Nanoencapsulation of rice bran oil increases its protective effects against UVB radiation-induced skin injury in mice

European Journal of Pharmaceutics and Biopharmaceutics 93 (2015) 11–17

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Research Paper

Nanoencapsulation of rice bran oil increases its protective effects against UVB radiation-induced skin injury in mice Lucas Almeida Rigo a,1, Cássia Regina da Silva b,1, Sara Marchesan de Oliveira b, Thaíssa Nunes Cabreira b, Cristiane de Bona da Silva c, Juliano Ferreira d, Ruy Carlos Ruver Beck a,⇑ a

Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752, 90610-000 Porto Alegre, RS, Brazil Programa de Pós-graduação em Ciências Biológicas: Bioquímica Toxicológica, Departamento de Química, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil Programa de Pós-Graduação e Ciências Farmacêuticas, Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Av. Roraima 1000, 97105-900 Santa Maria, RS, Brazil d Departamento de Farmacologia, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil b c

a r t i c l e

i n f o

Article history: Received 31 January 2015 Revised 12 March 2015 Accepted in revised form 16 March 2015 Available online 25 March 2015 Keywords: Rice bran oil Hydrogels Nanocapsules Skin UV-radiation

a b s t r a c t Excessive UV-B radiation by sunlight produces inflammatory and oxidative damage of skin, which can lead to sunburn, photoaging, and cancer. This study evaluated whether nanoencapsulation improves the protective effects of rice bran oil against UVB radiation-induced skin damage in mice. Lipid-core nanocapsules containing rice bran oil were prepared, and had mean size around 200 nm, negative zeta potential (9 mV), and low polydispersity index (<0.20). In order to allow application on the skin, a hydrogel containing the nanoencapsulated rice bran oil was prepared. This formulation was able to prevent ear edema induced by UVB irradiation by 60 ± 9%, when compared with a hydrogel containing LNC prepared with a mixture of medium chain triglycerides instead of rice bran oil. Protein carbonylation levels (biomarker of oxidative stress) and NF-jB nuclear translocation (biomarker of pro-inflammatory and carcinogenesis response) were reduced (81% and 87%, respectively) in animals treated with the hydrogel containing the nanoencapsulated rice bran oil. These in vivo results demonstrate the beneficial effects of nanoencapsulation to improve the protective properties of rice bran oil on skin damage caused by UVB exposure. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Excessive sunlight exposure causes skin damage, and may lead to sunburn, photoaging, immunosuppression, as well as cancer [1]. The severity of such complications depends mainly on sun exposure spans, genetic factors, and skin type [2,3]. One of the major alterations that lead to deleterious outcomes on the skin is caused by production of the highly unstable products, called reactive oxygen species (ROS), with the exposure to ultraviolet radiation (UVR) [4,5]. The harmful effects such as those that ROS may cause on healthy skin are well known [2,6,7]. Concerning the ultraviolet radiations in sunlight, both UVA (320–400 nm) and UVB (280– 320 nm) may promote skin damage, but UVB induces the most cytotoxic effects. Sunburn, characterized by erythema, edema, and skin sensibility, is caused by excessive UVB radiation. ⇑ Corresponding author at: Faculdade de Farmácia – UFRGS, Av. Ipiranga, 2752, 4° andar, 90610-000 Porto Alegre, Brazil. Tel.: +55 051 3308 5951; fax: +55 051 3308 5243. E-mail address: [email protected] (R.C.R. Beck). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.ejpb.2015.03.020 0939-6411/Ó 2015 Elsevier B.V. All rights reserved.

Additionally, although UVA may contribute to skin cancer indirectly, UVB radiation leads to direct DNA damage, and promotes various skin cancers [1]. Sunscreens are recommended for protection against UV lightinduced skin damage, but the use of pharmaceutical products containing antioxidant ingredients may also be a useful strategy in the prevention of UV-mediated cutaneous damage. There is a growing interest in the replacement for synthetic active molecules using natural ones in such formulations, fostering the scientific research on new sources of protective agents. Some reasons for this approach are the adverse effects, allergic reactions, and the increasing numbers of dermatitis cases caused by these sunscreen formulations, which are intended to protect the skin from UV radiation [8–11]. In this sense, naturally occurring compounds, especially phenolic substances, have recognized beneficial effects for the skin, such as anti-inflammatory, antioxidant, and DNA repairs [12]. Vegetable oils are composed by several bioactive compounds, such as polyphenols and tocopherols [13], and have been widely studied as natural sources to protect the skin against UVR [14–19].

