- 131 -
I. Le cas du dioxide de titane: Modifications of the bacterial
reverse mutation test reveals mutagenicity of TiO
2nanoparticles and byproducts from a sunscreen TiO
2-based
nanocomposite.
Résumé :
Recommandé par l’Organisation de coopération et de développement économiques (OCDE) le test de détection de mutation reverse bactérien a été récemment utilisé par un certain nombre d'équipes scientifiques dans le but d’évaluer le potentiel mutagène des nanoparticules. Lors de ces essais, le test d’Ames a produit des résultats négatifs alors que les autres tests de génotoxicité effectués sur les mêmes nanoparticules se sont avérés positifs. En effet, lorsque des tests in vitro sont réalisés avec des nanoparticules sur des cellules de mammifères, les réponses diffèrent de celle observées avec le test d’Ames.
Les travaux réalisés précédemment ont montré qu’une simple préexposition des bactéries à des nanoparticules de titane dans une solution présentant une faible force ionique, à savoir le NaCl 10 mM, et à un pH (pH= 5,5) inférieur au point isoélectrique des nanoparticules de titane (6,8), permettait de favoriser fortement les interactions électrostatiques entre ces deux éléments. Ainsi, nous avons supposé qu’en favorisant l’établissement des interactions entre les bactéries et les nanoparticules nous pourrions augmenter l’efficacité du test. Cette étude vise à déterminer le potentiel mutagène des nanoparticules de dioxyde de titane (TiO2-P25, TiO2-NA et TiO2-TLB) en utilisant le test d’Ames en version liquide, appelé également test en fluctuation. Ce test utilise les souches de Salmonella typhimurium TA97a, TA98, TA100 et TA102. Ces expérimentations ont montré que le test, lorsqu’utilisé de façon conventionnelle, n’est pas adapté à l’étude des nanoparticules. En effet, le milieu d’exposition utilisé dans le test présente une force ionique importante qui minimise les interactions électrostatiques entre les microorganismes et les TiO2-NPs conduisant le test à produire de faux-négatifs.
En modifiant la procédure de test en fluctuation, nous avons mis en évidence le potentiel mutagène des nanoparticules de dioxyde de titane. La souche de S. typhimurium TA 102 s’est également révélée être la plus sensible pour détecter ce potentiel mutagène, laissant à penser que l’effet mutagène implique un stress oxydatif malgré la conduite des expérimentations à l'obscurité afin de ce prémunir des effets photocatalytiques des nanoparticules.
ContentslistsavailableatSciVerseScienceDirect
Toxicology Letters
j o ur na l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / t o x l e t
Modificationsofthebacterialreverse mutationtest revealsmutagenicityofTiO
2nanoparticlesandbyproducts fromasunscreen TiO
2-basednanocomposite
StéphaneJominia,JérômeLabilleb,c,PascaleBaudaa,c,ChristophePagnouta,c,∗aLaboratoiredesInteractionsEcotoxicologie,Biodiversité,Ecosystèmes(LIEBE),UMR7146,CNRS-UPV-M,UniversitédeLorraine,rueduGénéralDelestraint,F-57070Metz,France
bCEREGEUMR6635CNRS/Aix-MarseilleUniversité,Europôledel’Arbois,13545Aix-en-Provence,France
cInternationalConsortiumfortheEnvironmentalImplicationsofNanotechnology(iCEINT),Europoledel’Arbois,F-13545AixenProvence,France
h i g h l i g h t s
◮TheAmestestisnotsuitablefornanoparticle(NP)genotoxicityassessment. ◮TheAmestestmediumpreventselectrostaticinteractionsbetweenbacteriaandNPs. ◮TheAmestestmediumstronglypromotestheaggregationofNPs.
