Co-encapsulation of lipophilic antioxidants into niosomal carriers: Percutaneous permeation studies for cosmeceutical applications

Colloids and Surfaces B: Biointerfaces 114 (2014) 144–149

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Co-encapsulation of lipophilic antioxidants into niosomal carriers: Percutaneous permeation studies for cosmeceutical applications Lorena Tavano a,b , Rita Muzzalupo a,∗ , Nevio Picci a , Bruno de Cindio b a Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria, Edificio Polifunzionale, 87036 Arcavacata di Rende, Cosenza, Italy b Dipartimento di Ingegneria Informatica, Modellistica, Elettronica e Sistemistica, Università della Calabria, Via P. Bucci Cubo 39/C, 87036 Arcavacata di Rende, Cosenza, Italy

a r t i c l e

i n f o

Article history: Received 27 May 2013 Received in revised form 26 September 2013 Accepted 30 September 2013 Available online 12 October 2013 Keywords: Niosomes Antioxidants Co-encapsulation Transdermal delivery Radical scavenging activity

a b s t r a c t Niosomal vesicular systems containing resveratrol, alpha-tocopherol and curcumin as single agents and in combination, were designed with the aim to develop novel cosmeceutical formulations. The effects of antioxidants co-encapsulation on the physico-chemical properties of the carriers, their antioxidant properties and in vitro percutaneous permeation profiles were evaluated. Results showed that the coencapsulation of resveratrol/curcumin and alpha-tocopherol/curcumin affected the physico-chemical properties of niosomes and the entrapment efficiencies values, respect to the formulations containing the single antioxidant. The antioxidants in vitro percutaneous permeations appeared to be controlled and improved respect to the corresponding free solutions used as control. Moreover the antioxidants combinations resulted in a promoted ability to reduce free radicals, due to a synergic antioxidant action. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The skin is continuously exposed to environmentally pollutants and UV irradiations increasing its risk of photo-oxidative damage caused by free radicals molecules [1]. Free radicals are reactive molecular species with unpaired electrons that oxidize other molecules to gain electrons and stabilize themselves. This reaction produces another free radical, initiating a domino effect of free radical stabilization and formation. Free radicals can oxidize macromolecules such as DNA, proteins, carbohydrates, lipids and are involved in several pathological disorders and skin damages such as photoaging, sunburn, photocarcinogenesis [2]. One of the pharmaceutical strategies to prevent or treat oxidant-induced cellular and tissue damage involves the use of antioxidants, organic substances which are able to avoid damage to a molecule [3]. Unfortunately some of these molecules do not easily penetrate the plasma membrane of cells and some of them have poor stability and short half-life, when administered through conventional delivery modes. Strategies to improve the effectiveness of antioxidants are focused on their chemical modifications, their coupling to affinity carriers and vehiculation by drug-delivery systems such as vesicles (liposomes and niosomes) [1].

∗ Corresponding author. Tel.: +39 0984 493173. E-mail address: [email protected] (R. Muzzalupo). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.055

Vesicular systems were discovered in 1963 and their use as systemic and topical drug delivery systems has attracted increasing attention [4]. The application in skin treatment is based on the similarity of their bilayer structure to that of natural membranes and to their ability to alter cell membrane fluidity and to fuse with cells [5]. In particular vesicular systems have been suggested as promising vehicles for many active molecules (AMs), enhancing their penetration into the skin, reducing irritation that is caused by some of these actives and enhancing therapeutic efficacies against oxidative stress-induced damage [6]. Vesicles have been successfully used for the vehiculation of water-soluble and lipid-soluble antioxidants [7–11]. Moreover, one of the most interesting properties of the vesicular systems, is the possibility to co-encapsulate two different AM into their structure. In fact it has been shown that the administration of vesicular systems containing more than one antioxidant is more beneficial in prolonging their circulation times, coordinating their release into the body and ameliorating oxidantinduced tissue injuries [7]. Some studies reported that vesicles co-encapsulation of antioxidant results in a desirable, synergistic action of the active molecules, achieving increased cosmeceutical activity [12]. Resveratrol, alpha-tocopherol and curcumin were used as antioxidants models. Resveratrol (trans-3,5,40-trihydroxystilbene) is a polyphenol and is widely distributed in plant foods such as grapes wine and peanuts. Its relatively simple molecular structure enables free radicals overproduced in disease conditions to

