Abstract
This paper describes a method to prepare indomethacin-loaded poly(butylcyanoacrylate) (PBCA) nanoparticles based on the anionic polymerization procedure, often used in the synthesis of poly(alkylcyanoacrylate) (PACA) nanoparticles for drug delivery. A detailed investigation into the capability of the polymeric nanoparticles to load this drug is discussed, along with the effect of the technique parameters on characteristics of the nanoparticles. The results indicated that indomethacin-loaded PBCA nanoparticles showed a particle size distribution that could be successfully exploited for the formulation of colloidal pharmaceutical systems. The particles were predominantly less than 200 nm in size with a negative charge, and rather stable when pH was adjusted to neutral. In addition, X-ray diffraction experiments revealed that the drug would be molecularly dispersed in the polymers in an amorphous state and crystalline with very small size. In vitro drug release revealed that indomethacin incorporation and/or adsorption led to a rapid drug release followed by a slower release in biological phosphate buffer and that the release rate decreased with increasing indomethacin content in the particle.
1. Introduction
As newer and more powerful drugs continue to be developed, greater attention is given to the administration methods of these active substances. During the past 30 years, controlled drug delivery has become an important area of research and development (1). Drug properties could be modified for medical therapy via a control release system, such as sustained or pulsatile drug release, natural targeting of drugs to different body compartments, and amplification of therapeutic effects or reduction of the therapeutic dose (2, 3). If the drug delivery could be precisely matched with physiological needs, controlled drug delivery would be the optimal way of administering drugs.
Considerable work demonstrates that biodegradable polymers are ideal as drug delivery carriers because of their biodegradability, stability, minimum toxicity, and immunological response under physiological conditions (4). In various biodegradable polymers, poly(alkylcyanoacrylate) (PACA) nanoparticles have been widely proposed as an effective drug delivery device (5). The PACA nanoparticles are generated from emulsion polymerization in acidic aqueous solutions in the presence of surfactants and are typically composed of a solid core with a highly porous inner structure (6). Various quantities of drugs, dissolved in the medium during or after polymerization, are entrapped within the nanoparticles' polymer network or covalently bound to the polymer for sustained and localized administration (7). It has been suggested that in chronic treatments rapidly degraded PACAs seem more adequate in avoiding the overloading of cells than slowly degrading polyesters (8, 9).
Indomethacin is a powerful non-steroidal anti-inflammatory drug (NSAID) that is effective in the management of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, and acute gout (10). Topical NSAIDs are also widely used to prevent intraoperative miosis and ocular inflammation (11, 12). However, their effectiveness is limited by various undesirable reactions, such as serious gastrointestinal reactions (even stomach perforation), central nervous system symptoms, liver function damage, inhibition of the hematopoeitic system, and allergic reaction (13); these reactions are dose-related and associated with long-term administration (14).
One feasible means of overcoming these shortcomings and increasing therapeutic effect is to encapsulate indomethacin into biodegradable polymers, such as spray-dried powders of polymeric nanocapsules (15), amphiphilic block copolymeric nanospheres (16), and core/shell-type nanoparticles (17). Nevertheless, comparing the processes to produce these biodegradable polymers with the emulsion process used to prepare PACA nanoparticle colloidal carriers, the presence of organic solvents is the more obvious disadvantage for therapeutic use. In addition, indomethacin is generally a highly crystalline and poorly water-soluble drug (18). Therefore, much interest is focused on improving its solubility by either increasing surface area or stabilizing the amorphous form of the drug within a polymeric matrix. Nanoparticles show an increased adhesiveness to tissue and may induce changes in the crystalline structure, increasing the amorphous fraction in the particle or even creating a completely amorphous particle (19).
The main aim of the present study was to prepare a new drug delivery carrier for indomethacin, taking advantage of nanoparticle engineering. High-intensity ultrasound can break the aggregation and reduce the size of the particles due to its dispersion, crushing, emulsifying, and activation effects, and thus has better control of the morphology of particles (20). The increment in dispersion of indomethacin in the reaction medium by ultrasound was also expected to promote the encapsulation of indomethacin into the poly(butylcyanoacrylate) (PBCA) nanoparticles. Particularly, we made a contribution to the characterization of indomethacin-loaded PBCA nanoparticles. The in vitro release profile of the nanoparticles was also evaluated.
