Hydrogel-thickened nanoemulsions based on essential oils for topical delivery of psoralen: Permeation and stability studies

Accepted Manuscript Hydrogel-thickened nanoemulsions based on essential oils for topical delivery of psoralen: Permeation and stability studies Thaís Nogueira Barradas, Juliana Perdiz Senna, Stephani Araujo Cardoso, Sara Nicoli, Cristina Padula, Patrizia Santi, Francesca Rossi, K. Gyselle de Holanda e Silva, Claudia R. Elias Mansur PII: DOI: Reference:

S0939-6411(16)30830-X http://dx.doi.org/10.1016/j.ejpb.2016.11.018 EJPB 12353

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

13 May 2016 10 October 2016 16 November 2016

Please cite this article as: T. Nogueira Barradas, J. Perdiz Senna, S. Araujo Cardoso, S. Nicoli, C. Padula, P. Santi, F. Rossi, K. Gyselle de Holanda e Silva, C.R. Elias Mansur, Hydrogel-thickened nanoemulsions based on essential oils for topical delivery of psoralen: Permeation and stability studies, European Journal of Pharmaceutics and Biopharmaceutics (2016), doi: http://dx.doi.org/10.1016/j.ejpb.2016.11.018

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Title Page

HYDROGEL-THICKENED NANOEMULSIONS BASED ON ESSENTIAL OILS FOR TOPICAL DELIVERY OF PSORALEN: PERMEATION AND STABILITY STUDIES Thaís Nogueira Barradas 1*, Juliana Perdiz Senna 1, Stephani Araujo Cardoso 1,, Sara Nicoli 3, Cristina Padula 3, Patrizia Santi 3, Francesca Rossi 4 , K. Gyselle de Holanda e Silva 2 and Claudia R. Elias Mansur 1 1

Instituto de Macromoléculas Professora Eloisa Mano, Universidade Federal do Rio de

Janeiro (IMA/UFRJ), Centro de Tecnologia, Bl. J, Avenida Horácio Macedo, 2030, Cidade Universitária, Ilha do Fundão – Rio de Janeiro – Brazil. 2

Departamento de Fármacos e Medicamentos, Faculdade de Farmácia, Universidade Federal

do Rio de Janeiro (UFRJ). Avenida Carlos Chagas Filho, 373, Cidade Universitária, Ilha do Fundão – Rio de Janeiro – Brazil. 3

Dipartimento Farmaceutico, Università degli Studi di Parma, Parco Area delle Scienze 27/A, 43100 Parma, Italy 4

Instituto IMEM-CNR, Parco Area delle Scienze 37/a, 43100 Parma, Italy

* Correspondent Author Thaís Nogueira Barradas Instituto de Macromoléculas Professora Eloisa Mano, Universidade Federal do Rio de Janeiro (IMA/UFRJ), Centro de Tecnologia, Bl. J, Avenida Horácio Macedo, 2030, Cidade Universitária, Ilha do Fundão – Rio de Janeiro – Brazil, CEP: 21941-598. [email protected]. Tel: (+55) (21) 2562-7209

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ABSTRACT Nanoemulsions (NE) have attracted much attention due to their as dermal delivery systems for lipophilic drugs such as psoralens. However, NE feature low viscosity which might be unsuitable for topical application. In this work, we produced hydrogel-thickened nanoemulsions (HTN) using chitosan as thickening polymer to overcome the low viscosity attributed to NE. The aim of this study is to develop and characterize oil-in-water (o/w) HTN based on sweet fennel and clove essential oil to transdermal delivery of 8-methoxsalen (8MOP). NE components (oil, surfactant) were selected on the basis of solubility and droplet size and processed in a high-pressure homogenizer (HPH). Drug loaded NE and HTN were characterized for particle size, stability under storage and centrifugation, rheological behavior, transdermal permeation and skin accumulation. Transdermal permeation of 8-MOP from HTN was determined by using Franz diffusion cell. Transdermal permeation from HTN using clove essential oil showed strong dependency chitosan molecular weight. On the other hand, HTN using sweet fennel oil showed an unexpected pH-dependent behavior not fully understood at the moment. These results need further investigation, nevertheless HTN revealed to be interesting and complex dermal delivery systems for poorly soluble drugs.

Keywords: hydrogel-thickened nanoemulsions, permeation studies, essential oils, clove essential oil, sweet fennel essential oil, eugenol, trans-anethole, psoralens, terpenes, 8MOP, Chitosan, transdermal delivery.

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1- INTRODUCTION Psoralens are naturally occurring tricyclic furocoumarins used, in association with ultraviolet light, in photochemotherapy (PUVA), a well-known treatment for psoriasis and vitiligo, which involves systemic or topical administration of psoralens and exposure of UVA light. PUVA can induce repigmentation by stimulation of melanogenesis, immunomodulation and activation of growth factors, though the exact mechanism is still unknown [1]. Among psoralens

(8-methoxypsoralen

(8-MOP),

5-methoxypsoralen

(5-MOP)

and

4,5’,8-

trimethylpsoralen (TMP)) 8-methoxsalen (8-MOP) is the most widely used in PUVA therapy [2]. Psoralens are lipophilic molecules with logP between 2 and 3, and are therefore, in principle, suitable for oral and topical administration. However, systemic treatment presents many drawbacks, such as liver carcinogenicity, tachycardia, herpes simplex, depression and insomnia, among others. The topical treatment, despite being a safer approach, has limitations related to intense local phototoxicity and slow therapeutic effect, due to insufficient drug penetration into the skin layers [3–5]. When activated by ultraviolet radiation (200 to 400nm), psoralens cause phototoxic reactions in the epidermis with an intensity which can vary according to the concentration and exposure time [6–8]. The oral therapy with psoralens is related to the high frequency of toxicity, such as liver carcinogenicity. The most frequent side effect is acute phototoxicity, similar to severe sunburn and may persist for days, followed by systemic effects such as tachycardia, herpes simplex, fever, nausea, headache, dizziness, depression and insomnia, unusual rashes with pain and deep distress in the skin. Psoralens can also reach the eyes and interact with proteins of retina, causing opacity, conjunctival changes and early cataracts, which makes this treatment unsuitable for children under 12 years. For this reason, the eye photoprotection must be emphasized during the treatment [5,9,10]. In the long term, phototherapy can cause photoaging, hepatotoxicity and liver damage, renal toxicity, hypertension, hyperlipidemia and immunosuppression. The risk of carcinoma and melanoma is by an average of 2.6 times higher than the rest of the population. Furthermore, the psoralens have the ability to inhibit enzymes of the P450 2A6 cytochrome family, responsible for the metabolism of numerous substances and drug [9–12]. In the intention to reduce the systemic effects of oral therapy, topical PUVA has become an alternative. Topical PUVA does not produce systemic effects, enhances the therapeutic effects of the treatment of various dermatoses and does not require prolonged ocular photoprotection [13,14]. 3

However, the local phototoxicity is intense and fast, being a dose-dependent phenomenon. Adverse effects are usually immediate such as erythema, edema and vesicles that occur due to uncontrolled psoralen photoreaction in the epidermis with radiation because the drug is freely available in the dosage form to react with the skin surface after topical application [15,16]. Besides the cases of toxicity, topical treatment with psoralen imposes complications since these drugs have low penetration capacity into the deeper skin layers, which requires the administration of higher doses and increased exposure time to UVA radiation, increasing the risk of serious adverse effects such as carcinogenesis [5,17,18]. Is well known that the vehicle in which a certain drug is included has a significant effect on its dermal or transdermal penetration. In general, it is possible through a careful selection of vehicle components to optimize skin penetration of an active molecule. Over recent years, several nanosystems have been proposed in order to increase the penetration of psoralens across the skin [19–22]. Among them, nanoemulsions are the most commonly used, for their ability to favor dermal drug penetration. In addition, several authors have demonstrated that it is possible to control drug release and skin permeation by adjusting the formulations, enabling dose reduction and, therefore, reducing adverse effects [5,23,24]. Another strategy to increase drug permeation through the skin is the addition of permeation enhancers, including terpenes, especially those of vegetable origin, which are described as a class of products with low toxicity, low irritation potential and listed in insurance excipients FDA [25,26]. One of the disadvantages of the use of nanoemulsions for topical application is the low viscosity, which limits their clinical application and makes their topical administration inconvenient. In this context, the development of a hydrogel-thickened nanoemulsion, by the introduction of a gel-forming polymer to the outer phase of the formulation, can solve this disadvantage and make the nanoemulsion suitable for topical application [27–30]. Such system can combine the characteristics of stability and skin penetration of nanoemulsions with the properties of sustained drug release of hydrogels thus optimizing the therapeutic effects and allowing a reduction of the dose [31,32]. One of the polymers more suitable for topical application is chitosan, a semisynthetic polymer capable of forming hydrogels by entanglement of polymer chains which entrap a large amount of water molecules, swelling in the presence of this solvent. In particular, chitosan hydrogels show mucus and bioadhesion properties, capable of increasing drug residence time on the skin and promote drug skin penetration. This is due to the cationic nature of chi4

tosan, which allows its polymeric chains to establish ionic interactions with the surface of the stratum corneum. Such characteristics make hydrogels based on chitosan good candidates for the development of dermal drug delivery systems [33–35]. This paper proposes the development of chitosan-thickened nanoemulsions containing essential oils rich in terpenes, for the topical application of 8-MOP. The effect of different surfactants (Pluronic and Cremophor) as well as of the presence and viscosity of chitosan was evaluated as well.

