European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 418–426

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Research paper

Resveratrol self-emulsifying system increases the uptake by endothelial cells and improves protection against oxidative stress-mediated death Ahmed Amri a,b, Solenn Le Clanche c, Patrice Thérond c,d, Dominique Bonnefont-Rousselot c,e, Didier Borderie c,f, René Lai-Kuen g, Jean-Claude Chaumeil a, Souad Sfar b, Christine Charrueau a,⇑ a

Laboratoire de Pharmacie Galénique EA 4466, Université Paris Descartes, Sorbonne Paris Cité, Paris, France Laboratoire de Pharmacie Galénique, Université de Monastir, Monastir, Tunisia Laboratoire de Biochimie EA 4466, Université Paris Descartes, Sorbonne Paris Cité, Paris, France d Service de Biochimie, Hôpital de Bicêtre AP-HP, Le Kremlin-Bicêtre Cedex, France e Service de Biochimie Métabolique, Groupe hospitalier Pitié-Salpêtrière-Charles-Foix AP-HP, Paris, France f Service de Biochimie Inter-Hospitalier Cochin-Hôtel-Dieu AP-HP, Paris, France g Plateforme Technique de l’IFR71/IMTCE, Imagerie Cellulaire et Moléculaire, Université Paris Descartes, Sorbonne Paris Cité, Paris, France b c

a r t i c l e

i n f o

Article history: Received 9 July 2013 Accepted in revised form 22 October 2013 Available online 31 October 2013 Keywords: Antioxidant Endothelial cells Nanoemulsion Oxidative stress-mediated death Polyphenol Atheroprotection

a b s t r a c t The aim of the present study was to develop and characterize a resveratrol self-emulsifying drug delivery system (Res-SEDDS), and to compare the uptake of resveratrol by bovine aortic endothelial cells (BAECs), and the protection of these cells against hydrogen peroxide-mediated cell death, versus a control resveratrol ethanolic solution. Three Res-SEDDSs were prepared and evaluated. The in vitro self-emulsification properties of these formulations, the droplet size and the zeta potential of the nanoemulsions formed on adding them to water under mild agitation conditions were studied, together with their toxicity on BAECs. An optimal atoxic formulation (20% MiglyolÒ 812, 70% MontanoxÒ 80, 10% ethanol 96% v/v) was selected and further studied. Pre-incubation of BAECs for 180 min with 25 lM resveratrol in the nanoemulsion obtained from the selected SEDDS significantly increased the membrane and intracellular concentrations of resveratrol (for example, 0.076 ± 0.015 vs. ethanolic solution 0.041 ± 0.016 nmol/mg of protein after 60 min incubation, p < 0.05). Resveratrol intracellular localization was confirmed by fluorescence confocal microscopy. Resveratrol nanoemulsion significantly improved the endothelial cell protection from H2O2-induced injury (750, 1000 and 1500 lM H2O2) in comparison with incubation with the control resveratrol ethanolic solution (for example, 55 ± 6% vs. 38 ± 5% viability after 1500 lM H2O2 challenge, p < 0.05). In conclusion, formulation of resveratrol as a SEDDS significantly improved its cellular uptake and potentiated its antioxidant properties on BAECs. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Resveratrol (3,5,40 -trihydroxystilbene) is produced by several plant species, such as grapes, mulberries and peanuts. It was first detected in the roots of white hellebore (Veratrum grandiflorum) [1]. Scientific studies have reported that resveratrol has a variety of desirable biological actions, including anti-inflammatory Abbreviations: BAEC, bovine aortic endothelial cell; DMEM, Dulbecco’s modified Eagle medium; FCS, fetal calf serum; HPLC, high performance liquid chromatography; NE, nanoemulsion; PI, polydispersity index; Res, resveratrol; ROS, reactive oxygen species; SEDDS, self-emulsifying drug delivery system; UV, ultraviolet. ⇑ Corresponding author. Laboratoire de Pharmacie Galénique, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, 4 avenue de l’Observatoire, 75006 Paris, France. Tel.: +33 1 53 73 95 85; fax: +33 1 53 73 99 52. E-mail address: [email protected] (C. Charrueau). 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.10.015

properties [2], hypoglycemic and hypolipidemic effects [3], cancer prevention [4] and prolongation of life span in several species [5,6]. One of the best-known benefits of resveratrol is for cardiovascular health [7–10]. It has been found to significantly increase the expression in human vascular endothelial cells of endothelial nitric oxide synthase, and to decrease the expression of the potent vasoconstrictor endothelin-1 [11], thereby decreasing risk of cardiovascular disease through reduced blood pressure. Other ways by which resveratrol could exert its cardioprotective effects include the inhibition of platelet aggregation, and its atheroprotective actions on cholesterol metabolism in cells of the arterial wall [12]. Many experimental studies have shown beneficial effects of resveratrol in atherosclerosis, the greatest contributor to cardiovascular diseases, involving reactive oxygen species (ROS) overproduction [13–17].