2. Materials and Methods
2.1. Chemicals
Indomethacin as anti-inflammatory drug was purchased from ShiJiaZhuang HuaSheng Pharmacy Co., Ltd. (Hebei, China). Butylcyacnoacrylate (BCA) used as the monomer for polymerization was obtained from ShunKang Medical-glue Co., Ltd. (Beijing, China). Pluronic F-68 and Dextran T-70 were obtained from Sigma (St. Louis, MO, USA). Deionized water was used. All other chemicals and solvents used were of analytical or pharmaceutical grade.
2.2. Nanoparticle Preparation
Indomethacin-loaded PBCA nanoparticles were prepared by emulsion polymerization according to the method described by Couvreur and Vauthier (7), with a few modifications. The technique involved the incorporating process followed by the adsorptive process (21).
The desired amount of indomethacin was dispersed into a polymerization medium (10 mL), composed of aqueous solution, acidified at 1.5, 2.0, 3.0, and 4.0 with HCl, containing Pluronic F-68 and Dextran T-70 at the ratio of 1:1 (w/w) and at the concentration of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 0.6%, respectively. An appropriate amount of BCA monomers (1%) was added dropwise over a period of 10 min under magnetic stirring (1000 rpm) to the above aqueous solution. After 2 h of stirring, this system was transferred to a sonicatior. The sample underwent ultrasonication for 3 min, and then was stirred for a further 2 h until the polymerization process was visually determined to be complete. The obtained colloidal suspension was neutralized with 0.5 N NaOH and stirred at 600 rpm (2 h) to reach equilibrium loading.
The resulting dispersions were then filtered through a 0.45-um sieve membrane to eliminate aggregated particles, and pale-green suspensions were obtained and stored at 4°C for further processing. Initial indomethacin content, pH of polymerizing mixture, and amount of emulsifiers were evaluated as variables to obtain the optimized formulation.
2.3. Particle Morphology Observation
The morphology of the nanoparticles was observed by a transmission electron microscope (TEM, JEOL TEM-2000EX, Tokyo,Japan). A sample was diluted with distilled water, and a drop of this dispersion was placed on a 300 mesh carbon-coated copper grid. After drying, the samples were negatively stained with an aqueous solution of phosphotungstate acid and analyzed at an electron voltage of 150 kV.
2.4. Analysis of Nanoparticle Size
Photon correlation spectroscopy (Malvern Instruments,Malvern, UK) was used to elucidate the particle size and size distribution of drug-loaded PBCA nanoparticles. Colloidal suspension of each nanoparticle sample were submitted to spectroscopic analysis. Each dispersion, diluted with filtered (0.22-μm), deionized water to a favorable concentration for better measurement, was kept in a 1-cm glass cell, without any preliminary filtration or centrifugation. The cell was put in the cell holder of a spectrometer and heated to 25°C. Measurements were recorded over the angular range of 20–140°. The obtained value is the average result of three experimental determinations and expressed as the value of z-average size ± standard deviation (SD). The polydispersity index (PI) was used as a measure of the width of the size distribution.
2.5. Zeta Potential Measurement
Drug-loaded PBCA nanoparticle suspensions at the same pH were diluted with an aqueous solution of NaCl (0.001 M) to ensure that the signal intensity was suitable for the instrument. The zeta potential was measured by laser Doppler Velocimetry (Malvern Instruments, Malvern,UK) at 25°C. Values are presented as mean ± SD from three replicate samples.
2.6. X-Ray Diffraction (XRD) Analysis
X-ray powder diffractometry analyses were performed to determine whether indomethacin was present in the nanoparticles in a crystalline or an amorphous state. A Philips PW diffractometer (beam 173 nm) was used; samples that were freeze-dried were then exposed to monochromatic Cu Kα radiation (0.45 kV × 20 mA, λ = 1.5406 Å). The diffraction pattern was determined in the area 10° < 2θ < 50°, using a stepwise method (0.2°/s).
