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Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency Cong Luo1, Yan Li1, Jin Sun1, 2*, Yan Zhang3, Qin Chen1, Xiaohong Liu1, Zhonggui He1* No. 59 Mailbox, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China 2 Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300193, PR China 3 Northeast Pharmaceutical Group Co., Ltd, Shenyang 110021, PR China *Correspondence: [email protected]; [email protected] 1
In the present work, nanocrystals were prepared to improve oral bioavailability of felodipine. Several important formulation factors and process parameters were explored, optimized and analyzed systematically. The prepared nanocrystals were characterized for morphology, particle size, zeta potential, possible crystallization changes, release behavior and oral absorption. The morphology of the obtained nanocrystals was found to be rod shape. The particle size and zeta potential were 140 ± 10 nm and -29.11 mV, respectively. The X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analysis indicated that felodipine had undergone crystal form transition. The dissolution rate of felodipine was significantly increased and the AUC0-t value of felodipine colloidal dispersion in beagle dogs was approximately 1.6-fold greater than that of the commercial tablets. Nanocrystals impose a faster dissolution rate and higher oral absorption efficiency than raw crystals, and show great potential as an effective strategy for improving oral bioavailability of poorly soluble drugs. Key words: Felodipine – Nanocrystals – Characterization – Dissolution rate – Oral bioavailability.
oral bioavailability without the support of the in vivo pharmacokinetic experiment data [17]. The present work is aimed at increasing the dissolution rate and oral absorption of felodipine by preparing stable colloidal dispersion systems. More comprehensive and systematic experiments, both in vitro and in vivo, were carried out. Several important factors involved in the preparation processing and formulation composition were investigated, such as stabilizers and their amount in the anti-solvent, the concentration of felodipine in the solvent, ultrasonic application power and time. The ready-made felodipine nanosuspensions were characterized utilizing transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Finally, the in vitro dissolution behavior and the oral bioavailability of felodipine nanosuspension tablets were also determined.
Poor water solubility has become a major hurdle in the development of the oral solid dosage forms for noticeable large amount of drug candidates due to their incomplete dissolution and low oral absorption [1, 2]. According to the Noyes-Whitney equation and the Ostwald-Freundlich equation [3], the enhanced aqueous solubility of these compounds can improve their dissolution rate, and thereby enhance oral bioavailability. Nanosuspensions, one of the stable colloidal dispersions, can dramatically improve the dissolution rate and oral absorption by reducing the particle size down to the nanoscale [4]. Two main approaches have been developed and widely applied to prepare nanosuspensions: the top-down approach (reducing the particle size of large crystals into nanocrystals) and the bottom-up approach (building up nanocrystals by precipitation of the dissolved molecules). Both methods are capable of distributing drug particles evenly in water as colloidal dispersion system. Typical “top-down” approaches include high-pressure homogenization [5], media milling [6] and supercritical fluid technologies [7, 8], etc. Even though frequently being used, “top-down” approach is difficult and expensive to scale up in the actual production due to its high energy-consuming and specific preparation devices. Anti-solvent precipitation, as a typical “bottom-up” approach [9], is an effective approach widely utilized to prepare nanosuspensions. Polymers and surfactants are usually used as stabilizers in the solvent or anti-solvent to inhibit the crystal growth by forming hydrogen bonds with the drugs and reducing the surface energy, i.e. hydroxypropylmethylcellulose (HPMC), Tween-80, polyvidone (PVP) and Pluronic F68 etc. [10, 11]. In recent decade, aiming at controlling the size and achieving monodispersion of nanocrystals, ultrasound has been widely used as a feasible and effective approach to regulate and control the nucleation and crystallization process [12-14]. Felodipine [15], as a dihydropyridine calcium channel antagonist, is a typical BCS II compound with low solubility and high permeability [1]. It has been reported that the absolute oral bioavailability of this drug is around 15 %. After oral administration, felodipine can be absorbed quickly, and the Tmax is around 1-2 h [16]. In view of its high lipophilicity, the dissolution is the rate-limiting process of oral absorption. Sahu and Das have prepared felodipine nanosuspensions by precipitation-ultrasonnication technique. However, they only focused on in vitro experiments, and speculated the possibility of improving
I. MATERIALS AND METHODS 1. Materials
Felodipine was purchased from Zhejiang Kelisian Pharmaceutical Factory (China). Felodipine commercial tablets were purchased from Jinan Limin Pharmaceutical Factory (China). Pluronic F68 and Tween 80 was generously supplied by BASF Co., Ltd. (Shanghai, China). Pregelatine starch was purchased from Colorcon Technology Co., Ltd. (Shanghai, China). MCC was purchased from Asahi Kasei Pharma Corporation (Japan). L-HPC was purchased from Yingkou Aoda Pharmaceutical Factory (China). HPMC E5 was purchased from Huzhou Zhanwang Pharmaceutical Factory (China). DMSO, mannitol and glycine were purchased from Tianjin Bodi chemical Co., Ltd. Acetonitrile and anhydrous ether of chromatographic grade were purchased from Concord Technology Co., Ltd. (Tianjin, China).
2. Experiments
2.1. Preparation of felodipine colloidal dispersions The precipitation-ultrasonication method was adopted to prepare felodipine nanosuspensions, and the whole preparation process is vividly shown in Figure 1. Briefly, Pluronic F68 was dispersed in DMSO under ultrasonic wave, and then added felodipine to it, heating until dissolved and cooling the solution to a room temperature as a series of organic solutions containing 10, 20, 40 mg/mL of drug. Different proportions of HPMC E5 and Tween 80 were dissolved in water to 173
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency C. Luo, Y. Li, J. Sun, Y. Zhang, Q. Chen, X. Liu, Z. He
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
2.2.4. X-ray diffraction (XRD) To further confirm the crystal form transition of felodipine during preparation, the raw crystals, blank excipients, physical mixture and nanocrystals of felodipine were analyzed by a Bruker AXS D8 discover (Bruker AXS, Karlsruhe, Germany) with Cu Kα radiation at a wavelength, generated at 200 mA and 40 kV. The scanning speed was 1.2°/min from 3 to 45° of 2θ. 2.3. Dissolution tests Dissolution tests were carried out using the USP Apparatus 2 setup (ZRS-8G; Tianda Tianfa Technology Co., Ltd., Tianjin, China). 0.1 % Tween 80 solution was selected as dissolution medium. The temperature was maintained at 37 ± 0.5 ℃, and the paddle speed used was 100 rpm. Felodipine nanosuspension tablets and its commercial tablets containing the equivalent of 5mg drug were put into the 250 mL dissolution medium, then, samples, each of 5 mL, were withdrawn at different times (2, 5, 10, 15, 30, 45, 60, 90 and 120 min) and passed through a 0.45μm syringe filter. Quantification of the samples was done using a UV spectrophotometer (Mode 1801, RaxLEIGH) at 361 nm.
