Ultrasonics Sonochemistry 21 (2014) 826–832
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Skin permeation of D-limonene-based nanoemulsions as a transdermal carrier prepared by ultrasonic emulsification Wen-Chien Lu a, Been-Huang Chiang b, Da-Wei Huang c, Po-Hsien Li a,⇑ a
Department of Medicinal Botanical and Health Applications, Da-Yeh University, No. 168, University Rd., Dacun, Changhua 51591, Taiwan, ROC Institute of Food Science and Technology, National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei City 10607, Taiwan, ROC c Department of Food and Beverage Management, China University of Science and Technology, No. 245, Sec. 3, Academia Rd., Nangang Dist., Taipei City 115, Taiwan, ROC b
a r t i c l e
i n f o
Article history: Received 9 August 2013 Received in revised form 2 October 2013 Accepted 15 October 2013 Available online 23 October 2013 Keywords: Nanoemulsions D-Limonene Transdermal delivery Franz diffusion cell Rat abdominal skin Mixed surfactant
a b s t r a c t Nanoemulsions can be used for transporting pharmaceutical phytochemicals in skin-care products because of their stability and rapid permeation properties. However, droplet size may be a critical factor aiding permeation through skin and transdermal delivery efficiency. We prepared D-limonene nanoemulsions with various droplet sizes by ultrasonic emulsification using mixed surfactants of sorbitane trioleate and polyoxyethylene (20) oleyl ether under different hydrophilic–lipophilic balance (HLB) values. Droplet size decreased with increasing HLB value. With HLB 12, the droplet size was 23 nm, and the encapsulated ratio peaked at 92.3%. Transmission electron microscopy revealed spherical droplets and the gray parts were D-limonene precipitation incorporated in spherical droplets of the emulsion system. Franz diffusion cell was used to evaluate the permeation of D-limonene nanoemulsion through rat abdominal skin; the permeation rate depended on droplet size. The emulsion with the lowest droplet size (54 nm) achieved the maximum permeation rate. The concentration of D-limonene in the skin was 40.11 lL/cm2 at the end of 360 min. Histopathology revealed no distinct voids or empty spaces in the epidermal region of permeated rat skin, so the D-limonene nanoemulsion may be a safe carrier for transdermal drug delivery. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Drug delivery through the transdermal route has many advantages over intravenous and oral administration. Transdermal delivery can avoid hepatic metabolism, and the treatment can be immediately withdrawn if necessary [1]. However, the stratum corneum (SC) of the epidermis is a barrier to the passage of molecules and reduces the efficiency of drug delivery through the skin. Many methods have been used to attempt to overcome the SC barrier, the most common being the use of permeation enhancers such as terpenes. Terpenes are not toxic to skin [2,3]; they can enhance skin permeation by interacting with SC lipids and keratin and increase the solubility of the drug into SC lipids [4]. However, when skin is treated with terpenes, the skin structure is changed because of disruption and extraction of SC lipid bilayers. Distinct voids and empty spaces are often visible in the epidermal region after terpenes treatment, and the extraction of SC lipids may also cause dehydration of the SC, with significant loss of moisture [3,5]. Nanoemulsions have droplet size between 20 and 200 nm that appear transparent or translucent to the naked eyes [6]. Because of ⇑ Corresponding author. Tel.: +886 4 8511655x6233; fax: +886 4 8511326. E-mail address: [email protected] (P.-H. Li). 1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2013.10.013
their small droplet size, nanoemulsions can offer high thermodynamic stability against aggregation, flocculation, coalescence and Ostwald ripening. Pharmaceutical phytochemicals can be incorporated into the nanoemulsions and transported through the cell membrane [7]. Nanoemulsions can be used as a vehicle for the transport of pharmaceutical phytochemicals through the SC barrier in skin-care products because of their stability and rapid permeation properties [8]. They have been used as carriers to deliver antifungal drugs, granisetron hydrochloride, ketoprofen, lecithin and meloxicam through the skin [9–14]. However, the droplet size of these carriers may be a critical factor affecting droplet permeation through skin [15]. As well, no reports have discussed the association of nanoemulsion droplet size and transdermal delivery efficiency. D-Limonene, a lipophilic terpene obtained from citrus fruits, has been found to effectively enhance skin permeation for delivering tamoxifen [16] and haloperidol [17]. Other compounds for which D-limonene can be used as a transdermal permeation enhancer includes butylparaben, sumatriptan succinate and nicardipine hydrochloride [18–20]. In our previous study, we used a mixture of the surfactants sorbitane trioleate and polyoxyethylene (20) oleyl ether to form D-limonene in water nanoemulsions with droplet size 140 s) might be attributed to the effect of over-processing of the emulsification, which lead to the coalescence of droplets [28].
