Abstract
Abstract:：To prepare ethosome loading simvastatin,an orthogonal test was applied to optimize the prescriptions, and the qualities of simvastatin ethosome were characterized by the shape, particle size, encapsulation efficiency (EE), and stability. The formation of 40% (v/v) ethanol, 0.02% (m/v) cholesterol, 2.0% (m/v) soy lecithin, and 5% (m/v) polyoxyethylene hydrogenated castor oil showed the maximal EE (69.3%). We observed the shape of simvastatin ethosome through TEM. The average size of the particles was 52.4 ± 3.24nm, which was detected by a N5 submicron particle size analyzer. After 120 days storage in 4℃ and at room temperature, the simvastatin ethosome had no significant change.
INTRODUCTION
Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. It has been shown to be especially effective in reducing low-density lipoprotein cholesterol (LDL) and improving other conditions that are influenced by lipid levels, including coronary artery diseases [Citation1]. Simvastatin has a high degree of selectivity in liver after oral administration. Almost 95% of simvastatin is metabolized by the liver and excreted from bile. Less than 5% of the active structures were found in the blood circulation after oral administration [Citation2]. Existing simvastatin formulations include tablets, capsules, dry suspension, orally disintegrating tablets, and particles without lubricant. They have low bioavailability after oral administration but cause a huge burden on the liver.
The ethosomal system is a vesicular system composed of phospholipids, ethanol, and water. Its difference from liposome is that it contains a large amount of ethanol, which give it a good transdermal property [Citation3,Citation4]. We prepared a transdermal formation, simvastatin ethosome, which can penetrate through the skin and into the blood circulation to avoid the first pass effect of the liver. Therefore, the transdermal administration of simvastatin ethosome could be expected to relieve the burden of the liver and a lesser dosage of simvastatin ethosome could be bioequivalent to the oral administration in which the bioactivity is 5%.
MATERIALS
Instruments
Agilent 1200 HPLC, Agilent; JEM 2010 TEM, JEOL; RCT B IKA magnetic stirring apparatus, Guangzhou Yike Corporation; Ultrasonic cleaner, Kedao Corporation; TDL-40C Centrifugal Machine, Anke Corporation; N5 submicron particle size analyzer, Beckman Coulter; Electronic balance, Shimadzu.
Reagents
Simvastatin, Zhejiang Hisun Pharmaceutical Co., Ltd; Soybean lecithin, Xinshidai Pharmaceutical Corporation; Cholesterol and bag filter, Beijing Solarbio Science & Technology Corporation; Ethanol absolute, Changzhou Hengguang Chemical Reagent Co., LTD; KH2PO4, Tianjin Basf Chemical Co., Ltd.; Acetonitrile, Sigma Aldrich. Other materials were all analytical or pharmaceutical grade.
METHODS AND RESULTS
Preparation of Simvastatin Ethosome
Operating Instructions. Soybean lecithin, simvastatin, and polyoxyethylene hydrogenated castor oil were dissolved in ethanol. Cholesterol was added under stirring; this mixture was heated to 40°C in a water bath. PBS solution (0.05M, pH7.0, 40°C)symbol is missing twice here, and once below; please insert was added slowly in consecutive drops to the lipid mixture with constant stirring at 1000rpm in a closed vessel. The mixing was stirred at 40°C for 5min, and maintained at room temperature for 30min. The resulting vesicle suspension was uniformed by passing through polycarbonate membrane of 0.45μm and 0.22μm three times, respectively [Citation5,Citation6].
Measurements of Encapsulation Efficiency (EE). The EE of simvastatin ethosome was determined using the dialysis technique for separating the non-entrapped drug from ethosome [Citation7,Citation8]. 2ml of drug-loaded ethosomal suspension was added into a dialysis bag (semi-permeable cellulose membrane with a cutoff of 10,000-14,000 Daltons); then the dialysis bag was put into a beaker (receptor cell) containing 100ml ethanol-0.01mol/L PBS (4:6) (pH7.0). Samples in the receptor cell were taken for determining content of the free drug after the homeostasis was reached; the EE was calculated according to the equation EE% = [(Qt-Qs)/Qt] × 100%, where EE is the entrapment efficiency, Qt is the total amount of simvastatin that was added, and Qs is the amount of simvastatin in the receptor cell.
