VOLUME 4, NUMBER 3, MARCH 2004

VOLUME4,NUMBER3,MARCH2004

©Copyright2004bytheAmericanChemicalSociety

Virus-BasedFabricationofMicro-andNanofibersUsingElectrospinning

Seung-WukLeeandAngelaM.Belcher*

DepartmentofMaterialsScienceandEngineeringandBiologicalEngineering,MassachusettsInstituteofTechnology,Cambridge,Massachusetts02139,and

ChemistryandBiochemistry,TheUniVersityofTexasatAustin,Austin,Texas78712

ReceivedOctober20,2003;RevisedManuscriptReceivedDecember18,2003

ABSTRACT

Longrod-shapedM13viruseswereusedtofabricateone-dimensional(1D)micro-andnanosizeddiameterfibersbymimickingthespinningprocessofthesilkspider.Liquidcrystallinevirussuspensionswereextrudedthroughthemicrometerdiametercapillarytubesinacross-linkingsolutionofglutaraldehyde.Resultingfiberswere10 20µmindiameter.AFMimagingverifiedthatthemolecularlongaxisofthevirusfiberswasparalleltothefiberlongaxis.M13virusesweresuspendedin1,1,1,3,3,3-hexafluoro-2-propanolandwerethenelectrospunintofibers.Afterblendingwithahighlywatersolublepolymer,polyvinylpyrolidone(PVP),M13viruseswerespunintocontinuousuniformvirus-blendedPVP(virus-PVP)nanofibers.Resultingvirus-PVPelectrospunfibersmaintainedtheirabilitytoinfectbacterialhostsafterresuspendinginbuffersolution.

Effortstomimictheuniquestructuresandspecificfunctionsofnaturalsystemshaveprovidedvarioususefultoolsandmaterialsinnanoscience.1-15Biosystemsproducehighlyprogrammed,self-assembled,self-templatedstructures.5-15Forexample,asmallpercentageofproteininabaloneshellnucleatesaCaCO3proteincompositethatis3000timestougherthanpureCaCO3.1Bymimickingthebiomineral-izationprocessinnature,ourgrouphasshownthatproteinsequences,selectedusingageneticallyengineeredpeptideviruslibrary,couldspecificallybindtoandnucleatedesiredmaterials.3,4,7,8,10Theone-potsyntheticrouteprovidedbythesegeneticallyprogrammedvirusesresultsintheself-assemblyofhighlyorderednanocrystallinecompositema-terials.4

Recently,muchresearchhasbeenfocusedonproducingnanosizedone-dimensionalmaterialssuchasnanorods,

*Correspondingauthor.E-mail:belcher@mit.edu.10.1021/nl034911tCCC:$27.50PublishedonWeb02/20/2004

©2004AmericanChemicalSociety

nanowires,andnanofibers.17-24Naturally,silkspidersandsilkwormsspinhighlyengineeredcontinuousfibersbypassingaqueousliquidcrystallineprotein(fibroin)solutionthroughtheirspinneret.17,18Oncethefibroinsolutionisreleasedtotheair,ithardensintoaflexibleandhighlyorientedsemicrystallinefiberthatisstrongerthananyotherpolymerfiberspun.17Amongsyntheticroutesinvestigated,nanofiberfabricationusingelectrospinninghasbeenanefficientmeansofgeneratinghighsurface-to-volumeratiosofmaterialsthatmaypossiblybeusedashighlysensitivesensorsandfunctionalmembranes.23-29Electrospinninguseshighelectricfieldsallowingforthefabricationofnarrowfiberswithdiametersrangingfromtensofnanometerstomicrometers.Thecontinuousfiberscanbeeasilyconvertedintononwovenfabrics,whichmaybeusefulforsynthesizingnovelmembranesduetotheirhighsurface-to-volumeratios.Herewereporttwomethodsoffabricatingvirus-based

Figure1.Aschematicdiagramillustratingthevirusfiberfabricationprocessusingwet-spinningand

electrospinning.

compositefibersthatmimicthespinningprocessofsilkspiders.M13viruseswerespunintomicro-andnanofibersusingwet-spinningandelectrospinning,respectively,whichareillustratedinFigure1.Theresultingfibersshowednematicorderedmorphologiesduetoflowingforces.Inaddition,theviruseswereblendedwithpolyvinylpyrolidone(PVP)toimproveprocessingability.Theresultingvirus-blendedPVPfiberswerecontinuousandweretransformedintononwovenfabricsthatretainedtheirabilitytoinfectbacterialhosts.Thesehybridmaterials,includingflexiblefibersandmats,havepossibleapplicationsincludingbio-medicalandtissueengineeringaswellastemplatestobiocontrolled-synthesisofelectronicoropticalmaterials.8,10Inaddition,furthergeneticengineeringofpVIIImajorandpIXminorcoatproteinsofM13virusmightprovidemultiplehybridfunctionsofvirus-basedfibers.34

