Desonide nanoencapsulation with açai oil as oil core: Physicochemical characterization, photostability study and in vitro phototoxicity evaluation

Journal of Photochemistry & Photobiology, B: Biology 199 (2019) 111606

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Desonide nanoencapsulation with açai oil as oil core: Physicochemical characterization, photostability study and in vitro phototoxicity evaluation

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Priscila Rosaa, Mariane Lago Friedrichb, Juliana dos Santosa, Daniele Rubert Nogueira Librelottoa, Luana Haselein Maurerc, Tatiana Emanuellic, Cristiane de Bona da Silvaa,b, ⁎ Andréa Inês Horn Adamsa,b, a

Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, Brazil Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, Brazil c Programa de Pós-Graduação em Ciência e Tecnologia dos Alimentos, Universidade Federal de Santa Maria, Santa Maria, Brazil b

ARTICLE INFO

ABSTRACT

Keywords: Nanoparticles açai oil Photostability Phototoxic activity Irritant potential

This study aimed to develop Eudragit® RL 100 nanocapsules loaded with desonide (DES) using açai oil (AO) or medium chain triglycerides (MCT) as oil core. Pre-formulation study showed that AO and MCT are suitable for nanocapsules preparation. The nanocapsules prepared with AO and MCT presented mean particle size around 165 and 131 nm, respectively; polydispersity index values < 0.20, positive zeta potential values, drug content close to the theoretical value (0.25 mg mL−1), and DES encapsulation efficiency around 81%, regardless of the oil core (AO or MCT). Considering the photoinstability reported to DES, photodegradation studies were performed. The UV-A (365 nm) and UV-C (254 nm) photodegradation studies revealed less DES degradation when associated to the nanocapsules containing AO in comparison to those with MCT. The in vitro release study showed a biphasic release profile for both nanocapsule suspensions: an initial burst effect followed by a prolonged DES release. In addition, the formulations were considered non-phototoxic at 0.5 mg mL−1 when tested on 3 T3 murine fibroblasts and HaCaT human keratinocytes using the MTT and NRU viability assays. The irritant potential of the prepared nanocapsules and DES in free form were evaluated by HET-CAM method. All formulations were classified as slightly irritant, including the non-associate DES. In conclusion, the nanocapsule formulations developed in this study may be promising for therapeutic applications.

1. Introduction Corticosteroids are drugs commonly used in dermatology, mainly for the treatment of inflammatory skin diseases, such as atopic dermatitis, psoriasis, contact dermatitis and some auto-immune topical disorders [1–3]. The anti-inflammatory activity of topical glucocorticoids (TG) has an immediate effect, it induces vasoconstriction consequently decreasing the edema and erythema and the effects mediated by the glucocorticoid receptor regulation [2–4]. In skin, TG's target cells are the keratinocytes and fibroblasts in the epidermis and dermis. However, the same mechanisms of action responsible for the therapeutic effect can cause adverse effects as skin atrophy, resulting from the antiproliferative effect on fibroblasts [3–5]. Desonide (DES) is a synthetic non-fluorinated corticosteroid classified as TG low potency (class VI) according to the World Health Organization [6]. It is commonly prescribed for the treatment of atopic dermatitis in sensitive areas as the face and intertriginous regions in ⁎

elderly and children. DES is being used for > 30 years for the treatment of steroid-responsive dermatoses and is already considered the most low potency corticosteroid prescribed by dermatologists in the USA [7]. Recently, some studies reported the desonide photoinstability in commercially available formulations after direct exposure to UV-A radiation [8–10]. The nanocapsules ability to protect substances from chemical and photodegradation has been well demonstrated by previous studies [11–16]. Polymeric nanocapsules (NCs) are vesicular systems, which have a polymeric wall surrounding a core, which is generally oily [17]. Regarding the oily core, medium chain triglycerides (MCT) are normally used as the oil for nanocapsules preparation. However, in the last years, vegetable oils were introduced as the oily core because they protect the encapsulated substance and improve the pharmacological effect [14,18]. In this context, studies with açai oil (AO), indicated the antiproliferative activity of the oil from fruit pulp extracts and the one from

Corresponding author at: Departamento de Farmácia Industrial, sala 1106, Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil. E-mail address: [email protected] (A.I.H. Adams).

https://doi.org/10.1016/j.jphotobiol.2019.111606 Received 7 January 2019; Received in revised form 16 August 2019; Accepted 24 August 2019 Available online 26 August 2019 1011-1344/ © 2019 Elsevier B.V. All rights reserved.

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açai fruit pulp also presented this activity in colon adenocarcinoma cells [19]. Favacho and co-workers (2011) [20] attributed the AO antiinflammatory effect to its composition, which is similar to other oils with the same activity, such as olive oil and fish oil. The AO genotoxic effect was evaluated through the comet assay and micronucleus test in an in vivo study in peripheral leukocytes, liver, bone marrow and testicular cells of rats. In these conditions, OA did not present a significant genotoxic effect [21]. Concerning the desonide use in nanostructured systems, Antonow and co-workers (2016) [22] described its incorporation into nanocapsules. The authors reported the development of desonide-loaded nanocapsules using Eudragit® S100, Eudragit® L100 and poly(ε-caprolactone) as polymers and MCT for the oily core. However, there are no studies about the desonide photostability associated to nanocarriers or the preparation of nanocapsules containing desonide associated to açai oil as the oily core. Thus, this study aimed to develop cationic desonide-loaded nanocapsules using Eudragit® RL 100, a biocompatible polymer. The formulations were prepared using AO and MCT as the oily core to evaluate the oil influence on the nanocapsules characteristics and photostability, and the in vitro irritant and phototoxic effects of such formulations were evaluated as well.