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be scavenged and the redox signaling pathways of the cells to be regulated [13]. Special attention has been devoted to the topical application of resveratrol in different physiological and pathological conditions, such as inflammatory, antimicrobial and antiviral disorders and skin cancer [14–16]. In addition resveratrol has been shown to act as an inflammatory, antimicrobial and antiviral agent to treat human skin diseases such as psoriasis. However, the low water solubility, stability and therapeutic index of resveratrol make it unsuccessful in clinical therapy. ␣-tocopherol, the major component of vitamin E, is a lipid-soluble hydrocarbon compound that partitions into lipid storage organelles and cell membranes. It is an efficient scavenger of lipid peroxyl radicals and, hence, it is able to break peroxyl chain propagation reactions in cellular membranes preventing lipid peroxidation [17]. Curcumin [1,7bis (4-hydroxy-3-methoxyphenyl)-1,6-hepadiene-3,5-dion] is a hydrophobic polyphenolic compound derived from turmeric, a dietary spice. The antioxidant activity of curcumin arises from scavenging of several biologically free radicals that are produced during physiological processes and possesses several pharmacological effects including anti-inflammatory, antioxidant, antiproliferative, and antiangiogenic activities [18]. The main drawback associated with the therapeutic potential of curcumin is its poor aqueous solubility and stability, which leads to poor bioavailability [18]. In this light we decided to develop niosomal formulations containing antioxidant molecules as single agent and in combination, and to evaluate the effect of the co-encapsulation on the physicochemical and antioxidant properties of the carriers. Niosomes (non ionic liposomes) are vesicular systems having a surfactant bilayer membrane. Nonionic surfactants are the major type of surface active agents used in drug delivery systems since their compatibility, stability and toxicity are quite significant compared to the cationic, anionic or amphoteric ones [19]. Among nonionics surfactants, Polysorbates are of great importance and find widespread applications in pharmaceutical and cosmetical fields. Polysorbates consist primarily of fatty acid esters of sorbitol-derived cyclic ethers (sorbitans and sorbides) condensed with approximately 20 mol of ethylene oxide per mole (polyethoxy sorbitan). The presence of ethylene oxide improves their water solubility, thereby expanding their applications as emulsifiers, defoamers, dispersants and stabilizers. These polysorbates are marketed under a variety of trade names and among these, Tween surfactants are one of the most used, because of their extremely interesting properties of biodegradability, biocompatibility and low toxicity. Tween surfactants have been tested in pharmaceutical fields as drug delivery systems and in particular they have been used to prepare highly stable niosomes [20,21]. In this study vesicular systems were prepared from Tween 60 as commercial surfactant and all formulations were compared in terms of dimensions, morphology and polydispersity index (P.I.). Resveratrol, alpha-tocopherol and curcumin were used as antioxidants and their encapsulation efficiency into niosomes were evaluated, together with the radical scavenging activity of the final formulations. In addition, with the aim to propose our preparations as novel cosmeceutical formulations, active molecules percutaneous permeation profiles in vitro were investigated by using the Franz diffusion cells.

2. Materials and methods 2.1. Material Tween 60, resveratrol, alpha-tocopherol, curcumin, 2,2diphenyl-1-picrylhydrazyl (DPPH) and Sepharose CL-4B gel were purchased from Sigma–Aldrich (Milan, Italy). Ethanol and chloroform were supplied from Sigma–Aldrich (Milan, Italy) and are of