Figure 1 - Graphical description of the precipitation-ultrasonication procedure for the preparation of felodipine colloidal dispersions.
obtain a series of anti-solvents with the concentrations of 0.8:0.8 %, 0.4:0.4 % and 0.6:0.8 % (w/v). The anti-solvent was cooled to below 3 ℃ in an ice-bath for 30 min. Then, 2 mL of organic solution was quickly added into 50 mL of the precooled anti-solvent at a stirring speed of 500 rpm. After the anti-solvent precipitation, the samples were immediately treated with an ultrasonic probe at different ultrasonic power (200, 300, 400 and 500W) for different ultrasonic application times (7.5, 15 and 30 min). An ice-bath was utilized to control the temperature of ultrasonication. Different formulation and process parameters were methodically investigated to explore the effects on the properties of felodipine nanocrystals, including the drug concentration in the solvent, the proportions of HPMC E5 and Tween 80 in the anti-solvent, the ultrasonic application power and time length. With the purpose of long-term stability and further dosage form formulation, the suspensions were freeze-dried using FDU-1100 (Eyela, Tokyo Rikakikai Co., Ltd., Japan) with 10 % (w/v) glycine and 10 % (w/v) mannitol as cryoprotectants. The freeze-drying processes were as follows: the samples were freezed at -70 ℃ in Sanyo VIP series - 45 ℃ ultralow-freezers (Sanyo Electric Co., Ltd., Tokyo, Japan) for 4 h, and subsequently lyophilized at a temperature of- 25 ℃ for 8 h, followed by a secondary drying phase of 8 h at 10 ℃. Then, with the purpose of comparing with its commercial tablets, the lyophilized powder of felodipine nanosuspensions was prepared into tablets by direct compression method: screened felodipine freezedried powder through a 100-mesh screen, after fully blended the felodipine freeze-dried powder, microcrystalline cellulose (MCC), pregelatine starch and low substituted hydroxyprepyl cellulose (LHPC), compressed into tablets directly. The content of felodipine was 5 mg. With the self-made tablets and commercial tablets of felodipine as the test and reference preparations respectively, pharmacokinetic studies were carried out in dogs.
2.4. Pharmacokinetic study 2.4.1. Animals and dosing According to the Guide for Care and Use of Laboratory Animals, six male beagle dogs were used for the pharmacokinetic study. These dogs were divided into two groups randomly, and a crossover-randomized experimental design was carried out with an interval period of seven days. The dogs were fasted for about 12 h prior to the experiments, and only given water freely. The tablets were administered orally at a single dose of 5 mg. Three millilitres of blood samples was got into a heparinized blood collection tube via a detaining needle at pre-dose, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.5, 6.0, 8.0, 12.0, 24.0, 36.0, 48.0 h, respectively. The plasma fraction was obtained by centrifuging the blood samples at 3000 rpm for 5 min, and was stored at - 20 °C. 2.4.2. Determination of felodipine in plasma The felodipine concentrations in plasma were determined by a validated ultra performance liquid chromatography-dual mass spectrometry (UPLC-MS/MS) method after liquid-liquid extraction by anhydrous ether with nimodipine as internal standard. The chromatographic separations were acquired on an Acquity UPLC system (Waters Corp., Milford, MA, United States) and BEH C18 column (50 mm × 2.1 mm, 1.7 µm; Waters Corp.) with a mobile phase composed of 70 % acetonitrile and 30 % water (containing 0.2 %, v/v formic acid). The compounds were analyzed by multiple reaction monitoring (MRM) of the transitions of m/z 384→338 for felodipine and m/z 419→343 for nimodipine, respectively.
2.2. Characterization 2.2.1. Particle size and zeta potential The laser diffraction (LD) method was used to determine the mean size and size distribution. The zeta potential of felodipine nanocrystals was investigated utilizing a Nicomp-380/ZLS Zeta potential analyzer (Beckman-Coulter Co., Ltd., United States).
2.4.3. Data analysis The maximum plasma concentration of felodipine (Cmax) and the time to reach Cmax (Tmax) were obtained directly from the drug concentration-time profiles. The area under curve (AUC), elimination rate constant (k) and the half life of the preparation (t1/2) were calculated using DAS 2.0 software. All pharmacokinetics parameters were shown as their mean ± SD (standard deviation). The t-test was utilized to conduct the statistical analysis, and the differences were considered significant at p < 0.05.
2.2.2. Morphology Transmission electron microscopy (TEM, H-600, Hitachi, Japan) was utilized to observe the morphology of the felodipine crystals.
II. RESULTS AND DISCUSSION 1. Influences of different proportions of HPMC E5 and Tween 80 on colloidal dispersity
2.2.3. Differential scanning calorimetry (DSC) To investigate the possible phase transition of felodipine crystals, the raw crystals, blank excipients, physical mixture and nanosuspensions of felodipine were analyzed by differential scanning calorimetry (DSC-60, Shimadzu Co., Japan) with a heating rate of 10 ℃/min from 28 to 250 ℃. Al2O3 was slected as reference and the whole experimental process was done under the protection of nitrogen.
