TE-Gel 30 min before IMQ application (62.5 mg per site for 6 consecutive days) inhibited macroscopic damage and the skin punch weight (P < 0.001), suggesting a reduction of skin edema (Fig. 8a). In accordance with macroscopic changes, IMQ-treated animals exhibited a massive neutrophilic infiltration compared with sham animals, evidenced by the increase of MPO activity, an established marker for inflammatory cell infiltration into the skin (P < 0.001) (Fig. 8b). Dex-TE-Gel application markedly diminished MPO activity compared with the IMQ-cream group (P < 0.001). Similarly, this marker was reduced by treatment with RA-Gel (P < 0.05); however, no significant changes were detected after RA-TE-Gel administration (Fig. 8b). Immune cell infiltration detected in the MPO measurement of the skin from IMQ-treated mice was correlated with increased levels of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-17, in comparison with the sham group (P < 0.05, P < 0.001, P < 0.001, and P < 0.05, respectively). The production of these cytokines was significantly reduced in animals treated with RA-Gel in comparison with IMQ group (P < 0.05, P < 0.05, P < 0.01, P < 0.05, respectively). As regards RA-TE-Gel, this formulation only was able to reduce TNF-α and IL-6 levels in a significant manner (Fig. 8c–f). DISCUSSION In this work, the use of transethosomes as drug delivery systems was mainly justified considering the ability of these nanocarriers to potentially increase the stability of labile drugs such as RA and to offer a sustained release of the drug, which enables dose optimization for psoriasis treatment. Besides, their vesicle sizes and the presence of SDC and ethanol in their composition would enhance their pass through the stratum corneum, 23 improving the drug access to the action site in the skin (16). To ensure the quality of formulations used for the in vivo study (RA-loaded and Dex-loaded transethosomes), a careful physicochemical characterization of these formulations was developed. Modified liposomes obtained in this work have resulted in deformable unilamellar anionic vesicles containing a relatively high concentration of ethanol in the formula, with a size less than 200 nm. As Verma et al. (35) postulated, vesicle size plays an important role in skin penetration of vesicular systems, being liposomes with sizes less than 300 nm able to deliver their contents to some extent into the deeper layers of the skin. Significant higher sizes obtained in RA-TE compared with Empty-TE (P<0.001) could be explained since the drug partially remained negatively charged into the aqueous space of liposomes (pH 7.4) because of electrostatic repulsion caused by ionic bilayer. From zeta potential values, we could also conclude that some interaction with the lipid bilayer exists (differences in zeta values between Empty-TE and RA-TE) in accordance with Fadel et al. (36). These authors evidenced, by using Langmuir monolayers, that although cholesterol decreases the penetration of RA into the bilayer in this type of liposomes, this repulsive effect gives rise to the increase in the PDE parameter and favors the maintenance of RA partitioned into the aqueous compartment. In the case of Dex, probably, a rearrangement of Dex into lipid bilayer was the cause for these dimensions, originated by the displacement of sterols such as cholesterol, from their positions in the lipid bilayer, as postulated by Tsotas et al. (37). Dex and RA displayed similar physicochemical properties in terms of molecular weight (392.46 g/mol for Dex, and 360.32 g/mol for RA), water solubility (very slightly soluble) and lipophilicity (log P 1.83 for Dex, and 1.82 for RA). However, Dex is a weak base (pKa 12.14) that is mainly positively ionized in the formulation (pH 7.4) while RA is negatively ionized at 24 this pH (pKa 3.57). In addition, as demonstrated by Pérez-Fons et al. (38), the incorporation of RA in phospholipid membranes had a minor effect on the perturbation of the lipid matrix and the decrease in the hydrophobic interactions between phospholipids and the drug was not effective compared with other antioxidant compounds. Hence, RA structure stays oriented to the aqueous phase, favoring the drug entrapping into the vesicles. However, in the case of Dex, this molecule tends to migrate to the lipid bilayer as a consequence of an electrostatic attraction and lipid nature, causing a displacement of structures in the bilayer, such as Ch, obtaining zeta potential values similar to Empty-TE (−15.9 mV). This effect may decrease PDE (37). Similar effects have been obtained for curcumin-loaded vesicles, in which negatively charged liposomes exhibited the highest encapsulation efficiencies compared to positively charged vesicles (39). Physicochemical parameters after 1 week of storing showed that sizes decreased (P<0.05) in Empty-TE and RA-TE, while no significant differences were obtained in Dex-TE. Rearrangement of components into the lipid bilayer together to the loss of RA over this time may contribute to this result since PdI was maintained. The modification in zeta potential values in RA-TE formulations may confirm this proposal. 