Journal of Biotechnology 160 (2012) 97 104

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology

jou rn al hom epage: www.elsev ier .com

Chitosa ctitargeti a 9

Gatesara th Uracha R la,b,

a Center of Exce ilandb Department oc National Nan (NSTDd National Cent y Deve

a r t i c l

Article history:Received 15 MReceived in reAccepted 23 AAvailable onlin

Keywords:Yellow head virus (YHV)RNA interference (RNAi)ChitosanQuaternized chitosanNon-viral vectSpodoptera fru

ng stlly dentinu

odoptgene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validatingnanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quar-ternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphologyand physicochemical properties were examined. When treated with chitosan and QCH4dsRNA com-plexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at24 h post infection with low cytoxicity. Inhibitory effects of chitosan and QCH4dsRNA complexes were

1. Introdu

Chitosantremendoutransfer annucleic acidionic interaable and bioet al., 2001charged chduced to opstudies demtive to carand in vitret al., 20082008). Howits insolubicity and t

CorresponBangkok 1040

E-mail add

0168-1656/$ http://dx.doi.oorsgiperda

comparable to that of dsRNA formulated with Cellfectin, a commercial lipid-based transfection reagent.The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specic dsRNAdelivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.

2012 Elsevier B.V. All rights reserved.

ction

and its derivatives as non-viral vectors have gaineds interest for their applications, especially for gened RNAi delivery systems. Chitosan can encapsulates, both DNA and RNA, and form nanopolyplexes viactions. The nanopolyplexes are low toxicity, biodegrad-compatibility (De Smedt et al., 2000; Koping-Hoggard). Chemical modications and the ratio of positivelyitosan and negatively charged nucleic acids are intro-timize stability of the nanopolyplex. Several previousonstrated the capacity of chitosan or chitosan deriva-ry and deliver plasmid DNA and RNA both in vivoo systems (Koping-Hoggard et al., 2001; Rojanarata; Techaarpornkul et al., 2010; Weecharangsan et al.,ever, applications of chitosan are still limited due tolity in neutral and basic pH. Moreover, its low speci-ransfection efciency of chitosan should be overcome

ding author at: Centex Shrimp, Faculty of Science, Mahidol University,0, Thailand. Tel.: +662 201 5870; fax: +662 354 7344.ress: vsaksmer@gmail.com (V. Saksmerprome).

for its use in clinical trials (Kim et al., 2007). Quaternized chitosan(QCH) was developed to improve solubility of natural chitosan atpH 7. It was prepared by using commercially available Quat 188under basic condition (Sajomsang et al., 2009a,b). The Quat 188is an aqueous solution of N-(3-chloro-2-hydroxypropyl) trimethy-lammonium chloride (Xiao et al., 2012). It is well-known as aquaternizing agent that introduce quaternary ammonium moietyinto the polymer backbone such as the one in starch (Heinze et al.,2004), cellulose (Hashem et al., 2003), and chitosan (Sajomsanget al., 2009a,b).

RNAi-based preventive approaches have shown promise againstshrimp viruses at lab-scale experiments. To date, intramuscularlyinjection of dsRNA appears to be the most effective delivery methodfor RNAi-mediated shrimp viral inhibition (Ongvarrasopone et al.,2008; Saksmerprome et al., 2009; Tirasophon et al., 2007). Thereremains a need to improve oral delivery method to make RNAi-mediated antiviral strategy feasible for shrimp farming. Due to thelack of availability of continuous shrimp cell line, mosquito andSpodoptera frugiperda 9 (Sf9) cell cultures have gained interest forthose who study shrimp-virus responses in molecular detail. Theyhave been reported to be immunopositive to white spot syndromevirus (WSSV) and yellow head virus (YHV), suggesting likelihood ofshrimp viruses persistently replicating in insect cells (Sriton et al.,

see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.jbiotec.2012.04.011n and its quaternized derivative as effeng shrimp virus in Spodoptera frugiperd