Polymers and surfactants are usually used together as effective stabilizers for the nanosuspensions. Tween 80 was found showing specific affinity towards felodipine during the formulation screening process, and HPMC E5 was selected as the co-stabilizer of Tween 80 for its relatively low viscosity in water. As shown in Table I, with the concentrations of 174
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency C. Luo, Y. Li, J. Sun, Y. Zhang, Q. Chen, X. Liu, Z. He
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
application power (200, 300, 400, 500W) was used for 15 min. The crystal size significantly decreased with the increase of ultrasonic power (below 400W). This phenomenon can be understood as the increased applied power effect on the surface of large crystals and crystal aggregates. However, the particle size did not show obvious difference between 400 and 500W. In addition, the particle size and size distribution were heavily influenced by ultrasonic application time. With the ultrasonic application power fixed at 500 W, the particle size of felodipine nanocrystals was reduced along with processing time increased to 15 min unexpectedly, a longer time (30 min) was not beneficial for further reducing the particle size at all, but lead to an uneven size distribution. Therefore, 500W and 15 min were selected as the optimal values of ultrasonic application power and time, respectively. It is no doubt that ultrasonic treatment helps to enhance the uniformity of felodipine colloidal dispersion prominently. During the crystallization process, ultrasound can enhance diffusion, strengthen the mass transfer and raise the nucleation rate, resulting in reduction and uniform distribution in crystal size during the crystallization process [21]. With fixed ultrasound intensity, the duration of ultrasonication dramatically influences the crystallization process. A short time ultrasound is not enough for molecular diffusion and mass transfer. But too long ultrasonication time does no benefit the uniform distribution of the colloidal dispersion, because too long ultrasonication time affects, adversely, the drug-polymer interaction, weakening the stabilizing effects of stabilizers.
Table I - Particle size and distribution of different amount of stabilizers combination. Stabilizers in water (mg/50 mL)
Particle description
HPMC E5
Tween 80
Particle size (nm)
Distribution
400 200 300
400 200 400
370 150 140
Polydispersion Polydispersion Monodispersion
HPMC E5 and Tween 80 ranging from 0.4:0.4 % to 0.8:0.8 % (w/v), the particle size increased from 150 to 370 nm, and they all showed polydispersion in size distribution. When the concentrations of HPMC E5 and Tween 80 altered from 0.4:0.4 % to 0.6:0.8 % (w/v), although the mean particle size had no obvious differences, the size distribution tended to be more uniform. This result indicated that the proportions of HPMC E5 and Tween 80 in anti-solvent solution were important formulation parameters influencing particle size and its distribution. An appropriate proportion of HPMC E5 and Tween 80 (0.6:0.8 % (w/v)) was screened to achieve small and uniform particle size. In this colloidal dispersion, as a nonionic polymer, complete adsorption of HPMC E5 on the growing crystal surface provides hydrodynamic layers surrounding the nanocrystals to prevent further growth and aggregation of crystals due to steric hindrance and repulsion between the neighboring nanocrystals [18, 19]. The hydrogen bonding has been reported in the literature as a major force driving the absorption of polymers on the crystal surface [20]. In this study, this strong drug-polymer interaction originated from two carbonyl groups of felodipine and the abundant hydroxyl groups of HPMC E5. Rapid crystal growth and aggregation would occur due to inadequate surface absorption on the surface of polymers. However, too high concentration of stabilizers resulted in the hindered conversion of ultrasonic energy to surface energy of the nanocystals during precipitation due to the enhanced viscosity. In addition, surfactants, absorbed on the particle surface, are another stabilizing factor to maintain the monodispersion by reducing the huge surface energy significantly.
4. Particle size, zeta potential and morphology
In the optimal formulation compositions and process conditions, the mean particle size and zeta potential of felodipine nanosuspensions were 140 ± 10 nm and - 29.11 mV, respectively. The morphology of felodipine nanocrystals is presented in Figure 2, and the nanocrystals were found to be rod in shape by TEM observation.
2. Drug loading
It is always expected to achieve higher drug loading when designing and preparing the desired drug delivery system (DDS), but this should be obtained on a stable and uniform DDS. Felodipine nanosuspensions were prepared at the optimal proportion of HPMC E5 and Tween-80 in the anti-solvent (0.6:0.8 % (w/v)) and at three drug concentrations in the organic solution (10, 20, 40 mg/mL) with the fixed ultrasonic application time and power at 15 min and 500 W, respectively. It was found that increasing the drug concentrations from 10 to 20 mg/mL had almost no effect on the particle size, but with the drug concentrations increased to 40 mg/mL, the nansuspensions presented as a polydispersion system with not evenly distributed size. This can be explained based on the crystallization theory. Two main stages make up the crystal formation process, namely nucleation and crystals growth, and the final particle size and size distribution depend on rates of the above two stages. The driving force is supersaturation, the growth rate of felodipine crystallization increased along with the degree of supersaturation. In our study, the particle size and its distribution of felodipine crystals at both low drug concentrations (10 and 20 mg/mL) and a high drug concentration (40 mg/mL) were investigated. Under the conditions of two low drug concentrations, the particle size was about 140 nm with a uniform distribution. Whereas, at the higher drug concentration, faster growth and aggregation rates occurred, leading to an uneven dispersed system due to greater supersaturation.
Figure 2 - Transmission electron microscopy micrographs of felodipine nanosuspensions, the scale bar represents for 100 nm.
5. DSC and XRD analysis
DSC and X-ray diffraction have been applied to explore potential transformations of the structure of felodipine crystals. Both results of DSC and X-ray diffraction indicated that there might be substantial crystalline change occurred in the felodipine nanocrystals. As shown in Figure 3, raw crystals showed a single sharp endothermic peak (146 ℃) ascribed to the melting of the drug, the blank excipients had two endothermic peaks (53 and 162 ℃), and the physical mixture just showed the above three endothermic peaks. However, the nanocrystals of felodipine displayed only one single sharp endothermic peak
3. The ultrasonic application power and time length
Fixing the drug concentration and the proportion of HPMC E5 and Tween 80 at 20 mg/mL and 0.6:0.8 % (w/v), different ultrasonic 175
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency C. Luo, Y. Li, J. Sun, Y. Zhang, Q. Chen, X. Liu, Z. He
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
Figure 4 - XRD curves of felodipine raw drug, blank excipients, physical mixture and nanocrystals.