2.7. Determination of Drug Content
A calibration curve for the validated UV assays of indomethacin was performed on five solutions in the concentration range 0.1 to 40 μg/mL, correlation coefficient >0.999. Indomethacin added to the blank PBCA nanoparticles was dissolved with ethanol and THF(Tetrahydrofuran) (1:1, v/v), and the indomethacin content was determined from the absorbance. The mean recovery was 97.61 ± 2.17 (n = 9) at high, intermediate, and low concentrations. The nanoparticle suspensions were sonicated and centrifuged at 4°C with a Beckman rotor model JA-2550 (16,500 rpm). The supernatant was discarded to remove free indomethacin, and the sediment was then lyophilized by a freeze-dryer system to obtain dried nanoparticle products. The freeze-dried nanoparticles, after being weighed accurately, were disrupted by ethanol and THF (1:1, v/v) and the amount of indomethacin entrapped was determined by measuring the UV absorbance at 319 nm. The indomethacin loading efficiency was expressed both as a drug efficiency (EE %) (eq 1) and drug content (LE %) (eq 2), calculated from these equations, respectively:
2.8. In Vitro Release Kinetics from Nanoparticles
The dialysis bag diffusion technique was used to study the release of indomethacin from nanoparticle suspensions produced at pH 2.0 with indomethacin concentration of 0.4 and 0.8%, w/v. An initial colloidal suspension (10 mL) was centrifuged and lyophilized as described above. After accurate weighing, half of the lyophilized nanoparticles were suspended in 5 mL of biological phosphate buffer. A portion (2.5 mL) of this suspension was placed into a dialysis tube, which was immersed in 25 mL of biological phosphate buffer solution (pH 7.4) and stirred at 50 rpm. The temperature was maintained at 37.0 ± 0.5°C during all the release experiments. Each experiment was performed in triplicate. At fixed time intervals, samples (3 mL) of the released solution were drawn and the concentration of indomethacin was analyzed at a maximum wavelength of 319 nm. After determination, 3-mL samples of the solvent were returned, and each time this was done a little buffer was added by weighing to maintain the release medium volume.
3. Results and Discussion
3.1. Particle Size and Morphology
Figure 1 shows transmission electron microscopy (TEM) photographs of the PBCA particles with a diameter between 100 and 200 nm. The sizes estimated from the photographs are 115 ± 25 nm (Figure 1b), 70 ± 10 nm (Figure 1c), and 275 ± 25 nm (Figure 1d). As observed, the PBCA particles are rather spherical and moderately monodisperse, slightly forming a network of spherical particles upon drying (Figure 1d). Figure 1a shows the particles synthesised when the initial monomer in the medium was 1%, and the picture clearly shows a polymer shell ≈17 nm thick. Concerning the thickness of the polymer coating, it should be noted that an excess of initially added monomer does not necessarily lead to a more efficient coverage, but lower monomer concentration in the medium (<0.3%, v/v) indeed is insufficient to the formation of spherical nanoparticles; small pieces were observed by TEM photographs. Thus, the changes observed in the surface texture of the drug after covering the nanoparticles with the polymer must reflect a true change in the physical properties of the surface of the drug upon sufficient coverage by the polymer.
TEM photographs of indomethacin-loaded PBCA nanoparticles using optimized method. A: pH = 3.0, Pluronic P-68 (0.3%), Dextran T-70 (0.3%), 100 μL BCA, indomethacin 0.8 mg/mL. B: pH = 2.0, Pluronic P-68 (0.2%), Dextran T-70 (0.2%), 100 μL BCA, indomethacin 0.8 mg/mL. C: pH = 2.0, Pluronic P-68 (0.2%), Dextran T-70 (0.2%), 100 μL BCA, indomethacin 0.2 mg/mL. D: pH = 4.0, Pluronic P-68 (0.2%), Dextran T-70 (0.2%), 100 μL BCA, indomethacin 0.8 mg/mL.
Considering the final potential use of these particles, it is interesting to comment on the significance of the sizes obtained for the particles. The particle size affects the biodistribution once it is injected into the body. According to Gupta and Gupta (22), particles with diameters in excess of 200 nm will easily be sequestered by the spleen and removed by the cells of the reticuloendothelial system. On the contrary, very small carriers (lower than 10 nm) will be rapidly cleared after their extensive extravasation. Therefore, the nanoparticles prepared under the condition in Figure 1b (pH = 2.0, Pluronic P-68 20 mg, Dextran T-70 20 mg, BCA 1%, indomethacin 0.2 mg/mL) might be the ideal size range for colloidal carriers to be spread systemically.
