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BiosensorsandBioelectronics

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Quantumdotsencapsulatedwithamphiphilicalginateasbioprobeforfastscreeninganti-denguevirusagents

Chung-HaoWang1,Yi-ShiouHsu1,Ching-AnPeng∗

DepartmentofChemicalEngineering,NationalTaiwanUniversity,Taipei,Taiwan

articleinfoabstract

Theincreasingthreatsofviraldiseaseshavegainedworldwideattentioninrecentyears.Quiteafewinfec-tiousdiseasesarestilllackingeffectivepreventionortreatment.Thepaceofdevelopingantiviralagentscouldbeexpeditedbytheavailabilityofquickandefficientdrugscreeningplatforms.Inthisstudy,quan-tumdot(QD),anemergingprobeforbiologicalimagingandmedicaldiagnostics,wasemployedtoformcomplexeswithvirusandusedasfluorescentimagingprobesforexploringpotentialantiviraltherapeu-tics.InorganicCdSe/ZnSQDssynthesizedinorganicphasewereencapsulatedbyamphiphilicalginatetoattainbiocompatiblewater-solubleQDsviaphasetransfer.Virusemployedforthisstudywasdengueviruswhichisanotoriousoneintropicalandsubtropicalregionsoftheworld.ToconstructaQD–virusimagingmodalitycapableofprovidingmeaningfulinformation,preservationofviralinfectivityaftertaggingviruswithQDsisofutmostimportance.InordertoformcolloidalcomplexesofQD–virus,electrostaticrepulsionforcegeneratedfrombothnegativelychargedvirusandQDswasneutralizedbyvariousconcentrationsofcationicpolybrene.ResultsshowedthatBHK-21cellsinfectedwithdenguevirusesincorporatedwithQDsexhibitedbrightfluorescenceintracellularlywithin30min.TodemonstratethepotencyofQD–viruscomplexesasbioprobesforscreeningantiviralagents,BHK-21cellswereincubatedforonehourwithallo-phycocyaninpurifiedfromblue-greenalgaeandtheninfectedwithQD–viruscomplexes.Basedonthedevelopedcell-basedimagingassay,allophycocyaninwithconcentrationof125␮g/mLledtoextremelyweakintracellularfluorescencepost-infectionofQD–viruscomplexesfor30min.Thatis,theefficacyofanti-dengueviralactivityofthealgaeextractwasclearlyillustratedbytheinorganic–organichybridplatformconstructedincurrentstudy.

©2008ElsevierB.V.Allrightsreserved.

Articlehistory:

Received16April2008

Receivedinrevisedform1August2008Accepted4August2008

Availableonline13August2008Keywords:

QuantumdotsDenguevirus

AmphiphilicalginateDrugscreeningAllophycocyaninPolybrene

1.Introduction

Unlikebacteria-basedinfectionwhichcanbecontrolledbyantibiotics,virusesfullyrelyingonhostcellsfortheirreplicationarenotsoreadilydealtwith.Theemergenceandspreadofviraldiseasesworldwide,particularlyHIV/AIDS,outbreaksofsevereacuterespi-ratorysyndrome(SARS)virus,andthescaresofpandemicavianinfluenzavirusseriouslyraisetheconcernthatanyvirusstrainhasthepotentialevolvingintoalife-threateningpathogen.Inthisregard,developingfastandefficientscreeningtechnologyhasitsmeritsofidentifyingpotentialdrugsagainstviraldiseasesthatstilllackofeffectivepreventionortreatment.Denguevirusisthetypicalonerepresentinganimportantemergingmosquitobornedisease

∗Correspondingauthorat:DepartmentofChemicalEngineering,NationalTaiwanUniversity,No.1,Section4,RooseveltRoad,Taipei106,Taiwan,ROC.Tel.:+886233663063;fax:+886223623040.