Figure 3 - DSC curves of felodipine raw drug, blank excipients, physical mixture and nanosuspensions.
(150 ℃), and this might indicate phase transformation in felodipine crystal structure. The results of X-ray diffraction further confirmed the crystal form and potential polymorphic conversion during the preparation process. The XRD patterns of crude felodipine, blank excipients, physical mixtures and felodipine colloidal dispersions are presented in Figure 4. Three new peaks (at 2θ of 9.64, 17.88 and 28.32°) appeared in the profile of colloidal dispersions, but could not be found in the other three groups, demonstrating felodipine had undergone a polymorphism transition from a stable crystal form to a metastable one during the precipitation process. Figure 5 - The ideal colloid structure of felodipine nanocrystals.
6. Colloidal structure and stability
Smaller size with huge surface energy makes colloidal dispersions thermodynamic unstable, nano-sized particles tend to aggregate together to reduce interfacial energy without the stabilization of various stabilizers, and this is a spontaneous process. A variety of stabilisation mechanisms have been observed for the polymer-coated nanoparticles [22]. In our study, there are mainly three factors contributing to the stability of felodipine nanosuspensions, as shown in Figure 5. First and foremost, steric hindrance and repulsion, deriving from HPMC E5 and Tween 80, play key roles in maintaining colloidal stability. HPMC E5, absorbed on the crystal surface, could effectively prevent further growth and aggregation of neighboring particles. Tween 80, with an amphiphilic structure, significantly reduced interfacial energy. Moreover, the zeta potential of felodipine nanosuspensions is - 29.11 mV, as a result, neighboring colloidal particles repel each other due to the similar surface electric charges, which is beneficial to keep the stability of the colloidal system. Finally, due to the hydration of stabilizers, the repulsion of hydrated shell also acts as a barrier to prevent particles aggregation. In summary, there existed three lines of defense guarding against the growth and aggregation of felodipine colloidal dispersion: steric hindrance, charge repulsion and hydrated shell.
Figure 6 - Dissolution profiles of felodipine nanosuspension tablets and its commercial tablets in 0.1 % Tween 80 solution (n = 3).
dose in dogs are presented in Figure 7. The main pharmacokinetic parameters of felodipine are shown in Table II. The Cmax and AUC0-t values of the nanosuspension tablets were approximately 1.2-fold and 1.6-fold greater than that of commercial tablets, respectively. The results showed that the nanosuspensions can obviously enhance the oral absorbability rate of felodipine and improve the bioavailability. There are at least three factors contributing to the improved oral absorption: i) the reduced particle size with increased surface area significantly enhance the dissolution performance of felodipine, which results in rapid absorption of drug molecules across the gastrointestinal membrane [23]; ii) due to the reduced size and a high degree of dispersibility, the bioadhesion between the nanocrystals and the intestinal epithelium was increased due to closer and longer contact between nanoparticles and the gastrointestinal membrane [24]; iii) most important, nanoparticles, including nanocrystals, may cross the gastrointestinal membrane via endocytosis, gain access into the mesenteric lymphatic systems and bypass the liver entering the blood circulation directly [5, 25]. And in this regard, we can better understand why nanosuspension tablets, with a faster dissolution rate, but a longer Tmax than the commercial formulation.
7. Dissolution rate
The dissolution profiles of the nanosuspension tablets and commercial tablets in 0.1 % Tween 80 solution are shown in Figure 6. Due to the higher surface area of nanocrystals available for dissolution, the nanosuspension tablets demonstrated a significantly improvement in dissolution rate with more than 80 % of the drug dissolved in 15 min. In contrast, the commercial tablets showed poor dissolution rate, and only around 30 % of the drug dissolved in 15 min.
8. Pharmacokinetics study
The accurate and sensitive UPLC-MS-MS method and the reliable liquid-liquid extraction process were applied to pharmacokinetic studies. The mean felodipine concentration-time curves of the nanosuspension tablets and commercial tablets following a single oral 176
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency C. Luo, Y. Li, J. Sun, Y. Zhang, Q. Chen, X. Liu, Z. He
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
7. 8.
9. 10. 11. 12.
Figure 7 - Average drug concentration-time profiles after oral administration of felodipine nanosuspension tablets and the reference tablets (means ± SD, n = 6).
13.
Table II - Pharmacokinetic parameters of felodipine nanosuspension tablets and commercial tablets in beagles (means ± SD, n = 6). Formulations
Tmax (h)
Cmax (ng/mL)
AUC0-t (ng·h/mL)
Nanosuspension tablets Commercial tablets
1.83 ± 0.58
23.06 ± 15.80
117.88 ± 65.74
1.00 ± 0.00
20.21 ± 14.27
73.13 ± 50.48
14.
15. 16.
No significant difference.
*
17.
Stable felodipine nanocrystals were prepared utilizing the combination precipitation-ultrasonication method. Process and formulation parameters could significantly affect the particle size and distribution of nanocrystals. With the optimum conditions, nanocrystals with size of about 140 ± 10 nm were successfully prepared. There was substantial crystalline conversion during the preparation of the nanocrystals, compared to the raw materials. The in vitro dissolution rate of felodipine was significantly increased by reducing the particle size. The pharmacokinetic studies demonstrated that the AUC0-t values of the felodipine nanosuspension tablets in beagle dogs was approximately 1.6-fold greater than that of the commercial tablets. To sum up, the nanosuspension delivery technology is an effective strategy for improving oral bioavailability of poorly soluble drugs.
18.
19.
20. 21. 22.
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ACKNOWLEDGEMENT This work was financially supported from the National Nature Science Foundation of China (No. 81173008), from the National Basic Research Program of China (973 Program, No. 2009CB930300) and from Project for Excellent Talents of Liaoning Province (No.LR20110028).
