Surface interactions of gold nanorods and polysaccharides: From clusters to individual nanoparticles

Gold nanorods (AuNRs) are suitable for constructing self-assembled structures for the development of biosensing devices and are usually obtained in the presence of cetyltrimethylammonium bromide (CTAB).

jo u r n al h om ep a g e :w w w e l s e v i e r c o m / l o c a t e / c a r b p o l

Heloise Ribeiro de Barrosa, Leandro Piovana, Guilherme L Sassakib,

Diego de Araujo Sabryb, Ney Mattosoc, Ábner Magalhães Nunesd, Mario R Meneghettid,

Izabel C Riegel-Vidottia,∗

a Grupo de Pesquisa em Macromoléculas e Interfaces, Departamento de Química, Universidade Federal do Paraná—UFPR, CxP 19081, CEP 81531-980,

Curitiba, PR, Brazil

b Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná—UFPR, CxP 19046, CEP 81531-980, Curitiba, PR, Brazil

c Departamento de Física, Universidade Federal do Paraná—UFPR, CxP 19044, CEP 81531-980, Curitiba, PR, Brazil

d Grupo de Catálise e Reatividade Química, Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Av Lourival de Melo Mota s/n, CEP

57072-970, Maceió, AL, Brazil

a r t i c l e i n f o

Article history:

Received 14 April 2016

Received in revised form 28 June 2016

Accepted 5 July 2016

Available online 5 July 2016

Keywords:

Gold nanorods

Sulfated chitosan

Surface interactions

Self-assembling

a b s t r a c t

Goldnanorods(AuNRs)aresuitableforconstructingself-assembledstructuresforthedevelopmentof biosensingdevicesandareusuallyobtainedinthepresenceofcetyltrimethylammoniumbromide(CTAB) Here,asulfatedchitosan(ChiS)andgumarabic(GA)wereemployedtoencapsulateCTAB/AuNRswith thepurposeofstudyingtheinteractionsofthepolysaccharideswithCTAB,whichiscytotoxicandis responsiblefortheinstabilityofnanoparticlesinbuffersolutions.Thepresenceofavarietyoffunctional groupssuchasthesulfategroupsinChiSandthecarboxylicgroupsinGA,ledtoefficientinteractions withCTAB/AuNRsasevidencedthroughUV–visandFTIRspectroscopies.Electronmicroscopies(HR-SEM andTEM)revealedthatnanoparticleclusterswereformedintheGA-AuNRssample,whereas individ-ualAuNRs,surroundedbyadenselayerofpolysaccharides,wereobservedintheChiS-AuNRssample Therefore,thepresentedworkcontributestotheunderstandingofthedrivingforcesthatcontrolthe surfaceinteractionsofthestudiedmaterials,providingusefulinformationinthebuilding-upofgold self-assemblednanostructures

©2016ElsevierLtd.Allrightsreserved

1 Introduction

Goldnanoparticles(AuNPs)haveincreasinglybeengiven

exten-sive attention due to their unique properties making those

materialsusefulincatalysis,nanoelectronicsand,more

interest-ingly, inoptical sensingand diagnosticsin thebiomedical field

(Garabagiu & Bratu,2013; Kopwitthaya et al., 2010; Mitamura,

Imae,Saito,&Takai,2007;Pierrat,Zins,Breivogel,&Sonnichsen,

2007)

AmongtheAuNPs,considerableattentionhasbeendedicated

togoldnanorods(AuNRs).Thecoherentoscillationoftheelectrons

alongtheshortaxis(transversalSPR)andthelongaxis(longitudinal

SPR)ofthenanorodscausestwosurfaceplasmonresonance(SPR)

bands.Atleastoneofthesebandscanbefoundinthevisible

spec-tra.ThetransversalSPRbandhasamaximumabsorptionaround

∗ Corresponding author.

E-mail addresses: izabel.riegel@ufpr.br , iriegel@gmail.com (I.C Riegel-Vidotti).

520nm(Rayavarapuetal.,2010), whereasthelongitudinalSPR bandisobservedintherangefrom650nm(shorterrods)to950nm (longer rods)(Eutis &El-Sayed, 2006; Rayavarapuet al.,2010)

AstransversalandlongitudinalSPRareshapeandsizedependent (Murphy&Jana,2002;Xieetal.,2011),theAuNRsareparticularly suitableforbuildingupself-assembledstructuresforthe develop-mentofbiosensors(Yuetal.,2014),nanodevices(Xieetal.,2011), andnon-invasiveprobes(Charanetal.,2012)

Regarding the applications of AuNPs in biological environ-ments,someimportantissuesariseconcerningthemaintenance

oftheirmorphologicalstability,cytotoxicity,andinteractionswith different organisms or theircomponents Therefore, the choice forappropriatestabilizingagentsisoftheutmostimportancein obtainingAuNRsthatarestableindifferentenvironmental condi-tions(pHandionicstrength)andthatexhibitlowtoxicity Theseedmediatedmethodinthepresenceofthesurfactant cetyltrimethylammoniumbromide(CTAB)isthemostcommonly employedproceduretoobtainAuNRs,althoughsomeother meth-ods have beenrecently proposed(da Silva, Nunes,Meneghetti, http://dx.doi.org/10.1016/j.carbpol.2016.07.018

0144-8617/© 2016 Elsevier Ltd All rights reserved.

