Preparation of a Capsaicin-Loaded Nanoemulsion for Improving Skin

Jan 7, 2014 - Capsaicin o/w nanoemulsions with enhanced skin permeation were successfully prepared by controlling the ratios of the surfactant mixture...
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Preparation of a Capsaicin-Loaded Nanoemulsion for Improving Skin Penetration Jee Hye Kim,†,‡ Jung A Ko,†,‡ Jun Tae Kim,§ Dong Su Cha,∥ Jin Hun Cho,⊥ Hyun Jin Park,*,† and Gye Hwa Shin*,† †

College of Life Sciences & Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Korea Department of Food Science and Technology, Keimyung University, Daegu 704-701, Korea ∥ Functional Food Research Center, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Korea ⊥ Coway Cosmetics R&D Center, Gasan-dong, Geumcheon-gu, Seoul 153-803, Korea §

ABSTRACT: Capsaicin o/w nanoemulsions with enhanced skin permeation were successfully prepared by controlling the ratios of the surfactant mixtures, oleoresin capsicum as the oil phase, and aqueous phase. Oleoresin capsicum contains 22.67 mg/g of capsaicin, which is an active and oil-soluble ingredient. Nonionic surfactants, Tween 80 and Span 80, were used to optimize the weight ratio of surfactant mixtures (85.98:14.02) by calculating the hydrophile−lipophile balance (HLB) value. The optimal processing conditions for stable nanoemulsions were investigated by using a ternary phase diagram. The mean droplet size of nanoemulsions ranged from 20 to 62 nm. Skin permeation studies were performed using a Franz diffusion cell. The permeation profiles and confocal laser scanning microscopy (CLSM) images supported that capsaicin nanoemulsion could well permeate all skin layers from the stratum corneum to the dermis. The selected nanoemulsions showed great potential as transdermal delivery carriers for enhancing the permeation of core materials. KEYWORDS: nanoemulsion, capsaicin, skin penetration, confocal microscopy, HLB value

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INTRODUCTION Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the primary active ingredient of chili pepper, is a pungent and lipid soluble compound.1 Capsaicin has been extensively used as a therapeutic agent for various diseases such as cancer, inflammation, cardiovascular disease, stroke, neurodegenerative disorder, and dermatological disease due to its excellent pharmacological activity.2−6 However, capsaicin has shown two contradictory physiological effects. First, capsaicin stimulates the nervous system and induces intense pain in mammalian tissues, but its strong pungency has limited its clinical applications. Second, capsaicin has great antinociceptive and anti-inflammatory effects even if it excites the desensitization of nociceptors by depleting the neurotransmitter substance P and other sensory neuropeptides.7 Especially, transdermal delivery system of capsaicin has received considerable attention in the pharmaceutical and medical fields due to the antinociceptive and anti-inflammatory functions of capsaicin.8,9 Compared to an oral drug delivery system, a transdermal delivery system provides various advantages such as avoidance of hepatic first-pass metabolism, reduction of side effects, and improvement in drug solubility.10 The challenge of skin penetration is to overcome the stratum corneum, (SC) which is highly hydrophobic and rich in proteins. Hydrophobic drugs, however, are not favorable to hydrophilic viable epidermis and dermis layer in deeper stage.11 To date, various transdermal delivery systems have been investigated for improving transdermal transport through the epidermal barrier. In order to enhance the skin penetration of drugs, nanocarrier delivery systems have been extensively investigated in various formulations such as nanoparticles, solid © 2014 American Chemical Society

lipid nanoparticle (SLN), nanostructured lipid carrier (NLC), liposomes, microemulsions, and noisomes.12−18 For improved transdermal delivery, carrier materials should be biocompatible, biodegradable, and easily excreted from the body as well as capable of sustaining the active core ingredient until reaching the target site without causing skin irritation during skin penetration. As an effective transdermal carrier systems, nanoemulsion appears an attractive and competitive system due to many benefits such as easy manufacturing, small droplet size (20−200 nm), high thermodynamic stability, and enhanced solubilization for hydrophobic ingredients.10,11 It has been shown that the active ingredients in an emulsion can enhance skin penetration through the large surface area of the emulsion system, the low surface tension of the whole system, and the low interfacial tension of the o/w droplets.19 Because of their small size (less than 100 nm), nanoemulsions can easily penetrate the skin surface.10 The aim of this study is to investigate the optimum condition of capsaicin-loaded nanoemulsion formulations for topical application. In order to compare the quantitative permeability of the capsaicin nanoemulsion with that of a capsaicin-free ethanol solution, skin permeation studies were conducted using a synthetic membrane similar to human skin. In addition, ex vivo permeation study was performed using pig skins to demonstrate that capsaicin can penetrate through the whole pig skin layers, which consist of the stratum corneum, epidermis, Received: Revised: Accepted: Published: 725