2- EXPERIMENTAL PART 2.1

Materials The materials used in this study were: the psoralen 8-methoxysalen (1, MW= 216.2

g/mol, m.p. = 142–148 ◦C, Xanthotoxin, 99% purity) was used as received Sigma-Aldrich, São Paulo, Brazil; Sweet Fennel, Peppermint, Orange, Lime, Buriti, Avocado, Clove and Copaiba essential oils obtained from Ferquima, São Paulo, Brazil; cellulose acetate membrane (pore size of 0.2 μm), acquired from Sigma-Aldrich, Missouri, USA; the poly(ethylene oxide)-poly(propylene oxide) block copolymer, Pluronic F68, obtained from Sigma-Aldrich, Missouri, USA; Cremophor RH 40 (PEG-40 hydrogenated castor oil), obtained from Oxiteno, São Paulo, Brazil and Chitosan of low (50,000 - 190,000 Da), medium (190,000-310,000 Da) and high (310,000 to 375,000 Da) molecular weight obtained from Sigma-Aldrich, Missouri, USA. All Chitosan grades were at least 75% deacetylated according to the supplier.

2.2

Methods

2.2.1 Screening of oil phase Due to its low water solubility, 8-MOP was subjected to preliminary solubility tests in various oil concentrations of 1 - 20% w/v. Therefore, we employed canola, almond, grape seed, avocado and buriti, copaíba, lemongrass, lime, sweet fennel, lemon balm, mint, clove (stem and button) and orange vegetable essential oils. For this purpose, we performed a preliminar selection of essential oils by visual observation of solubilization of 8-MOP using a qualitative criterion based on the United States Pharmacopeia (USP) in the chapter entitled “Description and Relative solubility” [36]. 1 mg of 8-MOP was added to 1 mL of each essential oil mentioned above. Then, the systems were subjected to ultrasonic cavitation to accelerate solubilization for 15 minutes, and solubility was assessed visually after 24 hours. To the oils that promoted solubilization,

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the above procedure was repeated consecutively until the observation of precipitation for each system. The essential oils in which 8-MOP had higher solubility classification were chosen to be evaluated quantitatively by Ultraviolet (UV) spectrophotometry. The determination of 8MOP concentration was performed in sweet fennel and clove essential oils. The samples for determining the solubility were prepared by adding an excess quantity of 8-MOP to a defined volume of each essential oil. These suspensions were kept under stirring for 24 hours at room temperature (25 ± 1 ºC), after which the suspensions were centrifuged at 3,000 rpm for 5 minutes. Then the supernatant was removed with the aid of a Pasteur pipette and each sample was filtered through cellulose filter element. An aliquot of each solution was diluted with ethanol and subsequent dilutions were prepared in ethanol. The absorbance of these solutions was determined using quartz cuvette (1 cm) and wavelength 300 nm, a calibration curve was prepared in ethanol.

2.2.2 Development of NE containing 8-MOP The formulations were produced by employing a high energy was performed by means of a high-pressure homogenizer (HPH). The concentration of 8-MOP in the formulation was 0.05% w/w in accordance with the systems produced by Lai et al., (2008) submited to sonication for 15 minutes to ensure complete solubilization of the drug. The proportion of oil phase was 5% w/w. All formulations were processed in high pressure homogenizer (HPH), EmulsiFlex C5, chosen as a high energy method to obtain NE. Samples were processed for 5 cycles and 1500 bar at 25 °C.

2.2.3 Preparation of hydrogel- thickened nanoemulsions (HTN) based on chitosan In this study, 2% w/w of chitosan with high, medium and low molecular weights were added to the outer phase of the NE, which had their pH adjusted with acetic acid solution 1% v/v. Sufficient amount of chitosan was slowly added to the systems under magnetic stirring until complete dispersion providing HTN based on chitosan. The NE, template for the production of these systems, have been selected according to the average droplet diameter and the physicochemical stability of the systems. The compositions of all samples produced in this study are shown in Table 3.

2.2.4 Hydrodynamic size and stability under storage

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The hydrodynamic size and polydispersity index of the processed formulations were determined by dynamic light scattering, in a Zetasizer Nano ZS (Malvern Instruments, UK). These measurements were performed on samples of approximately 10 μL of the dispersions, which were diluted in 1 mL of recently distilled and deionized water. The measures were conducted at room temperature (25 ºC), using a laser incidence angle in relation to the sample of 173º and a 5 mL quartz cuvette. The values reported correspond to the mean ± standard deviation (SD) of three measurements of each formulation. The polydispersity index, calculated by the device, reflects the homogeneity profile of the diameter of the sample’s droplets. A polydispersity index under 0.3 was considered satisfactory. The measurements were conducted for all the samples immediately after processing and again at intervals of 1, 2, 3, 4, 8 and 12 weeks to study the formulations’ stability.

2.2.5 Physical Stability Studies All formulations were subjected to different stability tests to assess their physical stability. The evaluation of organoleptic aspects was carried out subjectively by comparing all samples subjected to different storage conditions. It was defined as reference the sample kept at room temperature. For the evaluation of appearance, alteration levels observed were classified into (I) normal, without alteration; (II) slightly precipitated or slightly turbid and (III) phase separation, precipitate or cloudy. The degree of color and odor alterations observed in the formulation was classified according to the following criteria: (I) Normal without change; (II) slightly modified; (III) modified and (IV) intensely modified.

2.2.5.1

Stability under freeze/heating cycles: NE and HTN were subjected to alternate freeze and thaw cycles, 24 h each, for a pe-

riod of 12 days. The samples were stored at 4.0°C for 12 h then at 45.0°C for the same period of time. At the end, the samples were left over night to recover and equilibrate at room temperature. Signs of physical stability, for example, transparent appearance, pH and droplet size, were re-assessed.

2.2.5.2. Centrifugation test Formulations were heated for 24 hours and then, centrifuged first at 11.000 rpm for 30 min each time, and we evaluated phase separation by visual inspection.

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2.2.5.3

Stability under thermal stress Samples (triplicate) were placed in flasks with hermetically sealed cover. The total

volume of the flask was not completed to allow gas exchange possible. The samples were submitted to heating in stove at 45 ± 2 ° C for 90 days. Control samples were kept at room temperature for the same period of time. The evaluation of the samples was performed initially at time zero and after 15, 30, 60 and 90 days. We evaluated organoleptic (color, odor and appearance) and physicochemical (pH, droplet size and viscosity) parameters.

2.2.5.4

pH determination. The pH values of formulations were determined in triplicate directly in the samples

(Oakton Ion6 potentiometer).

2.2.5.5

Shear viscosity measurements Measurements were performed in a dynamic shear rheometer (HaakeRheoStress 600,

Thermo Electron Corporation) equipped with a cone-plate geometry with a cone diameter of 35 cm and angle of 1◦and a gap of 0.052 mm. Temperature (25◦C) was controlled by a Phoenix II cooling and heating system (±0.1◦C). The reliability of measurements was assured by obtaining the rheograms in triplicate. Flow curves were determined with two consecutive continuous shear rate ramps from 1 to 150 s−1 with ascending and descending cycles.

2.2.5.6 Quantification of 8-MOP by high performance liquid chromatography (HPLC) 8-MOP content in samples was determined using an HPLC system (Perkin-Elmer, Norwalk, CT) and a NovaPak® C18 column (150 × 3.9 mm, Waters, USA). Samples were eluted with a mobile phase consisting of methanol:water (65:35 v/v), The flow rate was 0.7 mL/min UV absorption was monitored at 300 nm and the run time was about 5 minutes using the method previously developed by Colombo et al., (2003) [37]. A stock solution was prepared by dissolving weighed amount of 8-MOP in methanol. Five solutions were obtained with appropriate dilution of the stock solution with methanol, to give concentrations in the 15.0, 25.0, 35.0, 45.0 and 50.0 μg mL-1. The analyses were conducted in triplicate.

2.2.8

Transdermal drug permeation studies

2.2.8.1 Skin samples preparation.

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Pig ears were obtained from a local slaughterhouse immediately after the death of the animal. Full thickness skin was excised from the outer region of the ear and separated from the underlying cartilage with a surgical blade. Hairs were trimmed carefully with scissors. After the removal of subcutaneous fat, the skin was wrapped in aluminum foil and stored at −20°C until use and used within 3 months.