◮Simplepre-exposurestepinanadequatemediumimprovetheaccuracyofthetest. ◮ModifiedAmestestshowedmutagenicityofNP-TiO2andNP-TiO2-basednanocomposite.
a r t i c l e i n f o
Articlehistory:
Received30May2012
Receivedinrevisedform8August2012
Accepted19September2012
Available online 28 September 2012 Keywords:
TiO2nanoparticles
Genotoxicity
Bacterialreversemutationtest
Fluctuationtest
Electrostaticinteractions
a b s t r a c t
Thebacterialreversemutationtest,recommendedbytheOrganizationforEconomicCo-operationand Development(OECD)todeterminegenotoxicityofchemicalcompounds,hasbeenrecentlyusedby sev-eralauthorstoinvestigatenanoparticles.Surprisingly,testresultshavebeennegative,whereasinvitro mammaliancelltestsoftengivepositivegenotoxicresponses.Inthepresentstudy,weusedthe fluctua-tiontestprocedurewiththeSalmonellatyphimuriumstrainsTA97a,TA98,TA100andTA102todetermine themutagenicpotentialofTiO2nanoparticles(NP-TiO2)andshowedthat,whenitisused convention-ally,thistestisnotsuitablefornanoparticlegenotoxicityassessment.Indeed,themediumusedduring exposurepreventselectrostaticinteractionsbetweenbacterialcellsandnanoparticles,leadingto false-negativeresponses.Weshowedthatasimplepre-exposureofbacteriatoNP-TiO2inalowionicstrength solution(NaCl10mM)atapHbelowthenanoparticleisoelectricpoints(pH5.5)canstronglyimprove theaccuracyofthetest.Thus,basedontheseimprovements,wehavedemonstratedthegenotoxicityof theengineeredNP-TiO2testedandaNP-TiO2byproductfromasunscreennanocomposite.Itwasalso shownthatstrainTA102ismoresensitivethantheotherstrains,suggestinganoxidativestress-mediated mechanismofgenotoxicity.
© 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Duetotheincreasingindustrializationofmanycountriesand resulting technological advances, environmental pollution has becomeaserioushealthissue.Manyeffortshavebeenmadeto pro-tecttheenvironmentandhumanhealth.Apriorityareaconcerns thedevelopmentofin vitroassaystoevaluatethetoxicological effectsofenvironmentalchemicalsandthenbuildprioritization modelsofinvivotoxicity.Overtheyears,multiplebioassayshave
∗ Correspondingauthorat:LaboratoiredesInteractionsEcotoxicologie,
Biodiver-sité,Ecosystèmes(LIEBE),CNRS-UMR7146,UniversitédeLorraine,rueduGénéral
Delestraint,F-57070Metz,France.Tel.:+33387378657;fax:+33387378512.
E-mailaddress:christophe.pagnout@univ-lorraine.fr(C.Pagnout).
beendeveloped utilizing many organisms. Microbialtests have severaladvantagesoverotherbioassays,includingrapidresponse timesduetothemuchshortermicrobiallifecycles,reproducibility oftestconditions,amenabilitytogeneticmanipulations,increased sensitivityandreducedcost(Davoren,2005).Inaddition, microor-ganismspossessthemajorityofthesamebiochemicalpathways presentin higherorganisms,theyexhibitsignificantmembrane structureorganizationandgenerallyelicittoxicresponsestomany chemicalsthroughmechanismssimilartothatofhigherorganisms (Qureshietal.,1984).
The bacterial reverse mutation assay is recommended by national and international environmental protection agencies for substance evaluations (e.g. Organization for Economic Co-operationandDevelopment(OECDtestguideline 471);andThe InternationalConference on Harmonization (ICH)) (Mortelmans
0378-4274/$–seefrontmatter © 2012 Elsevier Ireland Ltd. All rights reserved.
ofthetwoassaysrecommendedbyTheCommitteeon Mutageni-cityofChemicalsinFood,ConsumerProductsandtheEnvironment (COM)(Kirklandetal.,2011).Thebacterialreversemutationtest usesseveralstrainsofSalmonellatyphimuriumwithmutated his-tidinesynthesisgenes.Thetestprincipleisbasedonthefactthat reversemutationscausedbyexposuretomutageniccompounds canreactivatetheabilityofmutatedbacterialstrainsto synthe-sishistidine,therebyallowingthemtogrowintheabsenceofthis essentialaminoacid.Severalproceduresforperformingthe bac-terial reversemutation test have beendescribed. Among those commonlyusedare theplateincorporationmethod,commonly namedtheAmestest(Amesetal.,1972,1973a,b),the preincuba-tionmethod(MaronandAmes,1983;Aeschbacheretal.,1987), thefluctuationmethod(Greenetal.,1976;Hubbardetal.,1984; McPherson and Nestmann, 1990), and the suspension method (ThompsonandMelampy,1981).Overtheyears,manyvalidation studieshavebeenperformedtodeterminethesensitivityand cor-relationofthistestwithanimalcarcinogenicitystudies.Ithasbeen establishedthatthereisahighpredictivityofapositivemutagenic responseinthetestforrodentcarcinogenicityrangingfrom77% to90%(McCannetal.,1975;Tennantetal.,1987;Zeiger,1998). To date, there are thousands of research and testing laborato-riesthroughouttheworldusingthis assaytoscreenpotentially mutagenicdrugsandchemicals.Manycompaniesandregulatory agenciesusetheresultsfromthisassayaspartoftheirshort-term toxicologicaltestingprogramstodeterminechemicalsafety(Felton andWu,2003).