Mulvaney,2005).Ingeneral,themethodinitiallyproposedby

Mur-phy(Gole,Orendorff&Murphy,2004;Jana,Gearheart&Murphy,

2001a; Jana, Gearheart, & Murphy, 2001b; Jana, Gearheart, &

Murphy,2001c;Johnson,Dujardin,Davis,Murphy&Mann,2002;

Murphy&Jana,2002)andEl-Sayedgroups(Nikoobakht&El-Sayed,

2001)consists oftheformation of theAuNRs fromsmall sized

sphericalAuNPs(seedsolution),whichthenactsasnucleation

cen-tersintheAuNRssynthesis.InthepresenceofCTAB,theAuNRs

growthismainlyunidirectionalsincetheinteractionsbetweenthe

polarheadgroups of thesurfactant, i.e.thequaternary

ammo-niumbromidemoiety,andthecrystallographic{110}facetofthe

growingparticleispreferential,causingthegrowthinthe

longitu-dinaldirection,paralleltothe{001}planes(daSilva,Meneghetti,

Denicourt-Nowicki, & Roucoux, 2014; Meena & Sulpizi, 2013;

Nikoobakht&El-Sayed,2003).Therefore,thedifferentgrowthrates

ofthefacetsarethefactorsthatdeterminethefinalshapeofthe

nanoparticle(shortversuslongNRs).Inaddition,CTABis

responsi-bleformaintainingthecolloidalstabilitysincethebilayerstructure,

whichisformedbytheself-interactionofthealkylgroupsofCTAB,

promotesthesuitableprotectionagainstparticleagglomerationin

aqueousmediathroughelectrostaticandstericinteractions(Boca

&Astilean,2010).However,CTABiscytotoxicandcausesAuNRs

instabilityinbuffersolutions,whichthenrestrictstheirusein

bio-logicalapplications(Boca&Astilean,2010;Hamon,Bizien,Artzner,

Even-Hernandez,&Marchi,2014;Rayavarapuetal.,2010)

Therefore, when focusing on the obtention of AuNRs that

safelycan beusedin thebiomedicalfield, it isfundamental to

replacepartof CTAB or encapsulatethe CTAB/AuNRstoobtain

particles with reduced toxicity that are also stable under

dif-ferentenvironments.Kopwitthaya etal.(2010)and Rayavarapu

et al (2010) synthetized CTAB/AuNRs and showed that the

replacement of CTAB by thiolated poly(ethylene glycol)

(PEG-SH)producedparticleswithlowercytotoxicitycomparedtothe

as-preparedAuNRs(Kopwitthayaetal.,2010;Rayavarapuetal.,

2010) In addition, other molecules have beenused to replace

CTABintending to reduce thecytotoxicity,such aspolystyrene

sulfonate, polyethylene glycol (Rayavarapu et al., 2010),

1-Mercaptoundec-11-yl)hexa(ethyleneglycol) (EG6OH)(Xieet al.,

2011), thio-polyethylene glycols (Bogliotti et al., 2011),

poly-acrylicacid,poly(allylamine)hydrochloride(PAH)(Huang,Jackson

&Murphy,2012),and3-mercaptopropionicacid(MPA)(Garabagiu

&Bratu,2013)

Polysaccharidesarepartofaverypromisingfamilyofnaturally

occurringmoleculesthathavealsobeendescribedtointeractwith

goldnanoparticles.Thepresenceofavarietyoffunctionalgroups

intheirstructure assiststhefavorableinteractions betweenthe

AuNRsandthesurroundingmedia,whichisresponsibleforthe

AuNRsstabilizationand alsoprovidessites forfurtherchemical

modifications(Erathodiyil&Ying,2011;Liuetal.,2013).Inaddition

tobeingnaturalproducts,polysaccharideshaveinherent

proper-tiessuchasbiodegradability,biocompatibility,andlowtoxicity(Liu

etal.,2013).Surprisingly,thereisarelativelylownumberof

scien-tificworksreportingtheuseofpolysaccharidesasstabilizingagents

ofAuNRs(Wang,Chang&Peng,2011;Yuetal.,2014),although

manyworkshavereportedtheefficientstabilizationofspherical

goldnanoparticlesbypolysaccharides

Chitosan(Chi) is a linear polysaccharide extracted fromthe

exoskeletonof crustaceans,obtainedbydeacetylation of chitin

MediumandhighmolarmasschitosanisonlysolubleinwateratpH

lowerthan6.0(Williams&Phillips,2000,Chp.21).Chitosan(Boca

etal.,2011)anditsderivativeshavebeensuccessfullyemployedto

capAuNPsforphotothermaltherapy(Wang,Chang&Peng,2011;

Yangetal.,2015),foroptoacoustictomography(Wangetal.,2015),

amongotherapplications.Gumarabic(GA)isahighlybranched

naturalpolysaccharideexudedfromthetrunksandbarksofacacia

trees.Thispolysaccharidehasbeenextensivelyusedforthe stabi-lizationofsphericalAuNPs(Chandaetal.,2010;Kattumurietal., 2007;Wu&Chen,2010),displayingoptimalperformanceinawide

pHrange(Barrosetal.,2016)

In order to reduce the cytotoxicity inherent to CTAB stabi-lizedAuNRs andsimultaneouslyimprovetheAuNRsstability in physiologicalmedia,weusedasulfatedchitosan(ChiS)orGAto encapsulateCTAB/AuNRs Thenon-toxicityand biocompatibility

ofChiandGAwereevaluatedinpreviousworks(Bicho,Roque, Cardoso,Domingos,&Batalha,2009;Bocaetal.,2011).The chem-icalmodification of Chito obtainChiS is of interestbecause it doesnotonlykeeptheChimainchainbackboneintact,butitalso improvesitssolubilityinaqueousmedia(Jayakumar,Nwe,Tokura,

&Tamura,2007).Moreover,itcanpotentiallyinfernew functional-itiestothemodifiedChisincesulfatedpolysaccharides,likeheparin forexample,areknowntopresentimportantbiologicalfunctions suchasanticoagulantand/orantithromboticactions(Asifetal., 2016;Jayakumaretal.,2007;Maasetal.,2012)

HereinwedemonstratebyUV–vis(Ultraviolet–visible)andFTIR (FourierTransformInfrared)spectroscopies,andalsoby transmis-sionandscanningelectronmicroscopiesthatChiSandGAinteract differentlywiththeAuNRs.Thedifferencesarediscussedinterms

ofthedifferentfunctionalgroupspresentineachpolysaccharide thatleadstodistinctpolysaccharide/AuNRsstructures.Therefore, thisstudycontributestounderstandingandcontrollingthe self-assemblingbehaviorofAuNRs,mediatedbythecappingagent