3.2. Influence of pH Values on Indomethacin-loaded Nanoparticles
The polymerization of alkylcyanoacrylates occurred by the anionic and zwitterionic routes (9). The kinetics is governed by the relative amounts of the alcohol (
OH) groups of the surfactant, and hydroxide (OH−) from water dissociation. Meanwhile, the aqueous phase pH will influence the ionization of a drug and hence its solubility, which will in turn affect the maximum weight of the drugs that can be entrapped by PBCA nanoparticles. The solubility of highly hydrophobic indomethacin (pKa = 4.5) in acidic solution is poor. It would be largely unionized at pH 3.0 and fully ionized at pH 7.0. Therefore, it can be concluded that the H+ concentration determines both the polymerization rate and the drug encapsulation.
Experiments were carried out for pH values between 1.5 and 5 with 1% (v/v) monomer, 0.4% (w/v) Dextran T-70, 0.4% (w/v) Pluronic F-68, and 0.8% (w/v) indomethacin. It was observed visually that if a pH higher than 4 was used, an opalascent suspension was obtained with many macroaggregates and a comparatively large amount of solid polymer precipitates. On the other hand, no formation of PBCA nanoparticles was observed at pH below 3, as a translucent suspension was observed. Table I demonstrates that the pH of the polymerization medium greatly influenced the particle size. Obviously, as OH− concentration increased as pH increased from 2 to 4, the particle size increased. This can be attributed to the increase in the polymerization rates with the increase in the hydroxide concentration with increasing pH. An increase of particle size resulted in a decrease of the surface area per unit volume, which consequently prevented the total drug diffusion from the particles towards the suspending medium during the fabrication procedure, leading to a higher drug loading at pH 4 than that at pH 3. However, the smaller nanoparticle was obtained at pH 2.0, but not pH 1.5, with a particle size of 147.9 ± 2.8 nm. This probably was related to the longer polymerization time at low pH, resulting in longer chains and potentially larger particles.
Influence of pH Values of the Polymerization Medium on the Property of Indomethacin-loaded PBCA Nanoparticles
The zeta potential of the nanoparticles was also characterized. Commonly, zeta potential can be an index to the stability of the nanoparticles. The nanoparticles prepared in our study were negatively charged, similar to previous studies (23, 24), and strongly dependent on the pH of the system. Generally, the absolute value of zeta potential was enhanced by increasing the pH of the polymerization medium. The results corresponded to the acidic character of the molecule, of which the ionization degree was increased at higher pH with more negatively charged COOH−. The zeta potential was assumed to be due to the terminal carboxylic end groups of the polymeric chain and/or the adsorption of anions from the aqueous phase (24, 25).
The loading capability of the nanoparticles in different pH media is shown in Table I. The values increased when the pH of the nanoparticle suspension was elevated to 2.0, but reduced at pH 3 followed by a slight increase at pH 4. Such results lead us to think that a reduction of the ionic character of indomethacin might increase its affinity for oligomeric chains. Couvreur et al. (26) have also demonstrated that the adsorptive capacity of the nanoparticles was greater when the drug is non-ionized in the solution. In addition, it was proposed that the rapid polymerization rate might inhibit the diffusion of the water-insoluble drug from the suspending medium towards the particles, leading to a rapid reduction in loading efficiency at pH 3. The slight increase in loading efficiency at pH 4 could be caused by the increment in solubility of indomethacin at a higher pH.
3.3. Influence of the Emulsifier Concentration in a Medium
It is important to examine the effect of emulsifier concentration on particle size, size distribution, and drug loading capability of the manufactured nanoparticles. The average size of the nanoparticles characterized by laser scattering and their drug load capacity measured by UV are shown in Figures 2a and 2b, respectively. The Pluronic F-68 and Dextran T-70 (weight ratio of 1:1) as emulsifier are crucial to the generation of polymeric nanospheres, due to the combined action of initiating and stabilizing alkylcyanoacrylates by their hydroxyl group, which in some cases compete with polymerization initiated by the basic group in the drug molecule and decrease the drug loading capacity (27, 28). It is easy to understand that an insufficient amount of the emulsifier would fail to stabilize all the nanoparticles and thus some of them would tend to aggregate; as a result, nanoparticles with a large size would be produced. As indicated in Figure 2a, particle size decreased whereas the size polydispersity index increased as the concentration of Pluronic F-68 and Dextran T-70 increased from 0.1% to 0.4% (w/v). It was speculated that the stability of the particles increased with increasing coverage of their surfaces with emulsifier. However, the surplus of emulsifier did not help to further reduce the nanoparticles size when the concentration was up to 0.6%, perhaps because the fabricated nanoparticles were covered by the emulsifier molecules. The polydispersity index increased with the emulsifier concentration, perhaps because of excess surfactant.
