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Nanocarriers enhance the transdermal bioavailability of resveratrol: In-vitro and in-vivo study
Colloids and Surfaces B: Biointerfaces 148 (2016) 650–656
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Nanocarriers enhance the transdermal bioavailability of resveratrol: In-vitro and in-vivo study Ming-Jun Tsai a,b , I-Ju Lu c , Yaw-Syan Fu d , Yi-Ping Fang c , Yaw-Bin Huang c , Pao-Chu Wu c,∗ a
Department of Neurology, China Medical University Hospital, Taiwan, ROC School of Medicine, Medical College, China Medical University, Yuh-Der Road, Taichung city 404, Taiwan, ROC c School of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung city 807, Taiwan, ROC d Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung city 807, Taiwan, ROC b
a r t i c l e
i n f o
Article history: Received 6 July 2016 Received in revised form 27 September 2016 Accepted 28 September 2016 Available online 28 September 2016 Keywords: Resveratrol Topical application Transdermal amount Deposition amount Skin irritation Stability
a b s t r a c t The aim of this study was to develop and assess the potential of nanostructured emulsion carriers for resveratrol topical application. Different compositions of resveratrol-loaded nanostructured emulsions were prepared using different types and amounts of surfactants and oily phases (isopropyl myristate and caproyl 90). The produced nanostructured emulsions were within the nanosized range 23.4–422.2 nm with low viscosity range 2.15–17.53 cps. The transdermal amount and deposition amount in the skin after 24 applications of resveratrol-loaded nanostructured emulsion were significantly increased about 896.2-fold and 10.2-fold respectively, when compared to the drug-saturated solution-treated group. Nanostructured emulsion containing IPM and low amounts of mixed surfactant of Tween80/Span 20 showed highest permeation capacity. In vivo study showed that the plasma concentration of resveratrol could be maintained at high levels for a long time after topical application of drug-loaded nanostructured emulsion. The histological examination demonstrated that the free drug- and drug-loaded nanostructured emulsion demonstrated considerably less irritation than the standard irritation group (0.8% paraformaldehyde-treated). The residual contents of resveratrol in the tested formulations after 3 months of storage at 25 ◦ C and 40 ◦ C were more than 99.97 ± 3.90%. The results of present work confirm the high potential of nanostructured emulsion as carriers for drug topical application. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Resveratrol (trans-3,4,5-trihydroxystilbene), a component of a variety of common edible plants including grapes, nuts, fruits, and red wine, is a phytoestrogen [1,2]. Numerous studies have proven resveratrol possesses various systemic and local pharmacologic effects. For the systemic effects, resveratrol can prevent or slow the progression of various illnesses, including cardiovascular disease, cancer, and ischemic injuries [3–9]. For the skin topical protection effects, resveratrol can inhibit proliferation of keratinocytes (skin epidermal cells) and stimulate their differentiation [10,11]. It was also proven to inhibit melanin production by skin cells and to alleviate skin irritation that may be caused by alpha-hydroxy acids. Hence, it can be used to improve the appearance of wrinkled, flaky, lined, dry, aged or photo-damaged skin and ameliorate
∗ Corresponding author. E-mail address: [email protected] (P.-C. Wu). http://dx.doi.org/10.1016/j.colsurfb.2016.09.045 0927-7765/© 2016 Elsevier B.V. All rights reserved.
skin thickness, elasticity, flexibility, radiance, glow and plumpness [12–14]. Unfortunately, resveratrol is a poor water-soluble compound with lower bioavailability and short half-life owing to extensive metabolism [2], leading to an irrelevant in vivo effect with oral administration compared to its powerful in vitro efficacy. Topical application is a much less invasive, more comfortable and convenient route of administration, particularly for therapeutic substances with skin protection effect; therefore, the topically applied method has been shown as a potential administration route for resveratrol. Numerous dosage forms have been widely investigated for topical delivery of resveratrol, such as solutions, gels, emulsions, and liposomes [15–21]. Nanostructured dosage forms such as liposomes, microemulsions, niosomes and nanoparticles appear to be attractive strategies for facilitating drug permeation capability through the skin in recent years, because they can offer a better chance for adherence to biological membranes transporting therapeutic substances in a controlled manner. Nanostructured carriers are also capable of increasing drug-loading capacity, sustaining drug
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release, and demonstrate promising drug tissue-specific targeting, [15–20,22–26]. The present study aimed to develop a suitable nanostructured emulsion for resveratrol topical applications. With this purpose, different nanostructured emulsions were prepared by using different surfactants and oil phases. The physicochemical properties, in vitro permeation and deposition study, stability, skin irritation and in vivo pharmacokinetic study of drug-loaded formulations were conducted to confirm the potential of nanostructure emulsion as carriers for resveratrol topical applications. 2. Materials and methods 2.1. Materials Resveratrol (racemic form), naringenin and sorbitan monolaurate (Span 20) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Caproyl 90 was purchased from Gattefosse (Nanterre, Frence). Polyoxyethylene sorbitan monooleate (Tween 80), and propylene glycol (PG) was from J. T. Baker (Phillipsburg, USA). Pluronic® L44 and paraformaldehyde were purchased from SigmaAldrich (St. Louis, Missouri, USA). Polyoxyl 23 lauryl ether (Brij 35) was obtained from Acros Organic (Pennsylvania, USA). Isopropyl myristate (IPM) was purchased from Merck Chemicals (Darmstadt, Germany). All other chemicals and solvents were of analytical reagent grade. 2.2. Animals Sprague-Dawley rats weighing 250–300 g were purchased from BioLASCO Taiwan CO., Ltd. The hair of the abdominal skin was shaved with an electric clipper (Thrive® , Japan), then the abdominal skin was excised. The subcutaneous fat was removed. The skin was cleaned and examined for integrity. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University (Kaohsiung, Taiwan). The committee confirmed that the permeation experiment followed the guidelines as set forth by the Guide for Laboratory Fact Lines and Care. 2.3. Preparation of resveratrol-loaded nanostructured emulsions Caproyl 90 or IPM were used as oil phases, propylene glycol and ethanol were used as cosurfactant, and Tween 80, Span 20, Brij 35 and L44 were used as surfactant. Oil phase and surfactant were mixed well by a vortex at room temperature. The cosurfactant was dissolved in aqueous phase, and then was slowly added to the previous mixture by a vortex for 5 min. After the clarity and transparency of microemulsion were formed, resveratrol of 0.5% was dissolved in the nanostructured emulsions by a horizontal shaker for 10 min. All resveratrol-loaded nanostructured emulsions showed clarity without any precipitate. 2.4. Determination of viscosity Viscosities of resveratrol-loaded nanostructured emulsions were determined using a Brookfield, Model LVDV-II, cone-plate of viscometer (USA). Tested samples of 0.5 mL were placed into the cone-plate, and heated to 37 ◦ C by a thermostatic pump for three mins. Readings were recorded 30 s after measurement was made at a rate of 150 rpm. 2.5. Determination of average droplet size and polydispersity index The average droplet size and polydispersity index (PI) of resveratrol-loaded nanostructured emulsions were measured at
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25 ◦ C by Malvern, Zetasizer 3000HSA, using a computerized inspection system (UK) with a scattering angle of 90◦ . Testing samples of three milliliters were transferred in a cuvette and placed in the scattering chamber. The average droplet size and polydispersity index (PI) were obtained by software. Each tested sample was measured in triplicate. 2.6. In vitro skin permeation and deposition studies The rate and extent of skin permeation of resveratrol from tested nanostructured emulsions were determined using a modified diffusion cell fitted with excised SD rat skin. The skin with effective area of 3.46 cm2 was placed on the receiver chambers with the SC facing upward to the donor cell. The receptor cell was filled with pH 7.4 phosphate buffer saline containing 20% PG and 20% ethanol to maintain sink condition (solubility of 5.5 ± 0.1 mg/mL). The receptor cell was maintained at 37 ± 0.5 ◦ C by thermostatic pump and constantly stirred at 600 rpm throughout the permeation studies. At predetermined intervals, 1 mL of receptor buffer was withdrawn via the sampling port and equal volume of fresh buffer was replaced. The concentration of resveratrol was determined by a modified HPLC method [27,28]. After the 24-h experimental period, the donor cell was carefully removed and the drug-exposed skin was washed with deionized water three times to remove the residual resveratrol on the skin surface. The drug-exposed skin was dried with cotton wool and then the SC was removed from the rest of the treated skin by the tape-stripping method with 3 M, Scotch Book Tape no. 845, adhesive cellophane tapes (St Paul, MN, USA). The stripping tapes were placed in glass tubes. The epidermis layer was separated from the dermis layer by a heat method (at 80 ◦ C for 3 min) [29–31]. The residual amounts of drug in SC, epidermis and dermis layers were then extracted by methanol and determined by a modified HPLC. 2.7. Pharmacokinetics study Male SD rats weighing 250–300 g were used in the experiment. The hair on the abdominal area of rats was shaved using a hair clipper and an electric razor one day prior to the pharmacokinetics experiment. Rats were anesthetized throughout the whole investigation by intraperitoneally administered carbamic acid ethyl ester solution of 750 mg/kg and secured on their backs. The tested formulation (about 67 mg/kg) was applied to the shaven abdomen. To prove the sustained release property of tested formulation by topical application, resveratrol suspension of 30 mg/kg was orally administered by oral gavage. Blood was collected from the jugular vein at predetermined time intervals. Blood sample was centrifuged at 4 ◦ C and 1200g for 10 min. Plasma sample was extracted by liquid–liquid extraction using 1 mL of ethyl acetate. The sample was vortex-mixed for 2 min and centrifuged at 12,000g for 10 min. The supernatant was evaporated by the centrifugal vaporizer under reduce pressure and reconstituted with 100 L of methanol. The drug concentration was analyzed by a modified HPLC. 2.8. Chromatographic condition The Hitachi L-7100 series HPLC system was equipped with a prepacked LiChroCART® RP-18 column (125 4 mm I.D., particle size 5 m). The detect wave was set at 305 nm. The mobile phase of 2.0% acetic acid containing 27% acetonitrile was delivered at a rate of 1.0 mL/min. Naringenin was used as internal standard. The analysis method was successfully validated with coefficient of variation of 3.3%, relative error of 6.67% and a determination coefficient of 0.9995. The limit of quantitation was 0.01 g/mL.
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2.9. Skin irritation determination The safety of the tested formulations was evaluated by naked eye observation and histological microscopy. The animals were divided into four groups including negative control groups (untreated group), positive control group (treated with 0.8% paraformaldehyde) and tested groups (treated with drug-free and drug-loaded tested formulation) [32,33]. The hair on the abdominal region of the SD rat was shaved carefully with an electrical shaver one day before the drug administration. A glass ring with area of 3.46 cm2 was adhered to the skin with glue, then 0.5 mL of tested formulations were loaded and occluded by parafilm. After 24-h drug exposure, the rats were sacrificed and specimens of the exposed skin areas taken for histological examination. The skin pieces were immediately fixed in 4% buffered formaldehyde solution at least for 24 h and then the skin pieces were embedded in paraffin. The skin sample was sliced transversely of 20 m. The sliced section was rehydrated stepwise, stained with hematoxylineosin, and then observed using a Nikon microscope for an optical microscopic observation (Nikon Eclipse Ci, Tokyo, Japan).
were measured using one-way ANOVA by Winks software (Texasoft, Cedar Hill, TX, USA), followed by the Newman–Keuls test for comparisons between groups. The significance of differences among groups was set at p < 0.05. The significance was taken as 95% (probability < 0.05). 3. Results and discussion 3.1. Physicochemical characteristics Different types of surfactant such as Brij 35, L44 and Tween 80 were used to prepare nanostructured emulsions with oil phase (caproyl 90), aqueous phase and surfactant at ratio of 5, 75 and 20. The average droplet size of formulation with Brij 35, L44 and Tween 80 was 29.8 ± 6.9, 23.4 ± 3.0, and 31.2 ± 2.0 nm respectively. The viscosities of these formulations were 13.3 ± 3.7, 6.1 ± 1.8 and 17.5 ± 4.5 cps respectively. The results showed that the drugloaded nanostructured emulsions could be formed by using these three surfactants, and that the physical properties of drug-loaded formulations were affected by the incorporated types of surfactants [23,36,37].