Nanotechnology is a relatively new branch of science, her-alded as a technological revolution (The White House, 2000). Engineerednanoparticles have rapidlymoved from the labora-torytoindustryandarecurrentlybeingusedinmanyconsumer products. Currently, there are over 1000 products in the con-sumermarketplacethatincludenanomaterials(WoodrowWilson Database:http://www.nanotechproject.org),which isprojectedto substantially increasein thenearfuture. Thisphenomenon has arousedgreatconcernaboutpotentialhumanhealtheffectsand, onalargerscale,environmentaleffects(Neletal.,2006),giving birth toa newbiological field knownas“Nanotoxicology”. The RoyalSocietyandRoyalAcademyofEngineeringfirstraisedthis concernin 2003 (The RoyalSociety and theRoyalAcademy of Engineering,2003;TheRoyalSociety,2004), pavingthewayfor a rapid increase in investigational studieson nanoparticle tox-icity; in particular, genotoxicity studies, asmany nanoparticles werefoundtocausechromosomalaberrations,DNAstrandbreaks, oxidativeDNAdamage,andsubsequentgeneticmutations(Singh etal.,2009).Incommonlyusedinvitro(chromosomalaberrations, cometassay,micronucleus)andinvivomammaliantestcell sys-tems,nanoparticleshavebeenlargelyfoundtopromotepositive genotoxicresponses,whilenegativeresponseshavebeen gener-allyobtainedfor thesenanoparticleswith thebacterialreverse mutationtest(Doaketal.,2012).Itwasreportedthatwithin19 publishedstudies,wherethistestwasusedforthe genotoxicolog-ical analysis of nanoparticles, 17 showed negative mutagenicity. Thetworemainingstudiesonlyrevealedweakmutageniceffects. Therefore,thesestudiesseemedtohaveindicatedthatalthough theAmestestisexcellentfor testingchemicalmutagenic activ-ity,itdoesnotappeartobesuitablefornanoparticles.Thismight berelatedtothedegreeofnanoparticleuptakebybacterialcells, whichislikelytobelessthaninmammaliancells(Singhetal.,2009; Doaketal.,2012).Indeed,bacteriacannotperformendocytosisand theircellwallformsabarrieragainstsimplediffusionof nanopar-ticles.Thislackofuptakecouldpotentiallyleadtofalsenegative results.
Basedonourpreviouswork(Pagnoutetal.,2012),wethinkthat anotherplausiblehypothesis thatleadstofalse negativeresults
cellsduetotheuseofaninappropriatemediumduringthe expo-sure.Wealsothinkthatperforming thebacterialreversemutation testbythefluctuationmethodinsteadoftheplateincorporation method,withapre-exposurestepin alow-ionicstrength solu-tionatapHvaluebelowthenanoparticleisoelectricpoints(NaCl 10mM,pH5.5),couldimprovetheseinteractionsandmake the testmoreaccuratefortheassessmentofthenanoparticle geno-toxicity.Asaconsequence,inthepresentstudy,weassessedthe mutagenic potential of two engineered TiO2 nanoparticles and a TiO2-byproductderived froma nanocompositematerial com-monlyusedinsunscreenswiththeconventionalfluctuationtest andwithamodifiedversionofthistestaccordingtothe modifica-tionmentionedabove.TiO2nanoparticles(NP-TiO2)wereusedas amodelinthisstudyforthefollowingreasons:(i)these nanopar-ticlesarewidelyusedinconsumerproducts(e.g.paints,plastics, paper,ceramics,cosmetics,andsunscreens)withexpanded appli-cationsoverthelastdecade(Colvin,2003;Gleicheetal.,2006); (ii)in2006,TiO2wasreclassifiedfromUnclassifiableasto carcino-genicityinhumans(Group3carcinogen)toPossiblycarcinogenicto humans(Group2Bcarcinogen)basedonsufficientevidenceusing experimentalanimals(Ngetal.,2010);(iii)NP-TiO2wasrecently listedbytheOECDasoneoftheprioritynanomaterialsfor immedi-atetesting(OECD,2008);(iv)NP-TiO2nanoparticlesareminimally water-solubleandtheirpotentialcarcinogeniceffects cannotbe attributedtothereleaseoftitaniumionsinthemedium;and(v) severalstudiesshowednomutagenicity(Warheitetal.,2007;Pan etal.,2010)orveryweakmutagenicity(Kumaretal.,2011)caused byNP-TiO2withthebacterialreversemutationtest(plate incor-poration procedure), whereas NP-TiO2 hasbeen found tohave positivegenotoxicresponsesinotherinvitrocellulartestsystems (Balasubramanyametal.,2009;DiVirgilioetal.,2010;Osmanetal., 2010;Shietal.,2010).