2 Materials and methods

2.1 Materials Tetrachloroauricacid(HAuCl4·3H20,30%indiluteHCl,99,9%), CTAB(≥98%),GA(Mw=9.3×105gmol−1;uronicacidcontentof 17%)(Greinet al.,2013),chitosan (≥75% deacetylated),and sil-ver nitrate (AgNO3,>99%) were purchased from Sigma-Aldrich Sodiumborohydride(NaBH4,≥98%)waspurchasedfromNuclear (SãoPaulo,Brasil)andascorbicacid(AA,>99%)waspurchasedfrom Dinâmica(SãoPaulo,Brasil).Milli-Qgradewater(18.2Mcm, Mil-lipore,USA)wasusedinthepreparationofallsolutions.Priorto use,GApowderwassolubilizedinwater,leftovernightat4◦Cand subsequentlydialyzedfor48hagainstdistilledwater througha dialysismembrane(12–14kDacut-off)andfreeze-dried

2.2 Sulfationofacommercialchitosan Commercial chitosan (Chi) underwent thesulfation reaction according to Terbojevich, Carraro, and Cosani (1989)’s sulfuric acid:chlorosulfonicacidmethod.Briefly,1.00gofcommercial chi-tosanwasaddedtothepre-cooled(4◦C)reactionmixture(40mLof sulfuricacid(H2SO495%)and20mLofchlorosulfonicacid(HClSO3

98%)) Then, thereaction wascarried outat roomtemperature understirringfor1h.Thesulfationwasstoppedbypouring250mL

ofcolddiethylether(Et2O)intothereactionmixture.The precipi-tateformedwaswashedwithcoldEt2O,thensuspendedindistilled water,neutralized withsaturatedNaHCO3,dialyzedagainst tap waterthrougha3500kDacut-offmembrane,andfreeze-dried.The finalproductwasfullycharacterized(ChiS,Mw=1.4×104gmol−1;

SO4=48%;SO3 O–3=6.8%and SO3 O–6=41.2%)(Supplementary material—Fig.S1andTableS1)andresultedinapaleyellowpowder thatwasstoredinamoisturefreeenvironment

The surface charge wasobtained using a Zetasizer Nano ZS instrumentbysolubilizingthepolysaccharides(ChiSandGA)using Milli-Qwater

2.3 Synthesisofthegoldnanorods(AuNRs)

The AuNRs were synthetized by theseed-mediated method

accordingto daSilva etal (2013) andpreviously described by

Sau and Murphy (2004).In a typicalprocedure, the seed

solu-tionwaspreparedby5.0mLofaqueoussolution0.5×10−3molL−1

HAuCl4addedto2.5mLof0.20molL−1CTABsolution.Then,0.6mL

ofice-cold0.01molL−1 NaBH4 solutionwasadded.Thecolorof

thesolutionimmediatelychangedfromdarktobrownishyellow

Next,thesolutionwaskeptundergentlemixingfor2minandleft

torestforatleast2hpriortouse.Afterwards,thegrowth

solu-tionwasprepared bygentlemixing ofa 2.5mL of0.20molL−1

CTABsolution,5.0mLofaqueoussolution1×10−3molL−1HAuCl4

and150␮Lof4.0×10−3molL−1 AgNO3solution.Then,70␮Lof

80×10−3molL−1ascorbicacidsolutionwasaddedandthecolor

changedimmediatelyfromdarkyellowtocolorless.Lastly,12␮L

ofseedsolutionwasaddedbymixinggentlyfor 10s.Thecolor

changedslowlyfromcolorless topurple.Thefinal solutionwas

keptundisturbedfor at least4h UV–vis spectroscopy (Agilent,

model8453)wasusedtomonitortheAuNRsformationthrough

theobservationoftheSPRbands

2.4 PreparationofGA-AuNRsandChiS-AuNRs

TheAuNRsstabilizedbythepolysaccharides,eitherGA-AuNRs

orChiS-AuNRs,werepreparedbyasimplemethod.First,the

as-preparedAuNRswerecentrifuged(10.000rpm,15min) andthe

supernatantwasdiscardedtoremovetheexcessCTAB.The

precip-itatewasdispersedin0.5mLofwater.Then,itwasadded4.5mLof

GAorChiSsolution0.1wt%.Thefinalsolutionwaskeptundergentle

magneticstirring at roomtemperature(∼25◦C)for 24h.

After-wards,thesamplewasagaincentrifugedandthesupernatantwas

discardedtoremovetheunboundGAorChiSfromthesolution.The

precipitatewasdispersedin2.0mLofMilli-Qwater(18.2Mcm

at25◦C)andusedthereafter.UV–visspectroscopy(Agilent,model

8453)wasusedtoverifyanychangesintheSPRbandsasa

conse-quenceoftheinteractionsoftheAuNRswiththepolysaccharides

2.5 MicroscopicandspectroscopicinvestigationofAuNRs,

GA-AuNRsandChiS-AuNRs

Transmissionelectronmicroscopy(TEM)wasperformedusing

aJEOL1200EX-IImicroscopeworkingatanaccelerationvoltage

of80kV.Adrop(∼10␮L)ofthecolloidalsolutionwasdeposited

onto400meshcarbon-coatedgridsandair-dried.Highresolution

scanningelectronmicroscopy(HR-SEM)wasperformedusingaFEI

Quanta450FEGmicroscopeworkingatanaccelerationvoltageof

10kV.Analiquotof80␮Lofthecolloidalsolutionwasdeposited

onto a sample supportand air-dried FTIR measurementswere

performedusingaBIORADFTS-3500GXFTIRspectrometer.The

measurementsweremadeinthetransmissionmodeinaspectral

domainrangingfrom400to4000cm−1,usingKBrpellets

3 Results and discussion

InordertoensurethefullsurfacecoverageoftheAuNRs,the

concentrationofthepolysaccharides(GAorChiS)waskeptmuch

higher thanthe concentrationof theAuNRs inthe preparation

ofGA-AuNRsandChiS-AuNRs.Both,GAandChiSarenegatively

chargedinaqueoussolutionwithpH∼5.Thezetapotentialvalues

(␨)ofGAandChiSare−36.3mVand−28.6mV,respectively.The

negativechargeofGAisduetothepresenceofthe COO−groups,

whereasthenegativechargeofChiScorrespondstothepresence

of OSO3−.However,duetothecationicquaternaryammonium

headgroupofCTAB,theas-preparedAuNRsbearpositivesurface

400 500 600 700 800 900 1000 0,0

0,1 0,2 0,3

(a) (b)

