Hydrophobin-stabilized dispersions of PVDF nanoparticles in water

Hydrophobin-stabilized

dispersions

of

PVDF

nanoparticles

in

water

Claudia

Pigliacelli

a

,

Alessandro

D’Elicio

a

,

Roberto

Milani

b

,

Giancarlo

Terraneo

a

,

Giuseppe

Resnati

a,

**

,

Francesca

Baldelli

Bombelli

a,

**

,

Pierangelo

Metrangolo

a,b,

*

a

LaboratoryofNanostructuredFluorinatedMaterials(NFMLab),FondazioneCentroEuropeoNanomedicinac/oDepartmentofChemistry,Materials,and ChemicalEngineering‘‘GiulioNatta’’,PolitecnicodiMilano,ViaL.Mancinelli7,20131Milan,Italy

b

VTT-TechnicalResearchCentreofFinland,FI-02044VTT,Espoo,Finland

1. Introduction

Sinceitsdiscoveryandcommercialintroductioninthe1960s, poly(vinylidenefluoride)(PVDF)hasgainedagrowingattentionin bothindustrialandscientificfields,duetoitsuniquephysicaland chemicalproperties.Piezoelectric,pyroelectrical,andferroelectric behavioursofPVDF havewidelybeen studiedandcharacterized

[1–3],andpromotedtheuseofPVDFinelectricalandelectronic devices[4,5]. Thankstoitsfluorinationdegree,PVDFis character-izedbyhighthermalstability,mechanical,andchemicalresistance, makingitalsoanexcellentmembranematerial [6].Recentlythe synthesisofsilicananoparticlesfunctionalizedwithPVDFchains wasreportedinliteraturehighlightingpossibleinnovative applica-tions of PVDF in the production of nanocomposites [7–10]. Additionally, PVDF is widely employed in paints and coatings,

thankstoitslowsurfaceenergy,whichrendersPVDFanexcellent polymerforthesurfacefunctionalizationofmaterials,inorderto improvemetallicsubstratesresistancetoweather,corrosion,and chemicals[11]. Theseexcellentpropertiesandthewiderangeof applicationscontrastwithsomelimitationsinPVDFuseresulting from its poor water-solubility. In fact, due to its hydrophobic nature,theuseoforganicsolventsand/ormeltingprocessingofthe polymerarenecessaryforitsmanufactureanduses,increasingthe costsandpossibletoxicityimplicationsrelatedtosolventhandling. Awater-basedPVDFformulationmightfurtherpromotePVDFuse. Inthisregard,KynarAquatec1

,awater-basedPVDFformulation, represents a coatingemulsion thathas been showntoofferthe samematerialprotectionanddurabilityoforganicsolvent-based PVDFcoatings[12].Therefore,thedevelopmentofnewPVDF water-basedformulationsmightfurtherpromotetheapplicationofsuch versatilepolymerindifferentfields.Oneofthemostpromisingand sustainableapproachesforimprovingthesolubilityofpoorly water-solublematerialsisrepresentedbytheuseofbiosurfactants.Inthis regard, hydrophobins(HFBs) area classofsmall, highly surface-active proteins (7–10kDa) produced by filamentousfungi [13]. HFBsplayanimportantroleindifferentstagesofthefungalcell developmentand initsprotection.In particular,thanks totheir surfaceactivity,theycoatandprotectdifferentfungalstructuresand allowfungalattachmenttosurfaces.HFBs’structureischaracterized

JournalofFluorineChemistryxxx(2015)xxx–xxx

ARTICLE INFO

Articlehistory:

Received19November2014

Receivedinrevisedform6February2015 Accepted11February2015 Availableonlinexxx Keywords: PVDF Nanoparticles Hydrophobin Bio-nanocomposites Coatings ABSTRACT