W.-C. Lu et al. / Ultrasonics Sonochemistry 21 (2014) 826–832
3.2. Effect of HLB values on droplet size of D-limonene nanoemulsion In our study, we used the mixed surfactant system to form in water nanoemulsions by changing the ratio between the hydrophilic and hydrophobic surfactant. Droplet size decreased with increasing HLB value (Fig. 2). When the HLB values of mixed surfactant equaled 12, the droplet size was the smallest (23 nm) and emulsions were transparent to the naked eye. The other emulsions appeared translucent or opaque. Many studies have shown synergistic effects of mixtures of two surfactants on the formation of stability emulsions with identical headgroups but different hydrocarbon tails [29–32]. Sorbitane trioleate and polyoxyethylene (20) oleyl ether have similar hydrophobic hydrocarbon tails, but the amounts of head groups greatly differs. With the formation of an interfacial surface of mixed surfactant, the hydrocarbon chain of sorbitane trioleate approaches the D-limonene phase, and with polyoxyethylene (20) oleyl ether, the hydrophobic head and hydrophilic tail assisting the droplet formation. Furthermore, during the ultrasonic emulsification, cavitation helps disperse the D-limonene into the small droplet and then the mixed surfactant rapidly adsorbs onto the surface of newly formed droplets to accelerate the formation of D-limonene nanoemulsions.
829
fore, in small emulsion droplets, the possible covalent binding of core material with wall materials could modify their partition behavior.
D-limonene
3.3. Encapsulated ratio of D-limonene at different droplet sizes and HLB values Many studies have suggested that the emulsion droplet size has a significant effect on the encapsulation efficiency of different core materials [33–35]. These reports also show that reducing the emulsion droplet size can result in higher retention of encapsulated components in emulsion systems. However, the application of nanoemulsions in encapsulation of oils and flavors is rarely studied. The encapsulated ratio of D-limonene in nanoemulsions we obtained under different HLB values is in Fig. 2. The encapsulated ratio increased with increasing HLB value. At an HLB value of 2, the droplet size was 332 nm and the D-limonene encapsulated ratio was 71.7%; when the HLB value increased to 12, the droplet size was the smallest, at 23 nm, and the encapsulated ratio of D-limonene was 92.3%. In previous studies, the D-limonene retention was higher for smaller than larger droplets [8,15,34], and the D-limonene retention in all nanoemulsions formulations was >70%. Meynier et al. [34] proposed that the greater retention of flavor in small emulsion droplet was related to reduce volume surface mean diameter, which can enhance the modification of spherical interface organization and increase the interfacial area to govern the partition of aroma compounds in emulsions. There500
TEM was used to observe the D-limonene droplets in nanoemulsions. Phosphotungstic-acid-stained D-limonene droplets were clearly visible and droplet size agreed well with results from droplet size analysis by laser scattering analysis (Fig. 3). In addition, the D-limonene droplet was spherical and the gray parts of the droplet represented D-limonene precipitation incorporated in the emulsion system. According to the TEM results, we could describe the interfacial layer of the D-limonene droplet surface (Fig. 4). The interfacial layer of nanoemulsion droplet was composed of mixed surfactant, and the D-limonene was encapsulated in the droplet. Nanoemulsions were prepared at optimal hydrophilic–lipophilic surfactant combination, and emulsion droplets were stabilized by mixed surfactant. The adsorbing surfactant causes a lowering of the interfacial tension and provides the droplet steric and electrostatic repulsion to stabilize it against coalescence [36,37]. 3.5. Transdermal study of D-limonene nanoemulsion Transdermal study of the D-limonene nanoemulsions through the rat abdominal skin involved use of a Franz diffusion cell; we used the nanoemulsions formula at HLB 3, 5, 8 and 11 with droplet size 335, 226, 149 and 54 nm, respectively, with 10% D-limonene in hexane as a control. Fig. 5 shows the profiles of D-limonene nanoemulsions permeating the abdominal skin as cumulative concentration against time. D-limonene nanoemulsions showed sizedependent permeation, with maximum permeation with HLB 11; the concentration of D-limonene was 40.11 lL/cm2 at the end of 360 min. The permeation of D-limonene nanoemulsions increased with time. The cumulative concentration of permeated D-limonene in nanoemulsions increased with droplet size from 54 to 335 nm as compared with that in the micro-emulsion, with droplet size at 2741 nm, so the droplet size of emulsions affected the transdermal efficiency of entrapped D-limonene. Droplet size of nanodroplets along with the nature of the polymer, zeta potential, vehicle and coating with surfactants are the critical factors influencing droplet uptake of skin absorption cells [1,15,38–40]. In our study, the decrease in droplet size of nanoemulsions increased the permeation of D-limonene, and similar trends were observed by other investigators [14,15,41]. Furthermore, the mixed surfactants on the surface of emulsions play an important role in the permeation of D-limonene. The
100 Droplet size (nm)
450
Encapsulated ratio (%)
350
80
300 250
70
200 60
150
Encapsulated ratio (%)
90
400
Droplet size (nm)
3.4. Morphology by TEM
100 50 50 0
0 1
2
3
4
5
6
7
8
9
10
11
12
13
HLB value Fig. 2. Droplet size and encapsulated ratio of 10% D-limonene nanoemulsions at different hydrophilic–lipophilic balance (HLB) values.