Theerawanitchpana,b, Nattika Saengkrit c, Warayuuktanonchaic, Somsak Saesooc, Timothy W. Flegellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Bangkok, Thaf Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailandotechnology Center (NANOTEC), National Science and Technology Development Agencyer for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technolog

e i n f o

arch 2012vised form 20 April 2012pril 2012e 30 April 2012

a b s t r a c t

RNA interference (RNAi) is a promisiiments. Development of effective oraRNAi application at farm level. Since cooping a dsRNA-delivery system in Sp/ locate / jb io tec

ve long dsRNA carriers cells

Sajomsangc, Pattarapond Gonil c,d, Vanvimon Saksmerpromea,d,

A), Thailand Science Park, Pathumthani, Thailandlopment Agency (NSTDA), Thailand Science Park, Pathumthani, Thailand

rategy to combat shrimp viral pathogens at lab-scale exper-livered agents for double-stranded (ds)RNA is necessary forous shrimp cell lines have not been established, we are devel-era frugiperda (Sf9) cells for studying in vitro RNAi-mediated

98 G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97 104

2009; Gangnonngiw et al., 2010). The insect cells could be useful notonly for studying RNAi-mediated gene silencing of shrimp virus butalso for validating dsRNA carriers with promising inhibitory effectsprior to testing in shrimp.

In this stpared as pformulatedYHV RNA-dmations wethis study ischitosanviThe effectivwould havecontaining

2. Materia

2.1. Prepara

The chitpurchased degree of d94% by 1H 2-hydroxypobtained frtubing withMembrane to purify chacetic acid 1 g/l. QCchloro-2-hy(Sajomsang

2.2. Charac

All attenFTIR) spect(Thermo Cotroscopy (Smelement (IRtra were cwith 32 scwere measSwitzerlandthe pulse aD2O/CD3COing 5 mg ofmolecular wand Mw/Mngel permeaSeries generesolving ra(Mw 5.978were dissolVertiPure nCo., Ltd., Tha(acetate bufThen the in

2.3. Formatnanopolyple

Using thdsRNA was(Saksmerpr

QCH4 solution were mixed with 1 g of dsRNA solution. The com-plexes were formed through a self-assembly mechanism afterpipetting, and subsequently incubated at room temperature for15 min before use. Different weight ratios of chitosandsRNA and

dsRNnityorphed. Hotenrrelan (Ns wC. Fon, Qere p

of n25 Cicro

1018 kH

N/mcan rmin

ansfegen,

cell ancellsat 28ellfeclfectited aed fred (t a m/wellnd

wassfectls we

g , USAy atl RNt 50amplGCGTACGtin-Sf9-Rded iwed30 s,ion a5 minphor

epar

y mil(5 mliv./Gd toethyudy, chitosan and its quarternized derivatives are pre-reviously described (Sajomsang et al., 2009a,b), and

with bacterially expressed dsRNA (>100 bp) targetingependent RNA polymerase (RdRp) gene. Complex for-re analyzed by physicochemical methods. The aim of

to evaluate transfection efcacy and cell toxicity of theral specic dsRNA complexes in an insect cell system.e chitosan nanoparticles determined from this study

further applications in development of shrimp feedviral specic dsRNA.

ls and methods

tion of chitosan and QCH4

osan, with average molecular weights (Mw) of 276 wasfrom Seafresh Chitosan (lab) Co., Ltd. in Thailand. Theeacetylation (DDA) of chitosan was determined to beNMR spectroscopy (Lavertu et al., 2003). N-(3-Chloro-ropyl) trimethylammonium chloride (Quat 188) wasom the Dow Chemical Company in Thailand. A dialysis

Mw cut-off of 12,00014,000 g/mol from Cellu Sep T4,Filtration Products, Inc., (Segiun, TX, USA) were useditosan derivative. Chitosan was dispersed in 1% (w/v)to prepare the stock solution at a nal concentration ofH4 was synthesized by quaternizing chitosan with N-(3-droxypropyl) trimethylammonium chloride (Quat 188)

et al., 2009a,b).

terization of chitosan and QCH4

uated total reectance Fourier transform infrared (ATR-ra were collected with a Nicolet 6700 spectrometermpany, USA) using the single-bounce ATR-FTIR spec-art Orbit accessory) with a diamond internal reectionE) at the ambient temperature (25 C). These spec-ollected by using rapid-scan software in OMNIC 7.0ans and a resolution of 4 cm1. The 1H NMR spectraured on AVANCE AV 500 MHz spectrometer (Bruker,). All measurements were performed at 300 K, usingccumulation of 64 scans and LB parameter of 0.30 Hz.OD and D2O were used as the solvents for dissolv-

chitosan and QCH4, respectively. The weight averageeight (Mw), number average molecular weight (Mn),of chitosan and QCH4 were determined by using the

tion chromatography (GPC). It consists of Waters 600Eric pump, injector, ultrahydrogel linear columns (Mwnge 120,000 kDa), guard column, pollulans as standard8 kDa), and refractive index detector (RI). All samplesved in acetate buffer pH 4 and then ltered throughylon syringes lters 0.45 m (Vertical chromatographyiland). The mobile phases, 0.5 M AcOH and 0.5 M AcONafer pH 4), were used at a ow rate of 0.6 ml/min at 30 C.jection volume 20 l was used.