Wavelength (nm) (c)

Fig 1. UV–vis absorption spectra of (a) as-prepared AuNRs, (b) GA-AuNR and (c) ChiS-AuNRs.

charge(Rayavarapuetal.,2010).Therefore,thechoiceofusing neg-ativelychargedpolysaccharidesaidstheinteractionsbetweenthe AuNRsandthepolysaccharidesthroughelectrostaticattraction Theas-synthesizedAuNRspresenttypicalSPRbandscentered around␭1=515nmand␭2=740nm(Fig.1)thatallowthe determi-nationoftheparticleconcentrationusingtheextinctioncoefficient (Garabagiu&Bratu,2013;Orendorff&Murphy,2006).The esti-mated concentration of particles of the as-prepared AuNRs is

5×10−10molL−1 Theprofiles of theSPRbands of GA-AuNRsand ChiS-AuNRs are also shown in Fig 1 Slight shifts at the maximum wave-lengthsareobservedsincethepresenceofGAandChiSchanges thedielectricconstantofthesurroundingenvironment.Inthecase

ofChiS-AuNRs,itisevidenttheappearanceofastrongabsorption

atlongerwavelengths,possiblycausedbythechangesinthe sur-roundingenvironment,whichwillbeclarifiedintheTEMimages Furthermore,themaintenanceofthepositionoftheSPRbandsafter thepolysaccharidesadsorptionindicatesthataggregationhasnot takenplace,preservingthemorphologyoftheparticles

Through TEM images theAuNRs were observed to have an averagesize of45nm×15nm (aspectratio=3).Although some sphericalparticlesareseen,mostoftheobjectsarerod-like struc-tures,characterizingahighyieldsynthesis(Fig.2a).Also,theshape andsizeoftheAuNRswerepreservedinthepresenceofGAorChiS (Fig.2b–e).TheGA-AuNRsandChiS-AuNRsexhibitedaveragesizes

of47nm×15nmand43nm×14nm,respectively,corroborating whatwasobservedbyUV–visspectroscopy

ItisnoticeablefromtheTEMimagesthattheGA-AuNRssample resultedinnanoparticleclusters(Fig.2b).Thehighmagnification imagerevealed that theGAadsorbedmolecules arenot distin-guishablefromthesubstrate.However,thenanoparticlesinthe ChiS-AuNRsampleareseparatedfromeachotherbyadense struc-turethatissuggestedtobecomposedoftheChiSmolecules.This behaviorcouldbeassociatedwiththedifferencesobservedinthe shapeoftheUV–visabsorptionspectra.Forabetter understand-ingoftheorganizationofthepolysaccharidesaroundtheAuNRs, HR-SEMimageswereobtainedusingsecondaryandbackscattered electrons.Thecomparativeanalysisoftheimagesprovidesadeeper insightintotheinteractionsbetweenthepolysaccharidesandthe AuNRs

Fig.3 andbclearlyshowsthedifferencesintheAuNRs sur-roundingmediumduetothepresenceofGAorChiS,respectively Bothimageswereobtainedusingsecondaryelectronsignalthat providestopographic contrast.As observed byTEM, nanoparti-cleclusterswereseeninFig.3 (GA-AuNRs),whereasinFig.3b

(ChiS-AuNRs)theAuNRswereindividuallysurroundedbyChiSin

Fig 2.TEM images of (a) as-prepared AuNR, (b-c) after the adsorption of GA (GA-AuNRs) and (d-e) of ChiS (ChiS-AuNRs) on AuNR surface.

acloud-likearrangement.Asaresult,whenGAisusedasthe

sta-bilizingagent,anincreasedparticledensity(numberorparticles

perunitvolume)isattainedwhencomparedwithChiS.Fig.3cand

ecorrespondstolowmagnificationimagesthatwerecollectedat

thesameregionandobtainedbysecondaryandbackscattered

elec-trons,respectively.Thecontrastobtainedbybackscatteredelectron

signalisrelatedtotheelectrondensityofthematerial.Ascanbe

seen,noimportantdifferenceswereobservedsupposedlybecause

GAisarrangedasathinlayerontheSEMsupport.Sample

ChiS-AuNRswas analyzed similarly (Fig.3d and f) and in this case,

strikingdifferenceswereobserved.InthecaseofFig.3d,which

wasobtainedbysecondaryelectrons,theedgesofthestructure

arehighlighted,providingavolumeperspective(topographic

con-trast).InFig.3f,obtainedbybackscatteredelectrons,theelectron

densityandcontrastoftheorganicmatrixofChiSwiththemetallic

supportarestronglyevidencingthattheparticlesaresurrounded

byChiSmolecules,confirmingthatChiSactsasanefficient

encapsu-lating/wrappingagentforindividualAuNRs.Atlowmagnification,

thesharpdifferenceofcontrastinthistypeofsampleisveryuseful

toquicklyfindtheareatobestudiedatgreatermagnifications

ThedifferencesobservedbycomparingtheimagesofGA-AuNRs

andChiS-AuNRscanbeascribedtothedifferencesbetweenthe

electrondensities ofGA andChiS and tothedifferent

arrange-mentofthepolysaccharidesaroundtheAuNRs.ChiShashigher electron density when compared withGA due to thepresence

ofsulfategroups.Additionally,themoleculesaredenselypacked aroundthenanoparticlesduetotheirlowermolarmassandchain linearity,favoringtheformationofathree-dimensionalstructure

Ontheotherhand,GA,whichisahighlybranched,highmolarmass polysaccharide,bearsatomswithlowelectrondensity(mainlyC,

O,H),formingathinlayeronthesupport

Someother aspects can bediscussed to clarifythe HR-SEM observations, as follows It is widely known that GA presents surfactant-likeproperties and is highly soluble in water (Grein

etal.,2013).Therefore,whenGA-AuNRswerewashedtoremove theexcessofGA,themoleculesthatwereweaklyboundedonthe AuNRssurfacemostlikelywereremoved,loweringtheresulting finalconcentrationofGAaroundthegoldsurface.Conversely,itis knownthatChiexhibitsagglutinativeproperties(Lehr,Bouwstra, Schacht,&Junginger,1992)andsinceChiissolubleonlyinacidic media,thesulfationprocessimprovesitssolubilityinwater How-ever,GAis morewater soluble thanChiS,resulting ina higher concentrationofChiSthanGAmoleculesaroundtheAuNRs Thus, the adequate selection of the stabilizing agent can efficiently tunetheself-aggregationof AuNRs.Gold nanoparti-cleclustershave applicationsinphotothermal therapy (Zharov,

Fig 3. HR-SEM images of GA-AuNRs (a, c, e) and ChiS-AuNRs (b, d, f) obtained by secondary electrons (a, b, c, d) and backscattered electrons (e, f).