MANUSCRIPT Received 2 July 2013, accepted for publication 7 October 2013. 177
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency Cong Luo1, Yan Li1, Jin Sun1, 2*, Yan Zhang3, Qin Chen1, Xiaohong Liu1, Zhonggui He1* No. 59 Mailbox, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China 2 Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, 300193, PR China 3 Northeast Pharmaceutical Group Co., Ltd, Shenyang 110021, PR China *Correspondence: [email protected]; [email protected] 1
In the present work, nanocrystals were prepared to improve oral bioavailability of felodipine. Several important formulation factors and process parameters were explored, optimized and analyzed systematically. The prepared nanocrystals were characterized for morphology, particle size, zeta potential, possible crystallization changes, release behavior and oral absorption. The morphology of the obtained nanocrystals was found to be rod shape. The particle size and zeta potential were 140 ± 10 nm and -29.11 mV, respectively. The X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analysis indicated that felodipine had undergone crystal form transition. The dissolution rate of felodipine was significantly increased and the AUC0-t value of felodipine colloidal dispersion in beagle dogs was approximately 1.6-fold greater than that of the commercial tablets. Nanocrystals impose a faster dissolution rate and higher oral absorption efficiency than raw crystals, and show great potential as an effective strategy for improving oral bioavailability of poorly soluble drugs. Key words: Felodipine – Nanocrystals – Characterization – Dissolution rate – Oral bioavailability.
oral bioavailability without the support of the in vivo pharmacokinetic experiment data [17]. The present work is aimed at increasing the dissolution rate and oral absorption of felodipine by preparing stable colloidal dispersion systems. More comprehensive and systematic experiments, both in vitro and in vivo, were carried out. Several important factors involved in the preparation processing and formulation composition were investigated, such as stabilizers and their amount in the anti-solvent, the concentration of felodipine in the solvent, ultrasonic application power and time. The ready-made felodipine nanosuspensions were characterized utilizing transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Finally, the in vitro dissolution behavior and the oral bioavailability of felodipine nanosuspension tablets were also determined.
Poor water solubility has become a major hurdle in the development of the oral solid dosage forms for noticeable large amount of drug candidates due to their incomplete dissolution and low oral absorption [1, 2]. According to the Noyes-Whitney equation and the Ostwald-Freundlich equation [3], the enhanced aqueous solubility of these compounds can improve their dissolution rate, and thereby enhance oral bioavailability. Nanosuspensions, one of the stable colloidal dispersions, can dramatically improve the dissolution rate and oral absorption by reducing the particle size down to the nanoscale [4]. Two main approaches have been developed and widely applied to prepare nanosuspensions: the top-down approach (reducing the particle size of large crystals into nanocrystals) and the bottom-up approach (building up nanocrystals by precipitation of the dissolved molecules). Both methods are capable of distributing drug particles evenly in water as colloidal dispersion system. Typical “top-down” approaches include high-pressure homogenization [5], media milling [6] and supercritical fluid technologies [7, 8], etc. Even though frequently being used, “top-down” approach is difficult and expensive to scale up in the actual production due to its high energy-consuming and specific preparation devices. Anti-solvent precipitation, as a typical “bottom-up” approach [9], is an effective approach widely utilized to prepare nanosuspensions. Polymers and surfactants are usually used as stabilizers in the solvent or anti-solvent to inhibit the crystal growth by forming hydrogen bonds with the drugs and reducing the surface energy, i.e. hydroxypropylmethylcellulose (HPMC), Tween-80, polyvidone (PVP) and Pluronic F68 etc. [10, 11]. In recent decade, aiming at controlling the size and achieving monodispersion of nanocrystals, ultrasound has been widely used as a feasible and effective approach to regulate and control the nucleation and crystallization process [12-14]. Felodipine [15], as a dihydropyridine calcium channel antagonist, is a typical BCS II compound with low solubility and high permeability [1]. It has been reported that the absolute oral bioavailability of this drug is around 15 %. After oral administration, felodipine can be absorbed quickly, and the Tmax is around 1-2 h [16]. In view of its high lipophilicity, the dissolution is the rate-limiting process of oral absorption. Sahu and Das have prepared felodipine nanosuspensions by precipitation-ultrasonnication technique. However, they only focused on in vitro experiments, and speculated the possibility of improving
I. MATERIALS AND METHODS 1. Materials
Felodipine was purchased from Zhejiang Kelisian Pharmaceutical Factory (China). Felodipine commercial tablets were purchased from Jinan Limin Pharmaceutical Factory (China). Pluronic F68 and Tween 80 was generously supplied by BASF Co., Ltd. (Shanghai, China). Pregelatine starch was purchased from Colorcon Technology Co., Ltd. (Shanghai, China). MCC was purchased from Asahi Kasei Pharma Corporation (Japan). L-HPC was purchased from Yingkou Aoda Pharmaceutical Factory (China). HPMC E5 was purchased from Huzhou Zhanwang Pharmaceutical Factory (China). DMSO, mannitol and glycine were purchased from Tianjin Bodi chemical Co., Ltd. Acetonitrile and anhydrous ether of chromatographic grade were purchased from Concord Technology Co., Ltd. (Tianjin, China).
2. Experiments
2.1. Preparation of felodipine colloidal dispersions The precipitation-ultrasonication method was adopted to prepare felodipine nanosuspensions, and the whole preparation process is vividly shown in Figure 1. Briefly, Pluronic F68 was dispersed in DMSO under ultrasonic wave, and then added felodipine to it, heating until dissolved and cooling the solution to a room temperature as a series of organic solutions containing 10, 20, 40 mg/mL of drug. Different proportions of HPMC E5 and Tween 80 were dissolved in water to 173
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency C. Luo, Y. Li, J. Sun, Y. Zhang, Q. Chen, X. Liu, Z. He
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
2.2.4. X-ray diffraction (XRD) To further confirm the crystal form transition of felodipine during preparation, the raw crystals, blank excipients, physical mixture and nanocrystals of felodipine were analyzed by a Bruker AXS D8 discover (Bruker AXS, Karlsruhe, Germany) with Cu Kα radiation at a wavelength, generated at 200 mA and 40 kV. The scanning speed was 1.2°/min from 3 to 45° of 2θ. 2.3. Dissolution tests Dissolution tests were carried out using the USP Apparatus 2 setup (ZRS-8G; Tianda Tianfa Technology Co., Ltd., Tianjin, China). 0.1 % Tween 80 solution was selected as dissolution medium. The temperature was maintained at 37 ± 0.5 ℃, and the paddle speed used was 100 rpm. Felodipine nanosuspension tablets and its commercial tablets containing the equivalent of 5mg drug were put into the 250 mL dissolution medium, then, samples, each of 5 mL, were withdrawn at different times (2, 5, 10, 15, 30, 45, 60, 90 and 120 min) and passed through a 0.45μm syringe filter. Quantification of the samples was done using a UV spectrophotometer (Mode 1801, RaxLEIGH) at 361 nm.