In thisstudy, aqueousdispersionsof partiallycrystalline PVDF nanoparticles(NPs)wereobtained employinghydrophobin(HFB),anamphiphilicfilm-formingproteinabletofilmhydrophobicsurfaces. DynamicLightScattering(DLS)andTransmissionElectronMicroscopy(TEM)analysisofPVDF-HFBII aqueousdispersionsconfirmedtheHPBIIabilitytofilmPVDFhydrophobicNPs.Freeze-driedPVDF-HFBII bio-nanocompositeswereshowntobeeffectivelyre-dispersibleinwater.Anaqueousdispersionof PVDFNPsmayhaveanimpactontheapplicationsofthispolymerintheperspectiveofthedevelopment ofenvironmentallyfriendlycoatingmethods.

ß2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Correspondingauthorat:LaboratoryofNanostructuredFluorinatedMaterials (NFMLab),FondazioneCentroEuropeoNanomedicinac/oDepartmentof Chemis-try,Materials,andChemicalEngineering‘‘GiulioNatta’’,PolitecnicodiMilano,ViaL. Mancinelli7,20131 Milan,Italy.

** Co-correspondingauthors.

E-mailaddresses:giuseppe.resnati@polimi.it(G.Resnati),

francesca.baldelli@polimi.it(F.BaldelliBombelli),pierangelo.metrangolo@polimi.it (P.Metrangolo).

GModel

FLUOR-8510;No.ofPages8

ContentslistsavailableatScienceDirect

Journal

of

Fluorine

Chemistry

j ou rna l hom e pa ge : w w w. e l s e v i e r. co m/ l o ca t e / fl u or

http://dx.doi.org/10.1016/j.jfluchem.2015.02.004

0022-1139/ß2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

byaconservedpatternofeightcysteineresiduesthatform four disulfidebridges[14].HFBsareamphiphilicandtheself-assembling and filming abilities of these proteins are associated with the hydrophobicpatchcomposedofthealiphaticchainsofapartoftheir aminoacidsequences[15].Infact,HFBsareabletoformamphiphilic filmsatbothair/waterandhydrophobic/hydrophilicinterfaces[16]. TwoclassesofHFBshavebeenreportedintheliterature[13].ClassI HFBs form aggregates that show limited solubility in aqueous solutions,whileclassIIHFBsself-assembleinmorewater-soluble aggregates[17].HFBs’coatingabilitycanpotentiallybeexploitedin differentapplicationsandtheirpossibleuseinmaterials, nutraceu-tical,pharmaceutical, andnanomedicalfieldshaverecentlybeen regarded[15,17–19].HFBwas showntosignificantlyimpact the adsorptionofproteinsonthesurfaceofnanoparticles,oncetheyare exposedtobiologicalfluids,reducingtheformationoftheprotein corona [19], which has been proved to dramatically influence nanoparticlesbehaviourinthebiologicalenvironment[20]. More-over,Milanietal.reportedtheHFB’sabilitytostabilizefluorous dropletsinaqueousenvironment,provingitspotentialas fluorine-freefluorosurfactant[21].

Inthisstudy,wefocusedonthepossibledispersionofPVDFNPs inwaterthroughsurfacecoatingwithclassIIhydrophobinsHFBI andHFBII.HFBs’biocompatibility,theirabilitytoformrobustfilms, andreversesurfacewettabilitymakethempromisingcandidates forthisscope.ClassIHFBshavebeenproventofilmhydrophobic surfacesviatheformationofrodletstructures[22,23]. Asfaras classIIHFBsareconcerned,theirremarkableabilitytoincreasethe wettabilityofhydrophobicparticleshasbeenshownbyadsorption studies on Teflon1