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The major challenge now facing the successful development of resveratrol therapies for human patients is to enhance its very poor bioavailability, arising from a combination of several limiting factors including poor water solubility, limited chemical stability and high metabolism. Although there are promising perspectives for the development of pharmaceutical formulations geared specifically to resveratrol delivery, there is still no suitable delivery system that allows the use of resveratrol as an atheroprotective compound [18]. Among the pharmaceutical dosage forms particularly wellsuited to lipophilic substances, self-emulsifying drug delivery systems (SEDDSs) are of major interest. They consist of ternary mixtures of oils, surfactants and co-surfactants that have the ability to form fine oil-in-water emulsions, microemulsions or nanoemulsions upon gentle agitation following dilution in an aqueous phase [19]. SEDDSs offer the possibility of enhancing both the rate and the extent of drug absorption, and the reproducibility of the plasma concentration profile [20–23]. The present study was therefore performed to formulate, develop and characterize SEDDSs of resveratrol, to compare the cellular uptake of resveratrol with a resveratrol ethanolic solution, and to assess protection against H2O2-mediated endothelial cell death. Since endothelial damage is an early oxidative injury in several vascular diseases [24–27], endothelial dysfunction is considered as a target for the prevention of cardiovascular pathologies. Accordingly, BAECs were selected as a cellular model in the present study. 2. Materials and methods 2.1. Materials Trans-Resveratrol (Fig. 1) was purchased from Sigma–Aldrich (Saint-Quentin-Fallavier, France), polyoxylglycerides (LabrasolÒ, LabrafilÒ M 1944 CS), medium-chain triglycerides (LabrafacÒ WL1349) and propylene glycol monolaurate (LauroglycolÒ FCC) were a generous gift from Gattefossé (Saint-Priest, France). Medium-chain triglycerides (MiglyolÒ 812) were purchased from Sasol (Witten, Germany), isopropyl palmitate from Stéarinerie Dubois (Boulogne-Billancourt, France), polysorbate 80 (MontanoxÒ 80 VG PHA) from Seppic (Paris, France), 1,3-butanediol, glycerol, isopropyl myristate, ethanol 96% v/v and propylene glycol were purchased from Cooper (Melun, France). 2.2. Determination of the solubility of trans-resveratrol in various excipients All the experiments were conducted in a dark room to avoid isomerization of trans-resveratrol into cis-resveratrol. The solubility of resveratrol in the following excipients was determined: isopropyl myristate, isopropyl palmitate, MiglyolÒ 812, MontanoxÒ 80, LabrasolÒ, LabrafacÒ WL1349, LabrafilÒM 1944 CS, propylene glycol, 1,3-butanediol, ethanol 96% v/v, LauroglycolÒ FCC, and glycerol. An excess of resveratrol (approximately

Fig. 1. Chemical structures of trans- and cis-resveratrol.