Figure 1 - Graphical description of the precipitation-ultrasonication procedure for the preparation of felodipine colloidal dispersions.
obtain a series of anti-solvents with the concentrations of 0.8:0.8 %, 0.4:0.4 % and 0.6:0.8 % (w/v). The anti-solvent was cooled to below 3 ℃ in an ice-bath for 30 min. Then, 2 mL of organic solution was quickly added into 50 mL of the precooled anti-solvent at a stirring speed of 500 rpm. After the anti-solvent precipitation, the samples were immediately treated with an ultrasonic probe at different ultrasonic power (200, 300, 400 and 500W) for different ultrasonic application times (7.5, 15 and 30 min). An ice-bath was utilized to control the temperature of ultrasonication. Different formulation and process parameters were methodically investigated to explore the effects on the properties of felodipine nanocrystals, including the drug concentration in the solvent, the proportions of HPMC E5 and Tween 80 in the anti-solvent, the ultrasonic application power and time length. With the purpose of long-term stability and further dosage form formulation, the suspensions were freeze-dried using FDU-1100 (Eyela, Tokyo Rikakikai Co., Ltd., Japan) with 10 % (w/v) glycine and 10 % (w/v) mannitol as cryoprotectants. The freeze-drying processes were as follows: the samples were freezed at -70 ℃ in Sanyo VIP series - 45 ℃ ultralow-freezers (Sanyo Electric Co., Ltd., Tokyo, Japan) for 4 h, and subsequently lyophilized at a temperature of- 25 ℃ for 8 h, followed by a secondary drying phase of 8 h at 10 ℃. Then, with the purpose of comparing with its commercial tablets, the lyophilized powder of felodipine nanosuspensions was prepared into tablets by direct compression method: screened felodipine freezedried powder through a 100-mesh screen, after fully blended the felodipine freeze-dried powder, microcrystalline cellulose (MCC), pregelatine starch and low substituted hydroxyprepyl cellulose (LHPC), compressed into tablets directly. The content of felodipine was 5 mg. With the self-made tablets and commercial tablets of felodipine as the test and reference preparations respectively, pharmacokinetic studies were carried out in dogs.
2.4. Pharmacokinetic study 2.4.1. Animals and dosing According to the Guide for Care and Use of Laboratory Animals, six male beagle dogs were used for the pharmacokinetic study. These dogs were divided into two groups randomly, and a crossover-randomized experimental design was carried out with an interval period of seven days. The dogs were fasted for about 12 h prior to the experiments, and only given water freely. The tablets were administered orally at a single dose of 5 mg. Three millilitres of blood samples was got into a heparinized blood collection tube via a detaining needle at pre-dose, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.5, 6.0, 8.0, 12.0, 24.0, 36.0, 48.0 h, respectively. The plasma fraction was obtained by centrifuging the blood samples at 3000 rpm for 5 min, and was stored at - 20 °C. 2.4.2. Determination of felodipine in plasma The felodipine concentrations in plasma were determined by a validated ultra performance liquid chromatography-dual mass spectrometry (UPLC-MS/MS) method after liquid-liquid extraction by anhydrous ether with nimodipine as internal standard. The chromatographic separations were acquired on an Acquity UPLC system (Waters Corp., Milford, MA, United States) and BEH C18 column (50 mm × 2.1 mm, 1.7 µm; Waters Corp.) with a mobile phase composed of 70 % acetonitrile and 30 % water (containing 0.2 %, v/v formic acid). The compounds were analyzed by multiple reaction monitoring (MRM) of the transitions of m/z 384→338 for felodipine and m/z 419→343 for nimodipine, respectively.
2.2. Characterization 2.2.1. Particle size and zeta potential The laser diffraction (LD) method was used to determine the mean size and size distribution. The zeta potential of felodipine nanocrystals was investigated utilizing a Nicomp-380/ZLS Zeta potential analyzer (Beckman-Coulter Co., Ltd., United States).
2.4.3. Data analysis The maximum plasma concentration of felodipine (Cmax) and the time to reach Cmax (Tmax) were obtained directly from the drug concentration-time profiles. The area under curve (AUC), elimination rate constant (k) and the half life of the preparation (t1/2) were calculated using DAS 2.0 software. All pharmacokinetics parameters were shown as their mean ± SD (standard deviation). The t-test was utilized to conduct the statistical analysis, and the differences were considered significant at p < 0.05.
2.2.2. Morphology Transmission electron microscopy (TEM, H-600, Hitachi, Japan) was utilized to observe the morphology of the felodipine crystals.
II. RESULTS AND DISCUSSION 1. Influences of different proportions of HPMC E5 and Tween 80 on colloidal dispersity
2.2.3. Differential scanning calorimetry (DSC) To investigate the possible phase transition of felodipine crystals, the raw crystals, blank excipients, physical mixture and nanosuspensions of felodipine were analyzed by differential scanning calorimetry (DSC-60, Shimadzu Co., Japan) with a heating rate of 10 ℃/min from 28 to 250 ℃. Al2O3 was slected as reference and the whole experimental process was done under the protection of nitrogen.