, Kevlar1

, and other substrates[15]. In our study,fourincreasingconcentrationsofPVDFNPswere success-fullydispersedinHFBIIaqueoussolutions.ThepolymericNPswere filmedbyHFBIIandtheresultingstructureswereinvestigatedby TEM and DLS analyses and compared to pristine PVDF NPs dispersedinMethylEthylKetone(MEK).HFBIabilitytotransfer PVDFNPsinto water dispersions wasalso evaluated. Obtained dispersionsweresubsequentlyfreeze-driedandcharacterizedby

attenuated total reflectance infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), and powder X-ray diffraction(PXRD) analyses.Solid state analysiswasperformed forevaluatingthepossibleimpactofthedispersionprotocolonthe PVDFcrystallinitycontentandpolymorphism,andfor investigat-ingtheproteinsecondarystructurewhenadsorbedonthePVDF surface.Particularattentionwaspaidtothecharacterizationofthe freeze-driedPVDF-HFBIIbio-nanocompositesintheperspectiveto re-dispersetheminwater.Indeed,thiswouldallowthe prepara-tion of water-solublePVDF bio-nanocomposites, which to date havenotbeenreported,yet.

2. Resultsanddiscussion

2.1. PVDFdispersionsinorganicandaqueousenvironments

PVDFhasbeenshowntobedispersibleinMethylEthylKetone (MEK)[24].PVDFdispersionsinMEK(1mg/mL)werepreparedby

Fig.1.(a)TEMimageofPVDFpristineNPs,bar=200nm.(b)SizedistributionhistogramobtainedbysizeanalysisofseveralTEMimagesofparticles(about130NPs)toobtain meaningfulstatisticalresultsfortheparticlesize.(c)DLSintensity-weightedsizedistributionobtainedbyCONTINof1mg/mLPVDFNPsdispersioninMEK.

Fig.2.PVDF-HFBIIaqueousdispersionsattheconcentrationof1,2,3,and5mg/mL after24hatroomtemperaturefrompreparation.

simplemagneticstirringandanalyzedafter24h.AsshownbyTEM images (see Fig. 1a), PVDFdispersions in MEKafforded mono-dispersedround-shaped NPs ofan average size of22831nm (Fig.1b).DLSanalysisyieldedanaveragehydrodynamicdiameterof 2562nm(seethehydrodynamicsizedistributioninFig.1c).

PVDFdispersionsinaqueousHFBIIsolutionswerepreparedby ultrasonication. The provision of highly localized energy was essentialforthepolymerdispersionintheproteinsolution.Indeed, theuseofotherapproachessuchasmagneticstirring,vortex,and ultrasoundsbathfailedindispersingPVDFinHFBIIsolutions.PVDF concentrationsfrom1 to5mg/mLweresuccessfullydispersedin 0.1mg/mLHFBIIaqueoussolutions,formingopalescentdispersions

thatdidnotdisplaysignificantsedimentationover24h,asshownin

Fig. 2. PVDFdispersions at polymer concentrationshigher than 5mg/mL presented,instead,extensivesedimentation withinone hourfrompreparation(seeFig. S1inESI).

Theformationof amonolayerofHFBIIhasbeenreportedto correspond to a surfacecoverage in the200–250ng/cm2 _range

[25],andpreviousworkshowedafilmingabilityofHFBIItowards TE5069Teflon1

particlesof about125ng ofproteinpercm2 _of

particles[26]. ThesurfaceareaofourPVDFnanoparticlescanbe roughlyapproximatedas150cm2_{/mg,ascalculatedfora}

popula-tionofuniformlysizednanoparticleswitharadiusof114nmanda densityof1.75g/cm3.Accordingtothisapproximation,HFBIIhasa

Fig.3.(a)TEMimageofHFBIIcoatedPVDFNPs,bar=100nm.(b)SizedistributionhistogramobtainedbysizeanalysisofseveralTEMimagesofparticles(about110NPs)to obtainmeaningfulstatisticalresultsfortheparticlesize.(c)DLSintensity-weighedsizedistributionobtainedbyCONTINof1mg/mLPVDFNPsdispersedinasolutionofHFBII (0.1mg/mL)inwater.