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high performance liquid chromatography grade. Absorption spectra were recorded with a UV-Vis JASCO V-530 spectrometer using 1 cm quartz cells. 2.2. Preparation of niosomes Accurately weighed amounts of Tween 60 were dissolved in chloroform in a round-bottom flask. After this, solvent was evaporated under reduced pressure and constant rotation to form a thin lipid film [22]. The lipid film was then hydrated with 10 mL of distilled water to obtain empty vesicles, at 60 ◦ C for 30 min, to form large multilamellar vesicles (MLV), at 10 mM total lipid concentration. In order to obtain single antioxidant-loaded niosomes, 1.03 × 10−6 mol of resveratrol or 1.00 × 10−6 mol of alpha-tocopherol or 1.03 × 10−6 mol of curcumin were added to the initial chloroform mixture, so that the AM moles were constant. After evaporation, the film was hydrated with 10 mL of distilled water. Niosomes containing resveratrol/curcumin or alpha-tocopherol/curcumin were prepared adding the appropriate amount of each antioxidant solution to the initial chloroform mixture. Details on the niosomes preparation are reported in Table 1. After preparation, the dispersion was left to equilibrate at 25 ◦ C overnight, to allow complete annealing and partitioning of the AM between the lipid bilayer and the aqueous phase. Small unilamellar vesicles (SUVs) were prepared starting from MLV by sonication in an ultrasonic bath (10 min at 60 ◦ C, 3 cycles). The purification of niosomes from untrapped materials (AM) was carried out by a flow of niosomal suspensions across a Sepharose CL-4B gel [23]. After purification, niosomes were stored at 4 ◦ C in the dark, until needed in subsequent experiments. 2.3. Characterization of niosomes 2.3.1. Morphology Morphological analysis of vesicles was carried out by transmission electron microscopy (TEM), using a ZEISS EM 900 electron microscope at an accelerating voltage of 80 kV. A drop of the vesicular formulation was placed on a carbon-coated copper grid, and the sample excess was removed using a piece of filter paper. Then a drop of 2% (w/v) PTA (phosphotungstic acid solution) was applied to the carbon grid and left for 2 min. Once the excess of the staining agent was removed by absorption with the filter paper, the sample was air-dried and the thin film of stained niosomes observed under the TEM. Each experiment was carried out in triplicate. 2.3.2. Size and distribution Vesicles were also characterized by measuring the Brownian motion of the particles in samples using Dynamic Light Scattering (DLS) for mean size and polydispersity index (PI), a measure of the width of the size distribution) with a 90 Plus Particle Size Analyzer (Brookhaven Instruments Corporation, New York, USA) at 25.0 ± 0.1 ◦ C by measuring the autocorrelation function at 90◦ . The laser was operating at 658 nm. The PI was obtained by fitting data by the inverse “Laplace transformation” method and by Contin [24]. Each sample was measured three times and results are expressed as mean ± standard deviation. 2.3.3. Entrapment efficiency The purification of niosomes was carried out by a flow of niosome suspensions across a Sepharose CL-4B gel [23]. AM entrapment efficiency (E%) was expressed as the percentage of the AM entrapped into purified niosomes referred to the total amount of AM present in a non-purified sample. It was determined by diluting 1 mL of purified and 1 mL of non-purified niosomes in 25 mL of methanol, followed by the measurement of absorbance of these solutions at the corresponding AM wavelengths. Methanol allows

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Table 1 Composition of the vesicular systems used. Formulation name

Surfactant

Surfactant (mol)

Resveratrol (mol)

␣-Tocopherol (mol)

Curcumin (mol)

T60 T60-R T60-T T60-C T60-R-C T60-T-C

T60 T60 T60 T60 T60 T60

1 × 10−4 1 × 10−4 1 × 10−4 1 × 10−4 1 × 10−4 1 × 10−4

– 1.03 × 10−6 – – 1.03 × 10−6 –

– – 1.00 × 10−6 – – 1.00 × 10−6

– – – 1.03 × 10−6 1.03 × 10−6 1.03 × 10−6

the breaking of niosomal membranes and the release of encapsulated AM. The E% evaluation gave the possibility to calculate the AM moles loaded into niosomes, as reported in Table 2. Each experiment was carried out in triplicate and results are expressed as mean ± standard deviation. 2.3.4. In vitro permeation studies The experiments were carried out in the vertical Franz diffusion cells for 12 h at 37 ◦ C through rabbit ear skin, obtained from a local slaughterhouse [25]. The skin, previously frozen at −18 ◦ C, was pre-equilibrated in physiological solution at room temperature for 2 h before the experiments. A circular piece of this skin was sandwiched securely between the receptor and donor compartments with the dermal side in contact with the receiver medium and the epidermis side in contact with the donor chamber (contact area = 0.416 cm2 ). The donor compartment was charged with 0.5 mL of sample and the receptor compartment was filled with 5.5 mL of water/ethanol solution (1:1). During the study, the donor chamber was covered by parafilm. At regular intervals up to 12 h, the medium in the receiver compartment was removed and replaced with an equal volume of pre-thermostated (37 ± 0.5 ◦ C) fresh medium. The complete substitution of the medium was needed to ensure sink conditions and quantitative determination of the small amounts of AM permeated. The content of AM in the samples was analyzed by UV–vis spectrometry. Each experiment was carried out in triplicate, and the results were in agreement within ±5% standard error of mean (SE). 2.3.5. Antioxidant activity 2.3.5.1. DPPH radical scavenging activity assay. Radical scavenging activity was determined according to the technique reported by Wang et al. [10]. Briefly a concentration of 0.25 mM DPPH ethanol solution and stock solutions of each AM loaded niosomes were prepared. An aliquot of 1.5 mL of DPPH solution and 1.5 mL of niosomal solutions samples (containing 100, 200, 300, 400 and 500 ␮L of each initial niosomal stock solutions and ethanol up to 1.5 mL) were mixed. Mixtures were vigorously shaken and allowed to reach a steady state at room temperature for 30 min, in the dark. The bleaching of DPPH was determined by measuring the absorbance at 517 nm with UV–vis spectrophotometer. The DPPH radical scavenging activity was calculated according to the following equation (Eq. (1)): Scavenging activity (%) =