polymer in acetone followed by the solvent evaporation at room temperature. Portions of the films (around 25 mg) were accurately weighed and kept in contact with AO and MCT, in a quantity enough to cover the films. On the days 5, 7, 15 and 30 the films were removed, in sequence, they were carefully dried with absorbent paper and accurately weighed. The difference between the initial and final weight was calculated. 2.3.2. Determination of DES Solubility in the Oils To evaluate the DES solubility in MCT and AO, an excessive amount of DES was dispersed in 1.0 mL of each oil (n = 3). The samples were magnetically stirred over 60 min and then centrifuged at 10,000 rpm during 10 min. An aliquot of the supernatant was diluted in methanol and the DES content was determined through a previous validated HPLC method. 2.4. Nanocapsule Suspensions Preparation Nanocapsule suspensions were prepared by the interfacial deposition of preformed polymer method described by Fessi and co-workers [24]. Briefly, an organic phase containing the polymer (0.25 g), Span 80® (0.1925 g), AO at 2.5% (DES-NCAO) or MCT at 2.5% (DES-NCMCT), DES (0.0025 g) and acetone (67.5 mL for DES-NCAO and 135 mL for DES-NCMCT) were kept at 40 °C under moderate magnetic stirring for 60 min. This organic phase was injected under magnetic stirring at room temperature in an aqueous phase (135 mL) containing polysorbate 80 (0.1925 g). The mixture was homogenized during 10 min and then the acetone and part of the water were eliminated by evaporation under reduced pressure to achieve 25 mL of formulation corresponding to 0.25 mg mL−1 of desonide. For comparative purposes, formulations without the drug (B-NCAO and B-NCMCT) were also prepared. Additional nanocarriers (nanoemulsions and nanospheres) were prepared for further photostability evaluation. Nanoemulsions (DESNEAO and DES-NEMCT) were prepared by the spontaneous emulsification solvent diffusion method using the same aqueous and organic composition of the nanocapsule suspensions, except the polymer. DESnanospheres (DES-NS), that do not contain oil in their composition, were prepared following the same procedures for the nanocapsule suspensions preparation. All formulations were prepared in triplicate and stored in amber glass containers at room temperature (22 ± 2 °C).

2. Material and Methods 2.1. Materials Desonide (purity of 97.42% purity) was obtained from Fagron (São Paulo, Brazil). Polysorbate 80 and Span 80® (sorbitan monooleate) were bought from Sigma Aldrich (São Paulo, Brazil). Medium chain triglyceride was acquired from Delaware (Porto Alegre, Brazil) and açai oil (AO) was kindly donated by Beraca (Ananindeua, Brazil). Eudragit RL 100 was purchased from Evonik (Essen, Germany). Neutral red (NR) solution 3.3 g/L, 2,5-diphenyl-3,-(4,5-dimethyl-2-thiazolyl) tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS) solution, Earles balanced salt solution (EBSS), fetal bovine serum (FBS) and trypsin-EDTA solution (0.5 g porcine trypsin and 0.2 g EDTA.4Na/L of Hanks' Balanced Salt Solution) were obtained from Sigma-Aldrich (São Paulo, Brazil). Dulbecco's Modified Eagle's Medium (DMEM), supplemented with L-glutamine (584 mg/L) and antibiotic/ antimicotic (50 mg mL−1 gentamicin sulfate and 2 mg/L amphotericin B) were purchased from Vitrocell (Campinas, Brazil). Methanol and acetonitrile HPLC grade were obtained from Sigma Aldrich (São Paulo, Brazil). All other solvents and reagents were of analytical grade and used as received.

2.5. Desonide Quantitative Assay The quantitative analyses were performed by HPLC using a Shimadzu (Kyoto, Japan) LC system equipped with a LC 20AT pump, SPD M20A detector, a CBM 20A system controller, DGU degasser and SIL 20AHT autosampler. A mobile phase composed of a mixture of methanol, acetonitrile and ultrapure water adjusted to pH 4.0 with ortophosphoric acid 18% (60:10:30, v:v:v) was eluted at flow rate of 1.0 mL.min−1. The chromatographic separation was achieved using a Phenomenex Luna® (Torrance, USA) RP-C18 column (150 × 4.6 mm; 5 μm) coupled to a C18 guard column. Detection was performed at 244 nm and the injection volume was 20 μL. The analytical method was validated for the desonide assay in formulations according to the ICH guidelines (ICH, 2005). The method was specific, linear in the range of 0.5–100 μg mL−1 (y = 45,212× + 2408; r = 0.9999), precise, in levels of repeatability (RSD of 2.0% to DES-NCAO and 1.32% to DES-NCTCM) and intermediate precision (RSD of 1.67% and 1.61% to DES-NCAo and DES-NCMCT, respectively) and accurate (recovery of 100.83% to DESNCOA and 100.63% to DES-NCMCT). The desonide content in NCs was determined by the dilution of 1.0 mL of each sample in 5 mL of methanol followed by 10 min sonication. Then, the volume was filled up with the same solvent to 10 mL, providing DES theoretical concentration of 25 μg mL−1. The samples were filtered through a 0.45 μm nylon membrane filter.