2.10. Stability determination The stability of resveratrol-loaded nanostructured emulsions was evaluated by thermodynamic stability tests and accelerated stability test. For the thermodynamic stability, the sample was centrifuged at 3500 rpm for 30 min and 10,000 rpm for 10 min as well as being stored at different temperature cycles of 4 ◦ C and 45 ◦ C for 3 times; each temperature storage period was not less than 48 h [34,35]. The phase separation, creaming and cracking were evaluated. For the accelerated stability test, the resveratrol-loaded formulation was stored in dark-brown bottles for protection from light and placed at 25 ± 2 ◦ C/60 ± 5%RH and 40 ± 2 ◦ C/75 ± 5%RH for three months. The physicochemical characteristics of the tested formulation such as phase separation, clarity, precipitation of drug, color change and residual drug content after 3 months storage were evaluated. 2.11. Data analysis All data were presented as mean ± standard deviation (S.D.). To compare the difference of various groups, the statistical differences
3.2. In vitro skin permeation and deposition The cumulative amount of resveratrol in receptor fluid (transdermal amount) and deposition amount in SC, epidermis and dermis layer after 24 h application of resveratrol – loaded formulations are presented in Fig. 1. The results showed that resveratrol could deposit in different skin layers, particularly in the epidermis layer, after 24 h of treatment, which might be resulting in a reservoir effect [38–40]. The resveratrol-saturated aqueous solution was used as the control group to assess the potential of drug-loaded nanostructured emulsions. The transdermal amount (Q24h ) and total deposition amount in skin (D24h ) at 24th h application of tested formulation was 0.79 ± 0.78 and 4.26 ± 0.58 g/cm2 respectively, which was quite low, indicating that resveratrol is not easily transported through the skin. The result was in agreement with a previous study [20]. When using nanostructured emulsions containing caproyl 90 and different types of surfactants of Brij 35, L44 and Tween 80 as vehicle, the Q24h was non-detected and the D24h was significantly increased about 2.19 ∼ 3.25-fold (Fig. 1). The formulation containing Tween 80 showed a higher enhancement effect than that of the formulation containing L44. The result
Fig. 1. The transdermal amount and deposition amount in skin after 24 h treated with resveratrol saturated aqueous solution and drug-loaded nanostructured emulsions with caproyl 90 and different types of surfactant.
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showed that nanostructured emulsions could provide potential skin reservoir effect for resveratrol [32–34]. The emulsion system could be used as a promising vehicle for skin protection dosage of resveratrol. The enhancement mechanism of the nanostructured formulations might include, but is not limited to (1) the effect of adsorption and fusion of resveratrol-loaded vesicles onto the surface of the SC resulting in a high thermodynamic activity gradient of the drug–SC interface; and (2) the vesicles might impair barrier function of the SC leading to significant changes in drug permeation kinetics [41,42]. IPM is a potential penetration enhancer in transdermal formulations and is widely used as an oil phase to prepare microemulsions to enhance drug permeation capacity through the skin [23,43,44]. Hence, the oil phase (Caproyl 90) of the tested formulation was replaced by IPM. Unfortunately, the translucent formulation could not be formed. This phenomenon might be due to the hydrophiliclipophilic balance (HLB) of Tween 80 (15) and Brij 35 (16.9) being higher than the required HLB value (about 11.1) of IPM. Stable emulsion only formed at the HLB of the surfactant close to the required HLB of used oily substance [22,24,45]. Therefore, the mixed surfactant of Tween 80/Span 20 with HLB value of 11.16 was used to prepare a nanostructured emulsion containing IPM. As expected, a translucent formulation with average droplet size of 277 nm and viscosity of 5.82 cps was formed. The in vitro skin permeation and deposition study was conducted. The Q24h and D24h were 276.0 ± 42.3 and 29.4 ± 7.7 g/cm2 respectively, which was obviously higher than that of the formulation containing oily phase of caproyl 90. The result showed that the composition of formulation was an important influence factor for drug transportation through skin [32–34]. Previous studies [23,44] pointed out that microemulsions containing lower amounts of surfactant will result in higher thermodynamic activity of drug in the microemulsion and lead to a higher drug permeation rate. Hence, the surfactant amount of formulation was decreased from 20% to 15%. The average droplet size increased from 277.6 to 422.2 nm, and viscosity decreased from 5.82 to 2.15 cps, when emulsions had lower amounts of surfactant. This might be due to higher amounts of surfactant forming more stable emulsions with small size and higher viscosity. Although the droplet size of formulation with low amount of surfactant significantly increased, but the size still smaller than the desirable particle size of drug-carriers to penetrate the skin epithelium of ≤500 nm [46]. As expect, the Q24h and D24h of emulsion with 15% surfactant was further increased about 2.57-fold and 1.48-fold respectively
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(Fig. 2), indicating that average droplet size was not a superior factor in this study, whereas drug thermodynamic activity in the formulation might be a more important influencing factor. The result also demonstrated that appropriate nanostructured emulsion formulation might provide percutaneous absorption effect (Q24h increased about 896.2-fold) and local skin protection effect (D24h increased about 10.2-fold) after topical application of resveratrol. Fig. 3 shows the D24h after different application times of tested formulation containing 15% cosurfactant and saturated aqueous solution. It can be seen that using nanostructured emulsion as vehicle, the deposition amounts at different application times were significantly increased (p < 0.05). The drug deposition content in skin can be detected at 6 h after saturated aqueous solution application, whereas the drug level can be measured after 1 h with nanostructured emulsion applied. For drug-loaded nanostructured emulsion treatment, the deposition amount increased with application time to a maximum at 6 h. The D24h was increased about 10-fold when compared to the saturated aqueous solution-treated group. The result of in vitro study demonstrated that the used nanostructured formulation as carrier could accelerate the drug permeation rate and extent through skin, and enhance the deposit amount in skin, which confirms the potential of nanostructured formulation for resveratrol topical application. 3.3. In vivo pharmacokinetic study The pharmacokinetic studies of the topical application of drug-loaded nanostructured emulsion and oral administration of resveratrol dissolved in 60% PG were conducted. As shown in Fig. 4, the plasma concentration was rapidly decreased because of being extensively metabolized and excreted [27,47,48], whereas the plasma concentration of resveratrol could be detected 2 h after the tested formulation was applied, and maintained high levels for a long time after topical application of drug-loaded nanostructured emulsion. The relative bioavailability of topical administration was significantly increased as compared to oral administration. 3.4. Skin irritation Formulations might elicit primary skin irritation. The histological examination of skin specimen was conducted to assess the potential irritant effects of the developed tested formulation. The untreated rat skin and that treated with 0.8% paraformaldehyde
Fig. 2. The transdermal amount and deposition amount in skin after 24 h treated with resveratrol saturated aqueous solution (control group) and drug-loaded nanostructured emulsions with IPM and different amount of Tween 80/Span 20.