A0 − A1 × 100 A0

(1)

where A0 is the absorbance of the control (blank, without niosomes) and A1 is the absorbance in the presence of the niosomal formulation. Radical scavenging activity of empty vesicles was also performed to confirm the absence of antioxidant activity due to Tween 60 surfactant. Each experiment was carried out in triplicate, and the results were in agreement within ±5% standard error. 2.3.5.2. Statistical analysis. Data were expressed as the mean ± SD (standard deviation) of three independent experiments. Statistical significance was calculated by one-way analysis of variance (ANOVA) and Bonferroni-corrected p-value for multiple comparison test. The level of statistically significant difference was defined as p < 0.05. 3. Results and discussions In this research Tween 60 was used as surfactant and as reported in our previous work, was able to form vesicular systems without the presence of any membrane additive [20]. This is due to the fact Tween 60 possess an optimal critical packing parameter and space requirements of the hydrophobic and the hydrophilic parts of the amphiphiles, resulting in the formation of spherical aggregates. Empty niosomal were characterized by mean sizes of 455 nm and visually appeared as turbid and bluish dispersions, with the typical aspect of vesicular formulations. Moreover, transmission electron microscopy images (TEM) confirmed the formation of vesicular structures spherical and regular in shape (image not shown). In our study, resveratrol, alpha-tocopherol and curcumin were used as antioxidants models and were encapsulated in Tween 60-based vesicles as a single agent or in combination: the idea to make the association resveratrol/curcumin and alphatocopherol/curcumin arise from literature. In particular recent study reported that a combination of resveratrol and curcumin strongly reduced prostate and colon cancer and the effects were far superior to that seen following administration of each agent alone either in their free or vesicular form [26–28]. The synergistic antioxidative effect of alpha-tocopherol with other antioxidants, such as ascorbic acid (vitamin C), green tea polyphenols, resveratrol and quercetin have been well documented and proved due to the reduction of alpha-tocopheroxyl radical by the co-existent antioxidant to regenerate alpha-tocopherol, demonstrating the antioxidant synergism with alpha-tocopherol [1,29]. From these lights, the combination of alpha-tocopherol and curcumin could give similar results.

Table 2 Hydrodynamic diameter (nm), P.I. and moles of encapsulated antioxidants of vesicular systems at 25 ◦ C, (mean ± SD; n = 3). Formulation name

Hydrodinamic diameter (nm)

T60 T60-R T60-T T60-C T60-R-C T60-T-C

455 507 544 565 471 531

± ± ± ± ± ±

15 12 10 12 14 15

P.I. 0.248 0.166 0.156 0.107 0.122 0.188

Encapsulated resveratrol (mol) 5.60 × 10−7 – – 4.08 × 10−7 –

Encapsulated ␣-tocopherol (mol)

8.41 × 10−7

5.56 × 10−7

Encapsulated curcumin (mol)

Total antioxidants (mol)