E-mailaddress:chinganpeng@ntu.edu.tw(C.-A.Peng).1

Theseauthorscontributedequallytothiswork.0956-5663/$–seefrontmatter©2008ElsevierB.V.Allrightsreserved.doi:10.1016/j.bios.2008.08.009

worldwide.Ithasspreadfrombeingendemicinjust9countriesin1970to100countriesin2002,accordingtotheworldhealthreport(http://www.who.int/whr/2002/en).Apparently,globalpopulationgrowth,urbanization,andfrequentmoderntransportationhavecontributedtotheincreasedincidenceandgeographicspreadofdengueviruses.Thesediseasesoccurintropicalandsubtropicalareasoftheworld,where2.5billionpeopleareestimatedatriskfordenguevirusoutbreaks(GublerandClark,1995).Eachyear,tensofmillionsofcasesofdenguefeveroccur,particularlyinAsia,Africa,SouthAmerica,andPacific.Thefatalityrateisabout5%andthereisnovaccineavailablefordenguevirus.Controloftheprimaryvec-tor,Aedesaegypti,istheonlymethodcurrentlyemployedtopreventdenguevirusepidemics.

Thepaceofexploitingantiviralagentscouldbeexpeditedbytheavailabilityofquickandefficienthigh-throughputanti-dengueviralagentscreeningassays.Recently,acell-basedimmunofluores-cenceimagingtechnologyhasbeendevelopedtoidentifypotentialanti-dengueviraltherapeuticagents(ChuandYang,2007).Theyreportedthatinhibitorsofthec-Srcproteinkinasehindertheassemblyofdenguevirionsandmaythereforebeaneffective

C.-H.Wangetal./BiosensorsandBioelectronics24(2008)1012–10191013

therapy.Itwasanticipatedthatthenewassaycanbeusefulforidentifyingsmallmoleculeinhibitorsofdenguevirusinfectionandreplicationaswellasimprovingunderstandingofdenguevirus–hostinteraction.Althoughthedevelopedassayhasitsmer-its,theprocessofscreeningantiviralagentswasnotquickenoughbecausetheVerocellcultureplateaftertheadditionofdenguevirusandthenspecificproteinkinaseinhibitorhastobeincubatedfor3dayspriortotheperformanceofimmunofluorescencestain-ing.Moreover,anti-dengueEproteinmonoclonalantibodyandthesecondaryantibodyconjugatedwithfluorescentdyeFITChavetobeusedforlabelingthecellmonolayer.Thisisnodoubtatime-consumingandexpensiveprocess.Fluorescentdyehasbeenwidelyusedforvirallabelingexperimentsandimprovedourunderstand-ingofviralinfectionprocess.However,fluorophoresarenotoriousforphotobleachingandspectraloverlaps,andthereforecouldaffectthefluorescenceimagingqualityofdye-labeledvirussincetheobservationtimerangedfromonetotensecondsbeforephoto-bleachoccurs.Obviously,ahighfluorescencequantumyieldandalargenumberoffluorescentphotocyclesbeforephotobleachofthedyemoleculeoccursaretheprerequisitesforsuccessfullydetectingdye-labeledviralparticles.Moreover,afterlabelingviralparticleswithfluorescentdyes,thenumberofvirusesthatcaninfectacellmostlikelywillbeseverelydiminished.

Inviewofthedrawbacksofusingfluorescentdyes(ChanandNie,1998;Paraketal.,2005),colloidalsemiconductorquantumdots(QDs)wereinvestigatedinthisstudytoexplorethepotencyoftaggingQDstoviralparticles.Thankstoexcellentphotostabil-ity,broadadsorptionspectra,andnarrowemissionspectra,QDshaveattractedgreatinterestsinmanyareasofresearch,frommolecularandcellularbiologytomolecularimagingandmedicaldiagnostics(Michaletetal.,2005andreferencestherein).Tri-n-octylphosphineoxide(TOPO)-coatedQDsuspendedinorganicsolventwasfirstconvertedintowater-solubleQDemployingsyn-thesizedamphiphilicalginatesurfactants.ComplexesbetweenQDsanddenguevirus,bothpossessinganetnegativesurfacecharge,willbeformedbycolloidalclustering,facilitatedbypositivelychargedpolycationiccompound–polybrene.InordertotestifQD–viruscomplexescanbeharnessedasdiagnosticprobesforfastscreeningpotentialanti-dengueviraldrugcandidates,allo-phycocyanin(protein-boundpigment)purifiedfromablue-greenmicroalgaSpirulinaplatensiswasusedasthemodelagentsinceithasbeenreportedtoexhibitanti-EV71activities(Shihetal.,2003).OurresultsshowedthattheintensityofgreenQDimageswithinBHK-21cellswasdecreasedalongwiththedosageincreaseofallophycocyaninupto125␮g/mLprovidedonehourpriortotheadditionofQD–viruscomplexes.Itwasclearlyillustratedthatallo-phycocyaninpossessesanti-dengueviralactivityandthiscanbedeterminedwithinhalfanhouraftertargetcellsincubatedwithdenguevirusesincorporatedwithamphiphilicalginatecoatedQDinthepresenceof50␮g/mLpolybrene.