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The modulatory effect of ellagic acid and rosmarinic acid on ultraviolet-B-induced cytokine/chemokine gene expression in skin keratinocyte (HaCaT) cells. Biomed Res Int. 2014;2014:346793. 35 Fig. 1. Deformability of liposomal formulations evaluated. The deformability of RA- loaded liposomes (RA-L) and Dex-loaded liposomes (Dex-L) was statistically compared with Empty-L (Mean value was significantly different compared with empty-L (*P < 0.0501; Student t test, N=3). Fig. 2. Transmission electron micrographs (TEM) of samples at time 0 (left) and at time 7 (right). a, b Empty-TE; c, d RA-TE; e, f Empty-TE-Gel; g, h RA-TE-Gel; i Dex-TE. Diameters of vesicles in the TEM images were slightly lower than obtained by DLS technique due to the desiccation procedure at which the samples were submitted before visualizing them by TEM. Fig. 3. Confocal microscopy images of formulations. On the left, samples at time 0 days, on the right samples at time 7 days. a and b: RA-TE; c and d: Empty-TE-Gel; e and f: RA-TE-Gel. The inset shows the enlarged image of a vesicle (― scale bar 2 μm). Fig. 4. Viscosity (Pa s) as a function of shear rate (1/s) of gelled formulations. Fig. 5. a Oxidation index of Empty-TE and RA-TE over time. Data were extracted from UV absorbance spectra of samples solubilized in ethanol. b EC50 values obtained with the ABTS assay, taking Trolox as reference antioxidant compound. Measurements are expressed as Trolox equivalents. Values were compared with RA solution (RA-sol) (time 0 days, t-Student test; N=3; P<0.01: ++; P<0.05: +) (Time 7 days, t-Student test; N=3; P<0.05: #) (Difference between days, t-Student test; N=3; P<0.01: **; P<0.05: *). Fig. 6. In vitro release profiles of rosmarinic acid (RA, Fig. 5a) and dexamethasone (Dex, Fig. 5b) from free drug solution (RA-sol and Dex-sol), transethosomes (RATE and Dex- TE), gel (RA-Gel and Dex-Gel), and transethosomes in gel (RA-TE- Gel and Dex-TE- Gel). The study was carried out using dialysis bag method at 37°C, 100 rpm, and Hepes buffer solution (pH 7.4) was used as release medium. All data represent mean ± SD (N=3). RA profiles: t =0.5 h, * P <0.05 (RA-TE-Gel); t =1 h, ** P <0.01 (RA-TE, RA-Gel and RATE-Gel); t =2 h to t =24 h, *** P <0.001 (RATE, RA-Gel and RA-TE-Gel, except RA-TE- Gel which has not statistical significance at time 24 h). Dex profiles: t =0.5 to t =3 h, **P<0.01 (Dex-TE) and t=3 36 h to t=24 h, ***P<0.001. Dex-TE-Gel has **P<0.01 from t=4 h to t=6 h. All samples were compared with control solution (RA-sol or Dex-sol). Fig. 7. a In vitro release profiles of Rosmarinic acid (RA) from free drug solution (RA-sol and Dex-sol), transethosomes (RA-TE and Dex-TE), gel (RA-Gel and Dex-Gel), and transethosomes in gel (RA-TE-Gel and Dex-TE-Gel). The study was carried out using dialysis bag method at 37°C, 100 rpm, and Hepes buffer solution (pH 6.8) was used as release medium. All data represent mean ± SD (N=3). RA profiles: RA-TE (P<0.001 at t=2 h; P<0.05 at t=6 h and P<0.01 at t= 24 h). RA-Gel (P<0.05 at t=0.5 h, P<0.001 at t=2 to 4 h, P<0.01 at t=24 h). RA-TE-Gel (P<0.001 at t=0.5 to t=6 h, P<0.01 at t=24 h). b HPLC chromatograms corresponding to RA-TE-Gel samples at different times in the release study at pH 6.8. c Chromatogram of caffeic acid solution (0.1 mg/mL) and UV absorption spectrum. Fig. 8. The formulations with RA-loaded liposomes in gel (RA-TE-Gel) (100 mg per zone, 0.167% w/w) and RA in gel (RA-Gel) (100 mg per zone, 0.167% w/w) were topically administered 30 min before the IMQ application (62.5 mg per zone) during six consecutive days. Dexamethasone-loaded liposomes in gel (Dex-TE-Gel) were employed as positive reference compound (100 mg per zone, 0.067% w/w). a Macroscopic appearance of the skin at the end of the experiment. b Skin edema as punch biopsy; weight of skin was used as marker of inflammatory skin process. c Myeloperoxidase (MPO) activity in dorsal skin. d TNF-α levels (ng/mg tissue). e IL-1β levels (ng/mg tissue). f IL-6 levels (ng/mg tissue). g IL-17 levels (ng/mg tissue). Values are means with standard errors represented by vertical bars. Data are means ± SEM (N=6). Mean value was significantly different compared with the sham group (*P < 0.05, **P < 0.01***P < 0.001; Student t test). Mean value was significantly different compared with IMQ group (+P < 0.05, +++P < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test). 37 Figure 1 0 50 100 150 200 250 300 Elasticity (µg.mL-1) * Empty-TE RA-TE Dex-TE 38 Figure 2 (a) (b) B. RA-TE (c) (d) (e) (f) (g) (h) (i) 39 Figure 3 10 µ M 10 µ M 10 µ M 10 µ M 10 µ M 10 µ M 40 Figure 4