Fig. 3. Transmission electron micrograph (TEM) of droplets in D-limonene nanoemulsion at HLB 11.
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W.-C. Lu et al. / Ultrasonics Sonochemistry 21 (2014) 826–832
Fig. 4. Schematic representation of the interfacial layer at the droplet surface of D-limonene nanoemulsions.
Cumulative concentration (µl /cm2)
50
HLB 3 HLB 5 HLB 8 HLB 11 10% d-limonene Normal emulsion
40
30
20
10
0 60
120
180
240
300
360
Time (min) Fig. 5. Cumulative concentration of emulsions and different times.
D-limonene
at various HLB values of nano-
results in Fig. 5 show that the D-limonene cumulative concentration was higher in nanoemulsions than in controls under different times. Previous reports demonstrated that surfactants can enhance the rate of permeation by two possible mechanisms. First, the surfactants may penetrate the intracellular regions of SC lipid and increase fluidity and solubility of lipid components [11]. Second, permeation of the surfactant into the intracellular matrix followed by interaction or binding with keratin may facilitate the perme-
ation [40,42]. In our study, the mixed surfactant was thought to enhance the permeation of D-limonene via both lipophilic and hydrophilic molecular mechanisms. The mixed surfactant contains ethylene oxide and a long hydrocarbon chain, thus allowing it to partition between lipophilic substances and the hydrophilic domain. This activity would then allow D-limonene to permeate the skin. The flux (J), permeability coefficient (Kp), and enhancement ratio for each droplet size and HLB value according to the equation are in Table 1. All nanoemulsions used increased the skin permeation parameters at HLB 3, 5, 8 and 11 as compared with microemulsion. The rank order of enhancement effect for D-limonene was HLB 11 > 8 > 5 > 3 > micro-emulsion. The best permeation enhancer was HLB 11 with droplet size 54 nm, providing an almost threefold increase in steady state flux and enhancement ratio as compared with micro-emulsion. Thus, nanoemulsion as a transdermal permeation carrier had excellent permeability through skin. Some previous reports discussed the permeation-enhancing ability of nanoemulsions for transdermal delivery. Mou et al. [43] reported that small droplets of nanoemulsions may embed into the SC and the core materials could be delivered directly from the droplet into the SC. Some studies elucidated that the water phase in the nanoemulsions system could hydrate skin to promote the core material permeation [10,44]. Another suggestion was that the drug in the nanoemulsion system could permeate the skin in the form of a nanoscale droplet [14].
3.6. Histopathology Fig. 6a represents the untreated rat skin (control) and Fig. 6b skin treated for 360 min with D-limonene nanoemulsions under
Table 1 Delivery characteristics of D-limonene nanoemulsion under different HLB values. Sample type
HLB
Droplet size (nm)
Steady state flux (lg cm2 h1)
Kp (103 cm h1)
ER
Nanoemulsion Nanoemulsion Nanoemulsion Nanoemulsion Micro-emulsion
3 5 8 11 12
335a ± 22.1 226b ± 13.5 149c ± 3.1 54d ± 0.7 2741 ± 327
129.9c ± 11.3 152.5c ± 14.8 180.8b ± 9.4 214.7a ± 16.4 67.8 ± 5.8
0.18 ± 0.03 0.19 ± 0.04 0.22 ± 0.07 0.23 ± 0.05 0.11 ± 0.06
3.29 3.86 4.57 5.43 1.71
ER, enhancement ratio; Kp, permeability coefficient. Means with different letters within the same column differed significantly (p < 0.05). Each value is expressed as the mean ± SD (n = 3).
a-d
W.-C. Lu et al. / Ultrasonics Sonochemistry 21 (2014) 826–832
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Fig. 6. Light microscopy (100) of rat skin after no treatment (a) and 360 min after treatment with D-limonene nanoemulsions (b).