2.Materialsandmethods2.1.SynthesisofCdSe/ZnSQDs

TopreparesemiconductorCdSe/ZnSQDs,amixtureof25.7mgofcadmiumoxide(CdO,Sigma,USA),3.88gofTri-n-octylphosphineoxide(TOPO,Sigma),and2.41gofhexadecylamine(HDA,Acros,Belgium)washeatedto300–320◦Cunderadrynitrogenatmo-sphere.AftertheformationofaCdO–HDAcomplexasindicatedbythechangeofcolorfromreddishtocolorless,thetemperatureofthesolutionwascooleddownto260◦Candwaitedforinjection.Astocksolutionwith31.58mgofseleniumpowder(Se,Sigma–Aldrich)dissolvedin5mLoftri-n-butylphosphine(TBP,Showa,Japan)was

quicklyinjectedintothesolutionunderrigorousstirringtonucleateCdSenanocrystals.Afterinjection,thecoresolutionwasgrownat260◦Cforapproximately5swhenthecolorofthemixtureturnedfromcolorlesstoslightlyred.Thetemperaturewasthencooledto200◦Cforshellgrowth.Theshellsolutioncontaining379.4mgzincstearate(J.T.Baker,Netherlands)and12.8mgsulfurpowder(S,Sigma)dissolvedin5mLofTBPwasheatedto110◦Cfor30minandthencooledtoroomtemperatureforinjection.Theshellsolu-tionwasaddeddropwiseintothecoresolutionunderstirringoveraperiodof15min.Afteradditionofshellsolution,thecore-shellsolutionwascooleddownto120◦Ctoannealfor2h.Thecore-shellsolutionwascooledtoroomtemperatureafterwards.TheCdSe/ZnSnanocrystalswereprecipitatedbyanhydrousmethanol(Tedia,USA)andthenstoredinchloroform(Tedia)forfurtheruse.

2.2.Preparationofamphiphilicalginatesurfactant

Sodiumalginatesolution(Sigma)mixedwith2wt.%sodiumperiodate(Acros)inadark,coldchamber(4◦C)for24h.Exclu-sionoflightwasessentialforthepreventionofsidereaction.Toobtainoxidized2,3-dialdehydicalginate,thereactionmixturewasextensivelydialyzedagainstdistilledwaterandsubsequentlyfreeze-dried.Twomilligramsofoctylamine(Acros)dissolvedin8mLmethanolwasreactedwith0.276mg2,3-dialdehydicalginatedissolvedin10mLphosphatebuffersaline(PBS)solu-tioncontaining0.1gsodiumcyanoborohydride(NaCNBH3,Acros).Thereductiveaminationproceededfor12hwithrigorousstir-ringatroomtemperature.Thereactionmixturewasdialyzedandsubsequentlyfreeze-driedtoobtaintheamphiphilicalginatesurfactant.

2.3.EncapsulationofQDswithamphiphilicalginate

0.4mgofCdSe/ZnSsuspendedin5mLofchloroformwasaddedwith2mgofalginatesurfactant.Themixturewassonicatedinabathfor10min,andthenthechloroformwasremovedbyarotaryevaporator(Eyela,Japan)atroomtemperature.Attheendoftheoperation,themixturewasresuspendedwithPBSsolutionandfil-teredthrougha0.22-msyringefilter(Millipore,USA)toremoveaggregates.Thefiltratewasfreeze-driedtoobtaintheamphiphilicalginatecoatedquantumdotsinthepowderform.2.4.Particlesizeanalysis

QDsencapsulatedbyamphiphilicalginate(AA-QDs)werethor-oughlydispersedinaqueoussolutionbyasonicator(200W,40kHz;BransonUltrasonicsCorporation,CT,USA)for10minandthenpassedthrougha0.22-␮msyringefiltertocollectnon-aggregatedAA-QDswhichwereanalyzedformeanparticlesizeandsizedistri-butionbythedynamiclightscattering(ZetasizerNanoZS,MalvernInstruments,UK)equippedwithadiode-pumpedsolid-statelaseroperatingat633nmwavelengthasalightsource.Theparticlesizedistributionandtheaverageparticlediameterwereobtainedfromthecorrelationfunctionbyaregularizationmethodincludedinthedataanalysissoftwarepackage(DispersionTechnologySoftware5.02,MalvernInstruments,UK).2.5.Zetapotentialmeasurement