September 20, 2013 November 27, 2013 January 7, 2014 January 7, 2014 dx.doi.org/10.1021/jf404220n | J. Agric. Food Chem. 2014, 62, 725−732

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found to maintain isotropic, transparent properties during the observation period. Measurement of Droplet Size, Polydispersity Index (PDI), and Zeta Potential. The mean droplet size, PDI, and zeta potential of nanoemulsions were measured using nano size analyzer (Malvern Zetasizer Nano ZS; Malvern Instruments, Malvern, U.K.). Three milliliters of capsaicin nanoemulsion diluted in 40 times Milli-Q water was added to polystyrene latex cells, and the mean droplet size, PDI, and zeta potential were measured at 25 °C with a detector angle of 90°, and wavelength of 633 nm. Each sample was measured at least three times, and the average values were used. Morphology of Nanoemulsions. The morphology of the nanoemulsions was investigated using a TECNAI G2 F30 transmission electron microscope (Philips-FEI, Eindhoven, Holland). For transmission electron microscopy (TEM) sample preparation, approximately 10 μL of the nanoemulsion was dropped onto carbon-coated grids for 30 s and the excess was drawn off with membrane filter paper. This step was performed three times. The grid was stained with uranyl acetate and dried overnight at room temperature. The images were obtained using TEM in the high angle annular dark field (HAADF) micrograph mode. Skin Penetration Experiments. A synthetic membrane (Strat-M) was used to mimic human skin. The skin penetration studies were performed using a vertical modified amber glass Franz diffusion cell (Daihan Labtech, Gyeonggi-do, South Korea) with an effective diffusion area of 2.27 cm2 and a receptor volume of 15.9 mL. The skin sample was sandwiched between the donor and receiver compartments, which were then securely fastened with a clip. The receiver compartment was filled with 20% ethanol in PBS solution and continuously stirred at 600 rpm at 37 ± 1 °C for 1 h to equilibrate the skin. After equilibration, 1 mL of the nanoemulsion or the capsaicin in a 40% ethanol aqueous solution (control) was dropped slowly on the donor compartment to cover the nanoemulsions on the skin surface. The receptor medium (1 mL) was withdrawn at fixed intervals (1, 2, 3, 6, 9, 12, and 24 h) and analyzed by HPLC to evaluate skin penetration. To fill the withdrawn amount, 1 mL of hydro ethanolic solution was added to the receptor medium. HPLC Analysis of the Cumulative Amount of the Nanoemulsion Penetrating the Skin. The capsaicin content was analyzed by reversed phase HPLC using the more sensitive fluorometric detector rather than the photodiode detector so that even a trace of capsaicin permeating the skin into the receptor solution could be detected.21 For capsaicin, the excitation wavelength was 270 nm and the emission wavelength was 330 nm. The HPLC system consisted of a model 2690 pump (Waters, Milford, MA, USA), a model 717 auto sampler (Waters), a fluorescence detector (Waters), and a Symmetry C18 column (100 Å, 5 μm, 4.6 mm × 150 mm, Waters). The mobile phase was an isocratic acetonitrile−water mixture (70:30 v/v), and the flow rate was 1 mL/min at room temperature. Permeation Data Analysis. The cumulative amount of capsaicin permeation per unit of skin surface area (Qt/S) was calculated using eq 2:22,23

and dermis. Confocal laser scanning microscopy (CLSM) images were examined to verify that the capsaicin-loaded nanoemulsion fully penetrated the whole skin layers in both in vitro and ex vivo permeation studies.