HLB 11. Compared to controls, the treated SC remained intact. With the rat skin treated with terpenes, the disruption and dehydration of SC lipid results in distinct voids and empty spaces in the epidermal region [5]. In our study, the epidermal region showed no change on histopathology, so D-limonene nanoemulsions could be used as a transdermal carrier and can be a safe carrier for transdermal drug delivery. 4. Conclusion We found that HLB values can influence the droplet size and encapsulated ratio of D-limonene in nanoemulsion systems. The smaller the emulsion size, the better the encapsulated ratio. Permeation of D-limonene nanoemulsion through the rat abdominal skin depended on emulsion size. The permeation was higher with D-limonene nanoemulsion than micro-emulsion and controls. D-Limonene nanoemulsions may be interesting carriers for transdermal delivery systems. We provide initial evidence of the transdermal delivery of D-limonene nanoemulsions. Their application for transdermal delivery of phytochemical components needs further study. References [1] A. Kogan, N. Garti, Microemulsions as transdermal drug delivery vehicles, Adv. Colloid Interface Sci. 123–126 (16) (2006) 369–385. [2] C.S. Asbill, A.F. El-Kattan, B. Michniak, Enhancement of transdermal drug delivery: chemical and physical approaches, Crit. Rev. Ther. Drug Carrier Syst. 17 (6) (2000) 621–658. [3] B. Sapra, S. Jain, A.K. Tiwary, Percutaneous permeation enhancement by terpenes: mechanistic view, AAPS PharmSciTech. 10 (1) (2008) 120–132. [4] A. Williams, B.W. Barry, Terpenes and the lipid-protein-partitioning theory of skin penetration enhancement, Pharm. Res. 8 (1) (1991) 17–24. [5] M. Rizwan, M. Aqil, A. Ahad, Y. Sultana, M.M. Ali, Transdermal delivery of valsartan: I. Effect of various terpenes, Drug Dev. Ind. Pharm. 34 (6) (2008) 618–626. [6] C. Solans, P. Izquierdo, J. Nolla, N. Azemar, M.J. Garcia-Celma, Nano-emulsions, Curr. Opin. Colloid Interface Sci. 10 (3–4) (2005) 102–110. [7] Q. Huang, H. Yu, Q. Ru, Bioavailability and delivery of nutraceuticals using nanotechnology, J. Food Sci. 75 (1) (2010) 50–57. [8] A. Soottitantawat, F. Bigeard, H. Yoshii, T. Furuta, M. Ohgawara, P. Linko, Influence of emulsion and power size on the stability of encapsulated D-limonene by spray drying, Innov. Food Sci. Emerg. Technol. 6 (2) (2005) 107–114. [9] M.P. Youenang Piemi, D. Korner, S. Benita, J.P. Marty, Positively and negatively charged submicron emulsions for enhanced topical delivery of antifungal drugs, J. Controlled Release 58 (1999) 177–187. [10] Y. Yuan, S.M. Li, F.K. Mo, D.F. Zhong, Investigation of microemulsion system for transdermal delivery of meloxicam, Int. J. Pharm. 321 (1–2) (2006) 117–123. [11] S. Hoeller, A. Sperger, C. Valenta, Lecithin based nanoemulsions: a comparative study of the influence of non-ionic surfactants and the cationic phytosphingosine on physicochemical behavior and skin permeation, Int. J. Pharm. 370 (1) (2009) 181–186. [12] M.H.F. Sakeena, S.M. Elrashid, F.A. Muthanna, Z.A. Ghassan, M.M. Kanakal, L. Laila, A.S. Munavvar, M.N. Azmin, Effect of limonene on permeation enhancement of ketoprofen in palm oil esters nanoemulsions, J. Oleo Sci. 59 (7) (2010) 395–400. [13] H. Zhou, Y. Yue, G. Liu, Y. Li, J. Zhang, Q. Gong, Z. Yan, M. Duan, Preparation and characterization of a lecithin nanoemulsion as a topical delivery system, Nanoscale Res. Lett. 5 (1) (2010) 224–230.