2.2.8.2 Permeation experiments Permeation experiments were conducted in vertical Franz-type diffusion cells (Disa, Milan, I), with an exposed surface area of 0.6 cm2 using full-thickness pig ear skin as barrier. The donor compartment was filled with 500 μL of NEs prepared. As reference a 0.05% w/w 8-MOP solution in ethanol was used. Although ethanol is well known as skin permeation enhancer, it was used because it has been often the main vehicle for 8-MOP. The receptor phase was filled with 4 mL of 0.9% sodium chloride solution and 1% wt of β-cyclodextrin, thermostatted at 37 °C and magnetically stirred in order to prevent any boundary layer effects. At predetermined time intervals the receptor solution was sampled and analyzed by HPLC for the determination of drug permeated. Each permeation experiment lasted 6 h and was replicated at least 6 times using skin form different animals. At the end of this period of time, the excess amount of the formulation was removed and the skin surface was cleaned with dry paper and isopropyl alcohol. SC and epidermis were separated from dermis with heat application [38]. All samples were weighed and inserted in vials. 8-MOP was extracted from the skin layers using 1 mL of a methanol:water (60:40, v/v) mixture. The amount of 8-MOP was determined by HPLC. The extraction method was validated in blank experiments and by spiking with a known amount of 8-MOP. In all cases, no interfering peaks derived from skin samples were detected. The recoveries were above 90% from both epidermis and dermis. 8-MOP skin concentration was calculated by normalizing the amount of drug recovered in the stratum corneum + epidermis and dermis by the weight of tissues and expressed as μg of drug per mg of tissue. Each experiment was replicated 6 times. Permeation profiles obtained were then fitted to Fick’s Second Diffusion Law mathematical model (Equation 1) [39], where Q is the cumulative amount of drug permeated per unit area at time t, Cveh the concentration of the drug in the donor vehicle, K the stratum corneum/vehicle partition coefficient, D the diffusion coefficient and H the diffusion path-

9

length. The permeability coefficient P was calculated as the product between KH and D/H2. The fitting was performed using KaleidaGraph® 3.6.2 (Synergy Software).

2.2.8.3 Skin retention experiments To evaluate the amount of 8-MOP accumulated in skin layers (Stratum Corneum + epidermis and dermis) skin retention experiments were conducted in Franz’s type diffusion cells in similar conditions of permeation experiments. NEs were applied in finite dose conditions (10 μL/cm2) and left in contact with the skin for 2 h. At the end of this period of time, skin samples were treated and extracted as described above.

2.2.8.4 Transmission Electron Microscopy (TEM) The surface morphology of NEs were observed using an JEOL 2200-FS electron microscope operated at 80 kV. NEs samples were deposited on a copper mesh grid. After 60 sec the excess of fluid was removed with filter paper. Staining was obtained with 2% phosphotungstic acid aqueous solution. Samples were left to dry overnight and then observed.

2.2.8 – Data analysis All the statistical tests were repeated three times and expressed as the mean ± SE using Origin Pro 8 (OriginLab, USA) software. 3 – RESULTS AND DISCUSSION 3.1

Screening of oil phase 8-MOP (Log P = 1.93) [40] is a soluble drug in ethanol, and freely soluble in chloro-

form, sparingly soluble in water (< 4×10−4M concentration can be achieved). The maximum solubilization capacity of 8-MOP in each essential oil was studied and the results are shown in Table 1. For this, the oil phase of the NE produced was preselected by visual observation of solubilization of 8-MOP in different essential oils. According to apparent solubility, it was possible to choose the best components essential oils for constituting the oil phase: Clove (Eugenia caryophyllus) and sweet fennel (Foeniculum vulgare Mill.) essential oils (Table 1). Once these were oils with higher drug solubilization capacity in the study, we performed the quantitative evaluation regarding the ability of each one to solubilize 8-MOP. Clove oil

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proved to be more effective in promoting 8-MOP solubilization (4.42 ± 0.23 mg/mL) compared to sweet fennel essential oil (1.46 ± 0.13 mg/mL).

3.2 Production of NE The right selection of surfactants is essential, since the most critical problem related to nanoemulsion-based systems is the toxicity of their components which might cause skin irritation. In this paper we employed non-ionic surfactants and hydrophilic, and which are widely used for pharmaceutical purposes due to their low toxicity and greater stability against changes in ionic strength and pH in biological environments [41]. Furthermore, the HLB of the selected surfactant (> 10) provided the formation of nanoemulsions with hydrophilic outer phase. The composition of each formulation is shown in Table 2. The selection of the surfactant was based on the composition that produced samples with smaller droplet size after HPH processing and stabilization promoted by the lower amount of surfactant possible (data not shown). From preliminary studies, we selected Pluronic F68 at a concentration of 10% w/w for the formulations containing clove oil as oil phase, based on previous results obtained by our research group [42], and for formulations containing sweet fennel essential oil was used Cremophor RH40 at 3% w/w. . 3.3 Dynamic light scattering experiments According to Gupta et al., (2016) [43] droplet size distribution of an emulsion impacts many properties such as long-term stability, texture and optical appearance. Measurements of droplet size by dynamic light scattering were performed for NE containing both clove and fennel essential oils, after HPH processing and before adding chitosan. NE containing clove oil and Pluronic F68 as single surfactant were produced with the aid of a HPH for 5 processing cycles at 1500 bar pressure and showed kinetic stability for at least 3 months (Figure 1). The surfactant plays an essential role in decreasing droplets size and also in maintaining droplets size and steric stabilization of formulations [44]. Block copolymers as PEO-PPO-PEO, as used in this work, tend to self-organize into micelles brush shaped, being able to perform effective steric repulsion [45] . Other authors published their results when developing NE and microemulsions with clove oil [46–49]. However, they used mixtures of surfactants, co-surfactants and often small molecules such as Tween and Span as surfactants. Moreover, they obtained formulations with

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larger average droplet diameter compared to those obtained in this work. Besides, they reported changes in droplet size over time and also, a high surfactant/ oil proportion [46–49]. Previously, NE containing sweet fennel oil were produced for the most varied applications [50,51]. However, their work also used high concentrations of surfactants which may cause skin irritation problems. In this paper, we present an alternative to the production of NEF by HPH with the use of a single non-ionic surfactant (Cremophor RH40) at a concentration of 3% w/w. Such a low surfactant concentration for achieving stable NE is not common in the literature. However, this can be explained by the self-emulsification intriguing called "ouzo effect" [52] This phenomenon can act synergistically to the stable NE production process and favor the use of lower concentrations of surfactant, which explains the large difference surfactant concentration necessary for the production of NE containing clove and sweet fennel essential oils. Carteau et al., (2008) [52] reported the obtantion of spontaneously formed microemulsions produced by diluting solutions ethanolic contend trans-anethole without using surfactants. Samples were evaluated during 12 weeks in order to analyze size and PdI of dispersed droplets. After processing, all formulations displayed a relatively narrow size distribution range with PdI values less than 0.2, suggesting the high-energy method employed for the production of NE was well succeeded, which resulted in droplets of 68.1 ± 0.6 nm for sweet fennel-based NE and 91.3 ± 0.3 nm for clove-based NE during at least 3 months (12 weeks) (Figures 1 and 2).

3.4

Stability under freeze/heating cycles NE based on clove oil and sweet fennel essential oil (NEC and NEF, respectively)

formulated in the absence of chitosan and their HTN formulated with low, medium and high molecular weights of chitosan, were submitted to preliminary stability studies. The samples were examined macroscopically and subjected to pH, organoleptic parameters and droplet size. The results are shown in Table 3 and Figures 3 and 4. The organoleptic tests allowed assessing the samples by comparative analysis, in order to verify changes regarding to color, odor and appearance. It was found similarity of the formulations subjected to stability tests in relation to the newly developed, regardless of the conditions and storage periods. We performed centrifuge tests at 11,000 rpm to access samples behavior under increased centrifugal force. All formulations subjected to preliminary stability

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tests showed similarity with regard to color, odor, and appearance, compared to control formulation, regardless of storage conditions. Furthermore, the formulations submitted to centrifuge tests showed no visible turbidity and cremation or phase separation. All tested formulations appeared to maintain their initial pH characteristics under alternating storage conditions according to Figure 3. In general, it can be noted that the pH values obtained for formulations depended on the magnitude of molecular weight of chitosan used as a gelling agent. Lower pH values were needed to ensure complete solubilization of larger polymer chains. The average pH values showed no significant difference (P> 0.01) when analyzed by ANOVA, which demonstrates that the formulations feature physicalchemical stability when submitted to heating and cooling cycles and also centrifugation. Regarding the average droplet size, both NEF and NEC when subjected to centrifugation showed results comparable to that observed for the respective NE freshly prepared. Figure 4 depictures a gaussian distribution for both formulations (NEC and NEF), suggesting uniformity of droplet size under such conditions after the centrifugation test. However, the same was not observed for the NEC formulation after the heating and freezing cycles. A new population of droplets with average size of about 3000 nm was formed after heating and freezing cycles, which can be explained by the thermosensitive behavior attributed to PEOPPO-PEO triblock copolymers as Pluronic, due to the limited water solubility of PPO block, which is extremely sensitive to increasing temperature [53,54]. It is possible that this phenomenon has led to a decrease in surfactant solubility which is important to stabilize micelles in NEC. Such fact would induce the approximation of droplets with consequent spontaneous coalescence between them in order to reduce the extent of the oil/water interface. This destabilization initial sign was not detected macroscopically as commented previously. It is worth mentioned that HTN formulations were not evaluated as to the droplet size, since the presence of large polymeric chains of the chitosan prevented the proper dynamic light scattering measurement and probably would interfere in oil droplets size measures.

3.5

Stability under thermal stress Given the relative stability demonstrated in preliminary studies, the systems were sub-

jected to accelerated stability studies over a period of 90 days. It was observed up to 30 days at high temperature of 45 °C, the formulations showed no destabilization signs, color or odor

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alterations (Table 3). However, after 60 days exposed to high temperature, there was slight change in coloring of HTN formulations obtained from the clove essential oil, which became darker. At the end of 90 days all HTN containing clove oil showed strong brownish, suggesting oil oxidation. The same was not true for the NEC in the same time period, suggesting that under these conditions the interaction between chitosan and, NE constituents can speed up the process of destabilizing HTN containing essential oil of clove.