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MATERIALS AND METHODS

Chemicals. Span 80 and Tween 80, used as surfactant, were purchased from Samchun Pure Chemical (Seoul, Korea). Oleoresin capsicum was purchased in pungency ratings of 500,000 Scoville Heat Units (SHU) from EVESA (Madrid, Spain). Since oleoresin capsicum is an oil extraction from a fruit of Capsicum species and consists of mainly capsaicin, oleoresin capsicum was used as the oil phase. Strat-M membrane was purchased from Merck KGaA (Darmstadt, Germany), and Nile red was purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade methanol and acetonitrile were used for HPLC analysis. Skin. The flank area of a pig skin sample (700 μm thick as measured using dermatome) was obtained from Micropig (Medi Kinetics Co. Ltd., Pyeongtaek, Korea). All skin samples were prepared less than 1 mm thick to allow following confocal microscope study. The skin samples prepared were carefully washed with a phosphate buffered saline (PBS) solution, stored at −20 °C, and used within 1 week for the ex vivo skin permeation experiments. Capsaicin Contents in Oleoresin Capsicum. Oleoresin capsicum is an extract of chili pepper that contains capsaicin as a primary active ingredient. The accurate amount of capsaicin in the nanoemulsion was determined by measuring the capsaicin contents in oleoresin capsicum by high performance liquid chromatography (HPLC) consisting of a pump (model 2690, Waters, Milford, MA, USA), a photodiode array detector (model 996, Waters), and a Kromasil C18 column (250 mm × 4.6 mm; EKA Chemicals AB, Bohus, Sweden). The mobile phase was a mixture of isocratic methanol−water (70:30 v/v), and the flow rate was 1 mL/min at room temperature. The injection volume was 10 μL, and the column effluent was monitored at a wavelength of 280 nm for capsaicin. Preparation of Capsaicin Nanoemulsion. Capsaicin nanoemulsion was prepared using hot homogenization and high pressure homogenization. Tween 80 and Span 80 were mixed in various ratios, and the surfactant mixture was added to distilled water with magnetic stirring at 75 °C for 30 min. The surfactant and water mixing solution was added into oleoresin capsicum, which was preheated to 75 °C. The mixture of surfactant, water, and oil was homogenized using an Ultra-Turrax T 25 basic homogenizer (IKA-Werke, Staufen, Germany) at 12,000 rpm for 5 min, followed by size reduction using high pressure homogenization (model: M-110P Microfluidizer, Microfluidics, Newton, MA, USA) under 1,000 bar pressure. Screening of Hydrophile−Lipophile balance (HLB) Value. The HLB value of surfactant was screened to determine the best ratio of surfactant mixture.20 As nonionic surfactant, Span 80 (sorbitan monooleate, HLB value of 4.3) and Tween 80 (polyoxyethylene sorbitan monooleate, HLB value of 15) were chosen, and various mixtures of Span 80 and Tween 80 were prepared to determine the optimum ratio. The HLB values of the surfactant mixtures (HLBmix) were calculated using the weight fraction of Tween 80 and Span 80 according to the following equation:

HLBmix = fA HLBA + fB HLBB

t−1

Q t = VrCt +

∑ VsCi i=0

(2)

where Ct and Ci are the capsaicin concentrations of the receiver solution at each sampling time and for the ith sample, respectively, while Vr and Vs are the volumes of the receiver solution and the sample, respectively. The flux of capsaicin permeation at steady state (Jss) was calculated from the slope of the linear portion of the graph plotted between cumulative capsaicin permeation per unit of skin surface area (Qt/S) and time (t) as follows (eq 3):

(1)

where HLBA and HLBB are the HLB values for Tween 80 and Span 80, respectively, and fA and f B are the weight fractions of Tween 80 and Span 80, respectively. Phase separation and size distribution of the nanoemulsions were considered to determine the optimum condition. Ternary Phase Diagrams. To delineate ternary phase diagrams, the stability of the prepared formulations was monitored at room temperature for 1 month. The formulations, in which no phase separation, flocculation, and sedimentation were observed, were selected as stable nanoemulsions. In the selected formulations, the droplet size of nanoemulsions was not changed significantly over one month and the PDI value was also maintained less than 0.3. The formulations were

Jss =

ΔQ t Δt × S

(3)

The permeability coefficient (Kp) was calculated by dividing Jss by the initial concentration of capsaicin in the donor compartment (C0) as follows (eq 4): 726

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Jss C0

(4)

Enhancement ratio (Er) was calculated by dividing the Jss of the respective formulation with the Jss of the control formulation as follows (eq 5):

Er =

Jss of formulation Jss of control

(5)