Fig.4.(a)TEManalysisofpristinePVDFNPs(driedfromMEK);(b)TEManalysisofHFBIIcoatedPVDFNPs(driedfromwaterandstainedwithuranylacetate).Bar=50nm. TEMimage(b)showsthepresenceofHFBIIaggregatesinsolution(greyisletsonthecarbongrid),forwhichtheformationhasalreadybeendescribedintheliterature[28]. C.Pigliacellietal./JournalofFluorineChemistryxxx(2015)xxx–xxx 3 GModel

filmingabilityof133ng/cm2_{,inreasonableagreementwiththe}

abovementionedliteraturevalues.

TEManalysisofPVDF-HFBIIdispersionsshowedthepresenceof particleshavinganaveragesizeof22331nm,inagreementwith thesizeobtainedforthepristinePVDFdispersioninMEK(Fig.3aand b). HFBIImonolayeronhydrophobicsurfaceshasbeenshowntobe about2nmthick[26].Therefore,nosignificantimpactontheNPsize wasexpecteduponHFB surfaceadsorption.Differently fromTEM results,DLSanalysisindicatedaslightincreaseofthehydrodynamic radiusoftheparticles,yieldingasizeof2974nm.Thisislikelydue totheformation,drivenbytheproteinmonolayersurroundingthe polymericparticles,ofahydrationshellaroundtheNPsurface.Infact, itis wellknownthat HFBIIbinds to hydrophobicsurfacesvia its hydrophobicpatch,exposingthemorehydrophilicaminoacidstothe morepolarenvironment[25,27]. Thisabilityofturningthenatureof

asurfacefromhydrophobictohydrophilicthroughtheformationof an amphiphilic HFBIIfilm has beenexploitedhere fordispersing highlyhydrophobicPVDFNPsinwater.ThepresenceoftheHFBIIfilm can clearly be seenin the TEMimages reportedinFig. 4, which comparesPVDFparticleswithandwithoutHFBIIcoating.InFig.4b theHFBIIcoatedPVDFNPs’surfaceappearsmuchlesssmoothand regularthaninpristinePVDFNPs(Fig.4a).

SizedistributionsobtainedbyDLSanalysisofPVDF-HFBIIwater dispersions at different PVDF concentrations are shown in

Fig.5.ValuesreportedinthetableofFig.5showthattheaverage hydrodynamic size of theNPsincreases on increasing polymer concentrations. This size increase was also accompanied by a significantdecreaseinthepolydispersity(narrowerwidthofthe size distributions)of the systemthat might bedue to a lower concentrationoffreeHFBIIinthedispersionasthefreeprotein

Fig.6.ATR-FTIRspectraofthepristinePVDFnanostructuredpowder(lightbluecurve),solidHFBII(darkbluecurve),andofthefreeze-driedPVDF-HFBIIpowderobtained fromthe1mg/mLdispersion(redcurve).(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.) Fig.5.(a)DLSintensity-weighedsizedistributionsobtainedbyCONTINofPVDF-HFBIIwaterdispersionsatincreasingPVDFconcentrationsasshowninthelegend(HFBII concentration0.1mg/mL).(b)Averagedhydrodynamicsizescalculatedfromthesizedistributiongraphsreportedin(a),thereporteduncertaintyistheSDonthreedifferent measurements.

maypartlycontributetothescattering,increasingthe polydisper-sityofthesystem.

TheclassIIhydrophobinHFBI,whichhassimilarself-assembly features to those of HFBII, was also successfully tested for dispersing PVDF NPsin water. PVDF-HFBI aqueousdispersions (PVDF concentration 1mg/mL) yielded NPs having a size of 30615nm,slightlysmallerthanthoseobtainedwithHFBIIatthe samePVDFandproteinconcentrations(seeESIFig.S2).

BothHFBIandHFBIIwereefficientincoatingPVDFNPs,making themhydrophilicand dispersiblein water.Todate no environ-mentallyfriendlyPVDFwaterdispersionshavebeenreportedin literature.Duetotheeasypreparationmethodologyreportedhere andthehighreproducibilityoftheprocess,theuseofHFBmight opennewperspectivesintheapplicationsofPVDF.