3.5.1 pH determination The pH of a formulation should guarantee the stability of the formulation ingredients, also their effectiveness and safety as well as be compatible with biological tissues according to the intended route of administration [55]. The average pH values for formulations containing oil of fennel showed no significant difference (P> 0.01) when analyzed by ANOVA, suggesting that formulations feature reasonable physicochemical stability when subjected to 90 days of continuous exposure to heat (Figure 5). However, the evaluation during 90 days showed a significant decrease in pH of all formulations containing clove oil from the 30th day on. The change in pH value was greater for the formulations presented higher initial acidity, suggesting that reducing the pH of HTN to facilitate solubilization of chitosan can accelerate destabilization. This observation may reflect the oil phase oxidation phenomenon with hydroperoxide formation or the hydrolysis of triglycerides with the formation of fatty acids, responsible for the pH decrease of the formulations.

3.5.2 Shear viscosity measurements The evaluation of viscosity helps to conclude whether a product provides proper consistency and viscosity and can indicate if stability is adequate, i.e. can indicate product behavior over time [56]. Moreover, instabilities arising from variation in size, particle number and emulsifier orientation or migration over a period of time can be detected by changes in the apparent viscosity of the product . All HTN exhibited pseudoplastic flow (Figures 6 and 7), i.e. the viscosity decreasing with increasing applied shear stress. This behavior is desired in pharmaceutical formulations for preventing the mobility of the dispersed phase under low shear stress, being important that it exhibits free flow when shaken, showing low viscosity compared to high shear rates, these being reversible changes after certain time repose, delaying coalescence or creaming [43].

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The reduction in viscosity when increasing shear rate is associated with the entanglements or physical nodes, responsible for raising the viscosity of formulation, for obstructing the flow system. With increasing shear rate, such physical nodes are broken and polymer chains align in the direction of the applied strain, which is responsible for reducing the viscosity under these conditions [57]. Indeed, the addition of chitosan altered the rheological behavior of HTN compared to control (non-gelified NE) in all cases. NE showed low viscosity and newtonian behavior regardless the shear rate applied. In general, it was noticed an increase in viscosity of HTN, as the chitosan molecular weight increases. During this 90 days test, HTN showed marked decrease in apparent viscosity. These data are consistent with previous studies indicating decrease of viscosity of aqueous solutions and gels of chitosan as temperature increased [58,59]. Mironov et al., (2007) [60] reported a decrease in dynamic viscosity of solutions of chitosan in acetic acid during the storage, which would be related to polymer degradation phenomena with consequent reduction of its molar mass, which is reflected in the viscosity of chitosan based systems. Thus, temperature associated with the low pH of HTN, possibly caused degradation reactions of chitosan, which may explain the decrease in viscosity over time of study. In general, all the results observed in this study may be related. Oxidation oil phase could lead to the formation of acidic species which could be responsible for the accelerated degradation of chitosan polymer chains. This phenomenon was reflected in both the color alterations of HTN and also when assessing the viscosity of chitosan-based HTN. Thus, the accelerated stability study showed that the NE were able to maintain their initial appearance even facing extreme conditions of storage. No typical instability sign such as flocculation, creaming or phase separation were visible. The droplet size distribution of NE stored at 45°C is shown in Figure 8. The analysis of droplet size profile of NEF demonstrates a unimodal distribution of formulations submitted to heating.

Samples composed on clove essential oil

showed a bimodal distribution in droplet size distribution after 30 days of storage with a large peak at about 90 nm and a small peak at about 4000 nm, suggesting coalescence and characterizing instability of these NE submitted to these storage conditions. As previously discussed, this phenomenon is influenced by the thermosensitive behavior of this PPO block the surfactant Pluronic F68.

3.6 Permeation Studies

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The permeation of 8-MOP was studied in vitro using porcine skin, a well-known and accepted model of human skin. The formulations studied were constituted by a “control sample” (0.05 % w/v solution of 8-MOP in ethanol), the prepared NE and their correspondent hydrogel-thickened systems, containing either clove oil or fennel oil as dispersed phase and as penetration enhancer. Since oil phase constituents were different, formulations differed also regarding the surfactant used, i.e. Pluronic F68 vs. Cremophor RH40. Figure 9 shows that, in the case of NEC based on clove oil, the control sample provided a higher permeation profile compared to the plain NEC: It is interesting to observe that the difference between the control formulation and the plain NEC was only in the early time of the experiment, suggesting a similar permeability but a longer time-lag to reach steady-state for the NEC. This behavior was reversed in the fennel oil containing NEF: the control sample was almost superimposed with the plain NEF Both NEF and NEC contained different penetration enhancers, i.e. clove oil containing eugenol and sweet fennel oil containing trans-anethole. It is well known that enhancing properties of a given penetration enhancers depends on the permeant, however, there are reports in the literature that indicate a higher efficiency of trans-anethole compared to eugenol [61]. This is in agreement with the higher permeation of NEF compared to NEC observed in Figure 9. Another difference between NEC and NEF is the surfactant employed: Cremophor RH40, contained in the sweet fennel oil NE, is a non-ionic surfactant known in the literature for its ability to alter stratum corneum lipid arrangement: the presence of this enhancer can explain the slight, although not significant, increased permeation from such a formulation. Incidentally, it should be considered that clove oil-based NE show slightly larger droplet size compared to fennel oil-based formulations (68.1 ± 0.6 nm vs. 91.3 ± 0.3), although the relevance of this difference is not easy to predict. The effect of the presence of chitosan of different molecular weights was studied as well and it is depictured in Figures 10 and 11. The addition of chitosan further reduced 8-MOP permeation and skin accumulation in the case of clove oil containing formulations, probably because to the higher viscosity of the formulation with higher chitosan MW. On the other side, the

addition of chitosan increased 8-MOP skin penetration and accumulation, to reach the maximum level when high molecular weight chitosan was used.

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A previous research paper describes the interaction between the Pluronic and chitosan, and their thermo-responsive properties [62]. Thus, it is possible that the decrease of skin permeation observed in samples containing clove oil and stabilized by Pluronic (NHC), is due to interaction between the hydrophobic regions of the surfactant with chitosan. The formation of this complex would be influenced by the concentration and molecular weight chitosan and the sol-gel transitions would occur at temperatures around 35 °C [63]. Considering that the skin permeation experiments were performed at 37 °C in order to mimic the physiological conditions, it is possible that an increased interaction between the PPO blocks from Pluronic and chitosan had taken place, providing aggregates highly packaged, which hindered the diffusion of oil droplets containing 8-MOP. Such fact justifies the results observed in this work for NHC formulations. As expected, it is observed in Figures 10 and 11 that all formulations showed higher drug accumulation in epidermis as compared to dermis, since dermis has a higher hydrophilicity character than epidermis, which may represent a barrier to diffusion of many substances, particularly those with low partition coefficient and low water solubility [64]. Thus, very lipophilic substances like 8-MOP tend to accumulate in the stratum corneum and epidermis, either by affinity for these tissues, either by difficulty in crossing the dermis [37]. 8-MOP dermal penetration from NEC was more than two times higher than that observed for NHC formulations. This result confirms the ability to enhance drug skin penetration usually attributed to NE [65]. Concerning the formulations containing sweet fennel oil, NEF was able to promote increased drug dermal retention compared to the control. Moreover, the addition of chitosan was responsible for a significant increase in 8-MOP skin accumulation in both epidermis and dermis. Table 4 reports all permeation parameters calculated for all formulations. The permeation profiles of 8-MOP were generally not linear with time, suggesting that steady-state conditions were not achieved over the duration of the experiment. For this reason, the approximate solution of Fick’s second law [39] was applied, to calculate the relevant permeation parameters, such as partitioning parameter (KH), diffusive parameter (D/H2) and permeability coefficient (P): KH gives indications of the partitioning of the permeant between the stratum corneum and the formulation, whereas D/H2 gives indication of the overall diffusivity of the molecule. The lag time (T lag) values were determined from the x-intercept of the slope at a steady state.