3.50 × 10−7 4.18 × 10−7 5.83 × 10−7

5.6 × 10−7 8.41 × 10−7 3.5 × 10−7 8.18 × 10−7 1.13 × 10−6

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Size measurements of AM loaded niosomes are given in Table 2: the mean diameters ranged between 471 and 565 nm, and vesicles found to be smaller when both AM were co-encapsulated into the bilayer. Probably, this could be due to the increased amount of attractive interaction between the hydroxyl groups (H bonding) of AM and niosomal matrices that resulted in an increase of niosomal cohesion and then in a decrease of the vesicles size. The particle size distribution with a P.I. of less than 0.25 indicated a narrow size distribution of the niosomes and consequently homogeneous formulations. Generally, P.I. values lower than 0.3 were indicative of suitable measurements and good quality of colloidal systems [30]. TEM images confirmed that the encapsulation of AM molecules into niosomes did not affect vesicles morphology, respect to the empty formulation (Fig. 1). Since Tween 60 has a dual nature with part of the molecule exhibiting hydrophilicity and the other lipophilicity, derived vesicles provided a relatively more favorable environment to poor water soluble compounds in aqueous solution, compared to the other nonionic surfactants, indicating that they are more effective in solubilizing water insoluble compounds. Resveratrol, alphatocopherol and curcumin were successfully entrapped in all of the formulations based on Tween 60. In particular 8.41 × 10−7 mol were encapsulated into T60-T sample, while in the case of curcumin and resveratrol, 3.50 × 10−7 and 5.56 × 10−7 mol of antioxidant were loaded, respectively. The co-encapsulation of resveratrol/curcumin and alpha-tocopherol/curcumin led to an increase of the total amount of antioxidant molecules present into the vesicle. In particular, in T60-R-C formulation, the total amount of encapsulated antioxidants moles were 8.18 × 10−7 : in the case of resveratrol loaded moles decreased from 5.60 × 10−7 to 4.08 × 10−7 , while for curcumin, moles increased from 3.50 × 10−7

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to 4.18 × 10−7 . For T60-T-C samples, encapsulated moles were found to be 5.56 × 10−7 and 5.83 × 10−7 for alpha-tocopherol and curcumin respectively, corresponding to 1.14 × 10−6 total antioxidants: for alpha-tocopherol, encapsulated moles decreased from 8.41 × 10−7 to 5.56 × 10−7 mol, while for curcumin an increase from 3.50 × 10−7 to 5.83 × 10−7 mol was detected. From these results it appeared that the co-encapsulation of hydrophobic drugs, always resulted in a increment of the total antioxidant molecules loaded into the niosomal bilayer and that curcumin entrapment efficiencies always increased in T60-R-C and T60-T-C respect to the T60-C sample. 3.1. Antioxidant activity With the aim to evaluate the effect of antioxidants combinations on the radical scavenging activity of our niosomal formulations, DPPH test was performed, comparing equal volume of single antioxidant-loaded and double antioxidants-loaded vesicles. Details on the experimental procedure are reported in Table 2. Scavenging activity of empty vesicles was also performed to confirm the absence of antioxidant activity due to Tween 60 surfactant. Moreover, in order to evaluate a synergic effect between resveratrol and curcumin or alpha-tocopherol and curcumin, experiments were performed by using amounts of single antioxidant-loaded samples equal to the total antioxidants present in T60-R-C and T60-T-C (8.18 × 10−7 and 1.13 × 10−6 mol, respectively). DPPH (1,1-diphenyl-2-pircylhydrazyl) is a stable free radical widely used to test the ability of antioxidant molecules to act as free radical scavengers or hydrogen donors, and to evaluate the antiradical activity of an antioxidant matrix. Fig. 2a shows the percentage of DPPH scavenging activity of T60-R, T60-C and T60-R-C samples. It was observed that the color

Fig. 1. Typical TEM photomicrograph for: (a) T60-R sample; (b) T60-T sample; (c) T60-R-C sample; (d) T60-T-C sample.

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Fig. 2. (a) Scavenging effects of antioxidant formulations containing equal moles of total antioxidant on the DPPH free radical: (light gray) T60-R; (gray) T60-C; (white) T60-R-C; T60 (black) (mean ± SD; n = 3). (b) Scavenging effects of antioxidant formulations containing equal moles of total antioxidant on the DPPH free radical: (light gray) T60-T; (gray) T60-C; (white) T60-T-C; T60 (black) (mean ± SD; n = 3). *p < 0.05 vs T60-T-C.