ion of chitosandsRNA and QCH4dsRNAxes

e recombinant plasmid with YHV RdRp hairpin gene, prepared in E. coli HT115 (DE3) as previously describedome et al., 2009). One microgram of chitosan and

QCH4ing afand mexaminzeta pton Cotitratiosampleat 25

chitosaplex wstreamnet at force mrange with 21.41.8and a sto dete

2.4. Tr(Invitro

Sf9ing 1%5 105bated 5 l Cby CelincubaprepardescribYHV acopieschitosadsRNAto tran

Celat 2200(Rochetroscopof totaUSA) aRNA s(5-AC(5-TTGset, AcActin-Swas adas follo94 C, extens72 C, electro

2.5. Pr

Fiftwater 0.1 equadjuste(3-dimA complex were prepared to investigate dsRNA bind- by gel retardation. Physicochemical characterizationological analysis of the obtained nanoplexes were thenydrodynamic diameter, polydispersity index (PDI) and

tial of nanopolyplexes were determined by the Pho-tion Spectroscopy machine and electrophoretic mobilityanoZS4700 nanoseries, Malvern Instruments, UK). Theere obtained as the average of three measurementsr morphology investigation, 5 l of each of dsRNA,CH4, chitosandsRNA complex and QCH4dsRNA com-laced on a freshly cleaved mica surface, dried with aitrogen, and further dried in an electronic dry cabi-

for 30 min. All samples were determined by atomicscope (SPA400, Seiko, Japan) machine used the scanner000 nm area in tapping mode using a Micro cantileverz resonance frequencies and a constant force, range of. All images were recorded in air at room temperature

speed of 0.8 Hz and the phase image and topology usede the morphology and particle size.

ction of dsRNA into Sf9 cells using Cellfectin

USA) and chitosan nanopolyplexes

s were cultured in 2 ml of Sf-900 III SFM contain-tibiotic-antimycotic (pH 6.2) at a concentration of/ml for 6-well plate (Costar, Corning, USA) and incu-C overnight or until 6070% cell conuency. Usingtin, 2 g of dsRNA were transfected into Sf9 cellsn (Gibco Invitrogen, USA). The treated Sf9 cells weret 28 C for 12 h prior YHV infection. YHV stock wasom hemolymph of YHV-infected shrimps as previouslySaksmerprome et al., 2009). Sf9 cells were infected withultiplicity of infection (MOI) of 20 or around 2 107

by incubating on a shaker at 28 C for 6 h. In the case ofsRNA complex, 10 l of complex which contains 2 g of

diluted in 1 ml Sf-900 III SFM without antibiotics priorion.re harvested at 12 h after transfection by centrifugationfor 5 min. Total RNAs were extracted by Tri-pure reagent). RNA concentrations were determined by UV spec-

OD260. First strand cDNA was prepared from 150 ng/lA using Titan one tube reverse transcriptase (Roche,C for 30 min. Double-stranded RNA product in eache was monitored by specic primer YHV invert F salCGACGCATGTCCTGTTCTC-3) and YHV reverse R pstTCGAATTCTAGCCATGC-3). Actin-specic primerf9-Forward (5-GATATGGAGAAGATCTGGCAC-3) andeverse (5-ACGGGTCTGTTCCCTATGAAGCACCAC-3),n PCR reaction for internal control. PCR conditions were: the initial denaturing step at 94 C, 2 min; denaturing

annealing temperature depending on primers, 30 s,t 72 C, 30 s for 30 cycles; and a nal extension at. PCR products were analyzed using 1.5% agarose gelesis.

ation of the FITC-conjugated QCH4 and transfection

ligrams of QCH4 (0.3 mmol) were dissolved in deionized). Twelve milligram of uorescein isothiocyanate (FITC,lcN) was dissolved dimethylsulfoxide (DMSO, 3 ml) and

pH of 3.0 with 1 M HCl. Six milligram of 1-ethyl-3-laminopropyl) carbodiimide (EDC, 0.1 equiv./GlcN) was