Effect of emulsifier amount on particle size and polydispersity of indomethacin-loaded PBCA nanoparticles (a), and loading capacity of indomethacin-loaded PBCA nanoparticles (b). Condition: pH = 2.5, Pluronic P-68 (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%), Dextran T-70 (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%), 100 μL BCA, indomethacin 0.6 mg/mL.
The amount of emulsifier plays a fundamental role in determining not only the size of fabricated nanoparticles, but the drug loading capacity as well (Figure 2b). The drug loading and encapsulation efficiencies increased with increasing concentration from 0.1 to 0.2% and almost leveled off over the range of 0.2 to 0.4%, and decreased afterwards. At low surfactant concentrations, the PBCA nanoparticles with porous surfaces lose drugs from the surface into the suspension medium by diffusion (29). As the surfactant concentration increased, the porous surfaces of the PBCA nanoparticles gradually became smooth (30), and therefore minimized the loss of drug during synthesis (31). But a higher concentration of emulsifier (0.6 %, w/v) resulted in a reduction of encapsulation efficiency. This can possibly be explained by the fact that some drug molecules may bind to the excessive emulsifier molecules and thus cause the loss of drugs in the fabrication process. It can thus be concluded that the presence of an optimal amount of the emulsifier used in the fabrication process for the encapsulation efficiency is important.
3.4. Content of Indomethacin in the Medium
In this section, we describe our data about the role of changing indomethacin content on the particle characteristics. The size of the nanoparticles also depends on the drug content in the medium (Figure 3a). With constant monomer and emulsifiers, the size of nanoparticles increased with indomethacin content in the medium over the range of 0.2 to 1.2% (w/v) at pH 2.0. This slight increment of particle size might be attributed to the interaction of the drug with chain nucleation and monomer polymerization leading to the increase in particle size and molecular weight. The result reported by Van Snick et al. (32) suggested that a small amount of ionized indomethacin with negatively charged 
COOH− existing in an aqueous medium might act as an initiator to induce the polymerization, despite its poor solubility at a lower pH. When the amount of indomethacin enhanced the particle size, more indomethacin might be embedded onto the nanoparticles' surface with exposed negatively charged 
COOH−, contributing to an increment in absolute value of zeta potential.
Effect of indomethacin content on: (a) particle size and surface charge of indomethacin-loaded PBCA nanoparticles, and (b) loading capability of indomethacin-loaded PBCA nanoparticles. Condition: pH = 1.5, Pluronic P-68 (0.2%), Dextran T-70 (0.2%), 100 μL BCA, indomethacin 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1.0 mg/mL, 1.2 mg/mL.
With respect to drug loading capacity, the amount of nanoparticles dispersions increased with drug concentration up to 1.2 % (w/v) in the reaction medium (Figure 3b). Similar results have been reported from adsorption of hematoporphyrin onto PACA nanoparticles (25). Fawaz and Guyot (21) have also found that the association of ciprofloxacin with PIBCA nanoparticles prepared either by adsorption or by incorporation increased with the initial concentration of the drug in the reaction medium. However, it should be noted that this trend was contrary to that for the encapsulation efficiency, which dropped from 94.32 to 61.43% with increased content of indomethacin.