2.2. SolidstatecharacterizationofPVDF-HFBIIfreeze-dried dispersions

Attenuatedtotalreflectance-Fouriertransforminfrared spec-troscopy(ATR-FTIR)andX-raydiffraction(XRD)techniqueshave beenextensivelyusedfortheidentificationandcharacterizationof PVDFstructuralfeatureseitheraspurepolymerorblendedwith

otherpolymersorsmallmolecules[5]. ItiswellknownthatPVDF hasahighlypolymorphicbehaviourandcancrystallizeintofive possible forms, namely

a

,

b

,

g

,

d

, and

e

, depending on crystallization conditions [24]. The most common crystalline formisthenonpolar

a

-phasewiththeTGTGconformation.The

b

-and

g

-phase, which have TTT planar zigzag and TTTGTTTG conformations, respectively, are polar and thus responsible for thepiezoandferroelectricalpropertiesofthepolymer.Thetwo othercrystalphasesarerarelyobserved.

Weconsideredofinteresttoassesstheprevailingforminthe PVDF NPswe coated withHFB, in orderto studywhether the chosen protocol may affect crystallinity. Information on the structuralcharacteristicsofourpristinePVDFwasobtainedfrom detailedanalysisoftheIRregionbetween450 and1400cm 1

(Fig.6,top).Thepresenceofthe

a

forminourstartingPVDFNPs wasshownbytheidentificationofthebandsat1150cm 1_(CF

2

symmetric stretching mode) and at 976cm 1 _(CH

2 twisting

mode), which are exclusively present in this polymer phase

[24,29]. ThedetectionofadditionalCF2bendingmodes(490.9,

613.9 and762.7cm 1_{)andCHout-of-planedeformationbands}

(873.8cm 1_{) in thespectrum confirmed theidentity of the}

_a

phase.

Fig.7.PowderXRDpatternsforthepristinePVDFnanostructuredpowder(above)andthefreeze-driedPVDF-HFBIIpowderobtainedfromthe1mg/mLPVDFdispersion (below).Thecrystallographicplanesarelabelled.

C.Pigliacellietal./JournalofFluorineChemistryxxx(2015)xxx–xxx 5 GModel

Furtherproofofthe

a

phaseofthestartingPVDFwasgainedby powderXRDstudies.Thepowderpattern(Fig.7,top)showspeaks at 2

u

equal to 18.05, 19.95, 25.85, 33.05, 35.81, and 38.728, corresponding to the diffractions in planes (020), (110),(021), (130),(200),and (131),respectively,allcharacteristic ofthe

a

-phase[30].

DSC analysisofthepristinePVDFshoweda melting peakat 158.88C (see ESI, Table S1), as expected for the

a

-form. A comparisonbetween thearea of this peak and theenthalpyof fusionofa100%

a

-phasecrystallinePVDFreportedintheliterature (

D

Hfusion:104.6J/g)allowedforaroughestimationofthedegreeof

crystallinityforoursample,whichwasfoundtobearound40%. ATR-FTIRanalysiswassuccessivelyperformedalsoonsolidHFBII and freeze-dried PVDF (1mg/mL)-HFBII dispersions. The main peakvisibleintheHFBIIspectrumisrelatedtothestrongamideI (1624cm 1_{)signal,correspondingtotheC55Ovibrationsof the}

HFBIIpeptidebonds.Fig.6clearlyshowsabroadeningoftheamide Ibandtogetherwithashiftfrom1624.7cm 1_{forthepureHFBII,to}

1644.7cm 1_{forthefreeze-driedPVDF-HFBdispersion.Thisshift}

maysuggest a changein theproteinsecondary structure upon bindingtothePVDFsurface[31],and isinagreement withthe increaseof

a

-helixcontentdescribedintheliteratureforprotein assemblingatwater–hydrophobicinterfaces[32].