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50 mg) was placed in 0.5 ml of the vehicle in screw-capped glass vials, which were agitated for 48 h in a water bath at +20 °C, and then centrifuged at 2500g for 10 min to separate the undissolved drug. The supernatants were collected and diluted with methanol for quantification of resveratrol by reverse-phase high performance liquid chromatography (HPLC) with UV detection at 304 nm on a C18 KromasilÒ column (A.I.T, Houilles, France) (5 lm, 25 cm  4.6 mm internal diameter). The mobile phase, consisting of a mixture of methanol, distilled water and acetic acid (52/47.5/ 0.5, v/v/v), was pumped at a flow rate of 1 mL/min., and the column temperature was maintained at +40 °C [28–29]. The separation was performed in isocratic conditions. The concentration of resveratrol found in the sample was determined by external calibration. The resveratrol calibration curve was obtained by dilution of a stock solution of resveratrol (50 mM, 100% ethanol) in the mobile phase. Concentrations of resveratrol used were as follows: 0.1–0.2–0.3– 0.4–0.5–0.6–0.7–1–5–10 and 50 lM. The limit of resveratrol detection was 35 nM. The spectrophotometer operated at 304 nm, the maximum UV absorbance of trans-resveratrol. Results were expressed as mean solubilities ± SD (n = 3) in mg/mL and in mM. 2.3. Formulation of SEDDSs The excipients were selected for SEDDS formulation based on solubility results. Ternary phase diagrams were constructed for each system containing an oil, a surfactant and a co-surfactant, and 25 combinations were tested (Table 1), in a first step in the absence of resveratrol. The self-emulsifying regions were identified using a visual test adapted from Craig et al. [30] by dispersing each ternary mixture at the constant ratio of 50 lL SEDDS/40 mL water under gentle stirring at room temperature. In a second step, resveratrol was incorporated into each of the three optimal self-emulsifying systems selected by their droplet size and zeta potential. For that purpose, resveratrol was dissolved at the concentration of 200 mM in 96% v/v ethanol (stock solution) prior to mixing with the two other components of the self-emulsifying systems where resveratrol reached a concentration of 20 mM. 2.4. Droplet size and zeta potential determination The droplet size distribution and zeta potential values of the nanoemulsions resulting from the self-emulsification of the SEDDSs in water were measured by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Orsay, France). All measures were repeated three times, and the values of z-average diameters (nm), polydispersity index (PI), and zeta potential (mv) were recorded and expressed as mean ± SD. 2.5. Cell culture BAECs were cultured in 96-well plates (5  103 cells/well, 200 lL) for the evaluation of nanoemulsion cytotoxicity, in 75 cm2 flasks (3–4  106 cells/flask, 15 mL) for the evaluation of resveratrol cell uptake, and in 6-well plates (5  104 cells/well, 4 mL) for examination by confocal microscopy. The culture medium was Dulbecco’s Modified Eagle Medium (DMEM) (Sigma– Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen, Saint-Aubin, France), 1% 2 mM L-glutamine, 100 IU/mL penicillin (Sigma–Aldrich), and 100 lg/mL streptomycin (Sigma–Aldrich) at + 37 °C in a 5% CO2-humidified incubator, until they reached 80% confluence. Glucose concentration in this medium was 5.5 mM. The cells used in this study were between the fifth and tenth passage. Viability was assessed by the neutral red assay; cell viability greater than 95% was constantly required for performing experiments.

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Table 1 Ternary combinations tested for obtaining SEDDS.

Each combination numbered 1–25 was composed of an oil, a surfactant and a co-surfactant identified by a gray zone.

2.6. Evaluation of nanoemulsion cytotoxicity on BAECs in culture In order to ascertain the safety of the various nanoemulsion formulations on BAECs in culture, a neutral red assay in 96-well plate was performed. This evaluation was conducted with resveratrolfree nanoemulsions tested at two concentrations, X and 2X, able to solubilize 25 and 50 lM of resveratrol, respectively. Cells were incubated with 200 lL of each nanoemulsion previously formed in DMEM, at concentrations X and 2X, for 24 h at +37 °C in a 5% CO2humidified incubator. Ethanol (1%) in DMEM was used as a control as previously described [31]. After this incubation period, viability was assessed by the neutral red assay. For this, 100 lL of a solution of neutral red in distilled water (100 mg/L, Sigma–Aldrich) was added to each well. Cells were incubated for 3 h at +37 °C in a 5% CO2-humidified incubator. The plate was then emptied by reversal, and 100 lL of a solution of formol-calcium (37% formaldehyde plus 10 mL of 0.1 g/mL calcium chloride dihydrate, made up to 100 mL with distilled water) was distributed in each well and left in contact with the cells for 1 min. The plate was emptied again by reversal, and 100 lL of a solution of acetic acid (Sigma–Aldrich) in ethanol (1% v/v) was added to each well. The plate was then stirred for 5 min, and absorbance was measured at 540 nm on a microplate reader (MultiskanExÒ, Thermo Electron Corporation, Asnièressur-Seine, France). The results were expressed as mean viability ± SD (n = 3) in percentage. In order to determine the protective effect of resveratrol included in a self-emulsifying system independently of any effect of the excipients forming this system, the threshold of 95% viability was chosen as a total innocuity criterion. Therefore, when the viability was below 95%, the formulations were not further studied. Finally, the same experiments were carried out in the presence of 25 lM of resveratrol. 2.7. Evaluation of resveratrol location in BAECs 2.7.1. Treatments All cell treatments were implemented in DMEM supplemented as described above, except for FCS, whose concentration was reduced to 1%, and for ethanol (96% v/v) which was added at 1%