Polymers and surfactants are usually used together as effective stabilizers for the nanosuspensions. Tween 80 was found showing specific affinity towards felodipine during the formulation screening process, and HPMC E5 was selected as the co-stabilizer of Tween 80 for its relatively low viscosity in water. As shown in Table I, with the concentrations of 174
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency C. Luo, Y. Li, J. Sun, Y. Zhang, Q. Chen, X. Liu, Z. He
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
application power (200, 300, 400, 500W) was used for 15 min. The crystal size significantly decreased with the increase of ultrasonic power (below 400W). This phenomenon can be understood as the increased applied power effect on the surface of large crystals and crystal aggregates. However, the particle size did not show obvious difference between 400 and 500W. In addition, the particle size and size distribution were heavily influenced by ultrasonic application time. With the ultrasonic application power fixed at 500 W, the particle size of felodipine nanocrystals was reduced along with processing time increased to 15 min unexpectedly, a longer time (30 min) was not beneficial for further reducing the particle size at all, but lead to an uneven size distribution. Therefore, 500W and 15 min were selected as the optimal values of ultrasonic application power and time, respectively. It is no doubt that ultrasonic treatment helps to enhance the uniformity of felodipine colloidal dispersion prominently. During the crystallization process, ultrasound can enhance diffusion, strengthen the mass transfer and raise the nucleation rate, resulting in reduction and uniform distribution in crystal size during the crystallization process [21]. With fixed ultrasound intensity, the duration of ultrasonication dramatically influences the crystallization process. A short time ultrasound is not enough for molecular diffusion and mass transfer. But too long ultrasonication time does no benefit the uniform distribution of the colloidal dispersion, because too long ultrasonication time affects, adversely, the drug-polymer interaction, weakening the stabilizing effects of stabilizers.
Table I - Particle size and distribution of different amount of stabilizers combination. Stabilizers in water (mg/50 mL)
Particle description
HPMC E5
Tween 80
Particle size (nm)
Distribution
400 200 300
400 200 400
370 150 140
Polydispersion Polydispersion Monodispersion
HPMC E5 and Tween 80 ranging from 0.4:0.4 % to 0.8:0.8 % (w/v), the particle size increased from 150 to 370 nm, and they all showed polydispersion in size distribution. When the concentrations of HPMC E5 and Tween 80 altered from 0.4:0.4 % to 0.6:0.8 % (w/v), although the mean particle size had no obvious differences, the size distribution tended to be more uniform. This result indicated that the proportions of HPMC E5 and Tween 80 in anti-solvent solution were important formulation parameters influencing particle size and its distribution. An appropriate proportion of HPMC E5 and Tween 80 (0.6:0.8 % (w/v)) was screened to achieve small and uniform particle size. In this colloidal dispersion, as a nonionic polymer, complete adsorption of HPMC E5 on the growing crystal surface provides hydrodynamic layers surrounding the nanocrystals to prevent further growth and aggregation of crystals due to steric hindrance and repulsion between the neighboring nanocrystals [18, 19]. The hydrogen bonding has been reported in the literature as a major force driving the absorption of polymers on the crystal surface [20]. In this study, this strong drug-polymer interaction originated from two carbonyl groups of felodipine and the abundant hydroxyl groups of HPMC E5. Rapid crystal growth and aggregation would occur due to inadequate surface absorption on the surface of polymers. However, too high concentration of stabilizers resulted in the hindered conversion of ultrasonic energy to surface energy of the nanocystals during precipitation due to the enhanced viscosity. In addition, surfactants, absorbed on the particle surface, are another stabilizing factor to maintain the monodispersion by reducing the huge surface energy significantly.
4. Particle size, zeta potential and morphology
In the optimal formulation compositions and process conditions, the mean particle size and zeta potential of felodipine nanosuspensions were 140 ± 10 nm and - 29.11 mV, respectively. The morphology of felodipine nanocrystals is presented in Figure 2, and the nanocrystals were found to be rod in shape by TEM observation.
2. Drug loading
It is always expected to achieve higher drug loading when designing and preparing the desired drug delivery system (DDS), but this should be obtained on a stable and uniform DDS. Felodipine nanosuspensions were prepared at the optimal proportion of HPMC E5 and Tween-80 in the anti-solvent (0.6:0.8 % (w/v)) and at three drug concentrations in the organic solution (10, 20, 40 mg/mL) with the fixed ultrasonic application time and power at 15 min and 500 W, respectively. It was found that increasing the drug concentrations from 10 to 20 mg/mL had almost no effect on the particle size, but with the drug concentrations increased to 40 mg/mL, the nansuspensions presented as a polydispersion system with not evenly distributed size. This can be explained based on the crystallization theory. Two main stages make up the crystal formation process, namely nucleation and crystals growth, and the final particle size and size distribution depend on rates of the above two stages. The driving force is supersaturation, the growth rate of felodipine crystallization increased along with the degree of supersaturation. In our study, the particle size and its distribution of felodipine crystals at both low drug concentrations (10 and 20 mg/mL) and a high drug concentration (40 mg/mL) were investigated. Under the conditions of two low drug concentrations, the particle size was about 140 nm with a uniform distribution. Whereas, at the higher drug concentration, faster growth and aggregation rates occurred, leading to an uneven dispersed system due to greater supersaturation.
Figure 2 - Transmission electron microscopy micrographs of felodipine nanosuspensions, the scale bar represents for 100 nm.
5. DSC and XRD analysis
DSC and X-ray diffraction have been applied to explore potential transformations of the structure of felodipine crystals. Both results of DSC and X-ray diffraction indicated that there might be substantial crystalline change occurred in the felodipine nanocrystals. As shown in Figure 3, raw crystals showed a single sharp endothermic peak (146 ℃) ascribed to the melting of the drug, the blank excipients had two endothermic peaks (53 and 162 ℃), and the physical mixture just showed the above three endothermic peaks. However, the nanocrystals of felodipine displayed only one single sharp endothermic peak
3. The ultrasonic application power and time length
Fixing the drug concentration and the proportion of HPMC E5 and Tween 80 at 20 mg/mL and 0.6:0.8 % (w/v), different ultrasonic 175
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency C. Luo, Y. Li, J. Sun, Y. Zhang, Q. Chen, X. Liu, Z. He
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
Figure 4 - XRD curves of felodipine raw drug, blank excipients, physical mixture and nanocrystals.