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Fig. 3. The deposition amount in skin after different application time of resveratrol saturated aqueous solution and drug-loaded nanostructured emulsion.
Fig. 4. Mean plasma concentration-time curve (mean ± S.D., n = 3) of resveratrol after oral an aqueous solution and topical applied the tested formulation.
solution were used as negative control and standard irritant respectively [34,49]. As shown in Fig. 5a, the untreated skin showed well-defined epidermal and dermal layers. The paraformaldehydetreated group showed a slight edema exfoliation of the SC, and loosely textured collagen in the dermis (Fig. 5b). Non-significant edema and erythema were found in drug-free and drug-loaded nanostructured emulsion-treated skin samples (Fig. 5c and d), when compared to the negative control group, indicating that the experimental nanostructured emulsions appeared to be a safe carrier for transdermal delivery. 3.5. Stability The selected drug-loaded formulation was subject to thermodynamic stability tests, including centrifugation test and heating-cooling cycles test. No phase separation, creaming, precip-
itation or liquefaction was found, indicating that the drug-loaded formulation possesses good physical stability. The results agreed with previous reports [34,50] which pointed out that microemulsion with small droplet size and very low interfacial tension made the formulations thermodynamically stable. After 3 months of storage at 25 ◦ C/60% RH and 40 ◦ C/75%RH, the drug-loaded formulation showed no obvious change, and no drug crystal was found. The viscosities and average droplet sizes were 4.86 ± 0.87, 5.41 ± 0.61 and 5.12 ± 0.23 cps, and 577.1 ± 98.21, 585.6 ± 91.31 and 581.3 ± 83.2 nm respectively, at initial and 3 months of storage at 25 and 40 ◦ C. The viscosity and droplet size showed non-significant difference. The residual drug contents of drug-loaded formulations were 99.97 ± 3.48% and 100.76 ± 1.21% at 25 ◦ C and 40 ◦ C storage respectively, indicating that the experimental formulations were stable.
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Fig. 5. The Photomicrographs of a rat abdominal skin section, viewed under a light microscope (10 × 10). (a) Untreated-skin, (b) Paraformaldehyde treated, (c) Drug-free formulation treated, (d) Drug-loaded formulation treated.
4. Conclusions In this work, the resveratrol-loaded nanostructured emulsion showed its capability to go through the skin barrier, particularly when prepared by IPM. The Q24h and D24h significantly increased about 896.2-fold and 10.2-fold respectively. The deposition drug level could be detected after 1 h application of drug-loaded nanostructured emulsion, which is remarkably shorter than the saturated aqueous solution group of 6 h. In vivo pharmacokinetic study, the plasma concentration of resveratrol was maintained at high levels for a long time after topical application. The resveratrol-loaded nanostructured emulsion was also stable after three months of storage at 25 and 40 ◦ C. The drug-free and drug-loaded nanostructured emulsion showed less skin irritation than that of the standard irritant group. The result confirmed nanostructured emulsion was a potential vehicle for resveratrol topical application. Conflict of interests The authors declare that there is no conflict of interest regarding the publication of this paper. Acknowledgment This work was supported by grants from the Ministry of Science and Technology, Taiwan (NSC 102-2320-B-037-006-MY2 and MOST 105-2632-B-037-003). References [1] J.A. Baur, K.J. Pearson, N.L. Price, H.A. Jamieson, C. Lerin, A. Kalra, V.V. Prabhu, J.S. Allard, G. Lopez-Lluch, K. Lewis, P.J. Pistell, S. Poosala, K.G. Becker, O. Boss, D. Gwinn, M. Wang, S. Ramaswamy, K.W. Fishbein, R.G. Spencer, E.G. Lakatta, D. Le Couteur, R.J. Shaw, P. Navas, P. Puigserver, D.K. Ingram, R. de Cabo, D.A. Sinclair, Resveratrol improves health and survival of mice on a high-calorie diet, Nature 444 (2006) 337–342. [2] J.A. Baur, D.A. Sinclair, Therapeutic potential of resveratrol: the in vivo evidence, Nat. Rev. Drug Discov. 5 (2006) 493–506. [3] M. Nassiri-Asl, H. Hosseinzadeh, Review of the pharmacological effects of Vitis vinifera (Grape) and its bioactive constituents: an update, Phytother. Res.: PTR 30 (2016) 1392–1403. [4] D.Y. Yeh, Y.H. Fu, Y.C. Yang, J.J. Wang, Resveratrol alleviates lung ischemia and reperfusion-induced pulmonary capillary injury through modulating pulmonary mitochondrial metabolism, Transplant. Proc. 46 (2014) 1131–1134. [5] H. Ding, X. Xu, X. Qin, C. Yang, Q. Feng, Resveratrol promotes differentiation of mouse embryonic stem cells to cardiomyocytes, Cardiovasc. Ther. 34 (2016) 283–289. [6] C.Y. Huang, W.J. Ting, C.Y. Huang, J.Y. Yang, W.T. Lin, Resveratrol attenuated hydrogen peroxide-induced myocardial apoptosis by autophagic flux, Food Nutr. Res. 60 (2016) 30511. [7] L. Gliemann, M. Nyberg, Y. Hellsten, Effects of exercise training and resveratrol on vascular health in aging, Free Radical Biol. Med. 98 (2016) 165–176. [8] P. Jaisamut, K. Wiwattanawongsa, R. Wiwattanapatapee, A novel self-microemulsifying system for the simultaneous delivery and enhanced oral absorption of curcumin and resveratrol, Planta Med. (2016), Epub ahead of print DOI: 10.1055/s-0042-108734.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Nanocarriers enhance the transdermal bioavailability of resveratrol: In-vitro and in-vivo study Ming-Jun Tsai a,b , I-Ju Lu c , Yaw-Syan Fu d , Yi-Ping Fang c , Yaw-Bin Huang c , Pao-Chu Wu c,∗ a
Department of Neurology, China Medical University Hospital, Taiwan, ROC School of Medicine, Medical College, China Medical University, Yuh-Der Road, Taichung city 404, Taiwan, ROC c School of Pharmacy, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung city 807, Taiwan, ROC d Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung city 807, Taiwan, ROC b
a r t i c l e
i n f o
Article history: Received 6 July 2016 Received in revised form 27 September 2016 Accepted 28 September 2016 Available online 28 September 2016 Keywords: Resveratrol Topical application Transdermal amount Deposition amount Skin irritation Stability