HPLC Assay. The content of simvastatin was determined by HPLC. Chromatographic column: C18 column (250 mm × 4.60 mm, 5μm); mobile phase: 0.025mol/L NaH2PO4 (pH4.5) - acetonitrile (35:65); detection wavelength: 238nm; flow rate: 1ml/min; temperature of column: 25℃; injection volume: 20μl.
The concentration of simvastatin ranged from 0.5μg/ml to 20μg/ml and had a good linearity: Y = 58.7001X - 4.8219, R2 = 1.0000
Orthogonal Tests [Citation9, Citation10]. We designed an orthogonal test (L9(34)) of four main influence factors () A (percentage of ethanol); B (content of soybean lecithin); C (pH of PBS); and D (content of cholesterol). Each factor had three levels to prepare 10ml simvastatin ethosome, which contained 20mg simvastatin and 50mg polyoxyethylene hydrogenated castor oil. The encapsulation efficiency (EE) was the indicator for formulation optimization study. shows factors and levels of orthogonal tests. The results of the orthogonal tests are shown in <and .
Figure 1. Trends of levels in every factor. A, B, C means 3 different factors and 1, 2, 3 means 3 different levels.
Table 1. Factors and levels of orthogonal tests.
Table 2. Arrangement and results of orthogonal test according to L9(34).
Data analysis: The principal factor depended on the R(range) value; the factor sequence influencing the EE was A > B > C > D; the best combination of factors and levels was A1B3C3D2.
Characterizations of Simvastatin Ethosome
We prepared five batches of simvastatin ethosomes according to the optimal prescription and determined their shapes, particles size, EE, and physical stability.
Shapes of Simvastatin Ethosome. Ethosomal systems were diluted with 40% ethanol and dropped in a microscopic copper grid, negatively stained with 0.03% phosphotungstic acid (PTA) and dried, then the stained sample was observed through TEM [Citation11,Citation12]. The micrographs showed that the ethosomes were round or nearly round with estimated diameter at 50nm ().
Figure 2. Shapes of ethosomes through TEM.
Measurements of Particle Sizes. The simvastatin ethosomal suspensions were diluted with 45% ethanol solution and determined by N5 submicron particle size analyzer. The average particle size of 5 batches was (52.5 ± 3.91) nm ().
Table 3. Encapsulation efficiency (EE) and the particle size.
EE of Simvastatin Ethosomes. The EE of simvastatin ethosomes are shown in ; the maximum EE was 69.8%, and the average EE was 65.26 ± 0.05%.
Physical Stability of Simvastatin Ethosomes. Centrifuge test: samples were centrifuged at 2500rpm for 20min.
High-temperature test: samples were stored in a well-closed 10ml glass test tube, and then the tube was placed in an oven at 55°C for 3h. Low-temperature test: the samples were stored in a plastic tube, and the tube was then placed in a refrigerator (−20 ± 1°) for 6h.
Long-term test: samples were stored in a well-closed 10ml glass test tube at room temperature (22 ± 1°C) and in the refrigerator (4 ± 1℃) for 120 days.
There were no significant changes in particle appearance, particle sizes, and EE after the above-mentioned stability tests.
DISCUSSIONS
Injection method and film dispersion method are frequently used to prepare ethosome. Because of the existence of bubbles in the evaporation process caused by the small amount of surfactant, we couldn't get a uniform lipid film when we used the film dispersion method. Therefore we prepared the simvastatin ethosome with the injection method, which also could avoid residue of the organic solvent, such as chloroform and methanol.
On the simvastatin ethosome prescription study, we determined four factors that had greater influences on the particle size and shape of ethosome through the single factor tests, and then determined the optimal prescription and the weight factors by orthogonal tests. Results of the orthogonal tests showed that there were no differences at the preparation temperature of 30°C, 40°C and 50°C, and the ethosome particle size could be reduced by adding an appropriate amount of surfactant, which can reduce the surface-free energy of the ethosome.