ThesurfaceelectricchargeofAA-QDs,denguevirus,andQD–viruscomplexeswasmeasuredseparatelybythezetapotentiometer(ZetasizerNano-ZS,MalvernInstruments,UK)viadeterminingtheelectrophoreticmobility.Theelectrophoreticmobilityisobtainedbyharnessingmicroelectrophoresistechnique

1014C.-H.Wangetal./BiosensorsandBioelectronics24(2008)1012–1019

onthesampletomeasurethevelocityoftheparticlesusinglaserDopplervelocimetry.Theisoelectricpoint(pI)ofallophycocyaninwasdeterminedtobethepHatwhichitcarriesnonetelectriccharge(i.e.,zetapotentialequalstozero).

2.6.AbsorptionandphotoluminescencespectraofAA-QDs

AUV–visspectrophotometer(JascoModelV-570,Japan)andaphotoluminescencespectrophotometer(JascoModelFP-6000,Japan)wereusedtocharacterizeAA-QDs.UV–visabsorptionspec-trumofQDswasscannedinthewavelengthrangeof300–700nm.Thephotoluminescenceoftheparticleswasmeasuredinthewave-lengthrangeof400–700nm.Thespectrawereobtainedwiththedatapitchat5nmandthescanningspeedof500nmpermin.Allsampleswereplacedinquartzcuvettes(1-cmpathlength)andtheopticalmeasurementswerecarriedoutusingPBSasthereferenceforAA-QDs.

Fig.1.(a)SchematicdrawingofCdSe/ZnSquantumdotsencapsulatedbyamphiphilicalginatesurfactant;(b)particlesizedistributionofAA-QDsbydynamiclightscattering.Aftersonication,thedistributionwasnarroweddowntotherangebetween18.1and28.7nmwithameandiameterof23.1nm.

2.7.Transmissionelectronmicroscopy(TEM)analysis

TEMspecimensweremadebyevaporatingonedropofquantumdots/viruscomplexessolutiononcarbon-coatedcoppergrids.TEMmicrographsweretakenbyatransmissionelectronmicroscope(JEM-1230,JOEL,Tokyo,Japan)operatingat100kV.Phospho-tungsticacid(Sigma)wasusedasthenegativestainreagent.2.8.BHK-21cellsanddengueviruses

BHK-21cellsweremaintainedat37◦CinDulbecco’smodifiedEagle’smedium(DMEM;HyClone,UT,USA),supplementedwith10%fetalbovineserum(FBS;HyClone).Denguevirusserotype-2strainPL046waspropagatedinmosquitoC6/36celllinemaintainedat30oCinDMEM,supplementedwith10%FBS,

Fig.2.(a)PhotoluminescenceandUV–visspectraofAA-QDs.UV–visabsorptionspectrumofAA-QDswasdetectedintherangeof300–700nm.Thephotolumi-nescenceofAA-QDswasmeasuredintherangeof400–700nmwithanexcitationwavelengthof390nm.Thephotoluminescencewiththeemissionwavelengthpeakedat534nmindicatingbrightgreenfluorescenceofAA-QDsinaqueoussolu-tionshownintheinsetphotograph;(b)TEMimageoftheQD–viruscomplexesformedinthecationicpolybrenesolution.Thelight-colorparticleswiththesizearound40–50nmaredenguevirus(indicatedbyarrow).Thedark-colorparticlestaggedontheviralparticles(indicatedbyarrowhead)areAA-QDs(about10–15nm).(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)

C.-H.Wangetal./BiosensorsandBioelectronics24(2008)1012–10191015

Fig.3.PhotomicrographicalimagesofBHK-21cellsexposedseparatelytoAA-QDswithandwithoutpolybreneforvariousperiodsoftime.(a)–(c)ImagesofBHK-21cellsincubatedwithQDswithoutpolybrenefor30,60,and120min,respectively.QDwasnotdetectedintracellularlyunderfluorescentconfocalmicroscopyuntil120minpost-incubation;(d)–(f)imagesofBHK-21cellsincubatedwithQDsinthepresenceof10␮g/mLpolybrene.QDwasnotdetectedintracellularlyunderfluorescentconfocalmicroscopyuntil60minpost-incubation.(g)–(i)ImagesofBHK-21cellsincubatedwithQDsinthepresenceof50␮g/mLpolybrene.QDwasclearlydetectedintracellularlyunderfluorescentconfocalmicroscopyafter30minpost-incubation.Thenormalizedfluorescenceintensitiesperselectedcellareaof(b)–(i)basedonthefluorescenceintensityperselectedcellareaof(a)are1.02,1.06,0.94,2.55,3.65,1.70,3.93,and5.20,respectively.Scalebar=25␮m.