ThepowderX-raydiffraction(PXRD)patternofthefreeze-dried PVDF-HFBIIobtained fromthe 1mg/mL PVDFsample presents severalpeaksdistributedoverabroadbumpcharacteristicofan

amorphousstate(seeFig.7).Thispatternsuggestsareductionof theoverallPVDFcrystallinity,likelyduetotheprocedureadopted for dispersing thepolymer in the HFBII aqueous solution. The decreaseof crystallinityintheprotein-treatedsample wasalso confirmedbyDSCmeasurements,whichshowedadecreaseofthe PVDFcrystallinity,possiblyto30% fromthe40%of thepristine PVDF.

Moreover, the diffractogram, specifically the characteristic peaksat2

u

equalto18.3,19.85,35.87,and398,underlineshow theprevailingcrystallinephaseofthepolymerintheHFB-coated PVDF is the

a

-form asin the starting material. This result,in agreementwiththeFTIRdata,suggeststhattheformationofthe protein monolayer surrounding the PVDF particles does not influence the crystallinity of the polymer, which maintains largelyunchanged its bulkstructuralproperties.SimilarPXRD patterns were also observed when incremental amounts of polymerwereusedintheformationofthebio-nanocomposites, i.e.,2mg/mL,3mg/mL,and5mg/mL.Inallofthesamples,no changeinthecrystallinephaseofthepolymercomparedtothe 1mg/mLwasdetected(seeESI).

2.3. SolidPVDF-HFBIIbio-nanocompositeredispersioninaqueous environment

PVDF-HFBII dispersions were freeze-dried and stored in anhydrousenvironmentfora week.Freeze-dried powderswere subsequently re-dispersedinwater andleft forequilibration at room temperature for 24h. As shown by Fig. 8, no polymer sedimentation was observed after 24h. Therefore, the water dispersibilityofPVDFparticlesmediatedbyHFBIIwasnotaffected bylyophilization.ThisindicatesthattheHFBIImonolayeronPVDF NPsislikelytobekeptintactduringthelyophilizationprocessas alsosuggestedbythesolid-statecharacterizationofthe lyophi-lizedpowders.

Re-dispersedPVDF-HFBIIsampleswereanalyzedbyDLSand obtainedresults(Fig.9)areingoodagreementwithsizevalues obtainedforthefreshsamples,althoughaslightincreaseinsize canbeseenforallthePVDFstartingconcentrations.

The re-dispersibility of freeze-dried PVDF-HFBII powder renders the possible application of such systems even more convenient.Indeed,thepossibilitytostorethedispersionsintheir driedstateisexpectedtoimprovetheirstabilityavoidingpotential particles aggregation in solution over time as well as possible solutioncontaminations.

Fig.9.DLSintensity-weightedsizedistributionsobtainedbyCONTINofPVDF-HFBIIwaterre-dispersionsatincreasingPVDFconcentrationsasshowninthelegendreported intheinsetofthefigure(HFBIIconcentration0.1mg/mL).Thetableontheleftreportstheaveragehydrodynamicdiametersobtainedbythesizedistributionsreportedinthe figure.

Fig.8.PVDF-HFBIIwaterdispersionsobtainedbyre-dispersionoffreeze-dried powdersatdifferentPVDFconcentrationsaslabelledonthevials.

3. Conclusions

ThisstudyprovedtheabilityofclassIIhydrophobins,HFBIand HFBII,toreversethesurfacewettabilityofPVDFNPs.Inparticular, HFBII wasable to film PVDFsurface with a maximum filming capacity of approximately 133ng/cm2_{, yielding monodispersed}