Anti-streptavidinM13bacteriophage(virus),possessinganengineeredpeptidesequenceN′-WDPYSHLLQHPQ-C′asafusedproteinintothepIIIcoatprotein,wasusedasabasicbuildingblocktofabricatethemicro-andnanoscalefiberswithorwithoutconjugationwithR-phycoerythrin(eBioscience,CA).7TheM13virussuspension(～100mg/mLintris-bufferedsaline(pH7.5))wasextrudedthrougha

～20µmcapillarytubeintoa37.3%aqueousglutaraldehydesolution.Thiscross-linkedsuspensionwasair-driedandformedmicrofibers.Apolarizedopticalmicroscopy(POM)image(Figure2A),takenusinganOlympusIX51polarizedopticalmicroscope(Olympus,Japan),showedthefiberswerebirefringent,indicatingtheliquidcrystallineorderedstructureofthefibers.Scanningelectronmicroscopeimages(SEM;JEOL6320FEGSEM,Japan)(Figure2B),ofthefibersshowedthatthefibershad10-20µmdiametersandwerecomposedofseveralbundle-likefiberswhichwerepropa-gatedtothefiberlongaxis.TheparallelorientationoftheindividualvirusbuildingblockswasobservedusinganAtomicForceMicroscope(AFM;VeecoNanoscope,CA).AnAFMimage(Figure2C)showedclose-packedM13viruses,withalongaxisparalleltothelongaxisofthefibers.Duetotheflowfield,thesmecticorderedstructureinthesuspensionwasdisruptedandasmectic-to-nematictransitionoccurred.Becausethesuspensionwasimmediatelyexposedtoacross-linkingsolutionafterreleasefromthecapillary,thenematicorderedviruseswerecross-linkedtoeachotherandformednematicorderedfibers.Fluorescentviralmi-crofiberswerefabricatedafterbindingoftheanti-streptavidinviruswithR-phycoerythrinconjugatedstreptavidinpriortospinning.7Auniformfluorescentfieldobserved(Figure2D)throughoutthefiberssupportedthenematicorderedstructureobservedinPOMandAFM.

Variousvolatileorganicsolventsweretestedaselectro-spinningsolventsasapossiblemechanismtoelectrospinthelowviscosityM13virussuspension.Mostoftheelectro-spinningsolventstested,suchasmethanol,ethanol,DMF,acetone,andtrifluoroethanol,formedaslurry-likeaggrega-tionwhenaddedtoviruspelletsandsuspensions.However,1,1,1,3,3,3,hexafluoro-2-propanol(HFP;AlfaAeser,MA)successfullyformedahomogeneousvirussuspensionwhentheviruspelletsweredissolved.Tofabricatenanofibers,capillarytubes(20µLvolume,～0.5mmindiameter,6.5cmlong,DrummondScientificCo.,PA)werefilledwithvirussuspension(～92mg/mLinHFP),andagraphite

rod

Figure2.M13virusfiberfabricatedbywet-spinningprocess(A)POMimage(scalebar:100µm),(B)SEMimage(scalebar:20µm),and(C)AFMimageandschematicdiagraminsetofnematicorientedmorphologyofvirusfiber(scalebar:1µm).(D)fluorescencemicrographofvirus-phycoerythrinconjugatedfibersfabricatedbywet-spinning.

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Figure3.(A)POMimageofelectrospunvirus-onlyfibers,(B)SEMimageofelectrospunvirus-onlyfibers.(Scalebars:5µ

m).