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On first inspection it is evident that the formulations containing sweet fennel oil and Cremophor RH40 (NEF-NHF) showed higher permeability coefficients (P), whereas those containing clove oil and Pluronic F68 the opposite: the addition of chitosan reduced P in NEC whereas increased it in NEF. The analysis of the diffusion (D/H2) and partitioning parameters (KH) revealed that KH was the one modified, whereas the diffusive parameter remained more or less constant. The partitioning parameter (KH) increased slightly in the plain NEF, containing sweet fennel oil and Cremophor RH40, but such increase became more evident in the NHF formulation, containing chitosan (p<0.01 compared to the control). Notably, all NHC and NEC formulations produced with clove essential oil were able to reduce both P and D/H2. Furthermore, the addition of chitosan was responsible for a remarkable reduction in the permeation coefficient even if the differences observed for the samples NHC1, and NHC2 NHC3 are not significant (p> 0.05). The main mechanism to justify these results should be related to the high viscosity observed for the NHC which is reflected in the reduced diffusion coefficient. Finally, the skin retention experiments on NE and NH were replied in conditions similar to those used in therapy, i.e. in finite dose conditions for 2 hours of contact time (Figure 12): no control formulation as used, because in finite dose conditions ethanol evaporates quickly leaving the solid drug on the skin surface. Figure 12 presents 8-MOP skin acumulation from formulations based on clove (A) and sweet fennel essential oil (B) under finite dosing. As expected, the epidermis accumulation was higher than dermis accumulation in all cases. NEC formulation accumulated more 8-MOP in both tissues compared to the control formulation: this skin retention decreased in the presence of chitosan, following the same trend as the permeation profile. The higher retention from the plain NEC compared to control is in line with the higher partitioning parameter found; the same trend was found also for the presence of chitosan. Using NEF and NHF formulations the situation was completely reversed, because the retention increased with the presence of chitosan, as penetration did. The results obtained in finite dose conditions are similar to those obtained in infinite dose conditions showed in Figures 10 e 11, although generally lower. 8-MOP was found in the receptor compartment only the case of NHF2 and NHF3. In order to try to understand the results obtained, in particular the effect of chitosan TEM images of the prepared systems NEC and NEF were taken (after negative staining with phosphotungstic acid). The results are reported in Figure 13. Panel A depictures NEC formu-

18

lation containing clove oil and Pluronic F68 as surfactant. Such image is typical of NE, The nanoemulsions were presented as spherical structures of regular shape, and it is possible to notice the presence of small lipid droplets (dark stained), surrounded by a layer of surfactant (Pluronic F68) which stabilizes the micelle structure. On the contrary, the structure reported in Panel B, sweet fennel oil dispersed in water using Cremophor RH40 as surfactant (NEC), does not look like a typical NE but perhaps more as a bicontinuous NE, wherein small domains of oil and water are inter dispersed. Upon addition of water, bicontinous phases can generate NE, as suggested by the particle size measurement of the diluted sample by DLS. It has been reported that bicontinuous microemulsions are dynamic structures with continuous fluctuations occurring between the bicontinuous structure, swollen reverse micelle and swollen micelles and for this reason they may improve drug penetration across the skin [66]. The TEM images of chitosan containing formulation NHF show a significant change in the structure: the systems appears more like a very fine NE (note that in the presence of chitosan, phosphotungstenic acid binds to the polymer), which is probably more efficient in delivering 8-MOP to the skin. These changes may render 8-MOP somewhat more available for transdermal permeation. Such observation corroborates the results obtained in skin permeation studies. Furthermore, it is possible to notice the discrepancy from these results compared with data obtained by DLS measurements. These differences can be attributed to several factors such as aggregation or deformation as flattening of the micelles when deposited on the surface analysis. It is still necessary to consider that the drying process with the evaporation even partial of the external phase of NE might constitute a disturbance to the initial conditions of the samples, which can cause numerous structural changes, including aggregations, especially during the analysis of NE. Moreover, it is important to note that the hydrodanimc size were obtained by DLS upon dilution; this can be an artifact of sample preparation for TEM analysis (dehydration, droplet flattening).

4- CONCLUSIONS The incorporation of 8-MOP into two NE containing essential oils (either sweet fennel or clove essential oil) as oil phase lead to the development of formulations with which it was possible to modulate transdermal drug delivery and skin retention. In particular, the use of clove oil, associated with Pluronic F68, led to a reduced skin permeation and increased skin retention, also in the presence of chitosan as thickener. This formulation is very promis19

ing for the PUVA therapy, because it guarantees high efficacy and low systemic exposure to the drug. On the contrary, the formulation containing sweet fennel oil, associated with Cremophor RH40 and thickened with chitosan, allowed higher skin penetration and retention. The effect produced by addition of chitosan was completely unexpected and it is worth further studies to elucidate the exact mechanism of interaction. The NH proposed in this paper are complex systems, which does not allow the isolation of its components for perfect understanding of the phenomenon responsible for the results obtained in this work. It is true that, our data suggest that somehow the addition of chitosan led to significant changes in the transdermal permeation capacity of 8-MOP carried inside sweet fennel oil containing formulation. The full characterization of these systems is already being conducted and should be extremely important for a clear understanding of the results.

5- ACKNOWLEDGMENTS We thank the Office to Improve University Personnel (CAPES), of the Ministry of Education and the National Council for Scientific and Technological Development (CNPq) .

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Figure Captions

Figure 1: Average droplet diameter of nanoemulsions (NE) containing clove oil and 8-MOP Figure 2: Average droplet diameter of nanoemulsions (NE) containing sweet fennel oil and 8-MOP Figure 3: pH for NE and HTN based on clove and sweet fennel essential oils after heatingfreezing cycles and 11.000 rpm centrifugation. Figure 4: Average droplet diameter of NE containing sweet fennel oil (NEF) and clove oil (NEC) after heating-freezing cycles and 11.000 rpm centrifugation. Figure 5: pH for NE and HTN based on clove and sweet fennel essential after 15, 30, 60 and 90 days under 45oC. Figure 6: Flow curves under continuous shear rate ramps of 0.01 to 100 s-1 for formulations based on clove essential oil after 15, 30, 60 and 90 days under 45oC.

Figure 7: Flow curves under continuous shear rate ramps of 0.01 to 100 s-1 for formulations based on sweet fennel essential oil after 15, 30, 60 and 90 days under 45oC.

Figure 8: Average droplet diameter for formulations based on clove and sweet fennel essential oil after 15, 30, 60 and 90 days under 45oC

Figure 9: 8-MOP permeation profiles across pig ear skin from NEC, NEF and control sample (n=6). Mean values ± S.E.M. Figure 10: 8-MOP permeation profiles across pig ear skin (A) and skin acumulation from formulations based on clove oil under inifite dosing conditions (n=6). Mean values ± S.E.M. Figure 11: 8-MOP permeation profiles across pig ear skin (A) and skin acumulation from formulations based on sweet fennel oil under inifite dosing conditions (n=6). Mean values ± S.E.M. Figure 12: Transmission Electron Microscopy for NEC (A), NEF (B) and NHF1 (C). Magnification 2000x. Figure 13: 8-MOP skin acumulation from formulations based on clove (A) and sweet fennel essential oil (B) under finite dosing conditions (n=6). Mean values ± S.E.M.

28

Table Titles

Table 1: Results from Solubility Tests Table 2: Composition and pH of produced formulations Table 3: Results from accelerated stability studies of formulations regarding Color /Odor alterations Table 4: Permeation parameters and pH determined for formulations containing clove oil, and sweet fennel oil.

29

Table 1 Essential oil

8-MOP Solubility

Sweet Fennel

Freely Soluble (>20%)

Mint

Soluble (10%)

Bitter orange

Insoluble

Sweet orange

Insoluble

Lime

Soluble (10%)

Buriti

Insoluble

Avocado

Insouble

Clove

Freely Soluble (>20%)

Copaiba

Insouble

Table 2 Oil Phase (5%)

Clove oil

Sweet Fennel oil

Surfactant (wt%)

Chitosan (2%)

Sample

pH ± SD

-

NEC

5.39 ± 0.03

Low MW(2%)

NHC1

5.16 ± 0.08

Medium MW

NHC2

4.62 ± 0.07

High MW

NHC3

4.48 ± 0.04

-

NEF

5.43 ± 0.02

Cremophor RH 40

Low MW

NHF1

4.72 ± 0.1

(3%)

Medium MW

NHF2

4.45 ± 0.09

High MW

NHF3

4.26 ± 0.13

Pluronic F68 (10%)

30

Table 3 Sample

Color/odor

Color/odor

Color/odor

Color/odor

Color/odor

Initial

After 15 days

After 30 days

After 60 days

After 90 days

NEC

I

I

I

I

I

NHC1

I

I

I

II

II

NHC2

I

I

I

II

II

NHC3

I

I

I

II

II

NEF

I

I

I

I

I

NHF1

I

I

I

I

I

NHF2

I

I

I

I

I

NHF3

I

I

I

I

I

Table 4 Formulation

KH (cm)

D/H2 (h-1)

P (cm h-1) *103

Time lag (h)

Control

0.043 (0.021)

0.110 (0.104)

3.15 (0.36)

2.2 (1.0)

NEC

0.071 (0.023)

0.043 (0.004)

2.97 (0.89)

3.9 (0.3)

NHC1

0.011 (0.008)

0.082 (0.031)

0.73 (0.18)

2.4 (1.4)

NHC2

0.017 (0.008)

0.061 (0.030)

0.92 (0.28)

3.3 (1.4)

NHC3

0.009 (0.002)

0.065 (0.012)

0.55 (0.08)

2.7 (0.6)

NEF

0.090 (0.027)

0.088 (0.045)

7.29 (2.61)

2.3 (1.1)

NHF1

0.215 (0.079)

0.124 (0.082)

22.38 (5.00)

1.7 (0.6)

NHF2

0.269 (0.113)

0.094 (0.061)

21.78 (13.25)

4.0 (5.2)

NHF3

0.223 (0.093)

0.232 (0.180)

39.86 (6.06)

1.0 (0.4)

Note: and: Partitioning parameter (KH) gives indications of the partitioning of the permeant between the stratum corneum and the formulation, whereas the diffusive parameter (D/H2) gives indication of the overall diffusivity of the molecule. Permeability coefficient (P) is obtained as a product of KH*(D/H2). The lag time (T lag) values were determined from the x-intercept of the slope at a steady state.