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Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Skin permeation of D-limonene-based nanoemulsions as a transdermal carrier prepared by ultrasonic emulsification Wen-Chien Lu a, Been-Huang Chiang b, Da-Wei Huang c, Po-Hsien Li a,⇑ a
Department of Medicinal Botanical and Health Applications, Da-Yeh University, No. 168, University Rd., Dacun, Changhua 51591, Taiwan, ROC Institute of Food Science and Technology, National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei City 10607, Taiwan, ROC c Department of Food and Beverage Management, China University of Science and Technology, No. 245, Sec. 3, Academia Rd., Nangang Dist., Taipei City 115, Taiwan, ROC b
a r t i c l e
i n f o
Article history: Received 9 August 2013 Received in revised form 2 October 2013 Accepted 15 October 2013 Available online 23 October 2013 Keywords: Nanoemulsions D-Limonene Transdermal delivery Franz diffusion cell Rat abdominal skin Mixed surfactant
a b s t r a c t Nanoemulsions can be used for transporting pharmaceutical phytochemicals in skin-care products because of their stability and rapid permeation properties. However, droplet size may be a critical factor aiding permeation through skin and transdermal delivery efficiency. We prepared D-limonene nanoemulsions with various droplet sizes by ultrasonic emulsification using mixed surfactants of sorbitane trioleate and polyoxyethylene (20) oleyl ether under different hydrophilic–lipophilic balance (HLB) values. Droplet size decreased with increasing HLB value. With HLB 12, the droplet size was 23 nm, and the encapsulated ratio peaked at 92.3%. Transmission electron microscopy revealed spherical droplets and the gray parts were D-limonene precipitation incorporated in spherical droplets of the emulsion system. Franz diffusion cell was used to evaluate the permeation of D-limonene nanoemulsion through rat abdominal skin; the permeation rate depended on droplet size. The emulsion with the lowest droplet size (54 nm) achieved the maximum permeation rate. The concentration of D-limonene in the skin was 40.11 lL/cm2 at the end of 360 min. Histopathology revealed no distinct voids or empty spaces in the epidermal region of permeated rat skin, so the D-limonene nanoemulsion may be a safe carrier for transdermal drug delivery. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Drug delivery through the transdermal route has many advantages over intravenous and oral administration. Transdermal delivery can avoid hepatic metabolism, and the treatment can be immediately withdrawn if necessary [1]. However, the stratum corneum (SC) of the epidermis is a barrier to the passage of molecules and reduces the efficiency of drug delivery through the skin. Many methods have been used to attempt to overcome the SC barrier, the most common being the use of permeation enhancers such as terpenes. Terpenes are not toxic to skin [2,3]; they can enhance skin permeation by interacting with SC lipids and keratin and increase the solubility of the drug into SC lipids [4]. However, when skin is treated with terpenes, the skin structure is changed because of disruption and extraction of SC lipid bilayers. Distinct voids and empty spaces are often visible in the epidermal region after terpenes treatment, and the extraction of SC lipids may also cause dehydration of the SC, with significant loss of moisture [3,5]. Nanoemulsions have droplet size between 20 and 200 nm that appear transparent or translucent to the naked eyes [6]. Because of ⇑ Corresponding author. Tel.: +886 4 8511655x6233; fax: +886 4 8511326. E-mail address: [email protected] (P.-H. Li). 1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2013.10.013
their small droplet size, nanoemulsions can offer high thermodynamic stability against aggregation, flocculation, coalescence and Ostwald ripening. Pharmaceutical phytochemicals can be incorporated into the nanoemulsions and transported through the cell membrane [7]. Nanoemulsions can be used as a vehicle for the transport of pharmaceutical phytochemicals through the SC barrier in skin-care products because of their stability and rapid permeation properties [8]. They have been used as carriers to deliver antifungal drugs, granisetron hydrochloride, ketoprofen, lecithin and meloxicam through the skin [9–14]. However, the droplet size of these carriers may be a critical factor affecting droplet permeation through skin [15]. As well, no reports have discussed the association of nanoemulsion droplet size and transdermal delivery efficiency. D-Limonene, a lipophilic terpene obtained from citrus fruits, has been found to effectively enhance skin permeation for delivering tamoxifen [16] and haloperidol [17]. Other compounds for which D-limonene can be used as a transdermal permeation enhancer includes butylparaben, sumatriptan succinate and nicardipine hydrochloride [18–20]. In our previous study, we used a mixture of the surfactants sorbitane trioleate and polyoxyethylene (20) oleyl ether to form D-limonene in water nanoemulsions with droplet size 140 s) might be attributed to the effect of over-processing of the emulsification, which lead to the coalescence of droplets [28].