(0.5mmindiameter)wasinsertedintooneendofthecapillarytubeusingasyringepump(HarvardApparatus,Inc.,MA)atafeedingrateof3-6µL/min.A20-30kVpotentialwasappliedbyahighvoltagesourceacrossair(GlassmanHighVoltage,NJ).Electrospunfiberswerecollectedontometalplates(10cmindiameter),groundedandcoveredbyaluminumfoil.Thedistancebetweenthecapillaryandcollectorwas10-15centimeters.APOMimage(Figure3A)verifiedthatthesecontinuouselectrospunfiberswerehighlybirefringentunderthecrosspolars,indicatingthatfiberswerecomposedofhighlycrystallineorderedstructures.Arela-tivelybroaddistributionofdiameterwasobserved,rangingfromafewmicrometerstotensofnanometersindiameter.SEMimages(Figure3B)showedthatmostofthefibersandbundlesbranchedtoformsmallerfibers.DuetothetoxicityofHFPtotheM13virus,infectibilityofM13virusinHFPsolutionwasdramaticallydecreased,showingnoinfectibility.BecauseintactvirusstructureswererarelyobservedfromTEMobservation,virusfibersspunusingHFPcouldbecomposedofthefragmentofvirusanddissembledsubunits.Toimproveprocessingabilityandpreservetheintactviralstructureandinfectingability,theM13virussuspensionwasblendedwithahighlywatersolublepolymer,PVP(MW:1,300,000;AlfaAesar,MA).Thesuspensionswereblendedinratiosof1:1,1:2,1:3,and1:4betweenvirussuspensioninTBS(～100mg/mL)andPVPsolution(25%,(w/w))inwater.Duetothelowviscosityandhighsurfacetensionoftheaqueoussuspensions,1:1and1:2suspensions,whenelectrospun,depositeddropletsofvirus-blendedPVPsuspen-sion.Theelectrospinningofthe1:3suspensionresultedinsporadicfiberformation,withtypicalresultsbeingamixofbeadandstringtypefibers,whichisnormallyobservedinlowviscousorlowconcentrationsolutionsintheelectro-spinningprocess.29-31ContinuousM13virus-blendedPVPfiberswerefabricatedfromtheelectrospinningofthe1:4suspension.Aphotographofelectrospunfibers(Figure4A)showedthatelectrospunfiberscouldbetransformedtoanydesiredshapeofnonwovenfabricsusingamask.AnSEMimageshowedthattheresultingfiberswerecontinuousandformedhomogeneousropeshapes(Figure4B).Distri-butionofthediameterwasrelativelynarrowwiththediameterrangingfrom100to200nanometers.Theelectro-spunfibersobservedusingpolarizedopticalmicroscopyexhibitednematic-likebirefringency(Figure4C).Thefibersshowedmaximumbrightnesswhenthefiberswereorientedat45degreestothecrosspolarsandwereextinctwhenthe

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Figure4.ElectronspunfiberofM13virus-blendedwithPVP.(A)Photographofnonwovenfiberspunthroughthemaskinscribedwiththeword“NANO”,(B)SEMimage(scalebar:1µm)and(C)POMimage.(D)FluorescencemicrographsofPVPblendedwithvirus-phycoerythrinfibersfabricatedbyelectrospinning.

fiberswereorientedparallelwithcrosspolars.WhentheviruseswereconjugatedwithR-phycoerythrinusingastreptavidinlinker,fluorescenceimagescouldbeobservedusingfluorescencemicroscopy(Figure4D).Afterdissolvingtheelectrospunfibersin3mLofTBS,thesuspensionwastestedforinfectionabilityoftheM13virus.Theresuspendedvirussuspensionwasstillabletoinfectthebacterialhost.Thisresultsupportsthenotionthatboththeproteinstructureandgeneticinformationwerestableinthesevirus-basedfibers.Furthermore,webelievethisisthefirstelectrospinningofanorganismtoformorientedpseudo-infinitefiberstructures.TheincorporationofR-phycoerythrinintothevirusfibersdemonstratesthatanymoleculecouldbeincorporatedandorganizedinthese1Dfibers.Thissuggestedthatmanyothermaterialsincludingsmallmolecules,drugs,proteins,andsemiconductingandmagneticmaterialscanalsobeelectrospunintovirus-basedfibersandmatstoformcontinuousmaterialsoverapseudo-infinitelengthscale.Insummary,virus-basedmicro-andnanofiberswerefabricatedusingwet-spinningandelectrospinningprocesses.M13virusesinthewet-spunfiberswerealignedparalleltothefiberlongaxis.Electrospunfibers,composedoffrag-mentsofM13virusesanditssubunits,werealsofabricatedbysuspendingM13virusesinHFP.ByblendingtheviruseswithPVP,fiberswereformedwiththeintactstructureoftheviruses.Thesefibersdemonstratedthatnovelbiomaterialscanbefabricatedfromaprogrammedorganismtoextendthedimensionofengineeredvirusesintofibersusefulinnucleatingsemiconductornanofibersorinselectivelybindinganytypeofdesiredmaterials.3,4,7,10Additionally,uniformnanofibersfabricatedbyblendingnanomaterial-conjugatedM13viruswithPVPmightprovideusefulbiologicalfunc-tionsandhighlysensitivecatalyticfunctionsinfuturebiomedicalapplicationsand

biosensors.

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Acknowledgment.ThisworkwassupportedbytheARO,NSF,andInstituteforCollaborativeBiotechnologies.S.-W.LeethanksDr.EstherRyan,Dr.ChristineFlynn,andEricKraulandforassistanceineditingthispaperforclarity.References

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