(v/v). Cells were incubated for 20, 40, 60 or 180 min in 75 cm2 flasks containing 15 mL of either resveratrol-free DMEM or in DMEM containing 25 lM resveratrol as a nanoemulsion (obtained by 1/800 dilution of self-emulsifying systems in DMEM) or as an ethanolic solution (obtained by 1/8000 dilution of a 200 mM resveratrol ethanolic stock solution (96% v/v) in DMEM). DMEM containing the nanoemulsion was filtered on a 0.45 lm filter. After each time, cells were trypsinized, washed with ice-cold phosphatebuffered saline (PAA Laboratories, Les Mureaux, France) by centrifugation for 10 min at 2500g and at +4 °C, and harvested into 0.4 mL of lysis buffer (CelLyticÒM C2978, Sigma Aldrich). As previously described [31], lysates were centrifuged at 2500g for 10 min to separate cytosolic and membrane fractions, and protein concentration in the supernatants (whole cell extracts) was determined using the Bio-Rad protein assay kit (Marnes-la-Coquette, France). This protein concentration was used to express intracellular and membrane concentrations of resveratrol in BAECs (nmol/mg of proteins). 2.7.2. Determination of resveratrol intracellular and membrane concentrations by HPLC Measurements of resveratrol in intracellular and membrane fractions were determined by HPLC as described above. To determine membrane concentrations, 200 lL of mobile phase was added to 200 lL membrane fractions, and centrifuged for 10 min at 2500g. Then 100 lL of supernatant was injected into the column. To determine cytosolic concentrations, 100 lL of methanol was added to 100 lL of cytosolic fractions and centrifuged for 10 min at 2500g. Then 100 lL of supernatant was injected into the column. Concentrations of resveratrol in membrane and cytosolic fractions were expressed as means ± SD (n = 3) in nmol/mg of proteins. 2.7.3. Confocal microscopy A Leica scanning device, type TCS SP2 (Leica Microsystems, Heidelberg, Germany), was used for all the confocal microscopy examinations. Cells were incubated in resveratrol-free DMEM or in DMEM containing 25 lM resveratrol as a nanoemulsion or as an ethanolic

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solution (as described in Section 2.7.1.), for 1 and 3 h at +37 °C in a 5% CO2-humidified incubator. Cells were fixed by incubation with a 2% paraformaldehyde solution (VWR, Fontenay-sous-Bois, France) for 15 min at room temperature. For nuclear visualization, sections were then stained with the DNA marker TOPRO-3 (Molecular Probes, Interchim, Montluçon, France). Preparations were mounted using Dako mounting medium (Dako France S.A.S., Trappe, France). Images were acquired at 661 nm (FITC filter) after excitation at 642 nm. To precisely localize a positive fluorescence signal, cells were analyzed three-dimensionally, and a final merge was performed to confirm localization of resveratrol. The intrinsic fluorescent properties of resveratrol enabled us to localize it by fluorescence microscopy [32]. Images were acquired at 495 nm (FITC filter) after excitation at 488 nm.

Table 2 Solubility of resveratrol in various vehicles (mean ± SD). Vehicle

mg/mL

mM

Oils Isopropyl myristate Isopropyl palmitate MiglyolÒ 812

9.30 ± 0.30 8.20 ± 0.52 12.60 ± 1.10

40.7 ± 1.0 35.9 ± 3.0 55.2 ± 4.0

Surfactants MontanoxÒ 80 LabrasolÒ LabrafacÒ Labrafil ÒM 1944 CS

2.10 ± 0.20 0.42 ± 0.03 0.36 ± 0.04 0.39 ± 0.03

9.0 ± 0.8 1.8 ± 0.1 1.5 ± 0.1 1.7 ± 0.1

15.20 ± 1.40 13.30 ± 1.20 50.10 ± 3.60 0.42 ± 0.25 23.00 ± 3.50

66.6 ± 3.0 58.2 ± 2.0 219.0 ± 15.0 1.8 ± 0.9 98.5 ± 12.6

Co-surfactants Propylene glycol 1,3-Butane diol Ethanol Lauroglycol ÒFCC Glycerol

2.8. Resveratrol protection against oxidative stress-mediated death 2.8.1. Cell treatment For all experiments, BAECs were pre-incubated overnight with DMEM containing 25 lM resveratrol as a nanoemulsion or as an ethanolic solution (as described in Section 2.7.1.). Pre-incubation of BAECs in DMEM, DMEM with 1% ethanol, and DMEM with resveratrol-free nanoemulsion, was used as controls. After preincubation, all cells were treated by various concentrations of H2O2 (750, 1000 and 1500 lM) for 24 h. Absolute control cells were not treated with H2O2.