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Examples include rice bran oil (RBO), which presents considerable levels of phytochemically active compounds such as tocopherols and tocotrienols [20]. In addition, RBO presents V-oryzanol, which is a powerful antioxidant compound [21] with some important anti-inflammatory effects [22]. RBO has several health benefits, among which its role of antioxidant and anti-inflammatory agent, as demonstrated in in vitro as well as in vivo studies [23–25]. In this scenario, some studies report the development of pharmaceutical dosage forms containing RBO for topical use as sunscreen [26] and for the treatment of skin disorders [27]. Regarding the trends in the development of new pharmaceutical dosage forms, polymeric nanoparticles play a key role in the advancement of therapeutic systems, mainly due the ability to control the release of drugs and to improve drug stability [28,29]. Lipid-core nanocapsules (LNC) are formed by a lipid core prepared with a mixture of an oil and a solid lipid (sorbitan monostearate) surrounded by a polymeric wall [30–32]. These polymeric nanocapsules were developed in the last decade, and the promising applications of LNC have been widely demonstrated, especially for the treatment of several diseases, such as contact dermatitis [33], leukemia [32], tumors in the central nervous system [34,35]. LNC also increase antioxidant activity and photostability [36,37]. Moreover, in a recent study our research group demonstrated the environmental safety of LNC prepared with RBO as oily core through an in vivo protocol (Allium cepa test) [38]. In this context, this study specifically tests the hypothesis of improving the protective effect of the RBO against UVB radiationinduced skin injury by its encapsulation in LNC. The influence of nanoencapsulation on the anti-edematogenic and antioxidant effects of RBO was evaluated in vivo, after the development of a semisolid formulation (hydrogel) containing RBO-loaded LNC, making them suitable for cutaneous application. 2. Material and methods 2.1. Materials Poly(e-caprolactone) (PCL) (MW: 80,000) and sorbitan monostearate (Span 60Ò) were purchased from Sigma–Aldrich (São Paulo, Brazil). Rice bran oil and medium chain triglycerides mixture (MCT) were purchased from Irgovel (Pelotas, Brazil) and Brasquim (Porto Alegre, Brazil), respectively. Polysorbate 80 was supplied by Henrifarma (São Paulo, Brazil). Triethanolamine and Carbopol UltrezÒ were purchased from DEG (São Paulo, Brazil). Halothane was purchased from Cristália (São Paulo, Brazil). Antibodies anti-NF-jß polyclonal antibody and anti-rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless indicated otherwise, all other reagents were from Sigma (Sigma, St Louis, MO, USA) and were dissolved in the appropriate vehicle solutions. 2.2. Preparation of lipid-core nanocapsules The LNC formulation was developed as previously reported [38]. The formulation consisted of two phases, one organic and one non-organic. The organic phase was prepared with an acetone solution (67 ml) containing PCL (0.25 g), RBO (0.400 ml) and a solid lipid at room temperature (sorbitan monostearate) (0.957 g). The aqueous phase was composed by a surfactant with a low hydrophilic-lipophilic balance (HLB) value (polysorbate 80) (0.193 g) and water. After all compounds were solubilized, the organic phase was slowly injected in the aqueous phase with moderate magnetic stirring. Acetone was removed by evaporation (bath at 40 °C) under reduced pressure, and the final formulation adjusted to 25 ml. An LNC formulation containing medium chain triglycerides

(MCT) was prepared as control. This synthetic oil was chosen to replace RBO, because the former does not have any bioactive compound in its composition. LNC suspensions containing RBO or MCT mixture were coded LNC-RBO and LNC-MCT, respectively. All formulations were prepared in triplicate, stored at room temperature (25 ± 2 °C) and protected from light (amber glass flasks). 2.3. Evaluation of mean particle size and zeta potential The granulometric profiles of nanocapsule suspensions were determined by laser diffractometry (Mastersizer, Malvern Instruments, Worcestershire, UK) as volume-weighted (Dv,4.3). SPAN values were calculated using Eq. (1):