Mercer,Galitovskaya,&Smeltzer,2006),whereasindividual

func-tionalized gold nanoparticles can perform important biological

functionsvia specific signaling pathways(Li,Kawazoe &Chen,

2015;Nethietal.,2014)

FTIRspectroscopy waschosen toevaluatethenature ofthe

interactionsbetweenthepolysaccharidesandtheAuNRs.The

dis-placement, appearance or disappearance of bands in the FTIR

spectramaybeattributedtotheinteractionsthatoccurinthese

assemblies.First,thespectraoftheas-preparedAuNRsandneat

CTABispresented(Fig.4).Theassignmentsofthemainbandsare

inTable1.Itwasobservedthatthecharacteristicbandspresentin

CTABarepreservedintheAuNRs(Tang,Huang,&Man,2013).The

maintenanceofthebands,relativetosymmetricandasymmetric

stretchingvibrationof CH2 ofCTABchain(2918and2850cm−1),

indicatesthat thehydrophobictails ofCTABarenot interacting

withtheAuNRssurface.Itissuggestedthatthealkyltailsare

self-interacting,forminga bilayeronthegoldsurface thatdoesnot

restrainthestretchingvibrationalmodes.Accordingtothis

propo-sition,unboundandboundsurfactantheadgroupsarefoundinthe

goldsurroundings(Nikoobakht&El-Sayed,2001),thusrendering

someC N+groupsfreeforfurtherinteractions(Goleetal.,2004)

However,thebandscorrespondingtosymmetric and

asymmet-ricC HscissoringvibrationsofH3C N+moiety(1487,1473,1462

and1431cm−1),andthebandcorrespondingtoC N+stretching

(960cm−1)arerelativelylessintenseandslightlyshiftedinAuNRs

whencomparedtopureCTAB.Thisdecreaseinintensityindicates

Wavenumber (cm-1)

AuNR CTAB

18 28

Fig 4. FTIR spectra of as-prepared AuNRs and neat CTAB.

thatthehydrophilicportionofCTABboundtotheAuNRssurface Additionally,thebandscorrespondingtothe CH2chainrocking modedemonstrateimportantdifferences.PureCTABshowstwo bandsat719and731cm−1whereasAuNRsshowonlyonebandat

669cm−1.Thisfactisclearevidenceoftheconstrainmentsthatthe CTABalkylchainsaresubjectedtoduetotheinteractionswiththe particles,resultingintheformationofacompactlayeredstructure

4000 3500 3000 1500 1000 500

GA

AuNRs

Wavenumber (cm-1)

GA-AuNRs

18 2850

18 2850

Fig 5. FTIR spectra of GA-AuNR, as-prepared AuNR and GA.

aroundtheparticles.Therefore, it wasconfirmedbyFTIR

spec-troscopythatCTABremainedontheAuNRssurfaceevenafterthe

washingprocedure

TheFTIRspectrumofGA-AuNRsispresentedinFig.5

Impor-tantdifferencescanbeseenwhencomparingwiththeas-prepared

AuNRsspectrum.Theassignmentofthemainbandsforboth

sam-plesisshowninTable2.ThecharacteristicbandsofAuNRs,relative

tothesymmetricandasymmetricstretchingvibrationsof−CH2−

ofCTAB(2918and2850cm−1)weremaintainedintheGA-AuNRs

spectrum.Thus,itwasevidencedthatCTABisstillpresentonthe

AuNRssurface.Additionally,theabsenceofsomebandsattributed

toC N+moiety(1473,1462,1433and960cm−1)andtheabsence

ofthetwostrongbandsintheGAspectrumattributedtothe

asym-metricand symmetricstretchingvibration ofthe COO− group

(1608and1419cm−1).Theprofilechangeofthebands

correspond-ingtothestretchingoftheC O(1253,1143,1064and1031cm−1)

in the GA-AuNRs spectrum is an indication of the interaction

betweentheC N+headgroupofCTABwiththenegativelycharged

carboxylategroupsonGAstructure.Furthermore,theobservation

ofnewbandsaround1060and700–400cm−1(fingerprintregion)

intheGA-AuNRspectrumcouldbeassociatedtotheinteractions

thatoccurthroughGAadsorbedontheAuNRsurface

The presence of CTAB in ChiS-AuNRs samples was likewise

observed,asseenbytheFTIRspectrainFig.6.Thebandsat2918

and2850cm−1arepresent,whereasthebandsat1487–1433andin

960cm−1disappearedintheChiS-AuNRsspectrumincomparison

totheAuNRsspectrum.Furthermore,themainbandsobservedin

theChiSspectrumdonotdisappearwhenChiSisassociatedwith

theAuNRs,butinsteadexhibitanintensitydecreaseinthe

ChiS-AuNRsspectrum(1647,1542,1153,1072,1062and1004cm−1)

Theassignmentof themain bandsis shown inTable 2

There-fore,theinteractionbetweentheC N+fromCTABandChiSoccurs

throughamutualinteractionofthediversenegativecharge

func-tionalgroupspresentinitsstructure

4000 3500 3000 1500 1000 500

ChiS

AuNRs

Wavenumber (cm-1)

18 2

18 28

Fig 6.FTIR spectra of ChiS-AuNR, as-prepared AuNR and ChiS.