2.2. Açai Oil Fatty Acids Profile Açai oil was saponified in methanolic KOH solution and then esterified in methanolic H2SO4 solution [23]. Methylated fatty acids were analyzed using an Agilent Technologies gas chromatograph (HP 6890 N) equipped with a capillary column (DB-23 60 m × 0.25 mm × 0.25 μm) and flame ionization detector. The injector port temperature was set at 250 °C and nitrogen was the carrier gas (0.6 mL min−1). After injection (1 μL, split ratio 50:1), the oven temperature was kept at 150 °C for 1 min. Then, it was increased to 240 °C at 4 °C/min and kept for 12 min. Standard fatty acid methyl esters (37-component FAME Mix from Sigma, Saint Louis, USA) were analyzed under the same conditions and the subsequent retention times were used to identify the fatty acids. Fatty acids were expressed as percentage of the total ones identified. 2.3. Pre-Formulation Studies 2.3.1. Polymer Films Dissolution/Swelling Eudragit® RL 100 (EUD) films were prepared dissolving 1.0 g of the 2

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2.6. Nanocapsule Suspensions Characterization

2.5). The mathematical modeling was performed using the Scientist 2.0 software (MicroMath, USA) by fitting the data to mono (lnC = lnC0– kt) and bi-exponential eqs. (C = Ae-αt + Be-βt). The equation that better describes DES release was selected observing the highest model selection criteria (MSC) value and the correlation coefficient (r). The drug release mechanism was evaluated by fitting the data to KorsmeyerPeppas model (ft = atn) [25].

The presence of micrometric population of particles, size distribution and SPAN were evaluated by laser diffraction using the refraction index of Eudragit® RL 100 (1.38). The formulations were dispersed in distilled water directly in the equipment sampling apparatus (Mastersizer 3000, Malvern Instruments, UK). Mean particle size and polydispersity index (PDI) were determined by photon correlation spectroscopy (PCS) (Zetasizer, Malvern Instruments, UK) after samples dilution in ultrapure water (1:500, v:v). Zeta potential was determined by electrophoretic mobility (Zetasizer, Malvern Instruments, UK) after the samples' dilution (1:500, v:v) in 10 mM NaCl. The pH was evaluated in the suspensions using a calibrated potentiometer (Denver Instruments, USA). Desonide total content in nanostructures was determined by analyzing the samples at 25 μg mL−1, prepared as described in Section 2.5. The encapsulation efficiency (EE) was evaluated by ultrafiltration/ centrifugation technique in which an aliquot of 300 μL of the formulations was placed in a 10,000 MW centrifugal device (Amicon®, Millipore, USA) and centrifuged during 10 min at 3916 xg, to separate the free drug from the nanostructures. The ultrafiltrate, which contains the free drug, was analyzed by the analytical method described in Section 2.5. The EE (%) was calculated as the difference between total and free concentrations of desonide determined in the nanostructures and in the ultrafiltrate, respectively.

2.9. Phototoxicity Test The phototoxicity assay was carried out as described in the OECD Guideline for Testing Chemicals 432 [26], with some modifications. Cell lines 3 T3 and HaCaT were used as in vitro models to predict cutaneous phototoxicity. Two 96-well plates per cell line were seeded with cells (1 × 105 cells/mL for 3 T3 and 8.5 × 104 cells/mL for HaCaT) in 100 μL of DMEM medium supplemented with 10% FBS (v/v) and incubated at 37 °C, 5% CO2 for 24 h to allow the formation of a monolayer. In sequence, the cells were treated with DES, DES-NCAO, BNCAO, DES-NCMCT and B-NCMCT at 0.5 μg mL−1 in Earles Balanced Salt Solution (EBSS) and pre-incubated at 37 °C and 5% of CO2 for 1 h. One plate of each cell line was exposed to a UV-A irradiation of 1.5 J/cm2, while the other plate was wrapped in aluminum foil and then exposed to the same condition. UV-A irradiation was performed using a HPA 400/S (UV-Bravo, Germany) UV-A lamp with a spectral range of 300–400 nm. After irradiation, the treatment was replaced by a complete medium and the plates were again incubated under the same experimental conditions to complete 24 h of incubation. The cell viability obtained from each sample at 0.5 μg mL−1 in both cell lines was determined by MTT and NRU assays. The phototoxic potentials were estimated comparing the cell viabilities obtained in the presence and absence of UV-A radiation. Chlorpromazine (CPZ) at 5.0 μg mL−1 was employed as positive control because it is a known phototoxic chemical [26].

