КОЛЛОИДНЫЙ ЖУРНАЛ, 2010, том 72, № 2, с. 200-205
УДК 541.18
IN VITRO SKIN PERMEATION OF CUBOSOMES CONTAINING WATER SOLUBLE EXTRACTS OF KOREAN BARBERRY
© 2010 г. Taek Kwan Kwon*, Hyun Yong Lee*, Jong Dai Kim*, Won Cheol Shin**,
Seung Kyu Park***, Jin-Chul Kim*
*School of Biotechnology & Bioengineering and Institute of Bioscience and Biotechnology, Kangwon National University,
192-1, Hyoja 2 dong, Chunchon, Kangwon-do 200-701, Korea **Department of Bioengineering and Technology, Kangwon National University, 192-1, Hyoja 2 dong, Chunchon, Kangwon-do 200-701, Korea ***Department of Chemical Engineering, Hoseo University, 165, Sechul-ri, Baebang-myun,Asan-City, Choongnam, 336-795, Korea e-mail: jinkim@kangwon.ac.kr Поступила в редакцию 09.04.2009 г.
The monoolein (MO) cubic phases containing water soluble extract (WSE) from Berberis koreana (Korean barberry) were prepared by hydrating the molten MO with aqueous solutions ofWSE (0.5, 1.0, and 1.5%). The phase transition temperature of cubic phase containing WSE (~70°C) was almost the same as that of WSE-free MO cubic phase that indicates that WSE was immobilized in the water channels of the cubic phase and did not affect its structure. The release of WSE from the cubic phase fits the first order process. The cubosomes were obtained by micronizing the cubic phase in a sonicator using Pluronic F127 as a dispersant. The cubosomes were stable in size at the ethanol concentration <16%. When compared with WSE solution in phosphate-buffered saline (10 mM, pH 7.4), in vitro skin permeation of WSE in the cubosomes was enhanced by about two times.
INTRODUCTION
When biologically active substances are delivered transdermally, the reducing of the barrier properties of skin or enhancing permeation of such substances is of a great interest. Main pathways for the transdermal transport ofa drug are paracellular and intracellular routes. Because of the hydrophobic nature of extracellular lipid matrix of skin, lipophilic compounds are transferred by the paracellular route, whereas hydrophilic compounds are soluble in the inner space ofskin cells (corneocytes) [1, 2]. However, a water soluble drug has to be delivered into cells through the lipidic matrix of skin, in which corneocytes are embedded. Several strategies have been developed for efficient transdermal delivery of water soluble drugs [3— 5]. Recently, nanoparticles have been reported to penetrate into lipid space between corneocytes due to the small size of such transporters [6]. Other types ofcarriers, which can encapsulate water-soluble drugs, are microcapsules prepared using a double emulsion technique, liposomes, and cubosomes. The microcapsules cannot penetrate into skin due to their large size and strength of polymer wall that prevent such particles from the interactions with skin lipids [7]. Liposomes are phospholipids bilayer vesicles, which are biocompatible and non-toxic since phospho-lipids are the major components of biological membrane. Liposomes are known to enhance the permeation ofwater soluble compounds because they readily penetrate into skin due to their small size and membrane flexibility [7— 9]. However, water soluble compounds are, in most cases, encapsulated in liposomes with a low efficiency. More-
over, liposomes are not stable in solutions with low concentration ofethanol. Recently, the cubosomes (nanoparticles of cubic phase) have been proposed as drug carriers. Amphiphilic molecules with packing parameter larger than 1 have been reported to form bicontinuous inverted cubic phase in aqueous solutions [10—12]. Monoolein (MO) is a typical amphiphile that can form the cubic phase. Two intercrossing water channels pass through the cubic phase and they are separated by MO bilayers. Hy-drophilic compounds can be immobilized in the water channels and lipophilic ones can be loaded into bilayers. For instance, insulin was entrapped in the water channel ofcubosomes for oral administration [13—15]. The cubo-somes protect insulin from the severe conditions in stomach and promote the gastrointestinal absorption of the hormone. However, there are no published reports on the cubosomes used to enhance the skin permeation ofwater-soluble compounds. In this study, MO cubic phases containing water-soluble extract (WSE) ofbarberry were prepared by hydrating the molten liquid of MO with the aqueous WSE solution. The formation of cubic phase was confirmed by the measurements of phase transition temperature via differential scanning calorimetry. The cubo-somes were prepared by micronizing the cubic phase in a sonicator. To evaluate the potential promise ofcubosomes application in cosmetics, the stability of cubosome size in ethanol solution was evaluated. Finally, the effect ofcubosomes on the in vitro skin permeation was investigated using diffusion cells.