3.5. XRD of the Powder
The XRD spectra of indomethacin, virgin PBCA nanoparticle powder, and indomethacin-loaded PBCA nanoparticle powder prepared under the same condition are shown in Figure 4. Indomethacin exhibited several intense peaks at 2θ = 19°, 21°, 23°, 26°, and 29°. However, after the drug-incorporating process, XRD data clearly shows a notable decrease in crystallinity of the drug in the PBCA nanoparticles as compared to the XRD patterns of pure indomethacin. The characteristic peak at 2θ = 19° and 23° in the XRD data due to indomethacin crystals became weak, although it still can be observed, while other peaks were not observed in the XRD patterns from samples of indomethacin-loaded nanoparticles. The intensity of the XRD peak depends on the crystal size. Therefore, the diffractograms of the indomethacin-loaded PBCA nanoparticles indicated that the drug would be molecularly dispersed in the polymers in an amorphous state and crystalline with very small particle size.
XRD patterns of the powder: (a) indomethacin (b) indomethacin-loaded PBCA nanoparticles, (c) virgin PBCA nanoparticles.
3.6. In Vitro Release of Indomethacin
The in vitro release profiles of indomethacin from the PBCA nanoparticles into a biological phosphate buffer are presented in Figure 5. The release of indomethacin from nanoparticles was observed to occur as a biphasic process with an initial rapid release followed by a slower liberation. The percentages of drug release after 50 h are 89.03 and 84.78% (w/w) for indomethacin-loaded PBCA nanoparticles with indomethacin content in the medium of 0.4 and 0.8% (w/v), respectively.
In vitro cumulative indomethacin release profile for indomethacin-loaded PBCA nanoparticles prepared with constant monomer (0.1 mL) and indomethacin content in the polymerization medium of 0.4 and 0.8% (w/v) at pH 2.
This biphasic profile, typical of this polymer family, suggests that the major fraction indomethacin was entrapped into the core of the polymeric network, as previously observed in drugs of a different nature (30, 33). The initial rapid release probably represents the loss of surface-associated and poorly entrapped (adsorbed more deeply into the surface pores) indomethacin. The slower release phase might be caused by the drug from the inner core of the particle as a result of particle disintegration, of drug diffusion through the inner core of the nanoparticles, or both. The degradation of PACAs started with an initial attack of the hydroxyl ions leading to the formation of carboanions and formaldehyde (34). These products react with the hydroxylions to produce formaldehyde and corresponding alcohol as the final degradation products. Therefore, an increase in the hydroxyl ion concentration causes faster degradation of the PACA nanoparticles. It has been shown that the hydrophobic alkyl side chains would shield effectively against the hydroxyl ions attack on the ester groups of PACA and therefore decrease the disintegration rate of PACA (35). Accordingly, a larger content of indomethacin in PBCA would increase the barrier for water and hydroxyl ions to penetrate and degrade PBCA polymer, which would lead to the decrease of the hydrolytic rate of drug-loaded particles as well. Although there is not enough proof to confirm that the relative indomethacin release rate decreases with increasing indomethacin content in particles, it could be offered as a possible conclusion.
4. Conclusion
Indomethacin was successfully incorporated into PBCA nanoparticles by the polymerization emulsion method. Data presented indicate that the technique parameters, including the pH of the polymerization medium, the drug content, and the emulsifier concentration, affect characteristics like the particle size, size distribution, and surface charge. A small fraction of ionized indomethacin might act as the initiator during the polymerization, promoting the drug diffusion from an aqueous phase to the nanoparticles. The particles were predominantly less than 200 nm in size with a negative charge, and rather stable when the pH was adjusted to neutral. Moreover, the nanoparticles displayed a particle size distribution that could be successfully exploited for the formulation of colloidal pharmaceutical systems, especially for the formulation of topical ocular administration. The influence of the technique parameters on the overall drug loading were investigated by means of optical absorbance determinations, and the increased trend of drug loading and encapsulation efficiency can not be achieved simultaneously. XRD experiments revealed that the indomethacin was present in the nanoparticles, either dispersed molecularly in polymers in an amorphous state or distributed in a crystalline state with small crystal size to be detected. In vitro drug release revealed that indomethacin incorporation and/or adsorption led to a rapid drug release followed by a slower liberation in biological phosphate buffer and that the release rate decreases with increasing indomethacin content in the particle.
Acknowledgement
The authors wish to express their sincere appreciation to Professor John Martin, professor at ZheJiang University, for his valuable suggestions and careful proofreading of our manuscript.
Footnotes
- © PDA, Inc. 2009
References
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