W.-C. Lu et al. / Ultrasonics Sonochemistry 21 (2014) 826–832
3.2. Effect of HLB values on droplet size of D-limonene nanoemulsion In our study, we used the mixed surfactant system to form in water nanoemulsions by changing the ratio between the hydrophilic and hydrophobic surfactant. Droplet size decreased with increasing HLB value (Fig. 2). When the HLB values of mixed surfactant equaled 12, the droplet size was the smallest (23 nm) and emulsions were transparent to the naked eye. The other emulsions appeared translucent or opaque. Many studies have shown synergistic effects of mixtures of two surfactants on the formation of stability emulsions with identical headgroups but different hydrocarbon tails [29–32]. Sorbitane trioleate and polyoxyethylene (20) oleyl ether have similar hydrophobic hydrocarbon tails, but the amounts of head groups greatly differs. With the formation of an interfacial surface of mixed surfactant, the hydrocarbon chain of sorbitane trioleate approaches the D-limonene phase, and with polyoxyethylene (20) oleyl ether, the hydrophobic head and hydrophilic tail assisting the droplet formation. Furthermore, during the ultrasonic emulsification, cavitation helps disperse the D-limonene into the small droplet and then the mixed surfactant rapidly adsorbs onto the surface of newly formed droplets to accelerate the formation of D-limonene nanoemulsions.
829
fore, in small emulsion droplets, the possible covalent binding of core material with wall materials could modify their partition behavior.
D-limonene
3.3. Encapsulated ratio of D-limonene at different droplet sizes and HLB values Many studies have suggested that the emulsion droplet size has a significant effect on the encapsulation efficiency of different core materials [33–35]. These reports also show that reducing the emulsion droplet size can result in higher retention of encapsulated components in emulsion systems. However, the application of nanoemulsions in encapsulation of oils and flavors is rarely studied. The encapsulated ratio of D-limonene in nanoemulsions we obtained under different HLB values is in Fig. 2. The encapsulated ratio increased with increasing HLB value. At an HLB value of 2, the droplet size was 332 nm and the D-limonene encapsulated ratio was 71.7%; when the HLB value increased to 12, the droplet size was the smallest, at 23 nm, and the encapsulated ratio of D-limonene was 92.3%. In previous studies, the D-limonene retention was higher for smaller than larger droplets [8,15,34], and the D-limonene retention in all nanoemulsions formulations was >70%. Meynier et al. [34] proposed that the greater retention of flavor in small emulsion droplet was related to reduce volume surface mean diameter, which can enhance the modification of spherical interface organization and increase the interfacial area to govern the partition of aroma compounds in emulsions. There500
TEM was used to observe the D-limonene droplets in nanoemulsions. Phosphotungstic-acid-stained D-limonene droplets were clearly visible and droplet size agreed well with results from droplet size analysis by laser scattering analysis (Fig. 3). In addition, the D-limonene droplet was spherical and the gray parts of the droplet represented D-limonene precipitation incorporated in the emulsion system. According to the TEM results, we could describe the interfacial layer of the D-limonene droplet surface (Fig. 4). The interfacial layer of nanoemulsion droplet was composed of mixed surfactant, and the D-limonene was encapsulated in the droplet. Nanoemulsions were prepared at optimal hydrophilic–lipophilic surfactant combination, and emulsion droplets were stabilized by mixed surfactant. The adsorbing surfactant causes a lowering of the interfacial tension and provides the droplet steric and electrostatic repulsion to stabilize it against coalescence [36,37]. 3.5. Transdermal study of D-limonene nanoemulsion Transdermal study of the D-limonene nanoemulsions through the rat abdominal skin involved use of a Franz diffusion cell; we used the nanoemulsions formula at HLB 3, 5, 8 and 11 with droplet size 335, 226, 149 and 54 nm, respectively, with 10% D-limonene in hexane as a control. Fig. 5 shows the profiles of D-limonene nanoemulsions permeating the abdominal skin as cumulative concentration against time. D-limonene nanoemulsions showed sizedependent permeation, with maximum permeation with HLB 11; the concentration of D-limonene was 40.11 lL/cm2 at the end of 360 min. The permeation of D-limonene nanoemulsions increased with time. The cumulative concentration of permeated D-limonene in nanoemulsions increased with droplet size from 54 to 335 nm as compared with that in the micro-emulsion, with droplet size at 2741 nm, so the droplet size of emulsions affected the transdermal efficiency of entrapped D-limonene. Droplet size of nanodroplets along with the nature of the polymer, zeta potential, vehicle and coating with surfactants are the critical factors influencing droplet uptake of skin absorption cells [1,15,38–40]. In our study, the decrease in droplet size of nanoemulsions increased the permeation of D-limonene, and similar trends were observed by other investigators [14,15,41]. Furthermore, the mixed surfactants on the surface of emulsions play an important role in the permeation of D-limonene. The
100 Droplet size (nm)
450
Encapsulated ratio (%)
350
80
300 250
70
200 60
150
Encapsulated ratio (%)
90
400
Droplet size (nm)
3.4. Morphology by TEM
100 50 50 0
0 1
2
3
4
5
6
7
8
9
10
11
12
13
HLB value Fig. 2. Droplet size and encapsulated ratio of 10% D-limonene nanoemulsions at different hydrophilic–lipophilic balance (HLB) values.