G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97 104 99

Fig. 1. Chitosa hitosaefciency.

then addedN-hydroxystion mixturSubsequentture and stiwas dialyzeThe obtaineused to forQCH4-FITC:FITC-labelesame manncollected at

2.6. Evaluaanalysis

At 12 anhistochemimonoclonatein gp64 (M2003). Fluorwas quanti(FV1000, Ogroup wereone-way anhoc test witantigen ucalculated a

Favg = i=n

n

Fi = uorescpus FluovieJapan); i = 1

2.7. Cytotox

Toxicitytrypan bluewere collec

bluepliedable)

cells

ults

nthe

ternommyst ig waeratey ameophnium1H NndsRNA and QCH4dsRNA complexes at various weight ratios. At weight ratios of c

and stirred at room temperature for 1 h. Then 3.5 mg ofuccinimide (NHS, 0.1 equiv./GlcN) was added. The reac-e was stirred at room temperature for another 15 min.ly, the QCH4 solution was added into the reaction mix-rred at room temperature for 6 h. Finally, the mixtured against distilled water for 2 weeks and lyophilized.d FITC conjugated QCH4 was dissolved in water andm nanopolyplexes with dsRNA at the ratio of 10 l

2 g dsRNA. Transfection of FITC-labeled QCH4 andd QCH4dsRNA nanopolyplex were carried out, in theer as those of unconjugated chitosan complexes, and

6, 12, and 24 h after transfection.

tion of dsRNA transfection by immunohistochemistry

d 24 h after infection, cells were collected for immuno-stry assays. The experiments were carried out using

trypanwas ap(nonviViable

3. Res

3.1. Sy

Quawith ca catalyieldinily genprimara nuclammoUsingl antibodies (MAb) specic for the YHV structural pro-AbY18) as previously described (Soowannayan et al.,escence intensity representing level of YHV expressioned by Olympus Fluoview version 1.7 viewer programslympus, Japan). Approximately 50150 cells in each

analyzed. Statistic difference was determined usingalysis of variance (ANOVA), followed by an LSD posth signicance set at p-values of

100 G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97 104

Fig. 2. Physico(B) AFM imageratio of 0.2.

including smeasured chitosandsin the rangweight ratio

Fig. 3. RT-PCRweight ratios oRNA level. M: using DEPC wachemical property and morphology of complexes. (A) Size (), PDI () and zeta potentias of (a) naked dsRNA, (b and c) Chitosan and its derivative, QCH4, (d) chitosandsRNA co

ize, zeta potential and polydispersity index (PDI) wereby using Zetasizer Nano ZS. In Fig. 2A, the size ofRNA complexes at weight ratios 1.3, 1.5 and 1.7 weree of 350650 nm, while QCH4dsRNA complexes ats 0.16, 0.20 and 0.24 were approximately 150350 nm.

The zeta pof the comobserved a0.16 and 0.chitosand

analysis. YHV-specic dsRNA were transfected in Sf9 cells using chitosan and its derivaf a carrier:dsRNA. In the case of native chitosan complex, a = 1.3, b = 1.5, and c = 1.7. For QC2 log DNA marker, m: RNA from Sf9 cells without transfection, CF: RNA from dsRNA-transter instead of RNA template.l () of chitosan (QCH4)dsRNA complexes at different weight ratios.mplex at weight ratio of 1.5 and (e) QCH4dsRNA complex at weight

otential depends on the positive or negative chargeplexes. Electronegative values of zeta potential weret weight ratios of 1.3 of chitosandsRNA complex and2 of QCH4dsRNA complexes. At weight ratio of 1.5 ofsRNA and 0.24 of QCH4dsRNA, the zeta potential was

tive (QCH4) as dsRNA carriers. Symbols a, b and c represent differentH4:dsRNA, shrimp actin was used as internal control for normalizingfected cells using Cellfectin as a transfectant and -: negative control

G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97 104 101

approximately neutral while electropositive value was found inchitosandsRNA at weight ratio of 1.7.