2.7. Stability Study 2.7.1. Stability at Room Temperature The formulations stability was monitored for 30 days. Formulations were stored at room temperature in amber glass containers (22 °C ± 2 °C) and evaluated according to their desonide content, pH, zeta potential, mean diameter, polydispersity index and encapsulation efficiency. 2.7.2. Photostability Study For this purpose, 1.0 mL of a methanolic drug solution (0.25 mg mL −1 ), DES-NCAO (0.25 mg mL −1) and DES-NCMCT (0.25 mg mL −1) was disposed in UV transparent plastic cuvettes and exposed to UV radiation in a chamber with mirrored surfaces. The samples were exposed to UVA (365 nm, Ecolume, 30 W) and UV-C (254 nm, Philips, 30 W) radiation for 36 h and 90 min, respectively. At predetermined intervals, the exposure was interrupted and the samples were analyzed for desonide content, observing the procedure described in section 2.5. To discard thermal degradation, the samples protected from light (dark controls) were simultaneously exposed to UV radiation and then analyzed. The experiment was performed in triplicate. The DES degradation kinetics under UV-C radiation was determined according to zero order (C = C0-kt), first order (lnC = lnC0- kt) and second order (1/C = 1/C0 + kt) equations. The best correlation coefficient (r) indicated the reaction order and the parameters of t90 and t50 were estimated following the given model.

2.10. Nanocapsules Irritant Potential Evaluation The Hen's Egg Test Chorioallantoic Membrane (HET-CAM) method was employed to evaluate the irritant potential of the prepared formulations. The analyzed formulations were DES-NCAO, B-NCAO, DESNCMCT, B-NCMCT and the non-associated drug (dispersed in polissorbate 80 and water). The positive control was NaOH 0.1 M and the negative control was 0.9% NaCl. For the test, fertile chicken eggs incubated per 10 days were used. Initially, the eggshell around the air chamber was removed and the inner membrane was moisturized with 0.9% NaCl. In sequence, the membrane was carefully removed and 0.3 mL of formulations were directly applied on the chorioallantoic membrane (CAM) (n = 4/formulation). After 20 s, the formulations were removed using 0.9% NaCl and CAM was monitored during 300 s. The time (seconds) for each reaction was monitored and recorded. The observed endpoints were vasoconstriction, hemorrhage and coagulation. The irritation score (IS) was calculated according to Eq. (1).

2.8. In vitro Release Study

IS = 5 × (301

Desonide in vitro release from NCs was studied by the dialysis bag diffusion technique. For this, 2 mL of samples, DES-methanolic solution, (0.25 mg mL−1) or NCs (DES-NCAO and DES-NCMCT, 0.25 mg mL−1) was placed in the dialysis bag (10,000 Da, Spectra Pore), which were immersed in 70 mL of sodium acetate buffer pH 4.5 containing 0.5% of polysorbate 80 and maintained under constant moderate stirring at 37 °C. The release medium was chosen to obtain sink conditions. At predetermined intervals (0.5, 1, 2, 4, 6, 8 and 10 h), aliquots of 1.0 mL of the release medium were withdrawn and replaced by the same volume of fresh medium. The amount of drug released was determined by HPLC using the analytical conditions previously described (Section

where, h = hemorrhage time; v = vasoconstriction time, and c = coagulation time. According to IS values, the formulations were classified as non-irritant (0–0.9), slightly (1–4.9), moderate (5–8.9) and severe irritant (9–21) [27].

h)/300 + 7 × (301

v)/300 + 9 × (301

c)/300

2.11. Statistical Analysis Statistical analysis was carried out using one-way analysis of variance (ANOVA), post hoc Tukey's test and Student's-t-test at a significance level of 5%. 3

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values < 0.2, positive zeta potential and acidic pH. Table 2 presents the formulations physicochemical characteristics. Besides, DES content was close to the theoretical value (0.25 mg mL−1) and EE was around 82% for both formulations.

Table 1 Açai oil fatty acid composition. Fatty acid

% of total fatty acids identified

C16:0 (palmitic acid) C16:1n7 (palmitoleic acid) C18:0 (stearic acid) C18:1n9 (oleic acid) C18:1n7 (7-octadecenoic acid) C18:2n6 (linoleic acid) C18:3n3 (alpha-linolenic acid) Other fatty acids

21.78 3.26 2.17 57.42 3.24 11.08 0.59 < 0.5

3.4. Stability studies 3.4.1. Stability at Room Temperature The prepared nanocapsules did not show any changes in appearance during the storage period. Regarding the particle size (Fig. 1a), we verified that formulations maintained the initial characteristics (p > .05), as well as the polidispersity index, which did not show any significant difference (p > .05) at the end of the storage time. Considering the pH (Fig. 1b), after 30 days no differences were observed in relation to the initial values (p > .05), for both formulations. Concerning the zeta potential (Fig. 1c), DES-NCAO did not present any alteration in this parameter (p > .05), comparing the initial and final values. However, a significant increase (p < .05) in zeta potential was observed to DES-NCMCT from the seventh day of storage to the end. Desonide content was also evaluated. The results showed that at the end of 30 days, the drug content was in agreement with the most common specification (90–110%). DES content was 98.2 ± 1.1% for DES-NCAO and 94.8 ± 1.8% for DES-NCMCT. The encapsulation efficiency indicated no decay (p > .05) in the encapsulated amount in relation to the initial time for both NCs.

3. Results 3.1. Açai Oil Fatty Acid Profile Considering that several factors can affect the composition of vegetable oils, we developed chromatographic analyses to evaluate the quali/quantitative composition of the AO used in this study (Table 1). The most predominant fatty acids found were oleic, palmitic and linoleic, while palmitoleic and stearic acids were present in smaller levels. The fatty acids quali-quantitative composition present in the AO used is in agreement with the composition of Euterpe oleracea fruit oil reported by CIR, 2011 [28]; Nascimento et al., 2007 [29] and Pacheco-Palencia et al., 2008 [19].