CLSM Studies for Optical Images of ex Vivo Skin Penetration. Fluorescent dye (Nile red, 0.1% w/w) was incorporated in the selected nanoemulsions to study skin penetration using CLSM. Nile red could be mixed well with capsaicin due to its similar lipophilicity to capsaicin.32 The skin from the flank area of the pig was used for ex vivo skin permeation studies. The skin samples were thawed at room temperature and rehydrated in PBS solution for 30 min before the experiments. The skin permeation studies were performed for 24 h by using a vertical modified amber glass Franz diffusion cell as described above. After the ex vivo skin permeation experiments, the skin surface was gently rinsed with 50% ethanol and wiped. The skin was placed between a slide glass and a coverslip and examined using CLSM (LSM 5 Exciter, Carl Zeiss, Jena, Germany). The skin sample was scanned from the skin surface (0 μm) to a depth of 32 at 1 μm intervals. The skin was inspected under a 20× objective lens (EC Plan-NEOFLUAR 20x/0.50 M27). The detecting wavelength was 543 nm. CLSM images were recorded and further processed using LSM Image Browser software, version 4.2.0.121 (CarlZeiss Microimaging, Gottingen, GMBH). CLSM Images from Cryotome-Sectioned Skin. After the final sampling interval of 24 h for the ex vivo skin permeation experiments, the skin was washed with 50% ethanol and stored at −20 °C. The skin samples were cut into small pieces and sectioned vertically to a thickness of 30 μm by using a cryotome (Leica Cryostat CM 3505S, Wetzlar, Germany). The permeation of the fluorescence dye incorporated into the capsaicin nanoemulsion was qualitatively observed using CLSM (LSM 5 Exciter, Carl Zeiss, Jena, Germany). The skin was inspected under a 10× objective lens (EC Plan-NEOFLUAR 10x/0.30 M27). The images were obtained in differential interference contrast (DIC) mode, fluorescence mode, and merge mode. Statistical Analysis. All data were statistically analyzed using analysis of variance (ANOVA). Statistical Package for the Social Science (SPSS, Version 20.0, SPSS Inc., Chicago, IL, USA) was used for this analysis. Duncan’s multiple range tests were used to determine the statistical significance among the means at 95% significant level.

Figure 1. Droplet size and polydispersity index of nanoemulsions depending on the surfactant HLB value. Data are plotted as the mean ± standard deviation (n = 3).

size with the lowest PDI as shown in Figure 1. Nanoemulsions using higher hydrophilic surfactant showed smaller particle sizes than those using lower hydrophilic surfactant. According to our screening of the HLB value for oleoresin capsicum, the weight ratio of Tween 80 to Span 80 was fixed at 85.98:14.02. Ternary Phase Diagram. To determine the best formulation of capsaicin-loaded nanoemulsion, we constructed a ternary phase diagram that consisted of various ratios of water, oleoresin capsicum, and a fixed surfactant mixture of Tween 80 and Span 80 at a weight ratio of 85.98:14.02 (surfactantmix), respectively. In Figure 2, the area I indicates the optimal process conditions for fine and uniform sized nanoemulsions. The droplet size and zeta potential of nanoemulsions ranged from 20.26 to 62.98 nm and from −19.9 to −8.8 mV, respectively. Any phase separation was not observed until 30 days of storage for this region. Table 1 showed the formulations and characteristics such as droplet size, PDI, and zeta potential of nanoemulsions indicated in the area I. The droplet size of nanoemulsions was dependent on the ratio of oil to surfactant. In most case, droplet size of nanoemulsions decreased with lower oil to surfactant ratio. When the ratio of oil to surfactant was 1:1, the droplet size was around 63 to 64 nm and not statistically significant (P < 0.05) while the droplet size was around 20 nm when the ratio of oil to surfactant was 1:3. In contrast, area II nanoemulsions were not formed homogeneously and showed high PDI values (>0.3). For the remainder of the regions, immediate phase separation was obserbed. Our observations indicated that nanoemulsion regions were not identified when the aqueous phase was below 50%. The optimal nanoemulsion formulations were found to be in the range of more than 50% of water, less than 18% of oleoresin capsicum, and 15.4%−33.3% of surfactantmix with treatment of a high pressure homogenization. Optimal Formulation and Stability Study of the Nanoemulsions. Based on the HLB value and the phase study, the nanoemulsion formulations that had oil/surfactantmix ratios of 1:1, 1:1.3, 1:1.5, 1:2, and 1:3 were selected for the following penetration studies. Figure 3 represents the profile for the change in droplet size and PDI value of nanoemulsion formulations (NE-1 to NE-13) in which the oil and surfactantmix ratio ranged from 1:1 to 1:3. The formulations of the selected nanoemulsions were stable and showed a controlled droplet size ranging from 20.26 to 62.98 nm. As the surfactant concentration increased, the droplet size tended to become smaller, but PDI