2. Materialsandmethods
2.1. Evaluatednanomaterials
TiO2nanopowderAEROXIDE®P25(TiO2-P25)wasprovidedbyEvonikDegussa
GmbH (Frankfurt, Germany, Stock # 4168050298). These nanoparticles are
describedbythesupplierashavingaprimarysizeof25nmwithaspecificsurface
area(SSA)of50±15m2/gandaratioofanatase/rutileformsof80/20.TheTiO2-P25
stocksuspensionwaspreparedbydispersing100mgofNP-TiO2in10mLof
ster-ileultrapurewater(milli-Qwater,18.2Mcm).Theresultantsuspensionwasthen
probe-sonicated(SonicsVibra-cell750W,Sonics&Materials,Inc.,Newton,CT,USA;
frequency20kHz,3mmmicrotip,amplitude40%)for30minat4◦Ctohomogenize
andbreakthelargeragglomeratesapart(Pagnoutetal.,2012).
ThesecondtypeofNP-TiO2 usedinthisstudy(TiO2-NA)wasprovidedas
a15%(w/v)stablesuspensioninacidifiedwaterproducedbyNanostructured
&AmorphousMaterials, Inc.(Houston,TX,USA–Stock#7012WJWR).These
nanoparticlesaredescribedbythesupplierasbeing100%anatase,witha
pri-marysizerangingfrom5to30nm,aSSAof200–220m2/g,andapurity>99.5%.
Thestock suspension wasprepared to10g/Lby dilution insterileultrapure
water.
Thethirdnanomaterialusedinthisstudy wasabyproductobtainedafter
alterationofaTiO2-basednanocomposite,namelyT-LiteTMSF(BASF,Germany)
(TiO2-TLB), which is commonly usedin sunscreensas a UVblocker. The
T-LitenanocompositeconsistedofaTiO2rutilecore(5–10nmcross-sectionper
50–200nmlength)arrangedtogetherinlargeclusters,whichhadanaveragesize
of200nm.Theseclusterswereembeddedinanamorphouslayerofaluminum
oxide[Al(OH)3]andpolydimethylsiloxane(PDMS)(Labilleetal.,2010;Auffanetal.,
2010).Thebyproduct,resultingfromanacceleratedageingprocess,wasprovidedby
JérômeLabille(CEREGELaboratory,Aix-en-Provence,France).Briefly,thealteration
processconsistedofmixing100mgofTiO2-TLBin250mLofultrapurewater.The
mixturewasmagneticallystirredat690rpmunderawhitelight(400WPhilips®
114MasterHPI-TPlus)for48h.Afteralteration,thismixturewassettledfor48h
andthesupernatantcontainingstablealteredTiO2nanocomposites(byproducts)
wasobtained(Labilleetal.,2010;Auffanetal.,2010).Aspreviouslydescribedby
Bigorgneetal.(2011),thequantityofTiO2byproductswasmeasuredbyfiltering
analiquotofsuspension(20mL)througha25nmmembranefilteranddryingfilter
at105◦Cfor24h.TheconcentrationofTiO2byproductsobtainedwasadjustedto
Theshapesandprimarysizesofnanoparticlesweredeterminedbytransmission
electronmicroscopy(TEM),usingaCM20 Philipselectronmicroscopeat200kVat
theServiceCommundeMicroscopiesElectroniquesetdeMicroanalyses(SCMEM,
Nancy,France).Sampleswerepreparedbyevaporatingadropletof
nanoparti-clesuspensiononacoppergridatroomtemperature.Sizewasdeterminedusing
100randomlychosennanoparticles.Nanoparticlecrystallinestructureswere
deter-minedbyX-raypowderdiffraction(XRD).Electrophoreticmobilityandnanoparticle
sizedistributionmeasurementsinaqueousmediawereperformedwithaZetaSizer
3000HS(MalvernInstruments,Worcestershire,UK).
