Journal of Controlled Release 139 (2009) 146–152

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Journal of Controlled Release

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Preparation and characterization of chitosan/polyguluronate nanoparticlesfor siRNA delivery

Dong Wook Lee a, Kyoung-Soo Yun a, Hong-Seok Ban a, Wonchae Choe b,Sang Kyung Lee a,⁎, Kuen Yong Lee a,⁎a Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Koreab Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea

⁎ Corresponding authors. K.Y. Lee is to be contactefax: +82 2 2293 2642. S.K. Lee, Tel.: +82 2 2220 2344;

E-mail addresses: sangkyunglee@hanyang.ac.kr (S.K(K.Y. Lee).

0168-3659/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.jconrel.2009.06.018

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 June 2009Accepted 20 June 2009Available online 28 June 2009

Keywords:siRNAChitosanPolyguluronateNanoparticleGene silencing

Small interfering RNA (siRNA) has been widely investigated as a potential therapeutic for treatment of variousdiseases. However, the use of siRNA is limiteddue to its rapid degradation and low intracellular association in vitroand in vivo. Chitosan nanoparticles encapsulating siRNA were prepared using a coacervation method in thepresence of polyguluronate (PG), which is isolated from alginate and is strongly related to ionic interactions ofnegatively charged alginate. Various physicochemical properties of chitosan/PG nanoparticles, including size,surface charge,morphology, and interactionwith siRNA,were characterized. Themeandiameter of siRNA-loadedchitosan-based nanoparticles ranged from 110 to 430 nm, depending on the weight ratio between chitosan andsiRNA. Nanoparticles showed low cytotoxicity and were useful in delivering siRNA to HEK 293FT and HeLa cells.Chitosan/PG nanoparticles were considered promising for siRNA delivery due to their low cytotoxicity and abilityto transport siRNA into cells, which can effectively inhibit induction of targeting mRNA.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

RNA interference (RNAi) has proven to be a powerful mechanismwhereby small interfering RNA (siRNA) consisting of 21–23 nucleotidesdegrades target mRNA with the help of an RNA-induced silencingcomplex (RISC) and inhibits the synthesis of protein encoded by themRNA [1]. This approach has enormous therapeutic potential fortreating various diseases including genetic disorders, cancers, andinfectious diseases. However, the delivery of siRNA has raised severalissues, including rapid degradation by nuclease, low intracellularuptake, and limited blood stability. To overcome these drawbacks,siRNA has been combined with cationic polymers or liposomes forenhancing intracellular uptake and increasing stability against nuclease[2]. Two typical vectors have been used as a gene delivery carrier, viraland non-viral vectors. Although viral vectors have shown highertransfection efficiencies in most cells, the safety issue was raised inseveral clinical trials [3]. Non-viral vectors have recently attractedmuchattention due to their ease of synthesis and modification, lowimmunogenicity, and controllable size [4]. Non-viral delivery systemsusing cationic liposomes and polymers, such as polyethylenimine (PEI),poly(L-lysine) (PLL), and their various derivatives, have been used tocondense plasmid DNA or siRNA to form nanoparticles [5–7].

d at Tel.: +82 2 2220 0482;fax: +82 2 2220 1998.. Lee), leeky@hanyang.ac.kr

l rights reserved.

Chitosan is a naturally existing polysaccharide composed ofglucosamine and N-acetylglucosamine residues and can be derived bypartial deacetylation of chitin, which is generally obtained fromcrustacean shells (Fig. 1a) [8]. Chitosan is known to be biocompatible,low toxic, low immunogenic, and degradable by enzymes [9–11].Chitosan is a cationic polysaccharide and has beenwidely used in manydrugdelivery applications, especially in gene delivery systems, due to itspositively charged amines allowing electrostatic interactions withnegatively charged nucleic acids to form stable complexes [12–14].There have been many studies on the development of chitosan-basedcarriers for DNA delivery. A complex coacervation of chitosan and DNAwas formed and used to deliver plasmid DNA into cells [15]. Thetransfection efficiency of chitosan/DNA nanoparticles was evaluated fordifferent cell types, including mesenchymal stem cells [16]. Chitosanparticles were modified with lactobionic acid bearing a galactose groupfor enhanced hepatocyte specificity [17]. Folic acid-coupled chitosannanoparticles were found to promote the targeting efficiency to tumorcells and internalization into the cells, resulting in improved transfectionrates [18]. Many studies have shown an effective expression of reportergenes in the presence of chitosan in vitro and in vivo [19–21].