SPAN ¼ ðd0:9  d0:1 Þ=d0:5

ð1Þ

where d0.9, d0.5 and d0.1 are the particle diameters determined respectively at the 90th, 50th and 10th percentile of undersize particles. The SPAN value reflects the range of the size distribution, and smaller SPAN values represent narrower size distributions. Particle size and polydispersity index were determined by photon correlation spectroscopy (3 measurements/batch; 2 runs of 30 s/measurement, 25 °C) after adequate dilution of an aliquot of the suspensions in purified water (Zetasizer Nanoseries, Malvern Instruments, Worcestershire, UK). Zeta potentials were determined using the same instrument at 25 °C, after the dilution of the samples in 10 mM NaCl in aqueous solution (3 measurements/batch; 10 runs/measurement, 25 °C). 2.4. Preparation of hydrogels Hydrogels were prepared using a mortar and pestle. Carbopol UltrezÒ (acrylic acid polymer) was used for the preparation of the hydrogels due to its improved dispersion properties and wide range of applicability in the pharmaceutical and cosmetic fields [39]. Firstly, Carbopol UltrezÒ (acrylic acid polymer) (0.5%) was dispersed in part of LNC (12.5 ml) in order to swell the polymer chains. After, this dispersion was neutralized with triethanolamine (0.5 ml), and the rest of the LNC suspension (12.5 ml) was added. Hydrogels containing LNC prepared with RBO or MCT were called HG-LNC-RBO and HG-LNC-MCT, respectively. In order to evaluate the effect of encapsulation on the results, hydrogels containing non-encapsulated RBO (HG-RBO) or non-encapsulated MCT (HGMCT) were prepared at the same concentration as the LNC suspensions. Using a pestle and mortar, RBO or MCT were emulsified with polysorbate 80 (0.193 g) in one part of water (12.5 ml). This emulsion was added to Carbopol UltrezÒ (0.5%), the polymer chains were swelled, and the formulation was neutralized with triethanolamine (0.5 ml). Finally, the rest of the water (12.5 ml) was added. Three batches of each formulation were prepared. 2.5. Characterization of hydrogels The pH, rheological behavior, and spreadability of all hydrogel formulations were determined after preparation (first 48 h). 2.5.1. pH pH values of hydrogels were determined by the immersion of a calibrated potentiometer (MPA-210 Model, MS-Tecnopon, São Paulo, Brazil) in a dispersion of an aliquot of the formulation in ultrapure water (10% w/v). 2.5.2. Determination of spreadability The spreadability of formulations was evaluated according to the methodology previously described by Borghetti and Knorst [40]. The sample was introduced in a hole (1 cm) in the center of a glass mold plate. The mold plate was carefully removed and

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the sample was serially pressed with glass plates of known weights at 1-min intervals. Spreading areas reached by samples between each addition of a glass plate were measured in millimeters along the vertical and horizontal axes. Results represent the mean of three determinations and were expressed as the spreading area in function of the weight applied. The spreading area was calculated according to the following equation (Eq. (2)): 2

Si ¼ d  p=4

ð2Þ

in which Si is the spreading area (mm2 g1) after the application of a determined weight i (g) and d is the mean diameter (mm) reached by each sample. The spreading area was plotted against the plate weights to obtain the spreading profiles. The spreadability factor (Sf) was also calculated, and represents the spread a formulation is able to reach on a smooth horizontal surface when one gram of weight is added on top of it, under the conditions described in the methodology above. The following equation (Eq. (3)) was used to calculate the spreadability factor:

Sf ¼ A=W

ð3Þ

in which Sf (mm2 g1) is the spreadability factor, (A) is the maximum spread area (mm2) after the addition of the series of weights used in the experiment, and (W) is the total weight added (g). 2.5.3. Evaluation of rheological behavior Rheological analyses were carried out at 25 ± 1 °C using a rotational viscometer (LVDV II + Pro model, Brookfield, USA) with a SC4-25 spindle and a small sample adapter. The data obtained were analyzed with the Rheocalc software (V3.1-1 version, Brookfield, USA). The shear stress ramp was applied for 1200 s. Twenty different points were recorded using a shear rate interval of 0.05 s1. 2.6. Animals All experiments were performed using adult male Swiss mice weighing 25–30 g. Animals were housed under controlled temperature (22 ± 1 °C) on a 12 h light/12 h dark cycle and with standard laboratory chow and water ad libitum. The animals were acclimated to the experimental room for at least 1 h before the experiments. The present study was conducted in accordance with current guidelines for the care of laboratory animals and all procedures were approved by our Institutional Ethics Committee (Process number 130/2011). The number of animals and stimuli was the minimum necessary to demonstrate the consistent effects of treatments. Evaluation of inflammatory and oxidative stress parameters was performed blindly with respect to treatment administration. 2.7. UVB irradiation and experimental protocol The UVB source of irradiation consisted of a Philips TL40W/12 RS lamp (Medical-Eindhoven, Holland) mounted 20 cm above the table on which the mice were placed, and which emitted a continuous light spectrum between 270 and 400 nm with a peak emission at 313 nm. UVB output (80% of the total UV irradiation) was measured using a model IL-1700 Research Radiometer (International Light, USA; calibrated by IL service staff) with a radiometer sensor for UV (SED005) and UVB (SED240). The UVB irradiation rate was 0.27 mW/cm2 and the dose used was 0.5 J/cm2. Randomly chosen animals were divided into four groups of six animals. One hour before irradiation, each animal was topically treated on the ear surface with 0.06 g of the following semisolid formulations: Groups 1 and 2 were treated with hydrogel containing non-encapsulated MCT or RBO, respectively; Groups 3 and 4 were treated with

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hydrogel containing encapsulated MCT or RBO, respectively. Groups of non-irradiated (naive) (Group 5) and untreated irradiated animals (Group 6) were also included. Animals were anesthetized (90 mg/kg of ketamine plus 3 mg/kg of xylazine hydrochloride, intra-peritoneal) and submitted to ear irradiation as previously described [41]. 2.8. Ear edema measurement Edema was expressed as an increase in ear thickness and was recorded in lm. Ear thickness was measured before and 24 h after UVB irradiation using a digital micrometer (Starrett Series 734) in animals anesthetized with halothane [42]. The micrometer was applied near to the tip of the ear, just distally to the cartilaginous ridges. 2.9. Oxidative stress evaluation As an index of protein oxidative damage, carbonyl groups were determined based on a previous methodology described by Silva and co-workers [43]. After 24 h of UVB irradiation, the mice were euthanized and the ears were removed and disrupted and homogenized in ice-cold buffer. The proteins in the homogenate were precipitated by incubation with trichloracetic acid (TCA) 20% for 5 min on ice, and then centrifuged at 4000g for 5 min. The pellet was dissolved in NaOH 0.2 M and HCl 2 M (1:1), or 2,4-dinitrophenylhydrazine (DNPH) 10 mM in HCl2 for carbonyl groups derivatizing, respectively. DNPH in HCl2 was used as blanks. Samples were maintained for 30 min at room temperature. Next, proteins were washed three times with 1:1 ethanol:ethyl acetate, with 15-min standing periods to allow removing excess DNPH. Samples were dissolved in guanidine 6 M dissolved in KH2PO4 20 mM, pH 2.3. Absorbance was read at 370 nm. Carbonyl content (lmol/mg protein) was calculated using a molar extinction coefficient of 22,000 M/cm at 370 nm after subtraction of the blank absorbance. 2.10. Pro-inflammatory response assessment The cytosol to nucleus translocation of the transcription factor NF-rB was used as a pro-inflammatory response marker. The assay was carried out as described previously [44], with minor modifications. After 24 h of UVB radiation, mice were euthanized and ears were removed and sonicated in an ice-cold buffer containing protease and phosphatase inhibitors. The homogenates were chilled on ice for 15 min and then vigorously shaken for 15 min at 4 °C. The particulate fraction was precipitated by centrifugation at 14,000g for 45 min, and the supernatant reserved for analyses, as a fraction rich in cytosol. The pellet was resuspended in the same buffer, this time in the presence of Triton X-100, and incubated under continuous shaking at 4 °C for 30 min. The homogenate was centrifuged for 45 min at 14,000g and the supernatant was used. This procedure was repeated in the presence of glycerol, and the sample was considered rich in nuclei. The protein content was determined by the method of Bradford [45] using bovine serum albumin as a standard. Amounts of proteins (20 lg for nucleus and 50 lg for cytosol) were mixed in load buffer with 2.5% b-mercaptoethanol and 0.04% bromophenol blue and boiled for 10 min. Proteins were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride membranes, according to the manufacturer’s instructions (Millipore, Billerica, MA, USA). Ponceau staining was used as a loading control [46]. First, the membrane was blocked with 1% BSA in 0.05% Tween 20 in Tris–borate saline (TBS-T), then incubated for 10 min with specific primary antibody anti-NF-rB (1:1000). Membranes were washed three