2.3. Bacterialelectrophoreticmobility
Bacterial electrophoretic mobilitymeasurements were conducted using a
ZetaphoremeterIV(CADInstrumentations,LesEssartsleRoi,France)inaquartz
Suprasil®cell(HeraeusQuarzglasGmbH&Co.,Hanau,Germany)at24◦Cfromthe
reflectionofalaserbeambybacteriatrackedwithacharge-coupleddevice
cam-era.Imageanalysissoftwarewasusedtoprocessrecordedimagesinrealtimeto
calculatetheelectrophoreticmobilitiesfromthedisplacement(migrationmotion)
ofbacteriasubjectedtoaconstantdirect-currentelectricfield(800V/m).Different
cycleswererecordedtoyield100bacterialmobilitymeasurements.
2.4. Theconventionalfluctuationtest
ThebacterialreversemutationtestwasperformedusingS.typhimuriumstrains
TA97a,TA98todetectGCframeshiftmutations,strainTA100forGCbase-pair
substi-tutionmutationsandstrainTA102forATbase-pairsubstitutionmutations,without
S9metabolicactivation.ThesestrainswereagiftfromBruceAmes(Universityof
California,Berkeley,CA,USA).Thegeneticintegritiesofthesestrainswere
veri-fiedbeforethetestsforhistidine/biotin,rfamarkerandpKM101plasmid(Maron
andAmes,1983).Thetestwasconductedinliquidmediumin96-wellmicrotiter
plates,accordingtothefluctuationmethod(EnvironmentCanada,1993).Briefly,
bacterialstrainsweregrownovernightinOxoidbrothsupplementedwiththe
appropriateantibiotics(25�g/mLAmpicillinforstrainsTA97a,TA98,andTA100and
25�g/mLAmpicillin+2�g/mLTetracyclineforstrainTA102)at37◦Cwhileshaking.
Toavolumeof2.5mLoftheAmesreagentmixture(108mLof5.5×concentrated
Davis–Mingiolisalts)[38.5g/LK2HPO4,11.0g/LKH2PO4,5.5g/L(NH4)2SO4,1.375g/L
sodiumcitrate,0.55g/LMgSO4·7H2O–autoclaved15minat121◦C],24mLof40%
(w/v)d-glucose(6mLofd-biotin[0.1mg/mL],0.3mLofl-histidine[1mg/mL]and
12mLofbromocresolpurple[2mg/mL]),5�Lofanovernightcultureand17.5mL
ofsterileultrapurewatercontainingnanoparticles(1,10,100mg/Lorwaterasa
control)wereadded,givingfinalexposureNP-TiO2concentrationsof0,0.875,8.75,
87.5mg/L.A200�Lvolumeofthe mixturewasdispensedintowellsof96-well
microtiterplatesandincubatedfor5daysat37◦Cinthedark.Classical
posi-tivecontrols,9-aminoacridine(10�g/mL),2-nitrofluoren(0.4�g/mL),sodiumazide
(25ng/mL)andcumenehydroperoxide(3.5�g/mL),wereusedforTA97a,TA98,
TA100andTA102,respectively.Distillatedwaterwasusedasnegativecontrol.
Sterilitycontrols(i.e.sterilityofthemedia,sterilityoftheNP-TiO2andsterility
ofthepositivecontrols)werealsoincludedforeachtest.Thetestwasconsidered
validonlyifthepositivecontrolsinducedanumberofpositivewellssignificantly
higherthanthenumberobtainedwiththenegativecontrol(spontaneous
rever-tants),andonlyifthewellsofallthesterilitycontrolsstayednegativeduringthe
experimentation(0positivewellonthe96tested).