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RESULTS AND DISCUSSION Screening of Optimal Surfactant Mixtures. Before the screening of surfactant mixtures, the concentration of capsaicin in oleoresin capsicum was measured by HPLC. Oleoresin capsicum contains 22.67 mg/g of capsaicin (data not shown). The selection of the best emulsifier is really crucial to enhance the emulsion stability. The ratios of nonionic surfactant mixtures were studied to determine the most stable and size controllable nanoemulsion with a droplet size optimal for efficient topical delivery of capsaicin. In this experiment, various ratios of Span 80 (HLB value 4.3) and Tween 80 (HLB value 15) were selected to prepare the nonionic surfactant mixtures. The optimal HLBmix was determined when the nanoemulsion with a surfactant mixture having an appropriate HLB value also showed the smallest droplet size and a low PDI (less than 0.3). The droplet size distribution and phase separation were also considered. Results from a previous study suggested that surfactants with higher HBL values formed more stable o/w emulsion than surfactants with lower HLB values; thus, HLB values ranging from 9 to 15 were chosen to screen the optimum condition. We selected an HLB value of 13.5 for oleoresin capsicum because the nanoemulsion showed the smallest droplet 727

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Figure 2. Ternary phase diagram of nanoemulsions composed of oleoresin capsicum, the mixed surfactant (Tween 80/Span 80), and aqueous phase. Area I depicts the conditions in which fine and homogeneous nanoemulsions could be formed.

Table 1. Characteristics of the Nanoemulsion Formulationsa mixing ratio form no. NE-1 NE-2 NE-3 NE-4 NE-5 NE-6 NE-7 NE-8 NE-9 NE-10 NE-11 NE-12 NE-13

b

oil:surfactant 1:1 1:1.5

1:2

1:3

composition (% w/w) water

oil

surfactant

water

4 5 3 4 5 7 3 3.5 4 5 6 7 6

16.7 14.3 18.2 15.4 13.3 10.5 16.7 15.4 14.3 12.5 11.1 10.0 10.0

16.7 14.3 27.3 23.1 20.0 15.8 33.3 30.8 28.6 25.0 22.2 20.0 30.0

66.7 71.4 54.5 61.5 66.7 73.7 50.0 53.8 57.1 62.5 66.7 70.0 60.0

droplet size (nm) 62.98 64.27 48.20 47.16 48.31 41.71 31.70 34.56 30.92 25.13 23.46 24.62 20.26

± ± ± ± ± ± ± ± ± ± ± ± ±

0.49 0.10 0.80 0.15 1.64 0.76 4.27 2.68 2.21 2.40 0.10 0.65 0.43

ec e d d d c b b b a a a a

PDI 0.26 0.27 0.25 0.23 0.26 0.25 0.22 0.21 0.22 0.25 0.27 0.23 0.27

± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.04 0.01 0.00 0.00 0.02 0.01 0.00 0.01 0.01 0.03 0.02

zeta potential (mV) −15.0 −16.8 −12.7 −11.0 −9.2 −8.8 −18.8 −16.3 −11.7 −19.9 −16.9 −10.7 −12.7

± ± ± ± ± ± ± ± ± ± ± ± ±

1.48 2.17 0.44 0.87 1.46 1.02 3.70 1.03 2.15 4.20 0.82 0.47 2.55

abcd abc bcd cd d d ab abc cd a abc cd bcd

Data are plotted as the mean ± standard deviation (n = 3). bFormulation number. cDifferent letters indicate a significant difference at P < 0.05 by Duncan’s multiple range test. a

and zeta potential were not correlated with the amount of surfactant. A smaller droplet size might have a larger the surface area, so that more surfactant would be required to cover particles.24 When we fixed the ratio of oil and surfactant, the droplet size of nanoemulsion was decreased by adding more aqueous phase. Morphology of Capsaicin-Loaded Nanoemulsions. Figure 4 shows the TEM images of the nanoemulsions (NE-13). The droplet size of NE-13 was approximately 20 nm in TEM images, and this was consistent with our previous results obtained by using a size analyzer. The droplets were spherical and showed a uniform emulsion droplet distribution. Penetration Profiles of Different Formulation of Nanoemulsion. One of the main barriers for skin penetration is the lipophilic stratum corneum (SC) layer. It is well-known that it is difficult for hydrophilic substances and high molecular