a b s t r a c t The aim of this study was to develop and assess the potential of nanostructured emulsion carriers for resveratrol topical application. Different compositions of resveratrol-loaded nanostructured emulsions were prepared using different types and amounts of surfactants and oily phases (isopropyl myristate and caproyl 90). The produced nanostructured emulsions were within the nanosized range 23.4–422.2 nm with low viscosity range 2.15–17.53 cps. The transdermal amount and deposition amount in the skin after 24 applications of resveratrol-loaded nanostructured emulsion were significantly increased about 896.2-fold and 10.2-fold respectively, when compared to the drug-saturated solution-treated group. Nanostructured emulsion containing IPM and low amounts of mixed surfactant of Tween80/Span 20 showed highest permeation capacity. In vivo study showed that the plasma concentration of resveratrol could be maintained at high levels for a long time after topical application of drug-loaded nanostructured emulsion. The histological examination demonstrated that the free drug- and drug-loaded nanostructured emulsion demonstrated considerably less irritation than the standard irritation group (0.8% paraformaldehyde-treated). The residual contents of resveratrol in the tested formulations after 3 months of storage at 25 ◦ C and 40 ◦ C were more than 99.97 ± 3.90%. The results of present work confirm the high potential of nanostructured emulsion as carriers for drug topical application. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Resveratrol (trans-3,4,5-trihydroxystilbene), a component of a variety of common edible plants including grapes, nuts, fruits, and red wine, is a phytoestrogen [1,2]. Numerous studies have proven resveratrol possesses various systemic and local pharmacologic effects. For the systemic effects, resveratrol can prevent or slow the progression of various illnesses, including cardiovascular disease, cancer, and ischemic injuries [3–9]. For the skin topical protection effects, resveratrol can inhibit proliferation of keratinocytes (skin epidermal cells) and stimulate their differentiation [10,11]. It was also proven to inhibit melanin production by skin cells and to alleviate skin irritation that may be caused by alpha-hydroxy acids. Hence, it can be used to improve the appearance of wrinkled, flaky, lined, dry, aged or photo-damaged skin and ameliorate
∗ Corresponding author. E-mail address: [email protected] (P.-C. Wu). http://dx.doi.org/10.1016/j.colsurfb.2016.09.045 0927-7765/© 2016 Elsevier B.V. All rights reserved.
skin thickness, elasticity, flexibility, radiance, glow and plumpness [12–14]. Unfortunately, resveratrol is a poor water-soluble compound with lower bioavailability and short half-life owing to extensive metabolism [2], leading to an irrelevant in vivo effect with oral administration compared to its powerful in vitro efficacy. Topical application is a much less invasive, more comfortable and convenient route of administration, particularly for therapeutic substances with skin protection effect; therefore, the topically applied method has been shown as a potential administration route for resveratrol. Numerous dosage forms have been widely investigated for topical delivery of resveratrol, such as solutions, gels, emulsions, and liposomes [15–21]. Nanostructured dosage forms such as liposomes, microemulsions, niosomes and nanoparticles appear to be attractive strategies for facilitating drug permeation capability through the skin in recent years, because they can offer a better chance for adherence to biological membranes transporting therapeutic substances in a controlled manner. Nanostructured carriers are also capable of increasing drug-loading capacity, sustaining drug
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release, and demonstrate promising drug tissue-specific targeting, [15–20,22–26]. The present study aimed to develop a suitable nanostructured emulsion for resveratrol topical applications. With this purpose, different nanostructured emulsions were prepared by using different surfactants and oil phases. The physicochemical properties, in vitro permeation and deposition study, stability, skin irritation and in vivo pharmacokinetic study of drug-loaded formulations were conducted to confirm the potential of nanostructure emulsion as carriers for resveratrol topical applications. 2. Materials and methods 2.1. Materials Resveratrol (racemic form), naringenin and sorbitan monolaurate (Span 20) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Caproyl 90 was purchased from Gattefosse (Nanterre, Frence). Polyoxyethylene sorbitan monooleate (Tween 80), and propylene glycol (PG) was from J. T. Baker (Phillipsburg, USA). Pluronic® L44 and paraformaldehyde were purchased from SigmaAldrich (St. Louis, Missouri, USA). Polyoxyl 23 lauryl ether (Brij 35) was obtained from Acros Organic (Pennsylvania, USA). Isopropyl myristate (IPM) was purchased from Merck Chemicals (Darmstadt, Germany). All other chemicals and solvents were of analytical reagent grade. 2.2. Animals Sprague-Dawley rats weighing 250–300 g were purchased from BioLASCO Taiwan CO., Ltd. The hair of the abdominal skin was shaved with an electric clipper (Thrive® , Japan), then the abdominal skin was excised. The subcutaneous fat was removed. The skin was cleaned and examined for integrity. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University (Kaohsiung, Taiwan). The committee confirmed that the permeation experiment followed the guidelines as set forth by the Guide for Laboratory Fact Lines and Care. 2.3. Preparation of resveratrol-loaded nanostructured emulsions Caproyl 90 or IPM were used as oil phases, propylene glycol and ethanol were used as cosurfactant, and Tween 80, Span 20, Brij 35 and L44 were used as surfactant. Oil phase and surfactant were mixed well by a vortex at room temperature. The cosurfactant was dissolved in aqueous phase, and then was slowly added to the previous mixture by a vortex for 5 min. After the clarity and transparency of microemulsion were formed, resveratrol of 0.5% was dissolved in the nanostructured emulsions by a horizontal shaker for 10 min. All resveratrol-loaded nanostructured emulsions showed clarity without any precipitate. 2.4. Determination of viscosity Viscosities of resveratrol-loaded nanostructured emulsions were determined using a Brookfield, Model LVDV-II, cone-plate of viscometer (USA). Tested samples of 0.5 mL were placed into the cone-plate, and heated to 37 ◦ C by a thermostatic pump for three mins. Readings were recorded 30 s after measurement was made at a rate of 150 rpm. 2.5. Determination of average droplet size and polydispersity index The average droplet size and polydispersity index (PI) of resveratrol-loaded nanostructured emulsions were measured at