31

20

imediate 24 hours imediate hours 1 24 week week 2 1weeks weeks 3 23weeks weeks 1 1month month months 2 2months months 3 3months

Mean volume (%)

20

10

10

0 1

0

10

100

1000

10000

Diameter (nm)

1

10

100

1000

10000

Diameter (nm)

Figure 2 0imediate hours 24hours hours 24 week 11week 22weeks weeks 33weeks weeks 41weeks month 8 weeks 2 months 12 weeks

20

Mean Volume (%)

20

Mean volume (%)

Mean volume (%)

Figure 1

10

3 months

10

0

0

0,1

1

1

10

10

Size (d.nm)

100

100

1000

1000

10000

Diameter (nm)

Figure 3

32

1

Inicial After Heating-freezing cycles After Centrifugation test

6

pH

4

2

NEF NEFNHC1 after centrifugation test NHF1 NEC NHC2 NHC3 NEF 25 NEF after heating-freezin Sample cycles

0

2025

NEF 16 NEF after centrifugation test NEC NEF after heating-freezin cycles NEC after heating-freezinf cycle NECcentrifugation test NEC after NEC after heating-freezinf cycle NEC afterNEC centrifugation test NEC 16 NEC after heating-freezinf cycle NEC after heating-f NEC after8 centrifugation test NEC after centrifug

25

20

15

10

10

10

5

16 15

5

5

1

1000 0,1

0 0,1

1 1

10

10

100

100

10000 Size (d.nm) Size (d.nm) 10 100 1000

Size (d.nm) 10

100

Size (d.nm)

10000,1

1000

10000

10000 1000

8

0

0

1 0,1

100

16

5

0 0

16

10

8

5

0

(d.nm)

8

10

Mean Volume (%)

15

20

Mean Volume (%) Mean Volume (%) Mean Volume (%)

Mean Volume (%)

15

Mean Volume (%)

20

Mean Volume (%) Mean Volume (%)

NEF after heating-freezin cycles

0,1

1

0 0 0,1 10000 1

10

0,1 0,1 10000

Mean Volume (%)

25

NEF NEF after centrifugation test NEFNEF after heating-freezin cycles 15 NEF NEF after centrifugation test NEF after heatingcycles NEF after centrifugation test 16 freezin

Mean Volume (%)

25

NEF NEF after centrifugation NEF after heating-freezin

NHF3

20

Mean Volume (%)

NEF NEF after centrifugation Figure 4 test NEF after heating-freezin cycles

NHF2

8

0 1

1010

100 0 1 1 (d.nm) 10 10 Size

100

Size1000 (d.nm)

1000 10000 100 1000 0,1 10000

Size 100 100(d.nm) 10001000

Size (d.nm)

0,1 Size (d.nm) 1

1

10000 10000

10

8

0 1000 0,1

100

Size (d.nm)

33

1000

100

Figure 5 Inicial PH 15 dias PH 30 dias PH 60 dias PH 90 dias

6

5

pH

4

3

2

1

160

160

140 -20 Shear rate (1/s) 0 40 -20 120

1202040 140 0

2020

40

40

-20

0

20

Viscosity (mPas) Viscosity (mPas)

0

0

40

20

0

20

20

160

100

160-20

60 -20

80 0

22000 80

0

100 20 120 40 140

12000

10000 10000

0

8000 60 40 8000

Shear rate (1/s) 6000 6000 4000

4000 22000

2000

Viscosity (mPas)

Viscosity (mPas)

Viscosity (mPas)

8000

0

10000 -20 8000 80 6000 4000 2000 0

0

NEC 30 days120 Shear rate (1/s)(1/s) Shear rate NEC 60 days500 0 20 40 60 80 NEC 100 90 120days 140 100

20

20

100

1500 1000

40

500 20 0

Shear rate (1/s) 100 120

0

0 160-20

160

-500 160

140

NEC inicial 80 NEC 15 days NHC2 0 daysNEC25000 NHC2 0 days 30 days NHC2 15 days NHC2 15 days 60 NHC2 30 days NEC NHC2 30 days 60 days NHC2 60 days NEC 90 days NHC2 60 days NHC2 90 days NHC2 90 days 2000022000 40

0

40

40

60

60

80

100

Shear(1/s) rate (1/s) Shear rate

80

120

15000 140 160 100 10000

0

8000 0 10000

140

20

Viscosity (mPas)

NEC 15 days ShearN Shear rate (1/s) NHC3 00days NHC3 days 2500 4500 25000 NHC3 15 days NEC 30 days NHC1 3N NHC3 15 days NHC3 30 days NEC 60 days NHC1 6N 4000 NHC3 2000 30 days NHC3 60 days NEC 90 days NHC1 9N NHC3 90 days 3500 NHC3 60 days 20000 N 1500 NHC3 90 days 3000 1000 25000 NHC2 0 days NHC3 2500 15000 NHC2 0 days 25000 NHC3 500NHC2 15days days NHC3 0 days 2000 NHC2 15 NHC3 25000NHC3 15 days 30days days NHC2 30 1500 0NHC2 10000 NHC3 NHC3 30 days 20000 NHC2 60 NHC2 60days days 20000 1000 NHC3 500 NHC2 NHC3 60 days 90 days 90 days 20000NHC3 40 60 80NHC2 100 120 140 160 500 90 days 0 20 40 5000

20000 100

80 22000 15000 20000 60 10000 18000 40 5000 16000 14000 0 200 12000 0 10000-20 8000 600020

6000

4000

5000

2000

4000 22000

0

25000 120

NHC2 0 days 20000 NHC2 0 days NHC2 15 days 15000 20 18000 NHC2 15 days 25000 NHC2 0 days NHC2 30 days NHC2 30 days NHC2 15 days NHC2 60 days 16000 0 10000 NHC2 30NHC2 days60 days NHC2 90 days NHC2 days 20000 days90 40 60 NHC2 80 60100 120 140 160 14000-20 20 40 NHC2 60 90 days 80 100 Shear rate Shear rate (1/s)(1/s) 5000 12000

20000 8000 60

2500 2000 60

15 days Shear (1/s) 100NEC 60 rate80 120 140-20 160 0 20 -500 20 40 60 80 100 120 140 160 1000

Shear rate (1/s)

NHC26000 0 days 18000 4000 NHC2 15 days 40 16000 0 18000 16000 6000 NHC22000 30 days 16000 60 0 days -20 NHC2 0 14000 20 140004000 20 0 14000 2000 NHC2 90 days 12000 12000 0 20000

100

Viscosity (MPas)

20 10000 18000

Viscosity (mPas) (mPas) Viscosity

22000

80

14060 160 80

Shear rate (1/s)22000 20000 0 days20000 NHC2 120 18000 60 15 days NHC2 18000 16000 NHC2 30 days 100 16000 22000 14000 NHC2 60 days 40 14000 90 days 12000 NHC2 80 22000 20000 10000 12000

60

0

NHC1 3

Viscosity (mPas)

100

500 3000 0 20 160 2500 -500 2000 0 NEC 0 140 120 inicial 140 160 1500

20 0

20

0

20

40

Viscosity (MPas)

60800

60

60

40

Viscosity (mPas) Viscosity (mPas)

0

60

3500 3000 80

NEC 30 days

Viscosity (mPas)

20 20 80 160

NEC 60 days 1000 40 NEC 90 days 3500

NHC1 0 days

3000

Viscosity (mPas)

40

Viscosity (MPas)

40

100

4500

100 4000

3000 NEC inicial

5000 NEC 15 days NHC12500 0 days 45005500

3500

NH NH NH NHC1NH 0 NHC1NH 1

15 5000NHC12000 15days days NHC1 6 4000 NHC1 NHC1 30 days NEC 60 days 2500 NHC1 30 days NHC1 9 4500NHC1 60 days NEC 90 days 3500 NHC1 2000 NHC11500 90 60days days 4000 1500 3000 NHC1 90 days 1000 3500 1000 2500 5003000 500 2000 02500 5500 0 1500 -5002000 40 60 80 NEC inicial 100 1000 0 -500 20 40 60 5000 Shear rate1500 (1/s) NEC 15 days rate160 (1 500 40 6010004500 80 100 0120 Shear 14020 20 40 60 80 100 NEC 30 days 0 rate (1/s) Shear Shear rate 500 4000(1/s) NEC 60 days -500 20 40 600 80 100NEC 120 90140 160 0 20 40 days 60 3500 500 Shear rate (1/s) 5500 Shear NHC1 40 60 100 rate (1/s) 20 40 0 3000 0 80 NEC inicial 5000 NHC1 1

Viscosity (MPas)

60

5500

120 5000

Viscosity (mPas)

120

60

5500

NEC inicialinicial NEC 2500 30 days NEC 5000 NEC 15 days 2000 60 days 60 NEC 15NEC days NEC 30 days 4500 90 days NEC 60 days NEC 30NEC days 1500 NEC 90 days 4000

NEC inicial NEC 15 days 120 NEC 30 days 100 100NEC 60 days 90 days 80 80NEC

Viscosity (mPas)

80

140 120

5500

4000

140

Viscosity (MPas) (mPas) Viscosity

140

5000

--

160

Viscosity (mPas)