2.8.2. Cytotoxicity assay To determine the possible protective effect of resveratrol against oxidative stress, a neutral red assay was performed as described in Section 2.6. The results were expressed as mean viability ± SD (n = 4) in percentage.

3.2. Formulation of SEDDSs Several series of ternary combinations were prepared, and their stability and self-emulsifying properties were observed visually as previously described [30]. Three different behaviors were observed: (i) no stability of the ternary combination, whose self-emulsifying properties were consequently not evaluated, (ii) stability of the ternary combination, but without self-emulsifying properties, (iii) stability of the ternary combination with selfemulsifying properties (Table 3). Among the 25 ternary combinations tested, combinations 6, 13 and 22 were stable mixtures allowing self-emulsification. The phase diagrams of these combinations are shown in Fig. 2. Whatever the combination, we observed that emulsification was not efficient with less than 40% v/v of surfactant in SEDDSs. It has been reported that the drug incorporated into the SEDDSs may have some effect on the self-emulsifying performance [19]. In

2.9. Statistical analysis Statistical analysis was computed with SPSSÒ 16.0. (Statistical Package for the Social Sciences, SPSS Inc., Chicago, Illinois, USA). Student’s t-tests were performed to evaluate the significant differences. Values are reported as mean ± SD, and the data were considered statistically significant at p < 0.05 of three independent experiments for the determination of intracellular and membrane concentrations of resveratrol in BAECs, and four independent experiments for protection against oxidative stress-mediated cell death.

Table 3 Stability and self-emulsifying properties of ternary combinations.

3. Results 3.1. Solubility studies Solubility of resveratrol in various vehicles is presented in Table 2. Among the oily phases that were screened, MygliolÒ 812 provided the highest solubility of resveratrol, followed by isopropyl myristate and isopropyl palmitate. MontanoxÒ 80 displayed the highest solubilizing power for resveratrol among surfactants. The ability of co-surfactants to dissolve resveratrol was in the following decreasing order: ethanol > glycerol > propylene glycol > 1,3-butanediol. In spite of poor solubilization of resveratrol, LauroglycolÒ FCC was used in SEDDS formulation, since several studies reported its utility in self-emulsification [33–35].

Three different behaviors were observed: (i) no stability () of the ternary combination, whose self-emulsifying properties were consequently not determined (ND); (ii) stability of the ternary combination (+) without self-emulsifying properties (); (iii) stability of the ternary combination (+) with self-emulsifying properties (+) (gray zones).

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Fig. 3. Effect of surfactant concentration on mean droplet diameter of nanoemulsions formed in distilled water at room temperature from formulations containing constant cosurfactant concentration. The results are expressed as mean ± SD (n = 3).

Fig. 4. Effect of cosurfactant concentration on the mean droplet size of nanoemulsions formed in distilled water at room temperature from formulations containing constant surfactant concentration. The results are expressed as mean ± SD (n = 3).

Fig. 2. Ternary phase diagrams indicating the efficient self-emulsification region in distilled water at room temperature for combination 6 (isopropyl myristate, MontanoxÒ 80 and ethanol, Figure 2a), combination 13 (MiglyolÒ 812, MontanoxÒ 80 and ethanol, Figure 2b) and combination 22 (MiglyolÒ 812, LauroglycolÒ FCC and LabrasolÒ, Figure 2c). Key: The region of efficient self-emulsification is bounded by the solid line; the filled squares represent the compositions that were evaluated.

our study, and according to Balakrishnan et al. [36], no significant differences were found in self-emulsifying performance when compared with the corresponding formulations with resveratrol (data not shown).

3.3. Droplet size and zeta potential determination The effect of the surfactant concentration in the self-emulsifying systems, on the droplet size distribution is presented in Fig. 3. Increasing the surfactant concentration from 40% to 60% for combination 22 and from 40% to 70% for combinations 6 and 13, decreased the mean droplet size. Above these percentages, the droplet size was increased. Several studies have reported