EXPERIMENTAL
Materials
Monoolein (1-monooleoyl glycerol) was obtained free of charge from Danisco Ingredients A/S (monoglyceride content is approx. 95.7% and oleic acid content is approx. 90%). Berberis koreana (Korean barberry) was obtained from Seolim Co. (Gwancheon, Korea). Pluronic F127 and phosphotungstic acid were purchased from Aldrich Chemical co. (Milwaukee, USA). Water was distilled in a water purification system (Pure Power I+, Human Corporation, Korea) until the resistivity of 18 Mfi/cm was achieved. All other reagents were of analytical grade.
Extraction of Water-soluble Extract From Barberry
Bark ofBerberis koreana was micronized using a mixer. 100 g of a powder was mixed with 1 L of distilled water in a 3-neck round bottom flask equipped with a vertical cooling condenser. The extraction was carried out at 60°C for 12 h. The extraction mixture was filtered through a filter paper (HYUNDAI Micro Co., Ltd. Korea) and then the filtrate was concentrated under a reduced pressure using a rotary evaporator (DIGITAL WATER BATH SB-651, EYELA, Japan). Finally, the concentrated filtrate was lyophilized to obtain solid extract. The yield ofextrac-tion is defined as the percent of the mass of solid extract versus the input mass of bark powder. Hereafter, the extract is noted as water soluble extract (WSE).
Preparations of Cubic Phases and Cubosomes
2 g of MO were molten in a water bath at 60°C. WSE solution (0.5, 1.0 or 1.5%) in phosphate-buffered saline (PBS, pH 7.4) was layered over the molten MO; the content of aqueous phase is of 30%. The two-phase systems were kept at 25°C until clear gels (cubic phase) were obtained. To obtain the nanoparticles of cubic phase (so-called cubosomes), 200 ml of Pluronic F127 solution in a buffer (1.5 mg/ml) was added to the MO gel to achieve 15 : 100 ratio of the polymeric surfactant to MO. Then, the gels were micronized in a bath type sonicator (VC 505, Sonic & Materials, USA, 30% energy intensity, 30 s pulse on, 30 s pulse off) at room temperature for 15 min.
Differential Scanning Calorimetry
MO and MO gel prepared using WSE solution (1.5%) were thermally scanned on a differential scanning calorimeter (TA instruments DSC 2010). Each sample of10— 20 mg weighed into aluminum DSC pans and was scanned from 25 to 90°C at a heating rate of 3°C/min.
Release of WSE from Cubic Phases
Several samples of the cubic phase were prepared to determine the amount ofWSE released at various time intervals. PBS (10 mM, pH 7.4) of 5 ml was laid over the cubic phases contained in the glass tubes and they were
whirled at 120 rpm on a shaker (Jeico Tech SK-760A) at room temperature. The supernatant was assayed for WSE at predetermined time intervals. The amount released from the cubic phase was determined using HPLC at the predetermined time. The WSE assay was performed in a liquid chromatograph (M600E, M7725i/Waters, 996PDA) equipped with a UV detector (0.05 AFUS). A reversed phase column (4.6 x 250 mm, C18 5 ^m, XBridge™) was eluted with acetonitrile/phosphate buffer (10 mM, pH 5.2) (60/40, v/v) at a flow rate of 1.0 mL/min and a sample of5 |l was injected. The detection wave length was of254 nm.
Characterization of Cubosomes
The TEM images ofcubosomes were obtained with an electron microscopy (LEO-912AB OMEGA, LEO, Germany) by a negative staining technique [16]. The size distributions were obtained with a particle size analyzer (ZetaPlus 90, Brookhaven Instrument Co., USA) by a dynamic light scattering [17, 18]. To investigate the effect of ethanol on the size of cubosomes, the concentration of the alcohol in the suspension was varied from 0 to 16%. For the same purpose, the concentration of deoxycholate was varied from 0 to 0.16%.
In vitro Permeation
In vitro permeation of WSE-containing cubosomes was investigated in accordance to the procedure described elsewhere [19, 20]. The dorsal skins of female hairless mice (type SKH) aged 6 week were mounted onto Franz diffusion cells (0.636 cm2 surface area) having 5ml receptor compartment. PBS (pH 7.4) was used as the medium for the receptor, and the system was thermostated to 37°C under stirring. Cubosome suspensions containing WSE with concentrations of 0.5, 1.0, and 1.5%, respectively, were used for the experiments on skin permeation. As a control sample, the WSE solutions of the same concentrations in PBS (pH 7.4) were used. 200 |l of the WSE-containing cubosome suspensions or the WSE solutions was applied onto the skins and then the receptor solutions (300 |l) were assayed for WSE using HPLC at the predetermined time.
RESULTS AND DISCUSSION
Extractions of WSE from Barberry
The yield of extraction was about 7%. The retention time ofWSE on HPLC was 2.58 min. The equation ofcal-ibration curve for WSE was Y= 5288.49X + 124.96, where X is the concentration of WSE in PBS (10 mM, pH 7.4) and Yis the area ofpeak at the retention time.
Preparation of Cubic Phases
All the gels were transparent irrespectively of the WSE concentrations in the aqueous phase. The transparency is an evidence of the formation of cubic phases. (The MO
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