160

Viscosity (mPas) (mPas) Viscosity

16080 100

NEC inicial NHC14500 0 daysNEC 15 days NEC 30 days NHC1 15 days 5500 NHC14000 30 days NEC 60 days 5000 NHC1 60 days 3500 NEC 90 days 4500 NHC1 90 days

NHF1 NHF2 NHF3

NEC 30 days 3500 NEC 60 days NEC inicial 3000 80 NEC 90 days NEC 15 days

140

Viscosity (mPas)

120

Viscosity (mPas)

140

NEC inicial NEC 15 days NEC 306 days Figure NEC 60160days NEC 90 days

NEC inicial NEC 15 days NEC 305000 days 120 NEC 60 days 4500 NEC inicial NEC 904000 days NEC 15 days 100

NHC1 NHC2 NHC3 140 NEF 5500 Sample

Viscosity (MPas) (mPas) Viscosity

100

160

Viscosity (mPas)

Viscosity (mPas)

Viscosity (mPas)

120

NEC

5500

40

60

80

100

0 rate rate Shear (1/s) (1/s) Shear 15000 Sh 15000 -500 15000 40 0 60 80 100 120 140 160 00 20 20 40 40 60 60 Shear rate (1/s) Shear rate (1/ 10000 Shear rate (1/s) 60 10000 80 100 10000

Viscosity (MPas)

140

Viscosity (mPas) Viscosity (mPas)

0

Shear rate (1/s)

34

5000 5000

5000 0

160

0

8000 1000 6000 0

16000

Viscosity (mPas)

0

10000 2000

4000 22000 0 2000 20000 0 18000

0

1000

14000 12000 10000 8000

0

16000 2020 0 14000 0 12000 -20 10000 0 60

40 0

60 20

40

60 60 120

120

80

100

120

120

Shear rate (1/s)

Shear rate (1/s)

8000 rate (1/s) Shear 6000 4000

14000

0 4000 16000

4000

16000 40 2000

60

0

0 12000 8000 10000

140

160

-20

10000 6000 8000 4000 6000

2000 0

120

Shear rate (1/s)

60

120

12000 10000 8000 6000 4000

6000

2000

4000

12000 10000 8000 6000 4000

20

40

60

80

Shear rate (1/s)

0 0

20

40

60

Shear rate (1/s)

Viscosity (mPas)

4000

0

2000 20

0

40 120 120 40

40

20

Shear r 60 60 80 140 160 140 160

100 80

12 1

rate (1/s) ShearShear rate (1/s)

Shear rate(1/s) (1/s) Shear rate 10000

10000

-20 50000

20

40

5000 80

60

40

60

80

100

120

140

Shear rate (1/s) 100

60

0 Shear rate (1/s)

120

140

80

160

100

NHC2 NHC3 0 days Shear rate (1/s) 025000 20 0 days 40 60 80 NHC2 0 days NHC3 NHC2 15 days Shear rate (1/s) 15 days 0 NHC2 15 days NHC3 30 days NHC2 0 days 40 20 40 60 3030 80 20 NHC2 days NHC3 100 60 days Shear NHC2 days NHC3 90 days NHC2 20000 Shear20000 rate (1/s)6060days NHC2 9090 days NHC2 days 25000

12000 10000 8000 6000 4000

15000

10000

5000

15000

10000

2000

2000 100 100

Viscosity (mPas)

Viscosity (mPas)

Viscosity (mPas)

Viscosity (mPas)

2000

5000 0 20

0

0 80

0 Shear rate (1/s) 15000

6000

0 0

80

NHF3 90 days 0 Shear rate (1/s)

15000 -500 -2060 60 04000 100 20 2040 40 100 0 08080 20 20

0

2000

6000

-20 0 8000

4000 0 0 22000 -20 2000 220000 22000 Shear rate (1/s) 20000 NHC2 0 days 0 20 40 100 NHC2600 days 80 20000 20000 0 NHC2 15 days Shear rate (1/s) NHC2 15 days 18000 0 18000 20 40 NHC2 6030 30 100 018000 days NHC2 days80 16000 16000 16000 NHC2 days NHC2 60 60 days Shear rate (1/s) NHC2 90 days NHC2 90 days 14000 14000 14000 2000

Viscosity (mPas)

100

12000

22000 22000 4010000 10000 80 18000 8000 200008000 20000 60 20 16000 6000 180006000 18000

12000 14000 NHC2 days NHC2 9060days 2000 0200 -20 80 100 160 NHC2 120 90 days 14014000 -20 10000 12000

60 ShearShear rate (1/s) rate (1/s)

0

14000

140

80 100 120 140 160 0 days NEC Shear inicial rateNHC1 (1/s) 15 days Shear 3000 Rate (1/s) NHC1 NEC 15 days 16000 4500 2500 NEC 30 days NHC1 30 days NHF3 0 days NHC1 60 days 4000 NHF3 15 NEC days60 days 14000 NHF3 0 days NHF3 0 days 2000 NHC1 90 days NHF3 15 days NHF3 30 NEC days90 days 3500 NHF3 15 days NHF3 30 days 18000 12000 1500 NHF3 60days NHF3 30 days NHF3 60days 3000 NHF3 60days NHF3 90 days NHF3 90 days 16000 10000 1000 25000 NHC2 days 2500 NHC2 00days NHF3 90 days NHC3 NHC2 15 days 14000 8000 NHC2 15 daysNHF3 0 days0 days 2000 500 25000 NHC3 15 days NHC2 30 days NHF3 15 days 6000 NHC2 30 days 1500 12000 NHC3 30 days 0 NHC20 60 days NHF3 30 NHF3 days days 20000 NHC2 60 days NHC3 4000 1000 NHF3 15 days NHC2 90 days NHF3 60days60 days 10000 -500 20000 NHC3 90 days NHF3 30 days NHC2 90 days 90 days 500 60 20 8000 402000 80 100 0 120NHF3 140 160 40 0 20 40 60 80 NHF3 100 60days 120 140 160 20

Viscosity (MPas)

0

3000

40

6012000

Shear

120

20 Shear40 60 Rate (1/s)

60 0

5500 40 18000 5000

20

Viscosity (MPas)

12000

2000 18000

16000 120

20

-20

Viscosity (mPas)

Viscosity(mPas)

1000 4000

22000 60 20000 3000 4000

NEC 30 days NEC 60 days NEC 90 days

NEC inicial NEC 15 days NEC 30 days Shear rate (1/s) 4000 40 60 80 100 NEC 120 60 140days 160 20 40 60 80 100 -5000 Shear3500 rate (1/s) NEC 90 days

Viscosity (MPas)

1000

-20 14000

NHF2 0 days NHF2 NHC2150days days NHF2 0 days NHF2 30 days NHC2 15 NHF2 15 days NHF2NHC2 60 days 0days days NHF2 30 days NHF2 9030 days NHC2 days NHC2 15 days NHF2 60 days NHC2 30 days NHC2 60 days NHF2 90 days

80 5000

18000

14000

0

Viscosity (MPas)

2000

2000 5000

6000

0

140

80

NHF2 0 days

NEC 15 days NHF2 0 days NHF2 15 days NHF2 15 days NEC 30 days NHF2 30 days NHF2 30 days NEC 60 days NHF2 60 days NHF2 60 days NEC 90 days NHF2 90 days NHF2 90 days

-20

16000

Viscosity (mPas)

3000

Viscosity(mPas)

4000

Viscosity (mPas)

2000

160

850 800 NHF1 30 days 750 4000 NHF1 60 days 700 NHF1 90 days 650 3500 NHF1 0 days 600 NEC inicial NHF1 0 days 15days 5500 550 3000 NHF1 0 days NHF1 days days0 days NHF1 15days NEC 15 NHF1 30NHC1 500 NHF1 15days 5000 NHF1 30 days NEC 30 days NHC1 15 days 2500 450 NHF1 60 days NHF1 30 days NHF1 60 days NEC 60 days90NHC1 4500 400 NHF1 days30 days NHF1 90 days NHF1 200060 days 350 NEC 90 days NHC1 60 days 4000 NHF1 90 days 300 NHC1 90 days 1500 250 3500 200 3000 1000 150 100 2500 4050 60 80500 100 120 140 160 2000 0 Shear Rate (1/s)

850 800 750 700 650 600 550 500 450 400 350 20 300 55000 250 1500 -20 0 20 40 60 80 100 12 200 5000 -500 150 1000 Shear Rate (1/s) 20 0 10020 40 40 6060 8080 100 100 120 160 140 20 160 40 -20 120 0140 4500 500 50 0 Shear Rate (1/s) 0 0

850 800 120 750 700 650 100 600 550 500 80 450 400 350 60 300 250 0 200 40 150 100 20500

Viscosity (mPas)

7000 100

8000

3000

Shear rate (1/s) NHF2 0 days 100 120 140

Viscosity (mPas) (mPas) Viscosity

7000

16000 0 6000

3000

0

140

Viscosity (mPas)

120

7000

20 160

160

NEC inicial NEC 15 days Shear rate (1/s) NEC 30 days NEC 60 days NEC 90 days100160 20 40 60 80 100 120 140 18000