It is widely known that alkanethiols exhibit a preferential bindingonthesurfaceofgoldnanoparticles,promotedby ther-modynamicallyfavoredcovalentbonds(Karpovich,&Blanchard, 1994;Leff,Brandt,&Heath,1996;Templeton,Pietron,Murray,& Mulvaney,2000;Zakariaetal.,2013).Yet,thesulfurpresentin sul-fatefunctionalgroupsdoesnotpresentthesamecharacteristicsas alkanethiolssincethestabilizationandtheabsenceoffree elec-tronpairspromotedbytheelectrondelocalizationbetweenthe oxygenatomshinderstheoccurrenceofnewbindings.However, sulfategroupsmaystabilizenanoparticlesbyelectrostatic interac-tionlikehydroxyl,carbonylandaminogroupsthatareintrinsically inhibitorytoparticleaggregation

It was confirmed through FTIR analyses that CTAB was not entirelyremovedfromtheAuNRssurfacesincesulfateand car-boxylgroupsfromChiSandGA,respectively,exhibitpreferential interactionswiththepositivelychargedheadgroupsofCTAB.The interactionsofthepolysaccharidesandtheAuNRssurface, there-fore,occurviatheC N+ofCTABandcarboxylateorsulfategroups,

ofGAandChiS,respectively,asdepictedinFig.7

4 Conclusions

Withasimple,straightforwardmethodologywedemonstrated thatGAandChiSefficientlyinteractwiththesurfaceofCTABcoated AuNRs.Theresultingself-assembledstructureswerefully char-acterized Microscopyimagesshowedthat GAproduced AuNRs irregular clusters and ChiS acted as an efficient encapsulat-ing/wrappingagentresultinginindividualAuNRs,whichwerewell separatedbytheChiSmolecules.FTIRanalysesclearlyshowedthat

GAandChiSinteractwiththeAuNRsviachargedgroupsofCTAB

byelectrostaticinteractions,leavingthe CH2groupsintact Com-biningourresultswithdataalreadyavailableintheliterature,the toxicityofChiS-AuNRsandGA-AuNRsisexpectedtodecreasein relationtoCTAB-AuNRs.Therefore,byusinganewpolysaccharide

wepresentaninterestingstrategytoproduceindividuallywrapped

Fig 7. Schematic representation of the interaction of CTAB/AuNRs with (a) ChiS (CTA + ROSO − /CTA + /CTAB/AuNR) and (b) GA (CTA + RCOO − /CTA + /CTAB/AuNR).

Table 1

FTIR band assignments of CTAB and as-prepared AuNRs.

Symmetric and assymetric stretching of CH 2 of CTAB chain 2918 and 2850 2918 and 2850

Asymmetric and symmetric C H scissoring of H 3 C N + moiety 1487, 1473, 1462 and 1431 1487, 1473, 1462 and 1431

Rocking mode of the CH 2 chain ((CH 2 ) n , n > 4) 719 and 731 669

a Based on Nikoobakht & El-Sayed, 2001 ; Sui et al., 2006 ; Tang et al., 2013;Campbell et al., 2004 ; Innocenzi, Falcaro, Grosso & Babonneau 2006

FTIR band assignments of as-prepared AuNRs, GA, GA-AuNR, ChiS and ChiS-AuNRs.

Symmetric and asymmetric stretching of

C CH 2 of CTAB chain

2918 and 2850 – 2918 and 2850 – 2918 and 2850 Assymetric and symetric stretching of the

carboxilic acid salt COO

Asymmetric and symmetric C H scissoring

vibrations of CH 3 N + moiety

1487, 1473, 1462 and 1431 – 1487 – –

C O stretching – 1253, 1143, 1064 and 1031 – 1062 and 1004 1062 and 1004 Could be attributed to the interactions that

take place by GA-AuNRs interactions

Symmetric stretching of C O C bands – – – 1153 1153

a Based on Davidovich-Pinhas et al., 2014; Espinosa-Andrews et al., 2010; Tang et al., 2013

AuNRs,applicablewhenwelldispersednanoparticlesarerequired

Inaddition,theobservationofclustersorindividualAuNRsadds

informationtothepropermanipulationandusageofthese

polysac-charidefunctionalizednanoparticles

Acknowledgements

The authors acknowledge the support given by the

Brazil-ianNationalCounselofTechnologicalandScientificDevelopment

(CNPq)mainlythroughthegrants577232/2008-8,

477467/2010-5and564741/2010-8.H.R.Barros,D.A.SabryandA.M.Nunes

expresstheirgratitudetoCAPESfortheirfellowships.Theauthors

areverygratefultotheElectronMicroscopyCenterofUFPR

(CME-UFPR)fortheTEMimagesandtoSENAIPR-InstituteofInnovation

inElectrochemistryforthezetapotentialmeasurements

Appendix A Supplementary data

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.carbpol.2016.07

018

References

Asif, S., Ekdahl, K N., Fromell, K., Gustafson, E., Barbu, A., Le Blanc, K., et al (2016).

Heparinization of cell surfaces with short peptide-conjugated PEG-lipid

regulates thromboinflammation in transplantation of human MSCs and

hepatocytes Acta Biomaterialia, 35, 194–205.

Barros, H R., Cardoso, M B., de Oliveira, C C., Franco, C R C., Belan, D L., Vidotti,

M., et al (2016) Stability of gum arabic-gold nanoparticles in physiological

simulated pHs and their selective effect on cell lines RSC Advances, 6,

9411–9420.

Bicho, A., Roque, A C A., Cardoso, A S., Domingos, P., & Batalha, I L (2009) In vitro

studies with mammalian cell lines and gum arabic-coated magnetic

nanoparticles Journal of Molecular Recognition, 23, 536–542.

Boca, S C., & Astilean, S (2010) Detoxification of gold nanorods by conjugation

with thiolated poly(ethylene glycol) and their assessment as SERS-active

carriers of Raman tags Nanotechnology, 21, 235601.

Boca, S C., Potara, M., Toderas, F., Stephan, O., Baldeck, P L., & Astilean, S (2011).

Uptake and biological effects of chitosan-capped gold nanoparticles on Chinese

Hamster Ovary cells Materials Science and Engineering C, 31, 184–189.