3.4.2. Photostability Study Both NCs reduced DES photodegradation and the remaining contents after 36 h of UV-A exposure were 68.51% and 56.39%, for DESNCAO and DES-NCMCT, respectively. In the same conditions, a solution of DES in methanol (DES-MS) presented a residual content of 35.92% (Fig. 2). The protection effect against UV-A radiation was higher with AO NCs than with MCT NCs (p < .05). The dark controls assay showed DES content around 99%, indicating that degradation occurs without the influence of temperature. To verify the polymer effect and the influence of the oil used to prepare the NCs core on the DES photostability, nanospheres (NS) and nanoemulsions (NE) were also prepared. The results indicated that all the nanostructures were able to protect DES from UV-A degradation (Table 3) since drug residual content after 36 h of UV-A irradiation was greater in the nanostructures than in solution (p < .05). Considering the nanostructures, it can be observed that DES-NCMCT, DES-NEMCT and DES-NS did not differ on DES residual content (p > .05) at the end of UV-A exposure time (36 h), presenting drug remaining content of 56.39%, 54.41% and 54.39%, respectively. At the same time, DES-NCAO and DES-NEAO presented similar effect (p > .05) on DES photoprotection. Desonide residual content was 68.51% in DES-NCAO and 64.60% in DES-NEAO. These results suggest that the polymeric wall had no influence on the drug protection from photodegradation in nanostructures. The nanocarriers containing açai oil in their composition (NC and NE) showed higher effect against UV-A radiation than other structures (p < .05), indicating that the oil used in the NE and NCs core influenced DES photostability. DES-MS: desonide dissolved in methanol, at 0.25 mg mL−1; DESNEAO: DES-loaded nanoemulsion containing AO at 2.5%; DES-NEMCT:

3.2. Pre-Formulation Studies To verify a possible interaction between each oil (AO and MCT) and the polymer, Eudragit® RL100 films were kept submersed in the oils for 30 days. The initial mass of the polymer films in AO was 28.3 ± 5.0 mg and after 30 days of contact with oil, the film mass was 27.9 ± 4.9 mg. The initial mass of the polymer films immersed in MCT was 25.78 ± 2.9 mg and the final mass was 25.16 ± 3.7 mg. Statistical analysis (Student's-t-test) indicated that there was no significant change in both EUD films weight, showing that these oils are non-solvent for this polymer (p > .05). Regarding DES solubility, the results indicated approximately 291 μg mL−1 in AO and 792 μg mL−1 in MCT. Such values are considered adequate to prepare the nanocapsules suspensions in the experimental conditions of this study. 3.3. Preparation and Characterization After preparation, NC suspensions containing AO presented a slightly yellowish milky appearance and NCs prepared with MCT were white milky ones. Both NCs showed opalescent bluish reflection resulting from Brownian motion of the colloidal structures. The NC suspensions analysis by laser diffractometry demonstrated that 90% of the particles were lower than 429 ± 4 nm and 413 ± 2 nm for DES-NCAO and DES-NCMCT, respectively. Volume-weighed mean diameter (D4,3) were 284 ± 3 nm for DES-NCAO and 262 ± 4 nm for DES-NCMCT, with SPAN values < 2.0 for both formulations. The formulations presented size in the nanometric range, PDI Table 2 Nanocapsules suspensions physicochemical characteristics.

B-NCAO DES-NCAO B-NCTCM DES-NCTCM a

Mean diameter (nm)a

Polidispersity index

Zeta potential (mV)

pH

163 165 135 131

0.13 0.12 0.15 0.15

+14.9 ± 1.2 +13.8 ± 0.3 +9.3 ± 1.0 +6.9 ± 0.7

5.71 5.00 4.98 4.38

± ± ± ±

3 2 3 2

± ± ± ±

0.01 0.03 0.02 0.02

Determined by photon correlation spectroscopy (PCS) technique. 4

± ± ± ±

0.06 0.09 0.17 0.13

DES content (mg mL−1)

EE (%)

– 0.26 ± 0.004 – 0.26 ± 0.009

– 81.8 ± 1.8 – 81.6 ± 0.4

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Fig. 1. Mean diameter (a), pH (b) and zeta potential (c) of DES-NCAO and DES-NCMCT, during stability study. *Significant difference compared to zero time.

DES-loaded nanoemulsion containing MCT at 2.5%; DES-NS: DESloaded nanosphere. *Significant difference compared to zero time, in the same column; mean values followed by the same letter in each line do not differ (Tukey post-hoc test, α = 0.05). NCs showed a more prominent photoprotective effect against UV-C radiation. After 90 min of exposure, DES content was 44.11% for DESNCAO and 34.33% for DES-NCMCT, while DES residual content in methanol was 37.90% after 15 min of UV-C irradiation (Fig. 3). In this condition, DES degradation kinetics was estimated. For DES-NCAO and DES-NCMCT, the kinetic degradation followed first order, while DES-MS followed zero order (Table 4). 3.5. In Vitro Release Study Concerning in vitro DES release, NCs prepared with AO or MCT released 98.74 ± 0.05% and 96.58 ± 0.41%, respectively, after 10 h of experiment (Fig. 4). Conversely, DES-MS was totally released in 4 h (98.81 ± 1.42%). Data were fitted to zero, mono and biexponential equations. The release profile of both NCs is better described by the biexponential equation, since higher values of MSC and correlation coefficient were obtained to this model. Regarding DES-MS, the results

Fig. 2. Desonide residual content from nanocapsules and from solution, after UV-A radiation exposure. *Significant difference among desonide as a solution in methanol (DES-MS) and nanocapsules suspensions (DES-NCAO and DESNCMCT); #significant difference between DES-NCAO and DES-NCMCT.