Recentlychitosanhas foundpotential applications for siRNAdelivery.The formation of chitosan/siRNA nanoparticles was found to bedependent on the molecular weight and degree of deacetylation ofchitosan [22]. Chitosan was used to formulate easy-to-use freeze-driedsiRNA transfection reagents, which were used to store siRNA for2 months without loss of siRNA transfection activity [23]. Chitosan-sodium tripolyphosphate nanoparticles with entrapped siRNA had

Fig. 1. Chemical structure of (a) chitosan, (b) alginate, and (c) polyguluronate (PG).

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enhanced siRNA loading efficiency and considerable transfectionefficiency [24]. In addition, effective in vivo RNA interference wasachieved in bronchiole epithelial cells of transgenic EGFP mice afternasal administration of chitosan/siRNA nanoparticles, indicating thepotential application of chitosan-based nanoparticles in RNA-mediatedtherapy of systemic and mucosal disease [25].

Alginate has also been frequently used inmany applications of drugdelivery and tissue engineering due to its biocompatible, biodegrad-able, and non-toxic properties [26]. Alginate is a naturally occurringpolysaccharide composed of mannuronic acid (M) and guluronic acid(G) in a block wise manner (Fig. 1b). An inclusion of alginate intochitosan-based nanoparticles improved the transfection efficiency ofplasmid DNA in vitro, while maintaining biocompatibility and lowtoxicity [27,28]. However, the effect of alginate inclusion into chitosan-based nanoparticles encapsulating siRNAon the transfection efficiencyhas not been reported yet. In addition, direct complex formationbetween chitosan and alginate occurs instantly and often results in theformation of precipitates in an uncontrollablemanner, likely due to thehighmolecularweight of chitosan and alginate. Thus,we hypothesizedthat polyguluronate (PG), a block of G residues in the alginate back-bone, could be useful to control interactions with chitosan and to formstable nanoparticles due to its lowmolecular weight as well as its highcontribution to ionic interactions with cations.

In this context, we report a potential siRNA delivery system usingchitosan and an alginate derivative that are known to be biocompa-tible and non-toxic. Polyguluronate was isolated from alginate andused to cross-link chitosan chains to form stable nanoparticlesencapsulating siRNA. Nanoparticles were prepared from chitosanand PG by a coacervation method for siRNA delivery, and their variousphysicochemical properties such as size, zeta potential, morphology,and particle stability, were investigated. The toxicity, cellular associa-tion, and gene silencing efficiency of nanoparticles in various cellswere also evaluated.

2. Materials and methods

2.1. Materials

Chitosan glutamate (MW=470 kDa and DD=86%) and alginate(MW=200 kDa) were purchased from FMC Biopolymer (Norway).Sodium acetate was purchased from Sigma-Aldrich. Synthetic siRNAtargeting against pSV luciferase genes (sense: 5'-GGACAUUA-CUAGUGACUCATT-3', antisense: 5'-UGAGUCACUAGUAAUGUCCTT-3'),fluorescein isothiocyanate (FITC)-conjugated EGFP siRNA and diethyl-

pyrocarbonate (DEPC)-treated water were obtained from Samchullipharmaceutical (Korea). The pSV luciferase gene was purchased fromPromega (USA). The Endofree® plasmid Maxi kit was purchasedfrom Qiagen (USA). Dulbecco's modified Eagle's medium (DMEM),RPMI Medium 1640, phosphate buffered saline (PBS), penicillin-streptomycin, trypsin-EDTA, and fetal bovine serum (FBS) were pur-chased from Gibco (USA). Lipofectamine™ 2000 reagent waspurchased from Invitrogen (USA). The CellTiter 96R Aqueous OneSolution Cell Proliferation Assay kit was from Promega (USA) and usedfor MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. The water was distilled anddeionized using the Milli-Q System (USA). Other reagents were alsocommercially available and used without further purification.