Bogliotti, N., Oberleitner, B., Di-Cicco, A., Schmidt, F., Florent, J C., & Semetey, V.

research: functionalization, stabilization and purification Journal of Colloid and Interface Science, 357, 75–81.

Campbell, R A., Parker, S R W., Day, J P R., & Bain, C D (2004) External reflection FTIR spectroscopy of the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) on an overflowing cylinder Langmuir, 20, 8740–8753.

Chanda, N., Kan, P., Watkinson, L D., Shukla, R., Zambre, A., Carmack, T L., et al (2010) Radioactive gold nanoparticles in cancer therapy: therapeutic efficacy studies of GA- 198 AuNP nanoconstruct in prostate tumor-bearing mice Nanomedicine-Nanotechnology Biology and Medicine, 6, 201–209.

Charan, S., Sanjiv, K., Singh, N., Chien, F C., Chen, Y F., Nergui, N N., et al (2012).

Development of chitosan oligosaccharide-modified gold nanorods for in vivo targeted delivery and noninvasive imaging by NIR irradiation Bioconjugate Chemistry, 23, 2173–2182.

da Silva, M G A., Meneghetti, M R., Denicourt-Nowicki, A., & Roucoux, A (2014).

Tunable hydroxylated surfactants: an efficient toolbox towards anisotropic gold nanoparticles RSC Advances, 4, 25875–25879.

da Silva, M G A., Nunes, A M., Meneghetti, S M P., & Meneghetti, M R (2013).

New aspects of gold nanorod formation via seed-mediated method Comptes Rendus Chimie, 16, 640–650.

Davidovich-Pinhas, M., Danin-Poleg, Y., Kashi, Y., & Bianco-Peled, H (2014).

Modified chitosan: a step toward improving the properties antibacterial food packages Food Packing and Shelf Life, 1, 160–169.

Erathodiyil, N., & Ying, J (2011) Functionalization of inorganic nanoparticles for bioimaging applications Accounts of Chemical Research, 44, 925–935.

Espinosa-Andrews, H., Sandoval-Castilla, O., Vázquez-Torres, H., Vernon-Carter, E J., & Lobato-Calleros, C (2010) Determination of the gum Arabic–chitosan interactions by Fourier Transform Infrared Spectroscopy and characterization

of the microstructure and rheological features of their coacervates.

Carbohydrate Polymers, 79, 541–546.

Eutis, S., & El-Sayed, M (2006) Why nanoparticles are more precious than pretty gold: noble metal surface plasmon ressonance and its enhancement of the radiative and nonradiative properties of nanocrystals of different shapes Chemical Society Review, 35, 209–217.

Garabagiu, S., & Bratu, I (2013) Thiol containing carboxylic acids remove the CTAB surfactant onto the surface of gold nanorods: an FTIR spectroscopy study Applied Surface Science, 284, 780–783.

Gole, A., Orendorff, C J., & Murphy, C J (2004) Immobilization of gold nanorods onto acid-terminated self-assembled monolayers via electrostatic interactions Langmuir, 20, 7117–7122.

Grein, A., da Silva, B C., Wendel, C F., Tischer, C A., Sierakowski, M R., Moura, A B D., et al (2013) Structural characterization and emulsifying properties of polysaccharides of Acacia mearnsii de Wild gum Carbohydrate Polymers, 92, 312–320.

Hamon, C., Bizien, T., Artzner, F., Even-Hernandez, P., & Marchi, V (2014).

Replacement of CTAB with peptidic ligands at the surface of gold nanorods and their self-assembling properties Journal of Colloid and Interface Science, 424, 90–97.

Huang, J., Jackson, K S., & Murphy, C J (2012) Polyelectrolyte wrapping layers control rates of photothermal molecular release from gold nanorods Nano Letters, 12, 2982–2987.

Innocenzi, P., Falcaro, P., Grosso, D., & Babonneau, F (2006) Order-disorder

transitions and evolution of silica structure in self-assembled mesostructured

silica films studied through FTIR spectroscopy The Journal of Physical Chemistry

B, 107, 4711–4717.

Jana, N R., Gearheart, L., & Murphy, C J (2001a) Evidence for seed-mediated

nucleation in the chemical reduction of gold salts to gold nanoparticles.

Chemistry of Materials, 13, 2313–2322.

Jana, N R., Gearheart, L., & Murphy, C J (2001b) Seed-mediated growth approach

for shape-controlled synthesis of spheroidal and rod-like gold nanoparticles

using a surfactant template Advanced Materials, 13, 1389–1392.

Jana, N R., Gearheart, L., & Murphy, C J (2001c) Wet chemical synthesis of high

aspect ratio cylindrical gold nanorods The Journal of Physical Chemistry B, 105,

4065–4067.

Jayakumar, R., Nwe, N., Tokura, S., & Tamura, H (2007) Sulfated chitin and

chitosan as novel biomaterials International Journal of Biological

Macromolecules, 40, 175–181.

Johnson, C J., Dujardin, E., Davis, S A., Murphy, C J., & Mann, S (2002) Growth and

form of gold nanorods prepared by seed-mediated: surfactant-directed

synthesis Journal of Materials Chemistry, 12, 1765–1770.

Karpovich, D S., & Blanchard, G J (1994) Direct measurement of the

adsorption-kinetics of alkanethiolate self-assembled monolayers on a

microcrystalline gold surface Langmuir, 10, 3315–3322.

Kattumuri, V., Katti, K., Bhaskaran, S., Boote, E J., Casteel, S W., Fent, G M., et al.

(2007) Gum arabic as a phytochemical construct for the stabilization of gold

nanoparticles: in vivo pharmacokinetics and X-ray-contrast-imaging studies.

Small, 3, 333–341.

Kopwitthaya, A., Yong, K T., Hu, R., Roy, I., Ding, H., Vathy, L A., et al (2010).

Biocompatible PEGylated gold nanorods as colored contrast agents for targeted

in vivo cancer applications Nanotechnology, 21, 315101.

Leff, D V., Brandt, L., & Heath, J R (1996) Synthesis and characterization of

hydrophobic: organically-soluble gold nanocrystals functionalized with

primary amines Langmuir, 12, 4723–4730.