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25 ◦ C by Malvern, Zetasizer 3000HSA, using a computerized inspection system (UK) with a scattering angle of 90◦ . Testing samples of three milliliters were transferred in a cuvette and placed in the scattering chamber. The average droplet size and polydispersity index (PI) were obtained by software. Each tested sample was measured in triplicate. 2.6. In vitro skin permeation and deposition studies The rate and extent of skin permeation of resveratrol from tested nanostructured emulsions were determined using a modified diffusion cell fitted with excised SD rat skin. The skin with effective area of 3.46 cm2 was placed on the receiver chambers with the SC facing upward to the donor cell. The receptor cell was filled with pH 7.4 phosphate buffer saline containing 20% PG and 20% ethanol to maintain sink condition (solubility of 5.5 ± 0.1 mg/mL). The receptor cell was maintained at 37 ± 0.5 ◦ C by thermostatic pump and constantly stirred at 600 rpm throughout the permeation studies. At predetermined intervals, 1 mL of receptor buffer was withdrawn via the sampling port and equal volume of fresh buffer was replaced. The concentration of resveratrol was determined by a modified HPLC method [27,28]. After the 24-h experimental period, the donor cell was carefully removed and the drug-exposed skin was washed with deionized water three times to remove the residual resveratrol on the skin surface. The drug-exposed skin was dried with cotton wool and then the SC was removed from the rest of the treated skin by the tape-stripping method with 3 M, Scotch Book Tape no. 845, adhesive cellophane tapes (St Paul, MN, USA). The stripping tapes were placed in glass tubes. The epidermis layer was separated from the dermis layer by a heat method (at 80 ◦ C for 3 min) [29–31]. The residual amounts of drug in SC, epidermis and dermis layers were then extracted by methanol and determined by a modified HPLC. 2.7. Pharmacokinetics study Male SD rats weighing 250–300 g were used in the experiment. The hair on the abdominal area of rats was shaved using a hair clipper and an electric razor one day prior to the pharmacokinetics experiment. Rats were anesthetized throughout the whole investigation by intraperitoneally administered carbamic acid ethyl ester solution of 750 mg/kg and secured on their backs. The tested formulation (about 67 mg/kg) was applied to the shaven abdomen. To prove the sustained release property of tested formulation by topical application, resveratrol suspension of 30 mg/kg was orally administered by oral gavage. Blood was collected from the jugular vein at predetermined time intervals. Blood sample was centrifuged at 4 ◦ C and 1200g for 10 min. Plasma sample was extracted by liquid–liquid extraction using 1 mL of ethyl acetate. The sample was vortex-mixed for 2 min and centrifuged at 12,000g for 10 min. The supernatant was evaporated by the centrifugal vaporizer under reduce pressure and reconstituted with 100 L of methanol. The drug concentration was analyzed by a modified HPLC. 2.8. Chromatographic condition The Hitachi L-7100 series HPLC system was equipped with a prepacked LiChroCART® RP-18 column (125 4 mm I.D., particle size 5 m). The detect wave was set at 305 nm. The mobile phase of 2.0% acetic acid containing 27% acetonitrile was delivered at a rate of 1.0 mL/min. Naringenin was used as internal standard. The analysis method was successfully validated with coefficient of variation of 3.3%, relative error of 6.67% and a determination coefficient of 0.9995. The limit of quantitation was 0.01 g/mL.
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2.9. Skin irritation determination The safety of the tested formulations was evaluated by naked eye observation and histological microscopy. The animals were divided into four groups including negative control groups (untreated group), positive control group (treated with 0.8% paraformaldehyde) and tested groups (treated with drug-free and drug-loaded tested formulation) [32,33]. The hair on the abdominal region of the SD rat was shaved carefully with an electrical shaver one day before the drug administration. A glass ring with area of 3.46 cm2 was adhered to the skin with glue, then 0.5 mL of tested formulations were loaded and occluded by parafilm. After 24-h drug exposure, the rats were sacrificed and specimens of the exposed skin areas taken for histological examination. The skin pieces were immediately fixed in 4% buffered formaldehyde solution at least for 24 h and then the skin pieces were embedded in paraffin. The skin sample was sliced transversely of 20 m. The sliced section was rehydrated stepwise, stained with hematoxylineosin, and then observed using a Nikon microscope for an optical microscopic observation (Nikon Eclipse Ci, Tokyo, Japan).
were measured using one-way ANOVA by Winks software (Texasoft, Cedar Hill, TX, USA), followed by the Newman–Keuls test for comparisons between groups. The significance of differences among groups was set at p < 0.05. The significance was taken as 95% (probability < 0.05). 3. Results and discussion 3.1. Physicochemical characteristics Different types of surfactant such as Brij 35, L44 and Tween 80 were used to prepare nanostructured emulsions with oil phase (caproyl 90), aqueous phase and surfactant at ratio of 5, 75 and 20. The average droplet size of formulation with Brij 35, L44 and Tween 80 was 29.8 ± 6.9, 23.4 ± 3.0, and 31.2 ± 2.0 nm respectively. The viscosities of these formulations were 13.3 ± 3.7, 6.1 ± 1.8 and 17.5 ± 4.5 cps respectively. The results showed that the drugloaded nanostructured emulsions could be formed by using these three surfactants, and that the physical properties of drug-loaded formulations were affected by the incorporated types of surfactants [23,36,37].