Table 3 Desonide residual content (%) from nanostructures and non-associated drug observed in the UV-A (365 nm) photostability study. Time (h)

DES residual content (%) from nanostructures and solution (mean ± SD) DES-MS

DES-NCAO

DES-NCMCT

DES-NEAO

0 4 6 8 15 24 36

100 ± 1.20 85.42 ± 2.20⁎a 79.57 ± 1.90⁎a 74.63 ± 4.03⁎a 55.46 ± 3.43⁎a 42.05 ± 2.20⁎a 35.92 ± 2.72⁎a

100 ± 0.81 96.79 ± 0.72⁎b 93.83 ± 0.96⁎b 93.31 ± 0.70⁎b 86.26 ± 2.26⁎b 78.96 ± 1.26⁎b 68.51 ± 1.30⁎b

100 ± 0.79 92.85 ± 2.81⁎c 88.76 ± 3.39⁎c 84.81 ± 2.49⁎c 72.26 ± 1.13⁎c 56.04 ± 3.49⁎c 56.39 ± 1.39⁎c

100 ± 0.90 95.44 ± 0.89⁎b, 94.93 ± 1.20⁎b 91.98 ± 1.41⁎b 84.62 ± 2.65⁎b 76.66 ± 1.91⁎b 64.60 ± 1.92⁎b

5

c

DES-NEMCT

DES-NS

100 ± 0.85 93.56 ± 0.67⁎b,c 90.91 ± 1.05⁎b,c 86.61 ± 1.07⁎c 75.77 ± 3.19⁎c 67.38 ± 1.56⁎d 54.41 ± 0.19⁎c

100 ± 0.98 93.07 ± 1.49⁎c 90.94 ± 1.52⁎b,c 87.91 ± 0.84⁎c 81.60 ± 2.01⁎b 71.35 ± 2.02⁎d 54.39 ± 1.06⁎c

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70% of cell viability) was only observed in the combination of HaCaT cells and MTT assay. These results suggest that the keratinocytes are more sensitive to the NCs depending on the viability assay performed. Regarding these four different assessments (two cells lines vs. two viability assays), it was evidenced that the NCs are non-cytotoxic and they did not enhance the DES cytotoxic potential. 3.7. Formulations Irritant Potential The irritant potential of the prepared nanocapsules and DES in the free form were evaluated by HET-CAM method. For negative control (0.9% NaCl), irritation reactions were not observed, thus the irritant score (IS) could not be estimated. The positive control (0.1 M NaOH) was classified as severe irritant (IS of 17,20 ± 0,30), which is in agreement with the literature [27,30]. As it was expected, for negative control (0.9% NaCl), no irritant reactions were observed. Considering the DES-NCs, regardless of the oil used to compose the NCs core, the irritant scores (IS), 3.80 ± 0.22 for DES-NCAO and 3.78 ± 0.30 for DES-NCMCT, indicated that the formulations were slightly irritant. Blank nanocapsules (IS of 3.74 ± 0.02 to B-NCAO and 3.84 ± 0.32 to BNCMCT) and non-associate DES (IS of 2.96 ± 0.42) were also classified as slightly irritant.

Fig. 3. Desonide remaining content from nanocapsules and desonide dissolved in methanol, at 0.25 mg mL−1 after UV-C irradiation. *means significant difference between NCs formulations. Table 4 Parameters obtained in the UV-C degradation kinetics study. Formulation DES-NCAO DES-NCMCT DES-MS

Reaction order First First Zero

k −1

0.008 min 0.010 min−1 1.031 μg−1 mL−1 min−1

t90 (min)

t50(min)

13.2 10.6 2.4

86.6 69.3 12.1

4. Discussion

k: degradation constant.

Considering that nanocapsules are core-shell structures, choosing the appropriate polymer and oil to compose the core is essential for the maintenance of such structures [17,31]. Therefore, the chosen oil should have the drug solubility, toxicity and the compatibility with the polymer taken into consideration. In this context, capric/caprylic triglycerides are commonly used as the oil core in these systems because they solubilize a wide range of active substances and are compatible with different polymers [17]. The polymer selection must consider the biodegradability/biocompatibility characteristics, the intended route of administration and the method used in the preparation and application of the drug [32]. Our results indicated that AO is a suitable material for the development of Eudragit® RL100 NCs, since dissolution/swelling of polymer films was not observed. Furthermore, the DES solubility in AO indicated that this oil is appropriate for NCs preparation. In sequence, NCs were prepared by the deposition of preformed polymer, a commonly used method to prepare these nanostructures [17]. Initial tests were performed using AO at 3.0%; however, after 24 h the formulations presented a creaming aspect. Thus, the percentage of oil was reduced to 2.5% and more stable and homogenous systems were obtained. The results showed that DES-NCAO presented larger particle size in comparison to DES-NCMCT. Similar results were observed by Mora-Huertas and co-workers (2012) [31], who reported higher size of nanocapsules prepared with almond oil and corn oil, in comparison to the ones prepared with MCT. This effect was attributed to the high quantities of mono- and di-long-chain unsaturated fatty acids present in such oils. The AO used in our study presented high quantities of monoand di-long-chain unsaturated fatty acids (around 75%), which may explain the larger particle size obtained in these formulations. In relation to zeta potential, all formulations presented positive values (+6.9 to +14.9 mV). NCAO showed higher zeta potential values than those observed to NCMCT (p < .05). This parameter reflects the particle surface charge and it is related to the chemical nature of the polymer, the stabilizers present in the formulation and the medium pH [17,33]. Eudragit® RL 100 is a copolymer of polyethylacrylate, methylmethacrylate and chloro-trimethylammonioethyl methacrylate and contains 8.8 to 12% of quaternary ammonium groups, which confer a positive charge to the polymer [34,35]. The SPAN < 2.0 and PDI values, < 0.20, indicated a homogeneous size distribution, regardless of the oil type (p > .05). Regarding the desonide formulations photoinstability [8,10] and NCs advantages, which can improve drug photostability, we carried out