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penicillin(200U/mL)andstreptomycin(100␮g/mL).Denguevirus-containingsupernatantwasfirstcentrifugedat10,000rpm,andthenultracentrifugedat100,000×gat4◦Cfor3htopurifydenguevirions.ThetiteroftheviruswasevaluatedusingBHK21cellsbytheplaqueassay(Liuetal.,1997).

2.9.InhibitionofQD–virusinfectionbyalgaeextracts

AA-QDssuspendedin1mLDMEMmediumwereaddedinto1mLdenguevirus-containingmediumwithmultiplic-ityofinfection(MOI)of0.1,0.5,and1.0,respectively.Aftergentleshakingofthemixedmedia,polybrene(1,5-dimethyl-1,5-diazaundecamethylenepolymethobromide,MW=5000–10,000,Sigma–Aldrich)stocksolutionwaspipettedintothemixturetoreachafinalconcentrationof10and50␮g/mL,respectively.Then,themixedsolutionwasincubatedat4◦Cfor1htopreventdenguevirusfromlosinginfectivity.AftertheprocessofincorporatingviruswithAA-QDs,5×104BHK-21cellsintheexponentialgrowthphasewerereplacedwiththeQD–virus-containingmedium.Aftertreat-ingBHK-21cellswiththeQD–viruscomplexesforvariousperiodsoftime(30,60,and120min),thecellculturedishwaswashedwithPBSthreetimesandobservedunderSpectralConfocalandMultiphotonSystem(LeicaTCSSP5,Wetzlar,Germany)withthesettingsofobjectivelens63×,pinhole1.0,smartgain380mV,and

Fig.4.PhotomicrographicalimagesofBHK-21cellsexposedseparatelytoQD–viruscomplexesofdifferentMOIswith10␮g/mLpolybreneforvariousperiodsoftime.(a)–(c)ImagesofBHK-21cellsincubatedwithQD–viruscomplexesofMOIbeing0.1for30,60,and120min,respectively;(d)–(f)imagesofBHK-21cellsincubatedwithQD–viruscomplexesofMOIbeing0.5for30,60,and120min,respectively;(g)sixsequentialz-sectionimagesofselectedinfectedcellsweretakenbyaconfocalmicroscope,whichrevealingtheQD–viruscomplexeswereindeedinternalizedintoBHK-21cellsratherthanabsorbedoncellsurface.Thenormalizedfluorescenceintensitiesperselectedcellareaof(b)–(f)basedonthefluorescenceintensityperselectedcellareaof(a)are1.00,1.33,1.19,2.56,and3.54,respectively.Scalebar=25␮m.

C.-H.Wangetal./BiosensorsandBioelectronics24(2008)1012–10191017

405-nmlaserND3.ThefluorescenceintensityofQDsrevealedwithintheobservedBHK-21cellswasquantitativelyanalyzedbytheMetaMorph®imagingsoftware(Version7.5,MolecularDevices,USA).

InordertotestifQD–viruscomplexescanbeharnessedasdiag-nosticprobesforfastscreeningpotentialanti-denguetherapeuticagents.Allophycocyanin(FarEastBio-TecCo.,Taipei,Taiwan)withconcentrationof6.25,31.25,and125␮g/mLwasusedtotreatBHK-21cellsforonehour,andthenrinsedawaybyPBSsolutionforthreetimes.Afterallophycocyanintreatment,QD–viruscomplexeswereaddedseparatelyfor30,60,and120mintoexaminetheefficacyofdrugscreenassayproposedinthisstudy.3.Resultsanddiscussion