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times, with TBS-T followed by incubation with secondary antibody anti-rabbit IgG (1:3000) for 10 min. Membranes were dried, scanned, and quantified with Scion Image PC version of NIH image. The results were normalized for the control group (naive) densitometry values and expressed as the relative amount of NF-jB. 2.11. Statistical analysis Statistical analysis was carried out by one-way or two-way analysis of variance (ANOVA) followed by Tukey’s, Student– Newman–Keuls’ or Bonferroni posttests, when appropriate. P values 60.05 were considered significant. Results are expressed as mean ± SD (standard deviation) when appropriate. Analyses were run using the SigmaStat Statistical Program (Version 3.0, Jandel Scientific, USA) or GraphPad Software 4.0 (GraphPad, USA) and reported as means accompanied by their respective 95% confidence limits.

Table 1 Physicochemical characteristics of lipid-core nanocapsule suspensions containing rice bran (LNC-RB) or medium chain triglycerides (LNC-MCT) after preparation.

a

Formulation

Particle size (nm)

PDIa

Zeta potential (mV)

pH

LNC-RBO LNC-MCT

194 ± 3 195 ± 6

0.09 ± 0.04 0.09 ± 0.02

9.31 ± 0.32 9.98 ± 0.54

5.51 ± 0.11 5.62 ± 0.08

Polydispersity index.

Table 2 pH values and spreadability factors of hydrogels. Formulation

pH

Spreadability factor (mm2 g1)

HG-LNC-RBO HG-LNC-RBO HG-RBO HG-MCT

6.24 ± 0.05 6.23 ± 0.06 5.63 ± 0.08 5.87 ± 0.10

3.11 ± 0.11 3.23 ± 0.06 3.86 ± 0.10 3.66 ± 0.05

3. Results and discussion Nanoparticle development requires the use of suitable characterization tools in order to better understand the system and guarantee these particles’ nanotechnological behavior. The granulometry of both LNC-RBO and LNC-MCT formulations measured by laser diffractometry demonstrated a unimodal distribution and submicrometric particle sizes, regardless of the type of oil. The lower SPAN values of both LNC formulations (1.752 and 1.747 for LNC-MCT and LNC-RBO, respectively) indicate the narrow range of distribution profile of such nanoparticles. Physicochemical characteristics of the developed formulations were also evaluated by photon correlation spectroscopy (Table 1). All formulations presented particle sizes around 200 nm and low polydispersity index (below 0.10), demonstrating their particle size homogeneity. The slight acidic pH values could be explained by the presence of terminal carboxylic groups in the polymeric chains of poly(e-caprolactone) [47]. The slight zeta potential values (around 9 mV) are characteristic of the LNC due to the particle/emulsion coating by polysorbate 80, which in turn stabilizes such particles sterically at the particle/water interface [48]. Regarding the statistical analysis, the oil type did not have any influence on the physicochemical parameters of the formulations (p > 0.05), in agreement with our previous works [31,32,38,47–49]. In a subsequent step, semisolid formulations (hydrogels) were prepared using LNC in order to make possible their cutaneous administration. In fact, the properties of the hydrogels such as the good spreadability, biocompatibility, and biodegradability (regarding the nature of the polymer nature) [50–53], in combination with the unique features of nanoparticles, define a good strategy to develop drug delivery systems intended for treatment of skin disorders [33,54,55]. Regarding the physicochemical characteristics of hydrogels, all semisolid formulations had uniform color and pH values compatible with cutaneous application (Table 2), regardless of the presence of RBO or MCT (nanoencapsulated or non-encapsulated). However, hydrogels containing non-encapsulated RBO had lower pH values than those containing the nanoencapsulated oil (p 6 0.05). This result could be explained in light of the presence of free fatty acid lipid chains of the oils in the latter formulations. This hypothesis finds support in the comparison of the pH values of all hydrogels with the pH value of a hydrogel formulation prepared without LNC or any kind of oil (6.26 ± 0.06). Additionally, the presence of LNC influenced the spreadability factor (Fig. 1) (p 6 0.05). It is possible that the slight lower spreadability profile of the hydrogels containing LNC, when compared to those hydrogels without them, could reflect also the