We prepared simvastatin ethosomes with PBS whose pH were 5, 6.5, and 7; there was a little higher degradation of simvastatin at pH7 than pH5 PBS. Considering the skin irritation, we prepared the simvastatin ethosome with pH7 PBS.
Determination of EE needs a suitable dialysis media. Because of the bad water solubility of simvastatin, simple PBS couldn't be used as a dialysis medium. Two dialysis mediums, 0.01M NaH2PO4 buffer solution containing 0.5% sodium dodecyl sulfate and 0.01M NaH2PO4 buffer solution containing 40% ethanol, were used as dialysis mediums. Both solutions were adjusted at pH7 with NaOH. After dialysis for 16h, 0.24mg simvastatin was detected in the former medium, and 3.79mg was detected in the latter medium. Therefore we selected 0.01M NaH2PO4 buffer solution containing 40% ethanol as the dialysis medium.
Simvastatin ethosome suspension was unstable when the mixing speed was 700rpm; there were soybean lecithin sediment on the bottom of the suspension after putting in the room temperature for 1d, and it could not be resolved through the adjusting of the temperature and content of each component. When the mixing speed was increased to 1000rpm, there was no soybean lecithin sediment in simvastatin ethosome suspension after put in the room temperature for 3 months.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES
- Lagrost, L., Athias, A., Lemort, N., . (1999). Plasma lipoprotein distribution and lipid transfer activities in patients with type IIb hyperlipidemia treated with simvastatin [J]. Atherosclerosis, 143(2): 415–425.
- Krysiak, R., Okopien, B. (2010). Different effects of simvastatin on ex vivo monocyte cytokine release in patients with hypercholesterolemia and impaired glucose tolerance [J]. J Physiol Pharmacol, 61(6): 725–732.
- Barry, B.W. (2001). Novel mechanisms and devices to enable successful transdermal drug delivery [J]. Eur J Pharm Sci, 14(2): 101–114.
- Touitou, E., Dayan, N., Bergelson, L., . (2000). Ethosomes - novel vesicular carriers for enhanced delivery: Characterization and skin penetration properties [J]. J Control Release, 65(3): 403–418.
- Dayan, N., Touitou, E. (2000). Carriers for skin delivery of trihexyphenidyl HCI: Ethosomes vs. liposomes [J]. Biomaterials, 21(18): 1879–1885.
- Mura, P., Maestrelli, F., González-Rodríguez ML, . (2007). Development, characteration and in vivo evaluation of benzocaine-loaded liposomes [J]. Eur J Pharm Biopharm, 67(1): 86–95.
- Trotta, M., Peira, E., Debernardi, F., . (2002). Elastic liposomes for skin delivery of dipotassium glycyrrhizinate [J]. Int J Pharm, 241(2): 319–327.
- Foco, A., Gasperlin, M., Kristl, J. (2005). Investigation of liposomes as carriers of sodium ascorbyl phosphate for cutaneous photoprotection [J]. Int J Pharm, 291(1-2): 21–29.
- Du, B., Li, Y., Li, X., . (2010). Preparation, characterization and in vivo evaluation of 2-methoxyestradiol-loaded liposomes [J]. Int J Pharm, 384(1-2): 140–147.
- Tian, S., Zhou, X., Gong, H, . (2011). Orthogonal test design for optimization of the extraction of polysaccharide from Paeonia sinjiangensis K.Y. Pan [J]. Pharmacogn Mag, 7(25): 4–8.
- Zhou, Y., Wei, Y.H., Zhang, G.Q., . (2010). Synergistic penetration of eethosomes and lipophilic prodrug on the transdermal delivery of acyclovir [J]. Arch Pharm Res, 33(4): 567–574.
- Jain, S., Jain, P., Umamaheshwari, R.B., . (2003). Transfersomes: A novel vesicular carrier for enhanced transdermal delivery: development, characterization, and performance evaluation [J]. Drug Dev Ind Pharm, 29(9): 1013–1026.