3.1.QDsencapsulatedwithamphiphilicalginate

ColloidalnanocrystalQDsconsistingofaninorganiccore/shellstructure(e.g.,CdSe/ZnS)surroundedbyalayeroforganicligands(i.e.,TOPO)canbeconvertedintohydrophilicnanoparticlesbysev-eralapproaches(Bruchezetal.,1998;ChanandNie,1998;Dubertretetal.,2002;Mulderetal.,2006;Pinaudetal.,2004;Zhelevetal.,2006).Thecommonlyusedstrategyisbasedontheexchangeoftheoriginalorganiclayerwithhydrophilicligands,howeverthephysicalproperties(i.e.,quantumyield)areusuallydeterioratedthroughsuchsurfacemodification.Inthisstudy,wedemonstratedthefirsttimethatamphiphilicalginatecanencapsulateCdSe/ZnSbyintercalatingalginatesurfactant’shydrophobicpendantmoi-eties(i.e.,octylchains)intothehydrophobicsurfactantlayer(i.e.,TOPO)ontheQDsurface,therebyresultinginthephasetransferofhydrophobicQDsfromorganicsolventstoaqueoussolutionviahydrophilicbackbone(i.e.,alginate).Theschematicdrawingofthewater-solubleCdSe/ZnSQDsencapsulatedwithamphiphilicalgi-nateisshowninFig.1(a).Theaveragesizeofwater-solubleQDsencapsulatedbyalginatesurfactantswasdeterminedtobe23.1nmintherangeof18.1–28.7nmshowninFig.1(b).Aslongasthenum-berofpendantgroupsissufficientlyhigh,thelinkageofamphiphilicalginatetotheQDsurfacecouldbeverystableandtherebyleadtothephysicalpropertiesofQDintact.Theabsorptionandemis-sionspectraofQDencapsulatedwithamphiphilicalginategiven

inFig.2(a)revealhigherabsorbancebetween525and550nmandstrongphotoluminescencewiththeemissionwavelengthpeakedat534nmafterexcitedbyUV-light.TheinsetimageinFig.2(a)illustratesthebrightgreencolorofAA-QDinaqueoussolution.TodemonstratetheformationofQD–viruscomplexesviacolloidalclusteringofnegativelychargedQDanddenguevirusincationicpolybrenesolution,TEMimageofQD–viruscomplexeswastakenandshowedinFig.2(b).Intheimage,thelight-colorparticleswiththesizearound40–50nmaredenguevirus,andthedark-colorpar-ticlestaggedonthesurfaceofviralparticlesareAA-QDs(about10–15nm).

3.2.Cell–QDinteraction

Fig.3showedthephotomicrographicalimagesofBHK-21cellsincubatedwithAA-QDsatvarioustimeperiods.InFig.3(a)–(b),whentheAA-QDs(zetapotentialmeasurementof−7.18mV)addedincellculturedishwithoutpolybrenerespectivelyfor30and60min,AA-QDswerenotdetectedunderfluorescentmicroscopyafterremainedAA-QDswaswashedoffwithPBS.Thisisduetoelectricalrepulsionbetweenalginatemacromoleculeandcellsur-facewhicharebothnegativelycharged.However,after120minco-cultureofAA-QDsandcellswithouttheadditionofpoly-brene,greendotsrepresentingQDswereabletobepinpointedwithincellulardomain(Fig.3(c)).Itissurmisedthat,givenalongerincubationtime(i.e.,120min),sufficientamountofpro-teins(ingredientsfromfetalbovineserum)adsorbedontothesurfaceofnegativelychargedAA-QDstriggerednonspecificcellbindingandensuingendocytosis.While,inFig.3(d)–(f),AA-QDswerestartedtobedetectedintheintracellularspace60minpost-incubation,inthepresenceof10␮g/mLpolybrene.Thisisprobablybecausetheelectrostaticneutralizationeffectofpoly-cationicpolybreneledtononspecificadsorptionofnegativelychargedAA-QDsonnegativelychargedcellmembrane.Itisnoteworthythatpolybreneisacationicpolymerandhasbeenreportedtoactbyneutralizingnegativechargesonthesurfaceofcellsandvirionstopromotevirusattachmentandenhanceviraltransductionrate(KwonandPeng,2002).Oncethecon-centrationofpolybreneincreasedto50␮g/mL,AA-QDswereabletoenterthecellportswithinashortperiodoftimeand

Fig.5.PhotomicrographicalimagesofBHK-21cellsexposedtoQD–viruscomplexeswith50␮g/mLpolybreneat30,60,and120min,respectively.Thenormalizedfluorescenceintensitiesperselectedcellareaof(b)and(c)basedonthefluorescenceintensityperselectedcellareaof(a)are1.23and2.73,respectively.MOI=0.5andscalebar=25␮m.