2.5. Themodifiedfluctuationtest
BacterialstrainsweregrownovernightinOxoidbrothsupplementedwiththe
appropriateantibiotic(25�g/mLAmpicillinforTA97a,TA98,TA100and25�g/mL
Ampicillin+2�g/mLTetracyclineforTA102)at37◦Cwhileshaking.Avolumeof
2mLoftheseovernightculturesweretransferredto500mLErlenmeyerflasks
containing200mL of Luria–Bertani medium with the appropriate antibiotics
andincubatedat37◦Cwhileshakinguntiltheyreachedanopticaldensity(OD)
of0.5–0.6at600nm(exponentialgrowthphase).Thebacterialcellswerethen
centrifugedat8000×gfor10minat4◦C,washedtwicewithNaCl10mM(ultrapure
water–pH5.5),andresuspendedinthesamesalinesolutionatafinalODof4.0at
600nm.A200�Lvolumeofthesebacterialsuspensionswastransferredto100mL
flaskscontaining20mLofNaCl10mM(ultrapurewater–pH5.5)andnanoparticles
(1mg/L,10mg/Lor100mg/Lorwaterasacontrol).Afterashortpre-exposure
(0.1h),10h or20hat20◦Cunder agitation(150rpm), 500�Laliquotswere
transferredto2.5mLoftheAmesreagentmixture(previouslydescribed)and17mL
ofsteriledistilledwater,givingfinalconcentrationsofNP-TiO2inthefluctuation
testof0,0.025,0.25and2.5mg/L.A200�Lvolumeofthemixturethusobtained
wasdispensedinto96-wellmicrotiterplatesandincubatedfor5daysat37◦Cin
thedark.Experimentswereperformedindependentlytwiceandonemicroplate
wasusedforeachconcentration.Whentheseexperimentsgavecontradictory
results,theexperimentwasrepeatedathirdtime.Classicalpositive,negative,and
sterilitycontrolswerealsoincludedaspreviouslydescribedinSection2.4.
Transmissionelectronmicroscopy(TEM)observationswererealizedtostudy
NPsandbacterialinteractions.Afterexposure,bacterialsuspensionwascentrifuged
at10,000×gfor10minat4◦C.Then,thecellswerefixedwitha2.5%glutaraldehyde
solution,washedtwicewithultrapurewater,post-fixedwithosmiumtetraoxide
(OsO4),washedthreetimeswithultrapurewater,andthen,dehydratedingraded
concentrationsofethanol(Strumetal.,1971).Thepelletobtainedwasembedded
inEponandpolymerizedfor2hat38◦Cand2daysat60◦C.Ultra-thinsections
(90nm)werecollectedovercoppergridsandcounterstainedwithleadcitrateand
uranylacetate.ThesesectionswerethenobservedwithaCM20Philipselectron
microscopeat200kVattheServiceCommundeMicroscopiesElectroniquesetde
Microanalyses(SCMEM,Nancy,France).
3. Resultsanddiscussion 3.1. NP-TiO2characterization
TheNP-TiO2usedinthisstudyhasbeenthoroughly character-ized.AnalysesconfirmedthatTiO2-P25isamixtureofanataseand rutileforms(∼84%anataseand16%rutile)withanaverage pri-maryparticlesizeof23±4.9nm(SupportingInformationFig.S1). Dynamiclight scattering(DLS)measurementsrevealedthatthe averagehydrodynamicdiameterofthenanoparticlestock suspen-sionobtainedafterdispersioninmilli-Qwaterandprobesonication rangedbetween60and80nm(SupportingInformationFig.S1), meaningthatthenanoparticleswereagglomerated.Theisoelectric pointofTiO2-P25waspreviouslydeterminedtobeapproximately pH6.8(Pagnoutetal.,2012).
TiO2-NA,whichwasreportedtobe100%anatase,wasfound tobeamixtureofanataseandbrookiteforms(∼86%anataseand 14% brookite)withanaverageprimaryparticlesizeof5.7±1.9nm (Supporting Information Fig. S2). DLS measurements revealed thatthestocksuspensionprovidedbyNanoAmor,Inc.waswell dispersedwithanaveragehydrodynamicdiameterofthe nanopar-ticlesbetween5and10nm(SupportingInformationFig.S2).The isoelectricpointoftheTiO2-NAwasfoundtobearoundpH6.5(data