weight vesicles to penetrate the SC. On the other hand, hydrophobic substances easily get into the SC layer but they also have some limitation to penetrate the next epidermis layer which has a hydrophilic feature.25 Therefore, it needs to be delivered in both hydrophilic and hydrophobic barriers with special formulation vesicles to penetrate all layers of the skin. The effect of different compositions of nanoemulsions on skin permeation was investigated using the synthetic membrane that is considered the standard model for in vitro transdermal diffusion studies replacing human or animal skin. Reproducibility of results is enhanced by using this synthetic membrane as compared with using animal skin. Figure 5 shows the permeation profiles of capsaicin-loaded nanoemulsions and a control sample. The concentration of capsaicin in nanoemulsion ranged from 2.08 to 3.45 mg/g whereas the capsaicin concentration of control was 2.64 mg/g. All nanoemulsion samples continuously 728

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Figure 3. Droplet size profiles of nanoemulsion formulations for 30 days. Data are plotted as the mean ± standard deviation (n = 3).

sampling frequency, experiment duration, type of membrane sample, or membrane materials.24 It has been reported that infrequent sampling increases drug concentration in the receptor chamber and sink condition ( 0.05) compared to those of the control except NE-1 as shown in Table 2. The enhancement ratio (Er) of the nanoemulsions ranged from 0.849 for NE-13 to 1.658 for NE-1. The enhancement ratio of the nanoemulsions was statistically significant (P < 0.05) compared to that of the control except NE-13. The enhancement ratios of the nanoemulsions were also higher than that of the control sample, except for NE-13. It has been reported that the manner of skin penetration depends on various physiological properties including size, composition, and surface charge of the vesicles, the HLB value, and the type of surfactant, as well as the presence of a penetration enhancer.27 In general, the skin penetration should be fast and higher with smaller droplet size. However, the highest penetration flux was obtained with NE-1 containing larger mean droplet size of 62.98 nm and the lowest penetration flux was obtained with NE-13 containing smaller droplet size of 20.26 nm. The skin penetration might be highly dependent on the amount of surfactant because thermodynamic activity of core in nanoemulsion increased at the lower concentration of surfactant.28 NE-1 contains the least amount of surfactant in the formulations and showed the highest penetration flux. Based on the permeation profiles, NE-1 showed the best nanoemulsion

Figure 4. The morphology of the capsaicin-loaded nanoemulsion (NE-13) with TEM micrograph in the high-angle annular dark field (HAADF) micrograph mode.

permeated the membrane over time. NE-1 showed the highest cumulative capsaicin permeated amounts whereas NE-13 showed the lowest permeated amounts, which were not statistically significant (P > 0.05) with those of the control at the all sampling time as shown in Figure 5. Cumulative capsaicin permeated amounts of NE-4 and NE-9 were not statistically significant (P > 0.05) until 8 h and were significantly (P < 0.05) higher at 12 and 24 h compared to the control. Only NE-1 showed statistical significance (P < 0.05) in the capsaicin permeated amounts with other formulations including the control throughout all the time. Cumulative permeation of NE-1 was steeply increased for the initial time (until 6 h) and was slowly increased over time. This phenomenon could be explained by capsaicin-incorporated nanoemulsion being highly accumulated in the receptor fluid, which could affect the permeation flux and delay the capsaicin penetration since the sink condition limit might have been reached. However, many factors may affect the diffusion phenomenon for skin membrane permeation such as sink condition, 729

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Figure 5. Permeation profiles of capsaicin from capsaicin-loaded nanoemulsion formulations through a synthetic membrane similar to human skin. The control sample was a 40% ethanol solution of capsaicin (mean ± SD, n = 3). Different letters at the same time indicate a significant difference at P < 0.05 by Duncan’s multiple range test.