Fig. 4. DES release profile from DES-NCAO, DES-NCMCT and methanolic solution. *means significant difference between methanolic solution and NCs; #means significant difference between DES-NCAO and DES-NCMCT.

showed a good fit to zero order model. The mathematical modeling using Korsmeyer-Peppas model indicated correlation coefficients higher than 0.99 and release exponent (n) about 0.61, suggesting anomalous case as release mechanism for both NCs. 3.6. Phototoxicity Test The phototoxicity of non-associated DES and DES-loaded NCs was tested on 3 T3 murine fibroblasts and HaCaT human keratinocytes using the MTT and NRU viability assays. The phototoxic potential measurement was obtained from the comparison between the response from non-irradiated and irradiated plates in each treatment. Additionally, the data from non-irradiated plates provided the cytotoxicity effect of the treatment. To both cell lines, no phototoxic effect was observed in all evaluated treatments, since no significant effect was observed in the irradiated plates in comparison to the non-irradiated for the same treatment (p > .05) (Fig. 5). Additionally, it can be observed that the NC formulations and nonassociated DES displayed similar cell viability in non-irradiated condition (p > .05), except for the HaCaT cells when they were analyzed by the MTT endpoint (p < .05). Slight to moderate cytotoxicity (60 to 6

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Fig. 5. Phototoxicity of free DES and NCs formulations at concentration 0.5 μg mL−1, expressed as cell viability (%) on 3 T3 and HaCat cells line, measured by NRU and MTT assays. White bars = non-irradiated cells and black bars = UV-A 1.5 J/cm2 irradiated cells. Data are expressed as mean ± SEM.

a comparative photodegradation study. To perform the evaluation, aliquots of NCs and desonide solution with the same drug concentration were exposed to UV-A and UV-C radiation. Our findings showed that the association of DES with nanostructures had a positive effect on the drug photostability. The results indicated that both formulations significantly reduced the DES degradation. To elucidate the mechanisms involved in the photoprotection conferred by the NCs, nanoemulsions (NEs) containing AO or MCT and nanospheres (NS) were prepared and submitted to the same UV-A irradiation conditions. Concerning the structure, nanospheres do not present oil in their composition and can be considered matrix systems [17,36,37]. Nanoemulsions are considered kinetically stable systems due to their smaller particle size compared to conventional emulsions [38] and they have been associated to drug stability improvement [39,40]. The results showed that after 36 h of UV-A irradiation, formulations containing AO in their composition proved to be more photostable regardless of their structure (NE or NC). The residual drug content from other prepared nanostructures (DES-NS, DES-NEMCT and DES-NCMCT) was significant lower, no matter being matrix, core shell or nanoemulsion systems. A complementary photostability study, employing UV-C radiation (254 nm) was performed. Although most of this radiation is filtered by the ozone layer, the evaluation of the chemical and biological effects of UV-C radiation has received attention to better understand the photochemistry involved in these degradation reactions and know specific damages it may cause [41]. Considering that UV-C is the most energetic ultraviolet radiation and, this way, photodegradation reactions quickly occur, the use of this radiation source becomes interesting, since it exposes the sample to a more drastic degradation condition. The results showed that in this condition, the colloidal systems were able to improve DES photostability as well. The nanoencapsulation increased 5.5 and 4.4 times the t90 of desonide in DES-NCAO and DES-NCMCT, respectively, demonstrating that the oil used to compose the NCs core also influenced on the desonide photostability in this condition. Recently, Gehrcke and co-workers (2017) [13] reported the improvement of indole-3-carbinol (IC3) photostability associated to nanocapsules containing rose-hip oil or MCT as oily core. The authors found that the oil