2.2. Isolation of polyguluronate

Polyguluronate (PG) was isolated from sodium alginate by acidhydrolysis and collected at pH 2.85 [29]. The precipitate was dissolvedin distilled water and treated with activated carbon for furtherpurification. The solution was stirred thoroughly, filtered to re-move the activated carbon, precipitated by ethanol, and lyophilized(MW=6 kDa).

2.3. Preparation of nanoparticles

Chitosan glutamatewas dissolved in a sodium acetate buffer (0.1Msodium acetate, pH 5.5) to prepare a solution with different con-centrations, ranging from 70 to 280 µg/ml. Polyguluronate (PG) wasdissolved in sodium sulfate buffer (0.05 M). The solutions werefiltered through a 0.22-µm syringe filter (Millipore, USA), and gentlywarmed up to 55 °C. Nanoparticles were prepared by adding a chi-tosan solution to an equal volume of PG solution, and the nano-particles were incubated at room temperature for 30min before use orfurther analysis. For the preparation of siRNA-loaded nanoparticles,siRNA (14 µg/ml) in DEPC-treated water was first added to a PGsolution, followed by adding a chitosan solution. The weight ratio ofchitosan to PG was fixed at 20. The particles were then incubated atroom temperature for 30 min before use or further analysis.

2.4. Measurement of particle size and surface charge

The mean diameter and surface charge of nanoparticles weredetermined at 25 °C by Nano ZS Zetasizer (Malvern Instruments, UK).

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Three different samples were prepared, measured, and averaged(n=3).

2.5. Morphology

The morphology of nanoparticles was observed using atomic forcemicroscopy (AFM). An aqueous solution of nanoparticles was placedon a clean mica surface, and then washed with distilled water andpurged with nitrogen. The microscopic images were obtained by aNano-R2™ AFM (Pacific Nanotechnology, USA) in a scanned area of1.2×1.2 µm.

2.6. Gel retardation assay

The binding of siRNA with chitosan was tested by gel electrophor-esis using a 4% agarose gel. Samples with different weight ratios,defined as the weight ratio of chitosan to siRNA, were loaded into thegel, and electrophoresis was carried out at 55 V for 100 min runningwith a TBE buffer (4.45 mM Tris-base, 1 mM sodium EDTA, 4.45 mMboric acid, pH 8.3). Ethidium bromide was used to visualize siRNAbands using a UV transilluminator at 365 nm.

2.7. Determination of siRNA loading efficiency

The loading efficiency of siRNA in chitosan/PG nanoparticles wasobtained by determination of unbound siRNA concentration in thesupernatant recovered after particle centrifugation (20,000 ×g,20 min) using a UV spectrophotometer at 260 nm. The supernatantrecovered from chitosan/PG nanoparticles without siRNA was used asa blank. The siRNA loading efficiency (%) was expressed as thepercentage of bound siRNA (difference between the total amount ofsiRNA initially added for particle preparation and the amount ofunbound siRNA remaining in the supernatant after centrifugation) tothe total amount of siRNA initially added.

2.8. Serum stability of siRNA-loaded nanoparticles

Naked siRNA and chitosan nanoparticles loaded with siRNA wereincubated in the medium containing 20% fetal bovine serum at 37 °C.At each predetermined time point (3, 7, 16 and 24 h), the nano-particles were collected from themedium and stored at−20 °C until agel electrophoresis was performed. To detach siRNA from thenanoparticles, a phenol/chloroform extraction method was used be-fore the gel electrophoresis. The integrity of the siRNA was thenanalyzed by gel electrophoresis using a 4% agarose gel.

2.9. Cytotoxicity assay

HEK 293FT (human embryonic kidney cell line) and HeLa (humancervical carcinoma) cells were used to test the cytotoxicity ofnanoparticles. Cells were plated in 96-well tissue culture plates witha cell density of 1×104 per well and incubated at 37 °C under CO2

atmosphere overnight. After 24 h post-incubation with nano-particles at 37 °C, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox-ymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) wasadded to each well and then incubated for 2 h, followed bymeasurement of an optical density at 490 nm (Molecular Devices,USA). The cytotoxicity of Lipofectamine 2000™ and poly(L-lysine)(PLL), widely used non-viral vectors for gene delivery, was also tested.