Table 2. Permeability Parameters of Different Formulationsa form. no.b NE-13 NE-9 NE-4 NE-1 control

Jss (μg/cm2 h) 10.597 16.873 16.759 21.251 12.864

± ± ± ± ±

1.239 0.861 0.847 1.826 1.140

Kp (×102 cm/h) c

a b b c ab

0.509 0.811 0.805 1.021 0.618

± ± ± ± ±

0.060 0.041 0.041 0.088 0.055

a b b c ab

formulation for improved skin penetration and was, therefore, selected for optical image observation using CLSM. CLSM. Images derived from CLSM were used to show the permeation pathway of active compounds through the skin layer. This method provides multidepth images that parallel the skin surface without any physical sectioning.29 While the conventional method of studying skin penetration relies on classical sectioning processes that can destroy the skin membrane structures, CLSM is noninvasive and allows the observation of the skin penetration pathways of active compounds without

enhancement ratio (Er) 0.849 1.321 1.312 1.658 1.000

± ± ± ± ±

0.168 0.051 0.052 0.091 0.000

a b b c a

The control sample was 40% ethanol solution of capsaicin (mean ± SD, n = 3). bFormulation number. cDifferent letters indicate a significant difference at P < 0.05 by Duncan’s multiple range test. a

Figure 6. Confocal laser scanning microscopy (CLSM) images for penetration of capsaicin-loaded nanoemulsion (NE-1) into the pig skin. Nile red (0.1% w/w) was used as a fluorescence dye, and the detection wavelength was 543 nm. 730

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inflicting tissue damage.30,31 To obtain images using CLSM, the skin thickness was not more than 700 μm from SC to dermis so that the laser could penetrate all skin layers. The fluorescence dye, Nile red (0.1% w/w), was used as model compound for observation of capsaicin-loaded NE-1 permeation pathway. Nile red has the ability to emit strong fluorescence, and its lipophilicity (log P = 3.10) is similar to capsaicin (log P = 3.81).32 The fluorescence intensity indirectly indicates the amount of permeated capsaicin. Figure 6 shows 32 fluorescence images that parallel the skin surface (z direction images). The first skin surface image did not show strong fluorescence intensity compared with other images because of rinsing the skin sample with 50% ethanol in sample preparation. However, we could observe a clear fluorescence image of penetrated capsaicin nanoemulsion until 32 μm depth in other images. The strongest fluorescent spot was observed near the hair follicles. Capsaicin nanoemulsion can penetrate and accumulate in the skin well through the hair follicles as shown in Figure 6. Because the thickness of the stratum corneum is 10−20 μm, these images indicated that capsaicin-loaded NE-1 permeated stratum corneum and epidermis. CLSM Images of Cryotome-Sectioned Skin. Vertical sectioning of pig skin using the cryotome was performed to ensure and visualize the permeation profile through the skin layers. Figure 7 shows CLSM images of Nile red in 40% ethanol

potential as a transdermal delivery carrier for the effective penetration through all skin layers.

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AUTHOR INFORMATION

Corresponding Author

*Hyun Jin Park: tel, 82-2-3290-4149; fax, 82-2-953-5892; e-mail, [email protected]. Gye Hwa Shin: tel, 82-2-32904149; fax, 82-2-953-5892; e-mail, [email protected]. Author Contributions ‡

Authors contributed equally to this work.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2013R1A1A2008490) and Coway cosmetics R&D center. Notes

The authors declare no competing financial interest.

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REFERENCES

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Figure 7. Confocal laser scanning microscopy (CLSM) images of cryotome-sectioned pig skin of (a, b, c) Nile red in 40% ethanol and (d, e, f) capsaicin-loaded nanoemulsion (NE-1) permeation in differential interference contrast mode (a, d), fluorescence mode (b, e), and merge mode (c, f). Sample thickness is 700 μm. Images of the same area were obtained in fluorescence mode (420−720 nm and 580−720 nm).

(control) and capsaicin-loaded nanoemulsion (NE-1) permeating into pig skin layers consisting of stratum corneum, epidermis, and dermis. Nile red in 40% ethanol could not permeate even the stratum corneum layer, and any fluorescence intensity was not observed in Figure 7a. However, capsaicinloaded nanoemulsion (NE-1) could successfully penetrate the skin layers, and strong fluorescence intensity was observed through the whole skin layers from stratum corneum to dermis. The stratum corneum layer (10−20 μm thick) shows the strongest fluorescence intensity while the dermis layer shows less fluorescence intensity. Although it gradually decreased from stratum corneum to dermis, the red fluorescence intensity was observed through all skin layers up to 700 μm thick. This result indicates that capsaicin-loaded nanoemulsions have a great 731

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