NHF2 30 days NHF2 60 days NHF2 90 days

9000

8000

8000

80

140

Shear rate (1/s)NEC inicial 140 rate (1/s) NHF2 15 days Shear

9000

60

4000

60

0

120

Viscosity (mPas)

80

40

-20

100

Viscosity (mPas)

20

80

Viscosity (mPas)

0

9000

5000

40

80 60 60

100

5000

1000

5

Viscosity (mPas)

40

220006000 40 9000 20000 5000 8000 20 4000 18000 7000

6000

(mPas) Viscosity (mPas) Viscosity

50

100

Viscosity (mPas)

6000

40

(mPas) Viscosity Viscosity(mPas)

7000

Viscosity (mPas)

8000

NEF 0 days NEF 15 days NEF 30 days NEF 60 days NEF 90 days

120

160

450 400-20 350 0 Shear rate (1/s) 300 0 -20 0 40 20 40 60 80 100 120 140 160 0 140 250 140 0 Shear rate (1/s) 200 0 -20 0 20 0 4020 4060 60 80 100120 120 140 -20160 20 80 100 140 160 150 20 120 100 120 Shear rate (1/s) 50 -20 0 -20 0 40 60 80 100 120 140 160 0 160 160 20 100

0

9000 -20

10

60

2020 0160

-20

NEF 60 days NEF 90 days inicial 80 NEF NEC 0 days NEF 0 days NEF 15days days NEC 15 days NEF 15 NEF 30days days NEF 30 NEC 30 days60 NEF 60 NEF 60days days NEC 60 days NEF 90 days NEF 90 days NEC 90 days

140

10 5

Viscosity (mPas)

5

80

Viscosity (mPas)

0

160

Viscosity (mPas)

10

100

Viscosity (mPas)

5

7

Viscosity (mPas)

Viscosity (mPas)

10 Figure

Viscosity (mPas)

120

NEC inicial 140 NEC 15 days NEC 30 days 120 NEF 0 days NEC 60 days NEF 15 days NEC NEF90 30days days 100

Viscosity (mPas)

140

800 750 700 650 600 550 500 450 400 350 850 300 800 250 750 200 700 150 650 100 600 50 550 0 500

Viscosity (mPas)

NEF 0 days NEF 15 days NEF 30 days NEF 60 days NEF 90 days

Viscosity (mPas)

10

NHF1 0 days NHF1 15days 5500 NHF1 30 days NHF1 60 daysNEC inicial 5000 NHF1 90 daysNEC 15 days NHF1 0 days 4500 NHF1 15days

160 850

0

20

040

2060

Shear rate (1/s) 0 40 60 0

Shear rate (1/s)

4080

60100

80

1

Shear rate (1/s)

35

80 20

100 40

Shear r

10 1

1

10

0,1

0,1

Mean Volume (%)

20

Mean Volume (%)

Mean Volume (%)

20

9

10

10

0

0,1 0

1

100 0 1

Size (d.nm) 0 10

100

1000 10000

Size Size(d.nm) (d.nm)

Size (d.nm)

0,1 100

1000 1

Size (d.nm) 1

0,1 9 Figure

10

20

10

10

0,1

1

1 10 10

1

0,1

100

100 Size (d.nm) 1000 Size (d.nm) (d.nm) 1 10 Size

01

10

100

1000

0,1

1000Size (d.nm) 10000

100

10

1

100

10000 Size (d.nm) 10

10

1000 1000 10000

1000

10000 10000

0 100 10000 0,1

1

Size (d.nm) 100

1000

10000

Size (d.nm)

Size (d.nm)

20

NEF days0 days NEF 030 days NEF NEF 0 days NEF 15 60days days NEF 15NEF days NEF NEF 30 days 15 days 20 NEF 30NEF days 90 days30 days NEF NEF 60 days NEF 60 days NEF 90 days NEF 60 days NEF 90 days NEF 90 days

010000 0,1

0,1

1000010 100

30

NE NE NE NE NE

0

0

1000 0,1100000

10 100 10010000 1000

1000 10

NEC 30 days NEC 60 days NEC 90 days

18

0

20 2020

15

15

2

0,1

9

Permeated amount (g/cm2) Permeated amount (g/cm2)

001

9

30

30

30 NEC 15 days

Mean Volume (%)

0

9

10

Mean Volume (%)

9

2 amount((g/cm Permeatedamount g/cm )) Permeated

9

Mean Volume (%)

18

NEF 0 days NEF 15 days NEF 30 days NEF 600days NEF days NEF 9015 days NEF days

NEC 0 days

NEC NEC15 15days days NEC 15 days NEC 90 days NEC 30 NEC days NEC 30 days 30days NEC60 60days days 20 NEC 60NEC days NEC 90 days NEC 90NEC days 90 days

18 18

18

10

Volume MeanMean (%) Volume(%)

8

30

NEF 0 days NEF 15 days NEF 30 days30 NEF 60 days NEC 30 days NEC 20 0 days NEF days NEC 090 days NEC 60 days NEC 0 days

Figure 8

Mean Volume (%)(%) Mean Volume

Mean Volume (%)

20

NEC 0 days NEC 15 days NEC 30 days NEC 60 days NEC 0 days NEC 90 days NEC 15 days

Mean Volume (%)

30

10

10 5

0

5

1515

Control Control NEC NEC NEF

Control NEC NEF

NEF

10

10

5

0

0

Control NEC NEF

5

10

1

0

2

2

0

3 3 44 1Time 2 (hours) Time (hours)

5

6

3

6 7

7 4

5

6

Time (hours)

0

Figure 10

5

0

1

2

3

4

5

6

7

Time (hours)

36

7

2

1

2

1

1

0

1

0

1

1

0

00

2

0

0 2

1

0

2

0

1 31

2 24

335

44 6

55

3 4 5 Time(hours) (hours)6 Time (hours) Time

Time 1 (hours)2

0

7

6

0,6

0,4

0,2 0,2

7

3

0,6

0,4

6

B

0,0

0,4

B B

0,4

0,2

0,2

7 0,0

0,07

B

0,0

0,4

6 Epidermis

0,6

0,6

7

1

80

NHF 3

60 60

60

40

30

80

40 40

20

20

4

00

5

0 0 Time 1 020 (hours) 02 1 1 3 21 1

2

3

4

2

43

6

23

54 4

3

6 55

0,4

0,2

7

4 766 0,0

0,2

0,0 7

57

6 (hours) 7 (hours) Time Time Time5(hours) Time (hours)

1

2

3

4

Time (hours)

0,4

0,4

B

0,6

B

0,4

0,4

0,2

0,2

0,4

0,0

0,0

0,0 6

B

D

Control Control NEC NEC NHC1 NHC1 NHC2 NHC2 Control NHC3 NHC3 Control

B NEF NEF

NHF1 NHF1 NHF2 NHF2 NHF3 NHF3 NEF pHpH 4.2 4.2 NEF

Contr NEF NHF1 NHF2 NHF3 NEF

0,2

0,0

7

Epidermis

Epidermis Epidermis

Epidermis 5

Dermis

0,2

0,2

Epidermis

Time0(hours) 0

0,6

0,6

B

Dermis Dermis

Dermis

B

B

Acumulated amount (g/mg of tissue) amount (g/mg of tissue) Acumulated

2 80

NHF NHF 1 1 NHF 3 NHF 2 2 NHF NHF 3

100

Acumulated amount/mg of tissue

A

0,4

NHC2 NHC2 NHC3 NHC3

Epidermis

0,6

Acumulated amount (g/mg of tissue)

0

100

0,6

Acumulated amount (g/mg of tissue)

2

1

NEC NEC 120 NHC1 ANHC1 NEF 120 NHC2 NHF 1 NHC2 120 100 3 NEF NHC3A NHC3NEF NHF 2 2

2

NEF NHF 1 3 NEF NHF 2 NHF NHF 3 1 NHF 2 NHF 2 3

A

4

2 (g/cm amount Permeated ) (g/cm amount Permeated )

A

Permeated amount (amount Permeated g/cm2) (g/cm )

Permeated amount (g/cm )

2

4

Acumulated amount (g/mg of tissue)

5 Figure 11

Acumulated amount/mg of tissue

Time (hours) 5

Control NEC NHC1 NHC2 NHC3

0,2

Epidermis Epidermis Epidermis 0,0

5

4

Control NEC NHC1 Control BControl NHC2 NEC NEC NHC3 NHC1 NHC1

0,6

Acumulated amount/mg of tissue

AA

NHC1 3 3 NHC2 2 NHC3

3

2

NEC NHC1 NHC2 NEC NHC3 NEC NHC1 NHC1 NHC2 NHC2 NHC3 NHC3

Acumulated amount/mg of tissue

34

NEC5 NHC1 NHC2 4 4 3 NHC3 NEC

0,6

Acumulated amount/mg of tissue

A

4

of tissue amount/mg Acumulated mg of tissue amount/ Acumulated

5

A

5

Permeated amount (g/cm2)

4

2

A

Permeated amount (g/cm ) Permeated amount (g/cm2)

2

0,6

5

(2)g/cm ) amount Permeated (g/cm amount Permeated

5

6

7

0,0

Dermis Epidermis Dermis Dermis Dermis

Derm

Epidermis

37

Figure 12

Figure 13

Panel A: NEC

2µm 2µm

2µm

2µm Panel B: NEF

Panel C: NHF

38

Graphical abstract

39