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expressedgreenfluorescenceinBHK-21cellsafter30minoftreat-ment.

3.3.EffectofpolybreneoninfectivityofQD–virus

TheQDsencapsulatedwithamphiphilicalignatesurfactantswereemployedtoformcolloidalclusterswithdengueviruses(zetapotentialmeasurementof−6.04mV)inthepresenceofcationicpolybrene.Todeterminethesuitableviralconcentrationforthedevelopmentofquickandefficientcell-basedfluorescentimagingdrugscreeningplatform,variousMOIs(multiplicityofinfection)wereemployedrangingfrom0.1to1.OurresultsshowedthatQD–viruscomplexeswithanetpositivecharge(zetapotentialmeasurementof+2.84mV)wereinternalizedintoBHK-21cellsbyreceptor-mediatedendocytosis,andcanbedetectedincellu-larmilieu.AsshowninFig.4(a)–(f),greenfluorescentintensityexpressedwithinBHK-21cellsduetotheentranceofQD–viruswasaugmentedinproportiontotheincrementofMOI(from0.1to0.5).ToconfirmtheQD–viruscomplexeswereindeedinternalizedintoBHK-21cellsratherthanabsorbedoncellsurface,aseriesofpho-tomicrographstakenbyaconfocalmicroscopealongthez-axisofselectedinfectedcellsareshowninFig.4(g).Thegreenfluores-

cencewasfurtherenhancedwhentheconcentrationofpolybreneincreasedfrom10to50␮g/mLwithMOImaintainedat0.5(showninFig.5(a)–(c)).Sincepreservationofviralinfectivityaftertag-gingviruswithQDsisofutmostimportanceforQD–virusimagingmodalityofferingmeaningfulinformation,plaqueformingassayswereperformedwithBHK-21cellstreatedwithintactdenguevirusesandQD–viruscomplexes.Theresult(datanotshown)indi-catedthenumberofplaqueformingunitsnumeratedfromthecellsinfectedwithQD–viruscomplexeswasonlyslightlylowerthanthecontrolgroup(i.e.,dengueviruseswithoutQDstagging).ThisimpliesthatdengueviralinfectivitywasretainedusingthemethodofincorporationviruswithQDsinpolycationicsolution.Thisisconsistentwiththepreviousreportindicatingtheinfec-tivityofincompetentretrovirusasaviralgenedeliveryvehicledecreasesslightlywhenmixedwithQDinpolycationicsolution(Youetal.,2006).Incontrasttovirusescovalentlylabeledwithflu-orescentdyes,themethoddevelopedinthisstudyseemstobeasuperiorone.Inthecaseofadeno-associatedvirus(AAV),whichisarelativelysmallvirus(∼25nmindiameter),ithasbeenshownthatforadye-to-particleratiohigherthantwo,theinfectioustiterwasseverelyaffected,namelythenumberofviruseswhichwereabletoinfectacellwaslowered(Seisenbergeretal.,2001).It

Fig.6.PhotomicrographicalimagesofBHK-21cellstreatedwithandwithoutallophycocyaninforonehour,thenexposedtoQD–viruscomplexeswith50␮g/mLpolybreneforvariousperiodsoftime.(a)–(d)ImagesofBHK-21cellsincubatedwithQD–viruscomplexesfor30min,aftercellspre-treatedwithallophycocyaninof0,6.25,31.25,and125␮g/mL,respectively;(e)–(h)imagesofBHK-21cellsincubatedwithQD–viruscomplexesfor60min,aftercellspre-treatedwithallophycocyaninof0,6.25,31.25,and125␮g/mL,respectively.Thenormalizedfluorescenceintensitiesperselectedcellareaof(b)–(h)basedonthefluorescenceintensityperselectedcellareaof(a)are0.72,0.66,0.54,2.92,1.02,0.66,and0.52,respectively.MOI=0.5andscalebar=25␮m.