Fig. 1. Spreadability (mm2) of hydrogel formulations (HG) with rice bran (RBO) or medium chain triglycerides (MCT) oils in non-encapsulated (HG-RBO and HG-MCT) or nanoencapsulated form (HG-LNC-RBO and HG-LNC-MCT).

encapsulation of the oils into LNC (Table 2). Moreover, the formulations HG-LNC-RBO or HG-LNC-MCT did not feel greasy or sticky, when compared with those prepared with the non-encapsulated oils (HG-RBO and HG-MCT), making them sensorially more attractive. Rheological properties of the semisolid formulations were also evaluated, since they can help to predict product quality and to evaluate the influence of the formulation compounds on flow properties. Fig. 2 shows the rheograms (applied shear rate as a function of shear stress) of hydrogels. As can be observed, all semisolid formulations showed pseudoplastic non-Newtonian behavior, where the flow curves are non-linear [56], regardless of the presence of LNC. This characteristic is interesting during application of the product on skin, since the formulation may become less viscous when an external force is applied, with good coverage of the area to be treated. In addition, Newtonian systems such as emulsions or liquid formulations (including LNC suspensions) may fail to form a thin film on the skin, since they spread rapidly, possibly affecting the effectiveness of the product. In a subsequent step, a UVB irradiation-induced edema animal model was used to evaluate the influence of the nanoencapsulation of RBO (as hydrogel formulation) on UVB-induced skin inflammation. Concerning the results obtained from the pretreatment with the hydrogel formulations containing the oil used as control (HG-MCT and HG-LNC-MCT), it is possible to observe that these

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Fig. 2. Rheological behavior of the hydrogel formulations (HG) with rice bran (RBO) or medium chain triglycerides (MCT) oils in non-encapsulated (HG-RBO and HG-MCT) or nanoencapsulated form (HG-LNC-RBO and HG-LNC-MCT).

hydrogels did not reduce the ear edema induced by UVB-irradiation, when compared to untreated animals (Fig. 3A). On the other hand, the treatment with HG-LNC-RBO prevented the ear edema induced by UVB-irradiation by 60 ± 9% (Fig. 3B), suggesting that this formulation can inhibit some skin inflammatory responses caused by UVB exposure. Additionally, the results obtained for the hydrogel containing non-encapsulated RBO (HG-RBO) and HG-LNC-MCT were similar. These results suggest that LNC are not accountable per se for the anti-edematogenic property of HGLNC-RBO, and that the physical effect of the nanoparticles can be ruled out in the present study. UV radiation has been reported to generate ROS in a variety of cells, and intracellular ROS are thought to play an important role in UV-induced inflammatory responses in the skin [2]. Furthermore, although the human skin has endogenous defense

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mechanisms (enzymatic and non-enzymatic antioxidants), it is continuously exposed to sunlight and environmental oxidizing pollutants. This overexposure leads to the accumulation of ROS and their oxidized products, which over the years overloads this mechanism, resulting in progressive cellular damage that can affect the normal functionality of skin cells by DNA abnormalities caused by ROS [57–59]. Because the expression of protein carbonyl groups is one of the most commonly used parameters as a biomarker of oxidative stress caused by ROS, the possible effects of the formulations containing RBO or MCT on UVB-induced protein carbonyl formation were evaluated [60]. The UVB-irradiation induced a twofold increase in protein carbonylation (Fig. 4A), and pre-treatment with either HG-MCT or HG-LNC-MCT did not alter this production (Fig. 4A). On the other hand, HG-LNC-RBO prevented UVB-induced protein carbonylation by 81 ± 14% (Fig. 4B), suggesting the potential to reduce oxidative damage and showing interesting antiaging properties. In agreement with the results shown above, HG-RBO did not significantly alter the increased UVB-induced protein carbonylation. These results confirm that the nanoencapsulation is critical to improve the antioxidant activity of the RBO. When they act as different stimuli for the activation of signal transduction pathways, ROS involve nuclear factor kappa B (NFjB) activation [61]. Increased activation of NF-jB is often detected in both immune and non-immune cells, where it regulates the expression of genes that participate in pathways involving inflammation. Moreover, is believed that NF-jB induction exerts detrimental functions by inducing the expression of proinflammatory mediators that orchestrate and sustain the inflammatory response and cause tissue damage [62–64]. Additionally, NF-jB pathway is involved in UVB-mediated damage in skin including skin carcinogenesis, and regulation of NF-kB by antioxidants has emerged as a novel area with promising therapeutic implications [65,66]. In resting cells, NF-jB dimers are kept inactive by association with inhibitory proteins of the IjB family. Upon stimulation, IjB kinase