PVDFNPsaqueousdispersionsasshown byDLSanalysis.HFBII coatingon PVDF NPswasalsoimaged by TEManalysis. HFBII-coatedPVDFNPsresultedtobestableinaqueoussolutionsover time and easily re-dispersible after freeze-drying, showing the resistanceoftheHFBfilmtothisprocess.LyophilizedPVDF-HFB bio-nanocompositeswere studiedbyATR-FTIR, XRD,andDSC, whichindicatedproteinadsorptionontheNPsurfaceandminor changesofthecrystallinity(

a

-form)ofthepolymerduringthe coating protocol. The possibility of dispersing PVDF NPs in aqueoussolutions without significantly affecting their size or changing the polymer crystalline phase may open the use of PVDF to new applications. In fact, the obtained PVDF-HFBII bio-nanocomposites dispersions represent, to the best of our knowledge, the first environmentally friendly dispersions of PVDFNPsinwater.TheuseofsuchaqueousPVDFdispersionswill betested insome of thecurrent applicationsof thispolymer, which otherwise requires the use of organic solvents or high temperatureprocess,bringingforthamoresustainableapproach. Inparticular,thenewlydevelopedPVDFwaterdispersionsmight beemployed,uponHFBcalcination,assurfacecoatingmaterials. Furthermore, freeze-dried PVDF-HFBII bio-nanocomposites might potentially find application as polymer nanofillers for hydrophilicmatrices,afieldofresearchthatiscurrentlyunder investigationinourlaboratories.

4. Materialsandmethods

4.1. Chemicals

HFBIIwasobtainedasdescribed in[33]. PVDFwasobtained fromSolvaySpecialtyPolymers(Hylar1

301 F).Reagents were used without further purification. Milli-Q water (mQW) was obtainedbyaSimplicity(Millipore)instrument.

4.2. Preparationofthesamples

Accurately weighed PVDF was dispersed in mQ water and vortexedat30rpmfor1min.Threecyclesof1minultrasonication wereperformedusingaSONICVibracelloperatingat20V.Before measuring, samples wereleft at roomtemperature for 24h to allowequilibration.

4.3. TEManalysis

TEMimages were acquired by using a Philips CM200 TEM, equippedwithafieldemissiongunandoperatingat200kV.PVDF and PVDF-HFBII dispersions were prepared by dropping the sample solution on carbon-coated copper grids. Due to the intrinsiclow contrastof organic materials,negative stainingof PVDF-HFBIIsampleswasperformedbyusinguranylacetate(1%, w/v). Both samples were left drying overnight. TEM statistical analysiswasbasedonthemeasurementofabout100–150 NPs. Sizedistributionswerefittedbya GaussianequationusingIgor Pro4.02.

4.4. DLS

DLSexperimentswerecarried outusingaZetasizerNanoZS (Malvern Instrument, Malvern, Worcestershire, UK), equipped witha633nmredlaserandmeasuringthescatteredlightatan

angle of 1738. Samples were analyzed 24h after preparation. Measurementswereperformedat258Candeach measurement consistedof5runsandwasaveragedon3 replicates.

4.5. Solidstateanalysis

PVDF-HFBII dispersions were freeze-dried using a Edwars ModulyoEF4-1596(Edwards,Crawley,WestSussex,UK).

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) analysis was performed on freeze-dried samples using a Thermo Scientific Nicolet iS50 FT-IR spectrometer, equippedwithaiS50ATRaccessory(ThermoScientific,Madison, USA). 32 scans werecollected for eachsample at a resolution valueof2cm 1_.

ABrukerAXSD8powderdiffractometerwasusedforallpowder X-ray (PXRD) measurements. Experimental parameters are as follows:Cu-Karadiation(

l

=1.54056A˚),scanninginterval:6–608 2

u

. Stepsize:0.0168.Exposuretime1.5s/step.

Acknowledgments

Theauthorsgratefullyacknowledgethefinancialsupportfrom theAcademyofFinland(BioHalproject,fundingdecision260565) andRegioneLombardia(FondoperloSviluppoelaCoesione–FAS 2007–2013).WearealsogratefultoSolvaySpecialtyPolymersfor thegiftofasampleofPVDF(Hylar1

301F).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in the online version, at http://dx.doi.org/10.1016/j.jfluchem.2015. 02.004.

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