2.10. Cellular association of nanoparticles

Cells were plated on a 12-well culture plate (1×105 cells/well) inDMEM with 10% FBS, and the cells were treated with nanoparticlescontaining 50 pmol FITC-conjugated siRNA per well. After 4 h, freshmedia containing serum (1 ml) were added to the cells and incubated

at 37 °C under a 5% CO2 atmosphere for 18 h. Cells were then fixedwith 2% formaldehyde and analyzed by a flow cytometer (BectonDickenson FACS Caliber, USA) to determine the efficiency of cellularassociation of nanoparticles.

2.11. Gene silencing

HEK 293FT and HeLa cells expressing luciferase were generated bytransfection with plasmid DNA encoding luciferase using Lipofecta-mine 2000™ according to the manufacturer's instructions. Briefly,cells were seeded on a 12-well tissue culture plate at a density of1.0×105 per well, and incubated overnight in DMEM. On the day oftransfection, the mediumwas removed and replaced with fresh mediawithout serum. Lipofectamine/plasmid complexes (5:1 weight ratio,0.8 µg DNA per well) were added dropwise to each well, and cellswere treated with the complexes for 4 h. Media were then removedand the cells were washed with PBS, followed by replenishing freshmedia containing serum. The cells were incubated for 24 h at 37 °Cunder CO2 atmosphere. The variation of luciferase activity of each wellwas less than 4%. Next, a volume of 50 µl of chitosan nanoparticlescontaining 50 pmol siRNA was added to each well and incubated at37 °C under a 5% CO2 atmosphere for 24 h. Luciferase activity wasdetermined using an Orion II microplate luminometer (BertholdDetection Systems, USA). The total protein content of the samples wasmeasured using a BCA protein assay kit (Pierce, USA) and was used tonormalize the luciferase activity.

3. Results and discussion

3.1. Preparation of chitosan/PG nanoparticles

Chitosan nanoparticles were prepared using a coacervationmethod in the presence of either alginate or polyguluronate (PG).Although alginate has been frequently used to formulate micro- ornanoparticles, its interactions with chitosan are sometimes verystrong, and complex formation occurs instantly. Thus for this context,polyguluronate was tested to form stable chitosan nanoparticlesinstead of intact alginate. The mean diameter of nanoparticles pre-pared in the presence of either alginate or PG increased when theweight ratio increased from 5 to 20 (Fig. 2a). However, the size ofchitosan nanoparticles formed with PG was smaller than thosewith intact alginate, likely due to the relatively low molecularweight of PG (MW=6 kDa) compared with that of intact alginate(MW=200 kDa). This result indicates that PG was useful to formcomplexes with chitosan due to the high population of G-blockresidues which mainly contribute to ionic interactions with cations[26]. The surface charge of chitosan nanoparticles was about +10 mVin the range of weight ratios used (Fig. 2b). The particle size andpositively charged surface of nanoparticles are critical for effectivegene delivery [30,31], and chitosan/PG nanoparticles were consideredpromising as a gene delivery carrier.

3.2. Preparation of siRNA-loaded chitosan nanoparticles

The complex formation of chitosan with siRNA was confirmed bygel electrophoresis. Chitosan/siRNA and chitosan/(PG+siRNA) nano-particles were prepared at various weight ratios (Fig. 3). Movement ofsiRNA was substantially retarded at a weight ratio of 10 compared tocontrol siRNA. Over a weight ratio of 50, the siRNA band disappeared,indicating a complete complex formation between chitosan andsiRNA. The inclusion of PG did not alter the complex formation abilitybetween chitosan and siRNA. It was reported that DNA can becondensed at low weight ratios (N:P=4–6) [32–34]. This may indi-cate that chitosan/siRNA complexes may form different architecturecompared to that of chitosan/DNA complexes, even if electrostatic

Fig. 2. (a) Size and (b) zeta potential of chitosan nanoparticles prepared by acoacervation method in the presence of either alginate or polyguluronate (n=3).