type used to prepare the NCs influenced on this protection. Regarding the in vitro release profiles, the mathematical modeling indicated that both NCs formulations better fit the biexponential model. In this case, the release occurs in two different steps. The biphasic release profile presents the advantage of an initial rapid drug release followed by a prolonged release, which may maintain the drug effects for a longer time. The burst effect observed is probably related to the NCs encapsulation efficiency, which was around 80%. The release mechanisms were analyzed by Power Law model. According to it, for spherical drug release systems the limits for the release exponent are the following: n = 0.43 indicates Fickian diffusion; 0.43 < n < 0.85 corresponds to anomalous transport and n ≥ 0.85 implies in case II transport [25]. From the results obtained, both formulations showed release mechanism according to anomalous transport, indicating the polymer chain relaxation followed by the desonide diffusion. Over the past few years, several in vitro test methods have been suggested as alternatives to in vivo assays. HET-CAM represents a suitable method to evaluate eye and tissue irritation caused by topic applied substances [42]. The CAM has a large blood supply and due to its vascular mucosa, similar to the human eye, irritating effects become visible and endpoints like vasoconstriction, hemorrhage and coagulation can be observed [27,42,43]. In this study, we evaluated the DES irritant potential in free and nanoencapsulated forms. Results indicated the slightly irritant potential of all formulations. Similar findings were observed by Weber and co-workers (2015) [44] in the irritation assessment of betamethasone dipropionate lipid-core nanocapsules. It is well known that UV and visible radiation can induce the photochemical activation of certain chemicals [45]. The phototoxic reactions induced by drugs are caused after skin exposure to photoreactive drugs, triggered by UV-A and UV-B radiation [46,47]. Phototoxicity studies are generally performed on products that are exposed to solar radiation [48]. In this context, the in vitro 3 T3 NRU phototoxicity test is a validated alternative methodology for the evaluation of the phototoxic risk of pharmaceutical and cosmetic formulations [26,49]. In this study, we chose HaCaT and 3 T3 as cell lines model to evaluate the DES cutaneous phototoxic potential in both free and nanoencapsulated 7

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forms, using the NRU and MTT viability assays. The results obtained suggest that the treatments, at the tested concentration, are non-phototoxic. The decrease of the cell viability related to NCs, observed in the HaCaT/MTT assay, suggests that keratinocytes are more sensitive than fibroblasts to the nanostructured systems cytotoxic effects. This cytotoxicity was not detected by the NRU assay in the same cell line, which might be attributed to the different sensitivity of each viability endpoint. MTT and NRU assays are based on different mechanisms and, thus, show different sensitiveness in detecting cell damage. Moreover, the NRU is reported as a universal assay among cell types, independently of their nature, whereas MTT and other tetrazolium-based endpoints are more specific, since they measure the activity of intracellular enzymes whose localization, expression and activity depend on the cell type [50]. Therefore, the differences in cytotoxic responses observed here could be associated to some specific toxicological mechanism of the NCs, as well as to the characteristics and sensitivity of the cell lines. Nogueira and co-workers (2013) [51] also described different sensitivity of HaCaT and 3 T3 cells, using both MTT and NRU assays, to detect the phototoxicity and cytotoxicity of nanovesicles. Finally, it is worth mentioning that the cytotoxic responses associated to NCs could be related to the polymer and/or the presence of surfactants in these formulations. Similar effect was reported by Contri et al. (2016) [52] and Mendes et al. (2015) [53].

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5. Conclusion In this study, we showed the viability of developing desonideloaded nanocapsules with açai oil as oily core. The nanoencapsulation enhanced the desonide photostability against UV-A and UV-C radiations and reduced the drug release rate. However, NCs with AO presented a more promising effect in protecting desonide from photodegradation than the MCT nanocapsules. Besides, all formulations showed to be non-phototoxic in the tested concentration. Considering the results and the advantages of associating DES to nanocarriers, the nanocapsules developed can be considered promising and may be included in topical formulations aiming more appropriate cutaneous administrations. Declaration of Competing Interest None. Acknowledgments Priscila Rosa thanks the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 and Mariane L. Friedrich is thankful to Fundo de Incentivo à Inovação Tecnológica (FIT-UFSM) for their scholarships. References [1] H. Aubert-Wastiaux, L. Moret, A. Le Rhun, A.M. Fontenoy, J.M. Nguyen, C. Leux, L. Misery, P. Young, M. Chastaing, N. Danou, P. Lombrail, F. Boralevi, J.P. Lacour, J. Mazereeuw-Hautier, J.F. Stalder, S. Barbarot, Topical corticosteroid phobia in atopic dermatitis: a study of its nature, origins and frequency, Br. J. Dermatol. 165 (2011) 808–814, https://doi.org/10.1111/j.1365-2133.2011.10449.x. [2] V.S. Beltrani, F.A. Barsanti, L. Bielory, Effects of glucocorticosteroids on the skin and eye, Immunol. Allergy Clin. N. Am. 25 (2005) 557–580, https://doi.org/10. 1016/j.iac.2005.05.006. [3] A. Guichard, P. Humbert, M. Tissot, P. Muret, C. Courderot-Masuyer, C. Viennet, Effects of topical corticosteroids on cell proliferation, cell cycle progression and apoptosis: in vitro comparison on HaCaT, Int. J. Pharm. 479 (2015) 422–429, https://doi.org/10.1016/j.ijpharm.2014.12.066. [4] S. Wiedersberg, C.S. Leopold, R.H. Guy, Bioavailability and bioequivalence of topical glucocorticoids, Eur. J. Pharm. Biopharm. 68 (2008) 453–466, https://doi. org/10.1016/j.ejpb.2007.08.007. [5] U.R. Hengge, T. Ruzicka, R.A. Schwartz, M.J. Cork, Adverse effects of topical glucocorticosteroids, J. Am. Acad. Dermatol. 54 (2006) 1–18 (10.1016/ j.jaad.2005.01.010). [6] WHO, Model prescribing information, Drugs Used in Skin Diseases. (1997)

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