2.10. Stability determination The stability of resveratrol-loaded nanostructured emulsions was evaluated by thermodynamic stability tests and accelerated stability test. For the thermodynamic stability, the sample was centrifuged at 3500 rpm for 30 min and 10,000 rpm for 10 min as well as being stored at different temperature cycles of 4 ◦ C and 45 ◦ C for 3 times; each temperature storage period was not less than 48 h [34,35]. The phase separation, creaming and cracking were evaluated. For the accelerated stability test, the resveratrol-loaded formulation was stored in dark-brown bottles for protection from light and placed at 25 ± 2 ◦ C/60 ± 5%RH and 40 ± 2 ◦ C/75 ± 5%RH for three months. The physicochemical characteristics of the tested formulation such as phase separation, clarity, precipitation of drug, color change and residual drug content after 3 months storage were evaluated. 2.11. Data analysis All data were presented as mean ± standard deviation (S.D.). To compare the difference of various groups, the statistical differences
3.2. In vitro skin permeation and deposition The cumulative amount of resveratrol in receptor fluid (transdermal amount) and deposition amount in SC, epidermis and dermis layer after 24 h application of resveratrol – loaded formulations are presented in Fig. 1. The results showed that resveratrol could deposit in different skin layers, particularly in the epidermis layer, after 24 h of treatment, which might be resulting in a reservoir effect [38–40]. The resveratrol-saturated aqueous solution was used as the control group to assess the potential of drug-loaded nanostructured emulsions. The transdermal amount (Q24h ) and total deposition amount in skin (D24h ) at 24th h application of tested formulation was 0.79 ± 0.78 and 4.26 ± 0.58 g/cm2 respectively, which was quite low, indicating that resveratrol is not easily transported through the skin. The result was in agreement with a previous study [20]. When using nanostructured emulsions containing caproyl 90 and different types of surfactants of Brij 35, L44 and Tween 80 as vehicle, the Q24h was non-detected and the D24h was significantly increased about 2.19 ∼ 3.25-fold (Fig. 1). The formulation containing Tween 80 showed a higher enhancement effect than that of the formulation containing L44. The result
Fig. 1. The transdermal amount and deposition amount in skin after 24 h treated with resveratrol saturated aqueous solution and drug-loaded nanostructured emulsions with caproyl 90 and different types of surfactant.
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showed that nanostructured emulsions could provide potential skin reservoir effect for resveratrol [32–34]. The emulsion system could be used as a promising vehicle for skin protection dosage of resveratrol. The enhancement mechanism of the nanostructured formulations might include, but is not limited to (1) the effect of adsorption and fusion of resveratrol-loaded vesicles onto the surface of the SC resulting in a high thermodynamic activity gradient of the drug–SC interface; and (2) the vesicles might impair barrier function of the SC leading to significant changes in drug permeation kinetics [41,42]. IPM is a potential penetration enhancer in transdermal formulations and is widely used as an oil phase to prepare microemulsions to enhance drug permeation capacity through the skin [23,43,44]. Hence, the oil phase (Caproyl 90) of the tested formulation was replaced by IPM. Unfortunately, the translucent formulation could not be formed. This phenomenon might be due to the hydrophiliclipophilic balance (HLB) of Tween 80 (15) and Brij 35 (16.9) being higher than the required HLB value (about 11.1) of IPM. Stable emulsion only formed at the HLB of the surfactant close to the required HLB of used oily substance [22,24,45]. Therefore, the mixed surfactant of Tween 80/Span 20 with HLB value of 11.16 was used to prepare a nanostructured emulsion containing IPM. As expected, a translucent formulation with average droplet size of 277 nm and viscosity of 5.82 cps was formed. The in vitro skin permeation and deposition study was conducted. The Q24h and D24h were 276.0 ± 42.3 and 29.4 ± 7.7 g/cm2 respectively, which was obviously higher than that of the formulation containing oily phase of caproyl 90. The result showed that the composition of formulation was an important influence factor for drug transportation through skin [32–34]. Previous studies [23,44] pointed out that microemulsions containing lower amounts of surfactant will result in higher thermodynamic activity of drug in the microemulsion and lead to a higher drug permeation rate. Hence, the surfactant amount of formulation was decreased from 20% to 15%. The average droplet size increased from 277.6 to 422.2 nm, and viscosity decreased from 5.82 to 2.15 cps, when emulsions had lower amounts of surfactant. This might be due to higher amounts of surfactant forming more stable emulsions with small size and higher viscosity. Although the droplet size of formulation with low amount of surfactant significantly increased, but the size still smaller than the desirable particle size of drug-carriers to penetrate the skin epithelium of ≤500 nm [46]. As expect, the Q24h and D24h of emulsion with 15% surfactant was further increased about 2.57-fold and 1.48-fold respectively
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(Fig. 2), indicating that average droplet size was not a superior factor in this study, whereas drug thermodynamic activity in the formulation might be a more important influencing factor. The result also demonstrated that appropriate nanostructured emulsion formulation might provide percutaneous absorption effect (Q24h increased about 896.2-fold) and local skin protection effect (D24h increased about 10.2-fold) after topical application of resveratrol. Fig. 3 shows the D24h after different application times of tested formulation containing 15% cosurfactant and saturated aqueous solution. It can be seen that using nanostructured emulsion as vehicle, the deposition amounts at different application times were significantly increased (p < 0.05). The drug deposition content in skin can be detected at 6 h after saturated aqueous solution application, whereas the drug level can be measured after 1 h with nanostructured emulsion applied. For drug-loaded nanostructured emulsion treatment, the deposition amount increased with application time to a maximum at 6 h. The D24h was increased about 10-fold when compared to the saturated aqueous solution-treated group. The result of in vitro study demonstrated that the used nanostructured formulation as carrier could accelerate the drug permeation rate and extent through skin, and enhance the deposit amount in skin, which confirms the potential of nanostructured formulation for resveratrol topical application. 3.3. In vivo pharmacokinetic study The pharmacokinetic studies of the topical application of drug-loaded nanostructured emulsion and oral administration of resveratrol dissolved in 60% PG were conducted. As shown in Fig. 4, the plasma concentration was rapidly decreased because of being extensively metabolized and excreted [27,47,48], whereas the plasma concentration of resveratrol could be detected 2 h after the tested formulation was applied, and maintained high levels for a long time after topical application of drug-loaded nanostructured emulsion. The relative bioavailability of topical administration was significantly increased as compared to oral administration. 3.4. Skin irritation Formulations might elicit primary skin irritation. The histological examination of skin specimen was conducted to assess the potential irritant effects of the developed tested formulation. The untreated rat skin and that treated with 0.8% paraformaldehyde
Fig. 2. The transdermal amount and deposition amount in skin after 24 h treated with resveratrol saturated aqueous solution (control group) and drug-loaded nanostructured emulsions with IPM and different amount of Tween 80/Span 20.