similar results in droplet size by increasing the surfactant concentration in various self-emulsifying systems [30,37–38]. A decrease in the mean droplet diameter was observed with an increase in co-surfactant (ethanol and LauroglycolÒFCC) concentration from 5% to 10%, after which the mean droplet diameter was increased (Fig. 4). There was no significant difference between droplet size measured in water, DMEM or 0.9% NaCl (data not shown). The three formulations presenting the smallest droplet size (24 ± 7, 103 ± 14 and 198 ± 15 nm; PI 0.291 ± 0.062, 0.389 ± 0.051 and 0.402 ± 0.086, respectively for formulations 6, 13 and 22) were selected for further study. Compositions were 20% isopropyl myristate, 70% MontanoxÒ 80 and 10% ethanol for formulation 6, 20% MiglyolÒ 812, 70% MontanoxÒ 80 and 10% ethanol for formulation 13, and 30% MiglyolÒ 812, 60% LabrasolÒ and 10% LauroglycolÒ FCC for formulation 22. The charge of the oil droplets in the nanoemulsions was negative due to the presence of free fatty acids; the zeta potential of the nanoemulsions obtained from emulsification of formulations 6, 13 and 22 was 15.8 ± 2.6, 14.7 ± 2.0 and 12.2 ± 3.6 mV, respectively.

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Fig. 5. Evaluation of nanoemulsions cytotoxicity on BAECs in culture conducted with resveratrol-free nanoemulsions tested at two concentrations, X and 2X, able to solubilize 25 lM and 50 lM resveratrol, respectively. Cytotoxicity was assessed by the neutral red assay; the innocuity criterion was cell viability greater than 95%. The results are expressed as mean ± SD (n = 3).

3.4. Cytotoxicity toward BAECs A reduction in cell viability after 24 h of incubation with resveratrol-free nanoemulsions was observed. Formulations 6 and 22 even at the lower concentration (X), were toxic to BAECs, and irreversibly affected their viability. The formulation 13 at the higher concentration (2X, intended to solubilize 50 lM of resveratrol) was also toxic for BAECs. Only this formulation at the lower concentration (X, intended to solubilize 25 lM of resveratrol) allowed BAEC viability greater than 95% and was used for further study (Fig. 5). The concentration of 25 lM of resveratrol was consequently used throughout the study. 3.5. Determination of intracellular and membrane concentrations of resveratrol in BAECs A significant increase in the membrane concentration of resveratrol in BAECs was observed with the resveratrol nanoemulsion (formulation 13 at the lower concentration) (p < 0.05 vs ethanolic solution) after 20 and 40 min incubation (Fig. 6a). The intracellular concentration of resveratrol in BAECs was also significantly increased with the resveratrol nanoemulsion after 20, 40 and 60 min incubation (p < 0.05 vs. ethanolic solution) (Fig. 6b). Resveratrol concentration was significantly higher in cytoplasm than in membrane (p < 0.05 at all incubation times). 3.6. Visualization of resveratrol intracellular location Slides were prepared from BAECs pre-incubated with resveratrol for 1 or 3 h, and were examined by confocal microscopy. Resveratrol intrinsic green fluorescence allowed a direct visualization of its intracellular uptake (Fig. 7). While no green fluorescence could be observed in untreated cells, a green fluorescence was observed in all treated cells, mainly in cytoplasm. Intracellular fluorescence increased with the incubation time (Fig. 7b and d vs. Fig. 7c and e), and seemed more intense in BAECs incubated with resveratrol nanoemulsion than with an ethanolic solution of resveratrol (Fig. 7d and e vs. Fig. 7b and c). 3.7. Resveratrol nanoemulsion protection against oxidative stressmediated death BAEC viability after H2O2-induced oxidative stress is presented in Fig. 8.

Fig. 6. Membrane (6a) and intracellular (6b) concentrations of resveratrol in BAECs. Cells were incubated with either the resveratrol nanoemulsions formed in DMEM (25 lM resveratrol), or the ethanolic resveratrol solution (25 lM) for 180 min. Resveratrol concentration was determined by HPLC in intracellular and membrane fractions. Data are mean concentrations ± SD (n = 3) expressed in nmol/mg of proteins. ⁄p < 0.05 vs. ethanolic solution.