Fig. 3. Ear edema induced by UVB-irradiation (0.5 J/cm2) and anti-edematogenic effect of different hydrogel formulations. All formulations (0.6 mg/ear) were applied 1 h before irradiation with an UVB source. Edema was measured 24 h after irradiation. ⁄⁄⁄ P < 0.001 and ⁄⁄ P < 0.01 indicates significant differences in comparison with naive and # P < 0.05 indicates significant differences in comparison with HG-LNC-MCT treated mice. One-way ANOVA, followed by Dunnet post hoc tests.

Fig. 4. Effect of different hydrogel formulations on protein carbonylation content induced by UVB-irradiation (0.5 J/cm2). All formulations (0.6 mg/site) were applied 1 h before the irradiation with an UVB source. Protein carbonylation was measured 24 h after the ears irradiation. ⁄⁄⁄ P < 0.001 indicates significant differences in comparison with naive and ## P < 0.01 indicates significant differences in comparison with HG-LNC-MCT treated mice. One-way ANOVA, followed by Dunnet post hoc tests.

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injury in mice. Furthermore, the in vivo results support the remarkable advantages of RBO encapsulation in LNC, showing that this strategy is essential to improve the antioxidant and anti-edematogenic effects after cutaneous administration, as hydrogels. These results indicate that this strategy may be a suitable tool for the development of skin product containing vegetable oils intended to reduce skin damage produced by UV-light. Declaration of interest The authors report no conflict of interests. The authors alone are responsible for the content and writing of the article. Acknowledgments Lucas Almeida Rigo is grateful to CAPES/Brazil for his Master’s scholarship. Cássia Regina da Silva thanks to CAPES and CNPq/ Brazil. The authors thank the financial support from CNPq, FAPERGS, Secretaria de Inovação, Ciência e Tecnologia do RS and CAPES. The authors thank SS Guterres for the access to particle size analyses. Fig. 5. Western blot analysis showing NF-bj protein immunoreactivity in the nucleus of tissue samples obtained from naive mice ears and mice ears exposed to UVB-irradiation (0.5 J/cm2) and treated with 0.6 mg/site of HG-LNC-MCT or HG-LNC-RBO. One-way ANOVA, followed by Dunnet post hoc tests.

phosphorylates IjB proteins, triggering their ubiquitination and proteasomal degradation, which allows NF-jB dimers to accumulate in the nucleus and activate gene transcription [63]. To investigate the involvement of NF-jB pathway regulation by HG-LNCRBO treatment, NF-jB levels on ear tissue homogenates (obtained at the same time ear edema and carbonylation were measured) were analyzed. The UVB-irradiation induced a threefold increase in the NF-jB levels on nucleus, and nanoencapsulation of RBO prevented this increase by 86 ± 8% (Fig. 5). From these results it is possible to observe the clear effect of RBO nanoencapsulation on the improvement of the biological activities evaluated in the present study. Furthermore, the in vivo results suggest that it is possible to obtain a combined effect between protection (anti-edematogenic) and antioxidant using only one product (semisolid containing RBO-loaded LNC). This way, studies focused on the skin penetration of some phytochemically markers of rice brain oil, such as tocopherols and tocotrienols, are needed. However, the promising results obtained from HG-LNC-RBO formulation could be related with the ability of the hair follicles to act as a reservoir of nanoparticles, after the topical application, as previously reported [67]. Moreover, a recent work demonstrated that lipidcore nanocapsules remain retained at the outermost skin layers [68]. Concerning the nanoparticles safety, Paese and co-workers showed that the presence of polymeric nanocapsules prepared using PCL as polymer in hydrogels did not produce contact sensitization after the application in ear mice [69]. In addition, Fontana and co-workers reported that a hydrogel formulation containing clobetasol propionate-loaded lipid-core nanocapsules did not stimulate inflammatory or immune response in rats using a contact dermatitis model [33]. Taken together, these results enable the development of new therapeutic approaches based on the nanoencapsulation of RBO in LNC intended for the treatment of skin injuries triggered by UV irradiation.

4. Conclusions In this study we reported the role of the nanoencapsulation in the protective effects of RBO against UVB radiation-induced skin

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