of DPPH containing solution gradually changed from deep violet to pale yellow in the presence of niosomes, demonstrating a certain antioxidant activity. In particular, formulations containing the single antioxidants showed a cumulative percentage reduction of DPPH radical lower than that achieved in the case of T60-R-C. This results may be due both to the higher amount of total antioxidant molecules loaded into the T60-R-C niosomes (8.18 × 10−7 mol), but also to a probable synergic action between the active molecules. With the aim to evaluate this possibility, DPPH experiments were performed by using opportune volumes of T60-R and T60-C samples so that the amount of antioxidant moles were equal to those present into T60-R-C sample. Fig. 2a shows that the antiradical activity of T60-R-C samples was higher compared to that of T60R and T60-C, confirming that, using equal moles of antioxidants, the combinations of resveratrol and curcumin showed a synergistic effect on antioxidant activity against DPPH radical. Fig. 2b shows the percentage of DPPH scavenging activity of T60-T, T60-C and T60-T-C formulations. As reported, alpha-tocopherol-loaded formulation demonstrates a lower radical scavenging activity respect to the curcumin-based one. Formulations containing single antioxidants showed a cumulative percentage reduction of DPPH radical lower than that achieved in the case of T60-T-C and also in this case, experiments performed with the aim to confirm the synergic action between alpha-tocopherol and curcumin, showed the improved antioxidant capacity of the double antioxidants-loaded niosomal formulation. Moreover it clearly appeared that formulations based on alphatocopherol and curcumin possess higher radical scavenging activity (p < 0.05 vs T60-T-C) respect to the formulations based on resveratrol and curcumin (p > 0.05 vs T60-R-C). Generally it has been reported that the combination of vitamin E and carotenoids or vitamin C improve the antioxidant action at skin level, reducing skin photo-damage and preventing photoaging [31]. In our study we also demonstrated that in this case a synergistic antioxidant interaction takes place between the

curcumin and alpha-tocopherol. We hypothesized that the mechanism by which the synergic action between resveratrol/curcumin and alpha-tocopherol/curcumin occurred, may be based on the resveratrol or alpha-tocopherol regeneration reaction by curcumin, attenuating the propagation of free radical reactions, as proposed for other antioxidants combinations [32–34]. 3.2. Transdermal drug release Nonionic surfactants have long been recognized as those with the least toxicity and irritant potential. These properties make these compounds good candidates as potential penetration enhancers for use in transdermal delivery systems [35]. The cumulative amount of antioxidants permeated from different formulations were investigated for a period of 12 h: each sample was analyzed in triplicate. Fig. 3a shows the in vitro percutaneous permeation profiles of resveratrol and curcumin from all niosomal formulations. AM free solutions were used as controls (data not shown). As reported, the amount of resveratrol permeated in 12 h was found to be 93% and 71% from T60-R-C and T60-R samples respectively, whereas only 15% of the antioxidant permeated in the case of simple drug solution. In the case of curcumin the faster permeation was found for T60-C sample (about 35%), followed by T60-R-C one (19%), while the corresponding solution showed a very low permeation (only 5%). The permeation of both antioxidants from the corresponding solutions through the skin was lower compared to all niosomal formulations. This is due to the fact that the bilayer of vesicles is made up of surfactants that can behave as penetration enhancers thus allowing a temporary change in packing order of stratum corneum, enabling AM passage across the skin. In particular Tween 60, which is part of Polysorbates, is claimed to act as percutaneous permeation enhancer, because of its highly flexible and no bulky hydrocarbon chains and its large head group [36]. The different trends achieved for resveratrol and curcumin

Fig. 3. (a) Cumulative amount versus time of permeated antioxidants: () resveratrol from T60-R sample; (䊉) curcumin from T60-C sample; () resveratrol from T60-R-C sample; () curcumin from T60-R-C sample (mean ± SE; n = 3). (b) Cumulative amount versus time of permeated antioxidants: () alpha-tocopherol from T60-T sample; (䊉) curcumin from T60-C sample; () alpha-tocopherol from T60-T-C sample; () curcumin from T60-T-C sample (mean ± SE; n = 3).