BAEC exposure to H2O2 significantly decreased cell viability at all H2O2 concentrations tested (p < 0.01 vs. control cells not exposed to H2O2). Pre-incubation of BAECs with the nanoemulsion containing 25 lM of resveratrol was able to protect endothelial cells from H2O2-induced oxidative stress (p < 0.05 vs. all other groups exposed to H2O2). 4. Discussion Recent attention has been focused on the polyphenol resveratrol as an anti-atherosclerotic agent. It has been shown to possess complementary activities that fight against a critical event in atherogenesis, namely the oxidative stress especially involved in vascular alterations and inflammation [39,40]. Resveratrol could thus become an adjuvant treatment of major importance in cardiovascular diseases. However, despite the numerous attempts, resveratrol still lacks an adequate delivery system [18]. In this context, we chose to investigate a novel approach based on the formulation of SEDDSs to increase resveratrol endothelial cell uptake and to potentiate its protection against H2O2-mediated endothelial cell death. As we hypothesized, SEDDS successfully met both objectives. In the context of oxidative cardiovascular disorders, the antioxidant properties of unformulated resveratrol on cultured cells have already been shown on various cell lines. BAECs could be protected either from a 25 mM glucose-induced oxidative stress by an overnight pretreatment with 10 lM resveratrol [16], or from peroxynitrite-triggered injury by 14 h of pre-incubation with 50 lM resveratrol [41]. Likewise, rat cardiac H9C2 cells pretreated for 72 h with 25–100 lM resveratrol afforded a marked protection

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Fig. 7. Observation of resveratrol BAEC uptake by confocal microscopy. Cells were incubated either in DMEM (Figure 7a), in DMEM + resveratrol (25 lM) dissolved with 1% ethanol for 1 hour (Figure 7b) and 3 hours (Figure 7c), or in DMEM + resveratrol (25 lM) in nanoemulsions for 1 hour (Figure 7d) and 3 hours (Figure 7e), at +37 °C in a 5% CO2-humidified incubator. The slides were prepared as indicated in Materials and Methods, and examined by fluorescence. For nuclear visualization, sections were marked with the DNA marker TOPRO-3. Images were acquired at 642 nm (FITC filter) after excitation at 661 nm. The intrinsic fluorescent properties of resveratrol enabled us to observe it by fluorescence microscopy. Images were acquired at 495 nm (FITC filter) after excitation at 488 nm. Bar represents 23.91 lm.

against cytotoxic agents, i.e. the xanthine/xanthine oxidase combination, which generates both superoxide and hydrogen peroxide,

4-hydroxy-2 nonenal, a major aldehydic product of lipid peroxidation, and doxorubicin, which elicits severe cardiotoxicity, mainly

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Fig. 8. Resveratrol prevents H2O2-mediated apoptotic changes. BAECs were preincubated overnight with DMEM + resveratrol (25 lM) either dissolved with 1% ethanol, or in nanoemulsions. Pre-incubations of BAECs in DMEM, DMEM with 1% ethanol, and DMEM with resveratrol-free nanoemulsions, were used as controls. All cells were treated by various concentrations of H2O2 (750, 1000 and 1500 lM) for 24 hours, except for absolute control cells, which were not treated with H2O2. BAEC viability was assessed by the neutral red assay. The results are expressed as mean ± SD (n = 4). – p < 0.001 vs. control cells not exposed to H2O2. ⁄p < 0.05 vs. cells pre-incubated in DMEM with resveratrol nanoemulsions (25 lM).

through an oxidative mechanism [42]. Comparable cytoprotection was also observed in ventricular myocytes isolated from rats and pretreated with 30 lM resveratrol [43]. The authors concluded that this increased resistance to oxidative and electrophilic stress resulted from the upregulation of endogenous antioxidants (such as glutathione) and phase 2 enzymes induced by resveratrol within the cells [42,43]. It is therefore necessary for resveratrol to penetrate into the cells or to bind to cell membrane receptors to efficiently exert protective effects. Due to its poor water solubility (30 mgL1, 0.13 mM), resveratrol cannot be dissolved at a high concentration in aqueous culture media. However, none of the above-cited studies reported the method or the solvent used for dissolution. For example, in a study carried out on human keratinocyte HaCaT cells, resveratrol was dissolved with dimethylsulfoxide at a final concentration of 0.1% v/v in the culture medium [44]. In our study, the control DMEM medium containing 25 lM of resveratrol dissolved with 1% ethanol failed to promote the antioxidant activity of the polyphenol, probably because of a too low uptake by the cells. One way to improve the access of resveratrol to its intracellular sites of action is to develop a suitable pharmaceutical formulation. Several studies have been carried out to increase the cellular delivery of resveratrol using either liposomes or nanoparticles. Thus the incorporation of resveratrol in liposomes allowed its rapid cellular internalization by human-derived renal epithelial cells HEK293; compared with resveratrol dissolved with ethanol, resveratrol encapsulated into liposomes significantly improved cell response to the stress caused by UV irradiation [45]. In addition, liposomes provided a protection against the cytotoxicity induced by the highest concentration of resveratrol tested, i.e. 100 lM, through downregulating reactive oxygen species generation [45]. Delivering resveratrol by biodegradable nanoparticles also increased its uptake by rat cortical cell cultures, and significantly improved neuroprotection against hydrogen peroxide-induced oxidative stress versus free resveratrol at 1 lM concentration [46]. Likewise, resveratrol-loaded gelatin nanoparticles exhibited very rapid and more efficient uptake by NCI-H460 lung cancer cells than free resveratrol, and this improvement was associated with greater antiproliferative efficacy [47]. Beside these delivery systems, SEDDSs are reported here for the first time as promising formulations for resveratrol delivery, following the example of other antioxidants