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interaction is the driving force for both cases. This might be attributedto the short length of siRNA (21mer) and its linearity [24].

3.3. Size and surface charge of chitosan/(PG+siRNA) nanoparticles

Themean diameter and zeta potential of nanoparticles loadedwithsiRNA were next investigated. Although chitosan was useful to form adirect complex with siRNA (Fig. 3), the size of the chitosan/siRNAcomplexes was not able to be determined. This was attributed to thelack of stability and uniformity of the complexes due to uncontrollablecomplex formation. However, stable chitosan/(PG+siRNA) nanopar-

Fig. 3. Gel retardation assay of chitosan/siRNA and chitosan/(PG+siRNA) nanoparticles. C an50, 100, and 200 represent the weight ratio of chitosan to siRNA.

ticles were formed, likely due to the competitive binding of PG to thechitosan chain. The mean diameter of the chitosan/(PG+siRNA)nanoparticles ranged from 110 to 430 nm depending on the weightratio (Fig. 4a). The size of the nanoparticles increased in parallel withan increase of weight ratios. The zeta potential of the nanoparticlesranged from +10 mV to +15 mV, suggesting a net positive surfacecharge due to excess chitosan. The surface charge (i.e., zeta potential)of chitosan/(PG+siRNA) nanoparticles slightly increased withincreasing the concentration of chitosan up to the weight ratio of50. However, no significant increase was observed over the weightratio of 100. The image of the chitosan nanoparticles loaded withsiRNA (weight ratio=20) was obtained by AFM with a scanned areaof 1.2×1.2 µm (Fig. 4b), suggesting a round shape of siRNA-loaded,chitosan-based nanoparticles. Nanoparticles formed at a weight ratioof 20 were used for stability and gene silencing test, as the size ofnanoparticles significantly increased over the weight ratio of 50. Thesize of chitosan/(PG+siRNA) complexes was also smaller than that ofchitosan/(alginate+siRNA) complexes (data not shown).

3.4. Loading efficiency and stability of nanoparticles

The loading content of siRNA was measured right after thepreparation of nanoparticles. The nanoparticles were sunk down bycentrifugation (20,000 ×g, 20 min), and the free siRNA in the su-pernatant was determined by a UV spectrophotometer and comparedto the total siRNA initially added. The loading efficiencywasmore than60% in the range of weight ratios used in this study (Fig. 5).

Next, the stability of nanoparticles in physiological conditions wastested, as the nanoparticles were initially prepared in acidic condi-tions. The pH of the nanoparticle suspensions prepared in acidicconditions was neutralized by adding PBS to the solution. The size ofthe chitosan nanoparticles was monitored for 24 h and found to benearly constant over time, even in a neutralized buffer solution(Fig. 6). The serum stability of siRNA loaded in the nanoparticles wastested next in 20% serum-containing media for 24 h. Rapid degrada-tion of naked siRNA was observed after 30 min (Fig. 7a). By contrast,degradation of siRNA protected by the nanoparticles was processedslowly for 7 h (Fig. 7b). The more enhanced stability of siRNA in thepresence of serum might be essential for the improved in vitro and invivo transfection efficiency.

3.5. Cytotoxicity, cellular association, and gene silencing of nanoparticles

The cytotoxicity of nanoparticles was evaluated with HEK 293FTand HeLa cells. Although cationic liposomes and PLL have beenwidelyused as non-viral gene delivery carriers, their significant cytotoxicitywas reported [35]. Surprisingly, chitosan-based nanoparticles formedat different weight ratios displayed low cytotoxicity (Fig. 8), which

d M represent naked siRNA and DNAmarkers, respectively. The lanes marked with 1, 10,

Fig. 6. Size changes of chitosan/(PG+siRNA) nanoparticles in (a) original and(b) neutralized buffer solution for 24 h.