Polydispersity index of chitosandsRNA complexes at weightratios of 1.3, 1.5 and 1.7 were 0.56 0.04, 0.64 0.05 and0.61 0.09 and QCH4dsRNA complexes at weight ratios of 0.16,0.2 and 0.24 were 0.59 0.03, 0.52 0.06 and 0.56 0.08 respec-tively. This result indicated the narrow PDI which implied thenarrow size distribution of nanopolyplexes were obtained. Themorphology of each was monitored under AFM (Fig. 2B). The dsRNAwas condensated as rod-like structure at state equilibrium. Thestructures observed with AFM revealed that both chitosan andQCH4 depict with a brush-like conformation where aggregatescomprise a dense. AFM images of the chitosan complex at 1.5:1(w/w) and QCH4dsRNA complex at 0.2:1 (w/w) were shown inFig. 2d and e, respectively. In a narrow scan of the nanopolyplexes,

many spherical-shaped structures of similar size were observed.The diameter of QCH4dsRNA structure was within 100300 nm.The size of the chitosan/dsRNA structure was ranging between 300and 700 nm. The results suggested that the QCHdsRNA complexwas more tightly condensed than the native chitosandsRNA.

3.3. Evaluation of Cellfectin and chitosan nanoparticles asdsRNA-delivered reagents

Cytotoxicity of Cellfectin, chitosan and QCH4 in Sf9 cells wasinvestigated using trypan blue exclusion method. As shown inTable 1, untreated control, chitosan- and QCH4-treated groupexhibited comparable percentage viability (7580%), and the grouptransfected with the commercial liposome complex exhibited thelowest percent viability (65%).

Fig. 4. (A) Ave cells uat 12 (black ba ate ththe pair-wise tivelycells at 12 (B) e reactransfection; ( n thisrage YHV-antigen uorescence intensity from immunohistochemical staining of Sf9r) and 24 hpi (white bar) for determining the level of YHV infection. Error bars indiccomparisons relative to the YHV-infected group at 12 and 24 hpi (p < 0.01), respecand 24 (C) hpi using a monoclonal antibody against YHV structural protein. Positivc) chitosandsRNA; (d) QCH4dsRNA. (For interpretation of the references to color ising antibodies to structural protein of gp64 YHV. Cells were collectede standard deviation. * and ** indicate signicant differences from. A.U. arbitrary unit. Immunohistochemistry assay of YHV-infectedtions are shown in red. (a) YHV infection only; (b) Cellfectin-dsRNA

gure legend, the reader is referred to the web version of this article.)

102 G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97 104

Capabiliinto Sf9 cetreated cellucts of 40were detec(Fig. 3). Forresenting qprogram. Incomplexes Cellfectin

nicant (attreated by infection (Fvisualizing

Table 1Percentages opan blue exclu(p < 0.01).

Treatments

UntreatedCellfectin-dChitosandsQHC4dsRNFig. 4. (Continued ).

ty of Cellfectin, chitosan and QCH4 to deliver dsRNAlls was examined. Total RNAs were extracted froms after 12 h incubation with complexes. RT-PCR prod-0 bp, indicating the presence of YHV-specic dsRNA,ted in the cells incubated with all types of complexes

quantitative data, average uorescence intensity rep-uantity of YHV was calculated by FV10-ASW 1.7 Viewerhibitory effects by chitosandsRNA and QCH4dsRNAwere not as potent as to the lipid-based tranfectant,at 12 h after viral challenge (Fig. 4AB). However, sig-

least 50%) reduction in YHV expression in the groupsthe chitosan complexes became evident at 24 h postig. 4AC). We used confocal microscopy for directlycellular uptake of the FITC-labeled complex after

f cell viability under different treatments determined by the try-sion method. There are no signicant differences in all four groups

% cell viability (SD)78.4 (3.2)

sRNA 64.9(7.9)RNA 74.0 (3.2)A 73.9 (4.1)

transfectioncomplex wFig. 5, the gwere visiblcomplex clPCR and conantibody redelivery in

4. Discussi

In the prinfection w(2009) dempersistentlycells, and itmonoclona(gp64) of Yent from thmicroscopyshrimp virudsRNA carrshrimp. in Sf9 cells. FITC-labeled QCH4 was selected to formith dsRNA via electrostatic interaction. As shown inreen signals for the FITC-labeled QCH4dsRNA complexe throughout 624 h after transfection, and the labeledearly accumulated at 24 h after transfection. Both RT-focal immunouorescence microscopy using anti-YHVvealed utility of chitosan and QCH4 for successful dsRNASf9 cells for antiviral application.