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shouldbenotedthatthecellshapeofFig.5(b)turnedintolargeonesafterBHK-21cellswereinfectedwithQD–viruscomplexesfor60min.ThesituationgetsevenworseforcellstreatedwithQD–viruscomplexesupto120min(seeFig.5(c)).Wespeculatesuchcellmorphologicalchangeswerecausedbytheviraldam-ageofcellmachinery(e.g.,cytoskeleton),therebyleadingtocelllysis.

Inadditiontothemicroscopy-basedfluorescencetechnique,biochemicalmethodsarealsoavailabletoanalyzethebiologyofthevirus(Girodetal.,1999).Inmanyofthesemethods,viralgeneexpressionisdeterminedafteracertainlongincubationtime.Moreover,mostofthemethodsareverytimeconsumingandalargeamountofviruseshastobeused.Inviewoftheneedofpoly-breneforneutralizingelectrostaticrepulsionforcebetweenbothnegativelychargeddenguevirusandamphiphilicalginatecoatedQD,itisconjecturedthathavingQDscappedbypositivelychargedwater-solubleligands(orpolymers)todirectlyformcomplexeswithviruseswithouttheaidofpolybreneprobablyisafeasibleandmuchsimplemethod.

3.4.Antiviralactivityofallophycocyanin

AccordingtotheresultsshowninFig.5,itisreasonabletospeculatethat,ifanypotentialcompoundhasanti-dengueviralfeature,theintensityofgreenfluorescenceemittedfromQD–viruscomplexesinternalizedincellsshouldbedrasticallydiminished.InordertotestifQD–viruscomplexescanbehar-nessedasdiagnosticprobesforfastscreeningpotentialanti-denguedrugs,allophycocyaninwasselectedasthemodelcompoundforexploringtheefficacyofQD–virusimagingmodalityforscreen-inganti-dengueviraltherapeuticagents.AllophycocyaninhasbeenreportedtoneutralizeEV71-inducedcytopathiceffectinbothhumanrhabdomyosarcomacellsandAfricangreenmonkeykid-neycells.Antiviralactivitywasmoreefficientinculturestreatedwithallophycocyaninbeforeviralinfectioncomparedwiththatintheculturestreatedafterinfection(Shihetal.,2003).AsshowninFig.6(a)–(d),theintensityofgreenimageswithinBHK-21cellswasdecreasedalongwiththedosageincreaseofallophycocyaninprovidedonehourpriortotheadditionofQD–viruscomplexeswhichwereconstructedinthepresenceof50␮g/mLpolybrene.ItshouldbenotedthatcellsincubatedwithQD–viruscomplexesforonly30mincangiveafairlyclearfluorescentintensitydecreasewiththeconcentrationofallophyscocyaninupto125␮g/mL.Forcellstreatedwithallophycocyaninhigherorequalto31.25␮g/mLforonehourthenchallengedwithQD–viruscomplexesfor60min,thediminishofgreenfluorescenceisevenastonishingprominent.Comparedtootheranti-viralagentscreeningassays,thecell-basedQD–virusimagingmodalityexploitedinthisstudyindeedhasfast

andefficientfeaturesforscreeningantiviraltherapeutics.Since,thepIvalueofallophycocyaninwasmeasuredaround4.5,anetnegativechargeofallophycocyaninwillberevealedinthecellcul-turemedium.SuchnegativelychargedallophycocyaninmightbeassociatedwithpositivelychargedQD–viruscomplexesiffreeallo-phycocyaninremainedintheculturemedium.However,inourstudy,BHK-21cellswereonlytreatedwithallophycocyaninfor1handwashedthreetimeswithPBSsolution.ThereisnoconcernofallophycocyanininterferenceontheinteractionbetweenQD–viruscomplexesandcells.4.Conclusion

ToconstructQD–virusimagingmodalitycapableofprovidingmeaningfulinformation,preservationofviralinfectivityaftertag-gingviruswithQDsisofutmostimportance.Inthisstudy,wedemonstratetheinfectivityofQD–virusformedviacolloidalclus-teringinthepresenceofpolycationicpolybreneremainedintacttobeinternalizedbycellssusceptibletodenguevirus.Theefficacyofanti-dengueviralactivityofanalgalextractwasclearlyillustratedbytheconstructedinorganic–organichybridplatforminaquickandefficientmanner.Acknowledgements

TheauthorsthankDr.Huan-YaoLei(InstituteofBasicMedi-calSciences,NationalChengKungUniversity,Tainan,Taiwan)forprovidingdenguevirusanditsrelatedinformation.References

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