Fig. 3. Transmission electron micrograph (TEM) of droplets in D-limonene nanoemulsion at HLB 11.
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Fig. 4. Schematic representation of the interfacial layer at the droplet surface of D-limonene nanoemulsions.
Cumulative concentration (µl /cm2)
50
HLB 3 HLB 5 HLB 8 HLB 11 10% d-limonene Normal emulsion
40
30
20
10
0 60
120
180
240
300
360
Time (min) Fig. 5. Cumulative concentration of emulsions and different times.
D-limonene
at various HLB values of nano-
results in Fig. 5 show that the D-limonene cumulative concentration was higher in nanoemulsions than in controls under different times. Previous reports demonstrated that surfactants can enhance the rate of permeation by two possible mechanisms. First, the surfactants may penetrate the intracellular regions of SC lipid and increase fluidity and solubility of lipid components [11]. Second, permeation of the surfactant into the intracellular matrix followed by interaction or binding with keratin may facilitate the perme-
ation [40,42]. In our study, the mixed surfactant was thought to enhance the permeation of D-limonene via both lipophilic and hydrophilic molecular mechanisms. The mixed surfactant contains ethylene oxide and a long hydrocarbon chain, thus allowing it to partition between lipophilic substances and the hydrophilic domain. This activity would then allow D-limonene to permeate the skin. The flux (J), permeability coefficient (Kp), and enhancement ratio for each droplet size and HLB value according to the equation are in Table 1. All nanoemulsions used increased the skin permeation parameters at HLB 3, 5, 8 and 11 as compared with microemulsion. The rank order of enhancement effect for D-limonene was HLB 11 > 8 > 5 > 3 > micro-emulsion. The best permeation enhancer was HLB 11 with droplet size 54 nm, providing an almost threefold increase in steady state flux and enhancement ratio as compared with micro-emulsion. Thus, nanoemulsion as a transdermal permeation carrier had excellent permeability through skin. Some previous reports discussed the permeation-enhancing ability of nanoemulsions for transdermal delivery. Mou et al. [43] reported that small droplets of nanoemulsions may embed into the SC and the core materials could be delivered directly from the droplet into the SC. Some studies elucidated that the water phase in the nanoemulsions system could hydrate skin to promote the core material permeation [10,44]. Another suggestion was that the drug in the nanoemulsion system could permeate the skin in the form of a nanoscale droplet [14].
3.6. Histopathology Fig. 6a represents the untreated rat skin (control) and Fig. 6b skin treated for 360 min with D-limonene nanoemulsions under
Table 1 Delivery characteristics of D-limonene nanoemulsion under different HLB values. Sample type
HLB
Droplet size (nm)
Steady state flux (lg cm2 h1)
Kp (103 cm h1)
ER
Nanoemulsion Nanoemulsion Nanoemulsion Nanoemulsion Micro-emulsion
3 5 8 11 12
335a ± 22.1 226b ± 13.5 149c ± 3.1 54d ± 0.7 2741 ± 327
129.9c ± 11.3 152.5c ± 14.8 180.8b ± 9.4 214.7a ± 16.4 67.8 ± 5.8
0.18 ± 0.03 0.19 ± 0.04 0.22 ± 0.07 0.23 ± 0.05 0.11 ± 0.06
3.29 3.86 4.57 5.43 1.71
ER, enhancement ratio; Kp, permeability coefficient. Means with different letters within the same column differed significantly (p < 0.05). Each value is expressed as the mean ± SD (n = 3).
a-d
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Fig. 6. Light microscopy (100) of rat skin after no treatment (a) and 360 min after treatment with D-limonene nanoemulsions (b).