on

esent work, dsRNA-mediated inhibition of shrimp viralas investigated in Sf9 cells. Previous study by Sriton et al.onstrated that shrimp viruses, including YHV, can be

maintained in Sf9 insect cell line. YHV can infect Sf9s antigen is detected by immunohistochemistry usingl antibody (Y18) against antigen of envelope proteinHV. General morphology of infected cells is not differ-at of uninfected cells or mock cells under phase contrast. Taken advantages of reproducibility and convenience,s-infected Sf9 cells can be used to screen for effectiveiers, providing new approaches for RNAi applications in

G. Theerawanitchpan et al. / Journal of Biotechnology 160 (2012) 97 104 103

Fig. 5. Confoc ng timcontrast) imag FITC-color in this g

Natural plasmid DN2007) and gWeecharan(QCH4) waimprove wachitosan baefcient dsRuble in a wrelative to nstranded RN24 h after tQCH4dsRNcells withindetection mas QCH4, cacells.

Silencingand Cellfectassay. In aldelivered awas in agreet al., 2009structural geffects by dswere not pogroup transsilencing efthan that oof Cellfectinproperties. nucleic acidcomplexes nalization ochitosan cocondensed

In conclucells infect

i-mormuen sal cyanopan-bact cuxicitygatet of a

wled

autal images of Sf9 cells incubated with FITC-labeled QCH4dsRNA complex at varyies, and uorescent-FITC images, respectively. Red arrow indicates accumulation ofure legend, the reader is referred to the web version of this article.)

chitosan is shown to form complexes with siRNA andA for gene silencing (Howard et al., 2006; Liu et al.,ene therapy-related studies (MacLaughlin et al., 1998;gsan et al., 2008). In this study, chitosan derivatives prepared from quaternization of native chitosan toter solubility of chitosan by adding positive charges onckbone. Both chitosan and QCH4 are shown to mediateNA delivery into insect cells. QCH4 is more water sol-

ider range of pH, and it can be used in a lesser amountatural chitosan for efcient delivery of dsRNA. Double-A delivered by QCH4 was detected by RT-PCR withinransfection (Fig. 3). Confocal images of FITC-labeledA complex indicated the presence of the complex in

24 h post transfection (Fig. 5). The results from both

in RNAYHV, fhas beminimtosan nChitosin insecytotoinvestiopmen

Ackno

The

ethods demonstrate that chitosan nanoparticle, suchn be used for effective delivery of long dsRNA in Sf9

efciency of chitosandsRNA, QCH4dsRNA complexin-dsRNA was monitored by immunohistochemistryl cases, YHV-specic dsRNA targeting RdRp gene wasnd was able to reduce YHV infection (Fig. 4). The resultement with previous studies in shrimp (Saksmerprome; Tirasophon et al., 2005) that dsRNA targeting non-enes effectively inhibited YHV replication. InhibitoryRNA in cells treated with chitosan and QCH4 complexestent at 12 h post infection (hpi) when compared to thefected with Cellfectin complex. In contrast, at 24 hpiciency of the chitosan complexes was more prominentf the commercial lipoplex. Deviation of effective time and chitosan is probably resulted from their releaseCellfectin is a cationic-lipid formulated for binding

via adsorption on the lipid surface, while chitosanoccurred through electrostatic interaction. After inter-f complex into the cell, the release of dsRNA from themplex is probably more difcult because dsRNA is wellwith polymer to form particles.sion, this study demonstrates the effective use of insected with shrimp virus for selection of dsRNA carriers

(Srinakharigeting YHVfor cell cultfrom Thailaand Internatively.

Appendix A

Supplemfound, in th2012.04.01

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gments

hors would like to thank Dr. Paisan Sithikorngul

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Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9...1 Introduction2 Materials and methods2.1 Preparation of chitosan and QCH42.2 Characterization of chitosan and QCH42.3 Formation of chitosandsRNA and QCH4dsRNA nanopolyplexes2.4 Transfection of dsRNA into Sf9 cells using Cellfectin (Invitrogen, USA) and chitosan nanopolyplexes2.5 Preparation of the FITC-conjugated QCH4 and transfection2.6 Evaluation of dsRNA transfection by immunohistochemistry analysis2.7 Cytotoxicity test

3 Results3.1 Synthesis and characterization of QCH43.2 Physicochemical analysis and complex formation3.3 Evaluation of Cellfectin and chitosan nanoparticles as dsRNA-delivered reagents

4 DiscussionAcknowledgmentsAppendix A Supplementary dataReferences