Lehr, C M., Bouwstra, J A., Schacht, E T., & Junginger, H E (1992) In vitro

evaluation of mucoadhesive properties of chitosan and some other natural

polymers International Journal of Pharmaceutics, 78, 43–48.

Li, J J., Kawazoe, N., & Chen, G (2015) Gold nanoparticles with different charge

and moiety induce differential cell response on mesenchymal stem cell

osteogenesis Biomaterials, 54, 226–236.

Liu, X., Huang, H., Liu, G., Zhou, W., Chen, Y., Jin, Q., et al (2013) Multidentate

zwitterionic chitosan oligosaccharide modified gold nanoparticles: stability:

biocompatibility and cell interactions Nanoscale, 5, 3982–3991.

Maas, N C., Gracher, A H P., Sassaki, G L., Gorin, P A J., Iacomini, M., & Cipriani, T.

R (2012) Sulfation pattern of citrus pectin and its carboxy-reduced

derivatives: influence on anticoagulant and antithrombotic effects.

Carbohydrate Polymers, 89, 1081–1087.

Meena, K S., & Sulpizi, M (2013) Understanding the microscopic origin of gold

nanoparticle anisotropic growth from molecular dynamics simulations.

Langmuir, 29, 14954–14961.

Mitamura, K., Imae, T., Saito, N., & Takai, O (2007) Fabrication and self-sssembly of

hydrophobic gold nanorods The Journal of Physical Chemistry B, 111,

8891–8898.

Murphy, C J., & Jana, N R (2002) Controlling the aspect ratio of inorganic

nanorods and nanowires Advanced Materials, 14, 80–82.

Nethi, S K., Mukherjee, S., Veeriah, V., Barui, A K., Chatterjee, S., & Patra, C R.

(2014) Bioconjugated gold nanoparticles accelerate the growth of new blood

vessels through redox signaling Chemical Communication, 50, 14367–14370.

Nikoobakht, B., & El-Sayed, M A (2001) Evidence for bilayer assembly of cationic

surfactants on the surface of gold nanorods Langmuir, 17, 6368–6367

Nikoobakht, B., & El-Sayed, M A (2003) Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method Chemistry of Materials, 15, 1957–1962.

Orendorff, C J., & Murphy, C J (2006) Quantitation of metal content in the silver-assisted growth of gold nanorods The Journal of Physical Chemistry B,

110, 3990–3994.

Pérez-Juste, J., Pastoriza-Santos, I., Liz-Marzán, L M., & Mulvaney, P (2005) Gold nanorods: synthesis: characterization and applications Coordination Chemistry Reviews, 249, 1870–1901.

Pierrat, S., Zins, I., Breivogel, A., & Sonnichsen, C (2007) Self-assembly of small gold colloids with functionalized gold nanorods Nano Letters, 7, 259–263.

Rayavarapu, R G., Petersen, W., Hartsuiker, L., Chin, P., Janssen, H., van Leeuwen, F W., et al (2010) In vitro toxicity studies of polymer-coated gold nanorods Nanotechnology, 21, 145101.

Sau, T K., & Murphy, C J (2004) Seeded high yield synthesis of short au nanorods

in aqueous solution Langmuir, 20, 6414–6420.

Tang, J., Huang, J., & Man, S Q (2013) Preparation of gold nanoparticles by surfactant-promoted reductive reaction without extra reducing agent Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 103, 349–355.

Templeton, A C., Pietron, J J., Murray, R W., & Mulvaney, P (2000) Solvent refractive index and core charge influences on the surface plasmon absorbance

of alkanethiolate monolayer-protected gold clusters Journal of Physical Chemistry B, 104, 564–570.

Terbojevich, M., Carraro, C., & Cosani, A (1989) Solution studies of chitosan 6-O-sulfate Makromolecular Chemistry, 190, 2847–2855.

Sui, Z., Chen, M., Wang, X., Xu, L M., WC, Chai, Y C., et al (2006) Capping effect of CTAB on positively charged Ag nanoparticles Physica E, 33, 308–314.

Wang, C H., Chang, C W., & Peng, C A (2011) Gold nanorod stabilized by thiolated chitosan as photothermal absorber for cancer cell treatment Journal of Nanoparticle Research, 13, 2749–2758.

Wang, J., Xie, Y., Wang, L., Tang, J., Li, J., Kocaefe, D., et al (2015) In vivo pharmacokinetic features and biodistribution of star and rod shaped gold nanoparticles by multispectral optoacoustic tomography RSC Advances, 5, 7529–7538.

Williams, P A., & Phillips, G O (2000) Handbook of hydrocolloids Cambridge: Woodhead Publishing Limited.

Wu, C.-C., & Chen, D H (2010) Facile green synthesis of gold nanoparticles with gum arabic as a stabilizing agent and reducing agent Gold Bulletin, 43, 234–240.

Xie, Y., Guo, S., Ji, Y., Guo, C., Liu, X., Chen, Z., et al (2011) Self-assembly of gold nanorods into symmetric superlattices directed by OH-terminated hexa(ethylene glycol) alkanethiol Langmuir, 27, 11394–11400.

Yang, Z., Liu, T., Xie, T., Sun, Z., Liu, H., Lin, J., et al (2015) Chitosan layered gold nanorods as synergistic therapeutics for photothermal ablation and gene silencing in triple-negative breast cancer Acta Biomaterialia, 25, 194–204.

Yu, C., Zhu, Z., Wang, Q., Gu, W., Bao, N., & Gu, H (2014) A disposable indium-tin-oxide sensor modified by gold nanorod–chitosan nanocomposites for the detection of H 2 O 2 in cancer cells Chemical Communications, 50, 7329–7331.

Zakaria, H M., Shah, A., Konieczny, M., Hoffmann, J A., Nijdam, A J., & Reeves, M E (2013) Small molecule- and amino acid-induced aggregation of gold nanoparticles Langmuir, 29, 7661–7673.

Zharov, V P., Mercer, K E., Galitovskaya, E N., & Smeltzer, M S (2006).

Photothermal nanotherapeutics and nanodiagnostics for selective killing of bacteria targeted with gold nanoparticles Biophysical Journal, 90, 619–627.