Fig. 4. (a) Size and zeta potential of chitosan/(PG+siRNA) nanoparticles as a function ofweight ratio. (b) Atomic force microscopic image of chitosan/(PG+siRNA) nanoparti-cles (weight ratio=20).

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might be attributed to low toxicity of chitosan and alginate. Theseresults also indicate that the presence of PG does not increase thetoxicity of the nanoparticles. However, Lipofectamine™ 2000 and PLLshowed a substantial loss of cell viability, which was consistent withprevious findings.

Fig. 5. The loading efficiency of siRNA in chitosan/(PG+siRNA) nanoparticles preparedat various weight ratios.

Cellular associationwas next quantified by detecting the amount ofFITC-siRNA taken up by HeLa and HEK 293FT cells. The cellularassociation of chitosan/(PG+siRNA) nanoparticles was 28.6±7.2 and71.1±1.2% for HEK 293FT and HeLa cells, respectively. An increase ofchitosan content in the nanoparticles did not significantly change thecellular association (data not shown), likely due to a small increase inzeta potential (Fig. 4a).

The gene silencing effect of chitosan/(PG+siRNA) nanoparticleswas investigated in the presence of serum using HEK 293FT and HeLacells (Fig. 9). Lipofectamine and PLL were used as a positive control.The concentration of siRNA was kept constant at 50 pmol/well andweight ratios for Lipofectamine, PLL, and chitosan-based complexeswere 2, 4, and 20 respectively. A gene silencing effect was negligiblefor naked siRNA and complexes of Lipofectamine/mismatch siRNA

Fig. 7. Serum stability of (a) naked siRNA and (b) siRNA loaded in nanoparticlesincubated in 20% serum-containing media over time.

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complexes, indicating knockdown specificity. The gene silencing effectof chitosan/siRNA in both HEK 293FT and HeLa cells was enhanced byan inclusion of either alginate or PG. Surprisingly chitosan/(PG+siRNA) nanoparticles were most efficient to deliver siRNA into cells,compared with chitosan/siRNA and chitosan/(alginate+siRNA) nano-particles. This finding may indicate that gene silencing is clearlyrelevant to particle size and stability. Interestingly the gene silencingefficiency of siRNA delivered with chitosan-based nanoparticles wascomparable to that of Lipofectamine and PLL used as a control,indicating potential usefulness of chitosan-based nanoparticles innon-viral siRNA delivery.

4. Conclusions

Chitosan nanoparticles were prepared using a coacervationmethod in the presence of polyguluronate (PG) in order to developan efficient delivery carrier of siRNA. Chitosan/(PG+siRNA) nanopar-ticles had a mean diameter of about 110–430 nm, depending on theweight ratio. The surface of the nanoparticles was positively charged,and the loading efficiency of siRNA in the nanoparticles wasmore than60%. Chitosan/(PG+siRNA) nanoparticles were stable in neutral pHconditions and were effective in protecting siRNA from degradation in

Fig. 9. Gene silencing efficiency of chitosan/(PG+siRNA) nanoparticles in comparisonwith use of Lipofectamine™ 2000 and poly(L-lysine) (PLL) in (a) HEK 293FT and(b) HeLa cells (⁎Pb0.05, ⁎⁎Pb0.01).

Fig. 8. Cytotoxicity of nanoparticles tested with (a) HEK 293FT and (b) HeLa cells.

the presence of serum compared to naked siRNA. The cellular asso-ciation and gene silencing effects of nanoparticles were remarkable inserum conditions. Low cytotoxicity of chitosan-based nanoparticles,compared to commercially available liposome and PLL, supporteda promising characteristic as a non-viral vector. Moreover, chitosan/(PG+siRNA) nanoparticles showed high potential and were mostefficient as a non-viral delivery carrier of siRNA. This approach todeveloping delivery systemswith biocompatible and low toxic naturalpolymers may find useful applications in gene delivery for therapeuticpurposes.

Acknowledgments

This work was supported by the Korea Science and EngineeringFoundation (KOSEF) grant funded by the Korea government(MEST) (No. R01-2006-000-10506-0) and also by Korea Ministry of

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Knowledge Economy under the KORUS Tech Program (No. KT-2008-NT-APFS0-0001).

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