HLB 11. Compared to controls, the treated SC remained intact. With the rat skin treated with terpenes, the disruption and dehydration of SC lipid results in distinct voids and empty spaces in the epidermal region [5]. In our study, the epidermal region showed no change on histopathology, so D-limonene nanoemulsions could be used as a transdermal carrier and can be a safe carrier for transdermal drug delivery. 4. Conclusion We found that HLB values can influence the droplet size and encapsulated ratio of D-limonene in nanoemulsion systems. The smaller the emulsion size, the better the encapsulated ratio. Permeation of D-limonene nanoemulsion through the rat abdominal skin depended on emulsion size. The permeation was higher with D-limonene nanoemulsion than micro-emulsion and controls. D-Limonene nanoemulsions may be interesting carriers for transdermal delivery systems. We provide initial evidence of the transdermal delivery of D-limonene nanoemulsions. Their application for transdermal delivery of phytochemical components needs further study. References [1] A. Kogan, N. Garti, Microemulsions as transdermal drug delivery vehicles, Adv. Colloid Interface Sci. 123–126 (16) (2006) 369–385. [2] C.S. Asbill, A.F. El-Kattan, B. Michniak, Enhancement of transdermal drug delivery: chemical and physical approaches, Crit. Rev. Ther. Drug Carrier Syst. 17 (6) (2000) 621–658. [3] B. Sapra, S. Jain, A.K. Tiwary, Percutaneous permeation enhancement by terpenes: mechanistic view, AAPS PharmSciTech. 10 (1) (2008) 120–132. [4] A. Williams, B.W. Barry, Terpenes and the lipid-protein-partitioning theory of skin penetration enhancement, Pharm. Res. 8 (1) (1991) 17–24. [5] M. Rizwan, M. Aqil, A. Ahad, Y. Sultana, M.M. Ali, Transdermal delivery of valsartan: I. Effect of various terpenes, Drug Dev. Ind. Pharm. 34 (6) (2008) 618–626. [6] C. Solans, P. Izquierdo, J. Nolla, N. Azemar, M.J. Garcia-Celma, Nano-emulsions, Curr. Opin. Colloid Interface Sci. 10 (3–4) (2005) 102–110. [7] Q. Huang, H. Yu, Q. Ru, Bioavailability and delivery of nutraceuticals using nanotechnology, J. Food Sci. 75 (1) (2010) 50–57. [8] A. Soottitantawat, F. Bigeard, H. Yoshii, T. Furuta, M. Ohgawara, P. Linko, Influence of emulsion and power size on the stability of encapsulated D-limonene by spray drying, Innov. Food Sci. Emerg. Technol. 6 (2) (2005) 107–114. [9] M.P. Youenang Piemi, D. Korner, S. Benita, J.P. Marty, Positively and negatively charged submicron emulsions for enhanced topical delivery of antifungal drugs, J. Controlled Release 58 (1999) 177–187. [10] Y. Yuan, S.M. Li, F.K. Mo, D.F. Zhong, Investigation of microemulsion system for transdermal delivery of meloxicam, Int. J. Pharm. 321 (1–2) (2006) 117–123. [11] S. Hoeller, A. Sperger, C. Valenta, Lecithin based nanoemulsions: a comparative study of the influence of non-ionic surfactants and the cationic phytosphingosine on physicochemical behavior and skin permeation, Int. J. Pharm. 370 (1) (2009) 181–186. [12] M.H.F. Sakeena, S.M. Elrashid, F.A. Muthanna, Z.A. Ghassan, M.M. Kanakal, L. Laila, A.S. Munavvar, M.N. Azmin, Effect of limonene on permeation enhancement of ketoprofen in palm oil esters nanoemulsions, J. Oleo Sci. 59 (7) (2010) 395–400. [13] H. Zhou, Y. Yue, G. Liu, Y. Li, J. Zhang, Q. Gong, Z. Yan, M. Duan, Preparation and characterization of a lecithin nanoemulsion as a topical delivery system, Nanoscale Res. Lett. 5 (1) (2010) 224–230.
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