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Fig. 3. The deposition amount in skin after different application time of resveratrol saturated aqueous solution and drug-loaded nanostructured emulsion.
Fig. 4. Mean plasma concentration-time curve (mean ± S.D., n = 3) of resveratrol after oral an aqueous solution and topical applied the tested formulation.
solution were used as negative control and standard irritant respectively [34,49]. As shown in Fig. 5a, the untreated skin showed well-defined epidermal and dermal layers. The paraformaldehydetreated group showed a slight edema exfoliation of the SC, and loosely textured collagen in the dermis (Fig. 5b). Non-significant edema and erythema were found in drug-free and drug-loaded nanostructured emulsion-treated skin samples (Fig. 5c and d), when compared to the negative control group, indicating that the experimental nanostructured emulsions appeared to be a safe carrier for transdermal delivery. 3.5. Stability The selected drug-loaded formulation was subject to thermodynamic stability tests, including centrifugation test and heating-cooling cycles test. No phase separation, creaming, precip-
itation or liquefaction was found, indicating that the drug-loaded formulation possesses good physical stability. The results agreed with previous reports [34,50] which pointed out that microemulsion with small droplet size and very low interfacial tension made the formulations thermodynamically stable. After 3 months of storage at 25 ◦ C/60% RH and 40 ◦ C/75%RH, the drug-loaded formulation showed no obvious change, and no drug crystal was found. The viscosities and average droplet sizes were 4.86 ± 0.87, 5.41 ± 0.61 and 5.12 ± 0.23 cps, and 577.1 ± 98.21, 585.6 ± 91.31 and 581.3 ± 83.2 nm respectively, at initial and 3 months of storage at 25 and 40 ◦ C. The viscosity and droplet size showed non-significant difference. The residual drug contents of drug-loaded formulations were 99.97 ± 3.48% and 100.76 ± 1.21% at 25 ◦ C and 40 ◦ C storage respectively, indicating that the experimental formulations were stable.
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Fig. 5. The Photomicrographs of a rat abdominal skin section, viewed under a light microscope (10 × 10). (a) Untreated-skin, (b) Paraformaldehyde treated, (c) Drug-free formulation treated, (d) Drug-loaded formulation treated.
4. Conclusions In this work, the resveratrol-loaded nanostructured emulsion showed its capability to go through the skin barrier, particularly when prepared by IPM. The Q24h and D24h significantly increased about 896.2-fold and 10.2-fold respectively. The deposition drug level could be detected after 1 h application of drug-loaded nanostructured emulsion, which is remarkably shorter than the saturated aqueous solution group of 6 h. In vivo pharmacokinetic study, the plasma concentration of resveratrol was maintained at high levels for a long time after topical application. The resveratrol-loaded nanostructured emulsion was also stable after three months of storage at 25 and 40 ◦ C. The drug-free and drug-loaded nanostructured emulsion showed less skin irritation than that of the standard irritant group. The result confirmed nanostructured emulsion was a potential vehicle for resveratrol topical application. Conflict of interests The authors declare that there is no conflict of interest regarding the publication of this paper. Acknowledgment This work was supported by grants from the Ministry of Science and Technology, Taiwan (NSC 102-2320-B-037-006-MY2 and MOST 105-2632-B-037-003). References [1] J.A. Baur, K.J. Pearson, N.L. Price, H.A. Jamieson, C. Lerin, A. Kalra, V.V. Prabhu, J.S. Allard, G. Lopez-Lluch, K. Lewis, P.J. Pistell, S. Poosala, K.G. Becker, O. Boss, D. Gwinn, M. Wang, S. Ramaswamy, K.W. Fishbein, R.G. Spencer, E.G. Lakatta, D. Le Couteur, R.J. Shaw, P. Navas, P. Puigserver, D.K. Ingram, R. de Cabo, D.A. Sinclair, Resveratrol improves health and survival of mice on a high-calorie diet, Nature 444 (2006) 337–342. [2] J.A. Baur, D.A. Sinclair, Therapeutic potential of resveratrol: the in vivo evidence, Nat. Rev. Drug Discov. 5 (2006) 493–506. [3] M. Nassiri-Asl, H. Hosseinzadeh, Review of the pharmacological effects of Vitis vinifera (Grape) and its bioactive constituents: an update, Phytother. Res.: PTR 30 (2016) 1392–1403. [4] D.Y. Yeh, Y.H. Fu, Y.C. Yang, J.J. Wang, Resveratrol alleviates lung ischemia and reperfusion-induced pulmonary capillary injury through modulating pulmonary mitochondrial metabolism, Transplant. Proc. 46 (2014) 1131–1134. [5] H. Ding, X. Xu, X. Qin, C. Yang, Q. Feng, Resveratrol promotes differentiation of mouse embryonic stem cells to cardiomyocytes, Cardiovasc. Ther. 34 (2016) 283–289. [6] C.Y. Huang, W.J. Ting, C.Y. Huang, J.Y. Yang, W.T. Lin, Resveratrol attenuated hydrogen peroxide-induced myocardial apoptosis by autophagic flux, Food Nutr. Res. 60 (2016) 30511. [7] L. Gliemann, M. Nyberg, Y. Hellsten, Effects of exercise training and resveratrol on vascular health in aging, Free Radical Biol. Med. 98 (2016) 165–176. [8] P. Jaisamut, K. Wiwattanawongsa, R. Wiwattanapatapee, A novel self-microemulsifying system for the simultaneous delivery and enhanced oral absorption of curcumin and resveratrol, Planta Med. (2016), Epub ahead of print DOI: 10.1055/s-0042-108734.
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