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loaded into niosomal formulations is difficult to explain: in fact in the case of resveratrol, the higher amount of antioxidant permeated within 12 h was achieved from the sample containing both antioxidants, while in the case of curcumin, the best performance was achieved by using the single antioxidant formulation. In addition the cumulative drug permeations are much higher in the case of resveratrol respect curcumin (from 71% to 93% vs 19% to 35% of loaded antioxidants). Probably an important role was played by the antioxidants entrapment efficiencies into the vesicular systems, in fact a negative relationship between E% values and permeation profiles occurred. Resveratrol E% values decreased in the case of T60-R and T60-R-C samples, while cumulative AM permeated increased; conversely, curcumin E% increased for T60-C and T60-R-C formulations, while cumulative AM permeated decreased. However the highest total amount of antioxidants permeated across the skin within 12 h was achieved for the formulation containing both AM. Fig. 3b shows the in vitro percutaneous permeation profiles of alpha-tocopherol and curcumin from niosomal formulations; also in this case AM solutions were used as controls. As found for resveratrol/curcumin based formulations, the permeation of both antioxidants from the corresponding solutions through the skin was lower compared to niosomal samples and similar trends in terms of percutaneous permeation profiles across the skin were achieved. In particular the amount of alpha-tocopherol permeated in 12 h was found to be 94% and 83% from T60-T-C and T60-T formulations, respectively, whereas 22% of the antioxidant permeated in the case of the corresponding drug solution. In the case of curcumin the fastest permeation was found for T60-C sample (about 35%), followed by T60-T-C one (about 12%) and the corresponding solution (only 5%). The negative relation between encapsulation efficiency and permeation profile detected in the case of resveratrol/curcumin based formulations was maintained. In fact the alpha-tocopherol entrapment efficiency values decrease in the case of T60-T and T60-T-C samples, while cumulative permeated drug increased. At the same time curcumin E% increased for T60-C and T60-T-C formulations, while cumulative AM permeated decreased, also in this case the highest total amount of antioxidants permeated across the skin within 12 h was achieved for the T60-T-C formulation. Results showed that in the case of resveratrol/curcumin based formulations, the best performance in terms of percutaneous permeation was achieved by T60-R-C, respect to the single antioxidant-loaded samples. In the case of alpha-tocopherol/curcumin the highest cumulative alpha-tocopherol amount permeated across the skin was obtained from the T60-T-C formulation, while, unexpectedly, the highest curcumin amount permeated was obtained from the single antioxidant sample. Finally both combination of lipophilic antioxidants resulted in an enhanced percutaneous permeation of AM across the skin, in respect to the corresponding free solutions used as control. 4. Conclusion In this paper niosomal formulations containing resveratrol, alpha-tocopherol and curcumin as single agents and in combination were developed and the effects of their co-encapsulation on the physico-chemical properties of the carriers, their antioxidant properties and in vitro percutaneous permeation profiles were evaluated. Results showed that the co-encapsulation of resveratrol/curcumin and alpha-tocopherol/curcumin affected the physico-chemical properties of niosomes and the entrapment efficiencies values, respect to the formulations containing the single antioxidant. The antioxidants combinations resulted in a promoted ability to reduce free radicals, due to a synergic antioxidant action: in the