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such as coenzyme Q10 [35–37] or curcumin [48]. Several reasons, including an improvement of resveratrol dissolution in aqueous medium, a larger surface area provided by the nanometric droplet size, and a possible permeation enhancer effect of the surfactant, favor the use of SEDDSs for resveratrol delivery. In the present study, resveratrol concentrations were higher within the cytosolic compartment, where resveratrol could exert scavenging antioxidant activity, than in membranes. As previously shown for intracellular concentrations of resveratrol [32], membrane concentrations increased as a function of the incubation time of resveratrol initially added. The presence of resveratrol in cell membranes was consistent with the fact that resveratrol is a membrane-bound receptor ligand, as shown for estrogen receptors [49,50]. The main localization of resveratrol in cytoplasm was confirmed by fluorescence microscopy. A similar localization was reported in human hepatic cells with a cytoplasmic and nucleolar fluorescence distribution [32]. Such a distribution seems favorable to an efficient direct intracellular antioxidant activity of resveratrol by scavenging H2O2 and/or indirectly by preserving the antioxidant enzyme activities. Finally, it is important to underline a concern about the tolerance of the SEDDS formulation. Among the three formulations selected here, only one was safe at its lowest concentration for BAECs. The high proportion of surfactant required for SEDDS formulation could explain the toxicity observed on cultured endothelial cells. Even though the surfactants used were non-ionic (MontanoxÒ 80 and LabrasolÒ), and so less toxic than ionic surfactants, they may lead to reversible changes in the cell membrane permeability, and cause cell toxicity [51–53]. Because of this effect, it was not possible to test resveratrol concentrations above 25 lM, which might have afforded better antioxidant activity. More formulation studies, taking into account surfactant type-dependent toxicity on cell culture [54], may allow tolerance to be optimized and resveratrol delivery to cells to be increased, favoring the development of a pharmaceutical parenteral form. In conclusion, resveratrol was successfully formulated as a stable SEDDS formulation that allowed faster and greater uptake of resveratrol by BAECs, and significant improvement of the endothelial cell protection from H2O2-induced injury in comparison with resveratrol dissolved with ethanol. SEDDS formulation may therefore offer a novel and promising strategy to enhance the efficacy of resveratrol delivery. Acknowledgments The authors thank C. Tran Van Buu and E. Nubret for participation in SEDDS formulation and resveratrol measurements, respectively. Reference [1] B.C. Vastano, Y. Chen, N. Zhu, C.T. Ho, Z. Zhou, R.T. Rosen, Isolation and identification of stilbenes in two varieties of Polygonum cuspidatum, J. Agric. Food Chem. 48 (2000) 253–256. [2] B.S. Gordon, D.C. Díaz, M.C. Kostek, Resveratrol decreases inflammation and increases utrophin gene expression in the mdx mouse model of duchenne muscular dystrophy, Clin. Nutr. 32 (2013) 104–111. [3] H.C. Su, L.M. Hung, J.K. Chen, Resveratrol, a red wine antioxidant, possesses an insulin-like effect in streptozotocin-induced diabetic rats, Am. J. Physiol. Endocrinol. Metab. 290 (2006) 1339–1346. [4] M. Jang, L. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, C.W. Beecher, H.H. Fong, N.R. Farnsworth, A.D. Kinghorn, R.G. Mehta, R.C. Moon, J.M. Pezzuto, Cancer chemopreventive activity of resveratrol, a natural product derived from grapes, Science 275 (1997) 218–220. [5] K.T. Howitz, K.J. Bitterman, H.Y. Cohen, D.W. Lamming, S. Lavu, J.G. Wood, R.E. Zipkin, P. Chung, A. Kisielewski, L.L. Zhang, B. Scherer, D.A. Sinclair, Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan, Nature 425 (2003) 191–196.

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