Figure 3 - DSC curves of felodipine raw drug, blank excipients, physical mixture and nanosuspensions.
(150 ℃), and this might indicate phase transformation in felodipine crystal structure. The results of X-ray diffraction further confirmed the crystal form and potential polymorphic conversion during the preparation process. The XRD patterns of crude felodipine, blank excipients, physical mixtures and felodipine colloidal dispersions are presented in Figure 4. Three new peaks (at 2θ of 9.64, 17.88 and 28.32°) appeared in the profile of colloidal dispersions, but could not be found in the other three groups, demonstrating felodipine had undergone a polymorphism transition from a stable crystal form to a metastable one during the precipitation process. Figure 5 - The ideal colloid structure of felodipine nanocrystals.
6. Colloidal structure and stability
Smaller size with huge surface energy makes colloidal dispersions thermodynamic unstable, nano-sized particles tend to aggregate together to reduce interfacial energy without the stabilization of various stabilizers, and this is a spontaneous process. A variety of stabilisation mechanisms have been observed for the polymer-coated nanoparticles [22]. In our study, there are mainly three factors contributing to the stability of felodipine nanosuspensions, as shown in Figure 5. First and foremost, steric hindrance and repulsion, deriving from HPMC E5 and Tween 80, play key roles in maintaining colloidal stability. HPMC E5, absorbed on the crystal surface, could effectively prevent further growth and aggregation of neighboring particles. Tween 80, with an amphiphilic structure, significantly reduced interfacial energy. Moreover, the zeta potential of felodipine nanosuspensions is - 29.11 mV, as a result, neighboring colloidal particles repel each other due to the similar surface electric charges, which is beneficial to keep the stability of the colloidal system. Finally, due to the hydration of stabilizers, the repulsion of hydrated shell also acts as a barrier to prevent particles aggregation. In summary, there existed three lines of defense guarding against the growth and aggregation of felodipine colloidal dispersion: steric hindrance, charge repulsion and hydrated shell.
Figure 6 - Dissolution profiles of felodipine nanosuspension tablets and its commercial tablets in 0.1 % Tween 80 solution (n = 3).
dose in dogs are presented in Figure 7. The main pharmacokinetic parameters of felodipine are shown in Table II. The Cmax and AUC0-t values of the nanosuspension tablets were approximately 1.2-fold and 1.6-fold greater than that of commercial tablets, respectively. The results showed that the nanosuspensions can obviously enhance the oral absorbability rate of felodipine and improve the bioavailability. There are at least three factors contributing to the improved oral absorption: i) the reduced particle size with increased surface area significantly enhance the dissolution performance of felodipine, which results in rapid absorption of drug molecules across the gastrointestinal membrane [23]; ii) due to the reduced size and a high degree of dispersibility, the bioadhesion between the nanocrystals and the intestinal epithelium was increased due to closer and longer contact between nanoparticles and the gastrointestinal membrane [24]; iii) most important, nanoparticles, including nanocrystals, may cross the gastrointestinal membrane via endocytosis, gain access into the mesenteric lymphatic systems and bypass the liver entering the blood circulation directly [5, 25]. And in this regard, we can better understand why nanosuspension tablets, with a faster dissolution rate, but a longer Tmax than the commercial formulation.
7. Dissolution rate
The dissolution profiles of the nanosuspension tablets and commercial tablets in 0.1 % Tween 80 solution are shown in Figure 6. Due to the higher surface area of nanocrystals available for dissolution, the nanosuspension tablets demonstrated a significantly improvement in dissolution rate with more than 80 % of the drug dissolved in 15 min. In contrast, the commercial tablets showed poor dissolution rate, and only around 30 % of the drug dissolved in 15 min.
8. Pharmacokinetics study
The accurate and sensitive UPLC-MS-MS method and the reliable liquid-liquid extraction process were applied to pharmacokinetic studies. The mean felodipine concentration-time curves of the nanosuspension tablets and commercial tablets following a single oral 176
Felodipine nanosuspension: a faster in vitro dissolution rate and higher oral absorption efficiency C. Luo, Y. Li, J. Sun, Y. Zhang, Q. Chen, X. Liu, Z. He
J. DRUG DEL. SCI. TECH., 24 (2) 173-177 2014
7. 8.
9. 10. 11. 12.
Figure 7 - Average drug concentration-time profiles after oral administration of felodipine nanosuspension tablets and the reference tablets (means ± SD, n = 6).
13.
Table II - Pharmacokinetic parameters of felodipine nanosuspension tablets and commercial tablets in beagles (means ± SD, n = 6). Formulations
Tmax (h)
Cmax (ng/mL)
AUC0-t (ng·h/mL)
Nanosuspension tablets Commercial tablets
1.83 ± 0.58
23.06 ± 15.80
117.88 ± 65.74
1.00 ± 0.00
20.21 ± 14.27
73.13 ± 50.48
14.
15. 16.
No significant difference.
*
17.
Stable felodipine nanocrystals were prepared utilizing the combination precipitation-ultrasonication method. Process and formulation parameters could significantly affect the particle size and distribution of nanocrystals. With the optimum conditions, nanocrystals with size of about 140 ± 10 nm were successfully prepared. There was substantial crystalline conversion during the preparation of the nanocrystals, compared to the raw materials. The in vitro dissolution rate of felodipine was significantly increased by reducing the particle size. The pharmacokinetic studies demonstrated that the AUC0-t values of the felodipine nanosuspension tablets in beagle dogs was approximately 1.6-fold greater than that of the commercial tablets. To sum up, the nanosuspension delivery technology is an effective strategy for improving oral bioavailability of poorly soluble drugs.
18.
19.
20. 21. 22.
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ACKNOWLEDGEMENT This work was financially supported from the National Nature Science Foundation of China (No. 81173008), from the National Basic Research Program of China (973 Program, No. 2009CB930300) and from Project for Excellent Talents of Liaoning Province (No.LR20110028).
MANUSCRIPT Received 2 July 2013, accepted for publication 7 October 2013. 177