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case of resveratrol/curcumin formulation, the percentage of radical inhibition gave up to 40%, while the best performance was achieved by the alpha-tocopherol/curcumin sample (about 100%). However, the resveratrol/curcumin samples ensured an optimal performance in terms of cumulative amount of antioxidants permeated across the skin, regardless of the best formulation was the T60-T-C, which showed the best results in terms of percutaneous permeation and antiradical activity. For all these reasons all niosomal formulations showed a potential in the transdermal delivery of antioxidants molecules and may be useful in cosmeceutical field for the prevention of diseases caused by oxidative stress. Acknowledgements MIUR, the Italian Ministry for University, is acknowledged for financial support (Grants # EX-60%, PRIN 2010-11; Prot. N.2010H834LS 004). Moreover, the project has been co-funded with support from the Commission European Social Fund and Region of Calabria (Italy). The authors are grateful to Dr. Anna Internò for her assistance in English corrections. References [1] Z.E. Suntres, J. Toxicol. 2011 (2011) 152474. [2] J.M. McCord, Am. J. Med. 108 (2000) 652. [3] A.M. Papas, in: F.M. Clydesdale (Ed.), Contemporary Food Science. Antioxidant Status, Diet, Nutrition and Health, CRC Press, Boca Raton, FL, 1999. [4] R. Muzzalupo, L. Tavano, R. Cassano, S. Trombino, T. Ferrarelli, N. Picci, Eur. J. Pharm. Biopharm. 79 (2011) 28. [5] G.M. El Maghrabya, B.W. Barry, A.C. Williams, Eur. J. Pharm. Sci. 34 (2008) 203. [6] V.B. Junyaprasert, P. Singhsa, J. Suksiriworapong, D. Chantasart, Int. J. Pharm. 432 (2012) 303. [7] Z.E. Suntres, P.N. Shek, Biochem. Pharm. 52 (1996) 1515. [8] D. Pando, C. Caddeo, M. Manconi, A.M. Fadda, C. Pazos, J. Pharm. Pharmacol. 65 (2013) 1158–1167. [9] C. Caddeo, M. Manconi, A.M. Fadda, F. Lai, S. Lampis, O.D. Sales, C. Sinico, Colloids Surf. B 111 (2013) 327. [10] M. Chessa, C. Caddeo, D. Valenti, M. Manconi, C. Sinico, A.M. Fadda, Pharmaceutics 3 (2011) 497–509. [11] J. Kristl, K. Teskac, C. Caddeo, Z. Abramovic, M. Sentjurc, Eur. J. Pharm. Biopharm. 73 (2009) 253. [12] L. Barclay, J. Biol. Chem. 263 (1988) 16138. [13] M. Kelkel, C. Jacob, M. Dicato, M. Diederich, Molecules 15 (2010) 7035. [14] I. Scognamiglio, D. De Stefano, V. Campani, L. Mayol, R. Carnuccio, G. Fabbrocini, F. Ayala, M.I. La Rotonda, G. De Rosa, Int. J. Pharm. 440 (2012) 179. [15] M. Ndiaye, C. Philippe, H. Mukhtar, N. Ahmad, Arch. Biochem. Biophys. 508 (2011) 164. [16] C. Caddeo, K. Teskac, C. Sinico, J. Kristl, Int. J. Pharm. 363 (2008) 183. [17] G.W. Burton, Annu. Rev. Nutr. 10 (1990) 357. [18] B.B. Aggarwal, A. Kumar, A.C. Bharti, Anticancer Res. 23 (2003) 363. [19] D.G. Hall, in: M.J. Schick (Ed.), Nonionic Surfactants: Physical Chemistry, Surfactant Science Series, vol. 23, Marcel Dekker, New York, N.Y., 1987. [20] L. Tavano, P. Alfano, R. Muzzalupo, B. De Cindio, Colloids Surf. B: Biointerfaces 87 (2011) 333. [21] J. Jiao, Adv. Drug Deliv. Rev. 60 (2008) 1663. [22] A.D. Bangham, M.M. Standish, J.C. Watkins, J. Mol. Biol. 13 (1965) 238. [23] L. Tavano, M. Vivacqua, V. Carito, R. Muzzalupo, M.C. Caroleo, F. Nicoletta, Colloids Surf. B: Biointerfaces 102 (2013) 803. [24] S.W. Provencher, Comput. Phys. Commun. 27 (1982) 229. [25] L. Tavano, L. Gentile, C. Oliviero Rossi, R. Muzzalupo, Colloids Surf. B: Biointerfaces 110 (2013) 281. [26] N.K. Narayanan, D. Nargi, C. Randolph, B.A. Narayanan, Int. J. Cancer 125 (2009) 1. [27] A.P. Majumdar, S. Banerjee, J. Nautiyal, B.B. Patel, V. Patel, J. Du, Y. Yu, A.A. Elliott, E. Levi, F.H. Sarkar, Nutr. Cancer 61 (2009) 544. [28] V.B. Patel, S. Misra, B.B. Patel, A.P. Majumdar, Nutr. Cancer 62 (2010) 958. [29] P. Pedrielli, L.H. Skibsted, J. Agric. Food Chem. 50 (200) (2013) 7138. [30] D.D. Verma, S. Verma, G. Blume, A. Fahr, Int. J. Pharm. 258 (2003) 141. [31] S. Trombino, R. Cassano, T. Ferrarelli, R. Muzzalupo, L. Tavano, N. Picci, Vitamin E: Nutrition, Side Effects and Supplements, Nova Science Publishers, New York (USA), 2010. [32] X. Chen, R.M. Touyz, J.B. Park, E.L. Schiffrin, Hypertension 38 (2001) 606. [33] S.U. Mertens-Talcott, S.S. Percival, Cancer Lett. 218 (2005) 141. [34] J. Chen, D. Wanming, D. Zhang, Q. Liu, J. Kang, Pharmazie 60 (2005) 57. [35] P.P. Sarpotdar, J.L. Zatz, Drug Dev. Ind. Pharm. 13 (1987) 15. [36] A. Lopez, F. Llinares, C. Cortell, M. Herraez, Int. J. Pharm. 202 (2000) 133.