Assessment of gold nanoparticles on human peripheral blood cells by metabolic profiling with 1H

19 Pages·2017·5.77 MB·English

RESEARCHARTICLE
Assessment of gold nanoparticles on human
peripheral blood cells by metabolic profiling
1
with H-NMR spectroscopy, a novel
translational approach on a patient-specific
basis
MartinaPalomino-Scha¨tzlein1*,HermenegildoGarc´ıa2,PatriciaGutie´rrez-Carcedo3,
a1111111111 AntonioPineda-Lucena1,4,Jose´RaulHerance3*
a1111111111
a1111111111 1 LaboratoriodeBioqu´ımicaEstructural,CentrodeInvestigacio´nPr´ıncipeFelipe,Valencia,Spain,2 Instituto
UniversitariodeTecnolog´ıaQu´ımicaCSIC-UPV,Valencia,Spain,3 GrupdeRecercaenImatgeMèdica
a1111111111
Molecular,Valld’HebronResearchInstitute,CIBBIM-Nanomedicine,DepartamentdeMedicina,Universitat
a1111111111
AutònomadeBarcelona,Barcelona,Spain,4 UnidaddeDescubrimientodeFa´rmacos,Institutode
Investigacio´nSanitariaLaFe,HospitalUniversitarioiPolite´cnicoLaFe,Valencia,Spain
*[email protected](JRH);[email protected](MPS)
OPENACCESS
Abstract
Citation:Palomino-Scha¨tzleinM,Garc´ıaH,
Gutie´rrez-CarcedoP,Pineda-LucenaA,HeranceJR
(2017)Assessmentofgoldnanoparticleson Humanperipheralbloodcellsarerelevantexvivomodelsforcharacterizingdiseasesandeval-
humanperipheralbloodcellsbymetabolicprofiling uatingthepharmacologicaleffectsoftherapeuticinterventions,astheyprovideaclosereflec-
with1H-NMRspectroscopy,anoveltranslational
tionofanindividualpathophysiologicalstate.Inthiswork,anewapproachtoevaluatethe
approachonapatient-specificbasis.PLoSONE12
impactofnanoparticlesonthethreemainfractionsofhumanperipheralbloodcellsbynuclear
(8):e0182985.https://doi.org/10.1371/journal.
pone.0182985 magneticresonancespectroscopyisshown.Thus,acomprehensiveprotocolhasbeenset-up
includingtheseparationofbloodcells,theirinvitrotreatmentwithnanoparticlesandtheextrac-
Editor:AbhijitDe,AdvancedCentreforTreatment
ResearchandEducationinCancer,INDIA tionandcharacterizationofmetabolitesbynuclearmagneticresonance.Thismethodwas
appliedtoassesstheeffectofgoldnanoparticles,eithercoatedwithchitosanorsupportedon
Received:March15,2017
ceria,onperipheralbloodcellsfromhealthyindividuals.Aclearantioxidanteffectwasobserved
Accepted:July27,2017
forchitosan-coatedgoldnanoparticlesbyasignificantincreaseinreducedglutathione,that
Published:August9,2017
wasmuchlesspronouncedforgold-ceriumnanoparticles.Inaddition,theanalysisrevealed
Copyright:©2017Palomino-Scha¨tzleinetal.This significantalterationsofseveralotherpathways,whichwerestrongerforgold-ceriumnanopar-
isanopenaccessarticledistributedunderthe
ticles.Theseresultsareinaccordancewiththetoxicologicaldatapreviouslyreportedforthese
termsoftheCreativeCommonsAttribution
materials,confirmingthevalueofthecurrentmethodology.
License,whichpermitsunrestricteduse,
distribution,andreproductioninanymedium,
providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation Introduction
files.
Bloodcellsareinterestingexvivomodelstostudythepathophysiologicalstateofdiseases,and
Funding:Thepresentworkwassupportedby
topredictthebeneficialortoxiceffectpromotedbynewtherapies,withahightranslationality
grantsCP13/00252andPI16/02064fromCarlosIII
toclinicalstudiesbyconsideringthepatient’sspecificcharacteristics.Thereasonisthefact
HealthInstituteandbytheEuropeanRegional
thatbloodcellsarealteredindiseaseandcanreflecttheconditionandstateofdifferentorgans
DevelopmentFund(ERDF)andthegrantSAF2014-
53977-RfromtheMinistryofEconomy,Industry andtissues[1].Thus,thestudyoftheinteractionofbloodcellswithmedicinescanprovideus
PLOSONE|https://doi.org/10.1371/journal.pone.0182985 August9,2017 1/19
MetabolomicsofgoldnanoparticlesonhumanbloodcellsbyNMRastranslationalresearchapproach
andCompetitiveness.Inaddition,JRHisrecipient withanearlyindicationoftheeffectofacertaintherapyonthehumanbody.Forinstance,
ofaMiguelServetIcontractfromtheMinistryof erythrocytesorredbloodcells(RBCs)havebeenusedasdiseasemodelsforassessingdrugsas
HealthoftheCarlosIIIHealthInstitute.Thefunders
theyaresensitivetomanydisorders,includingdiabetes,Wilson’sdiseaseandAlzheimer’sdis-
hadnoroleinstudydesign,datacollectionand
ease[2–5].Moreover,neutrophilshavebeenanalysedtoobtaininformationaboutthediagno-
analysis,decisiontopublish,orpreparationofthe
sis,mechanismofactionandtherapyofdifferentdiseases,suchastuberculosis,malaria,
manuscript”insteadof“Thepresentworkwas
supportedbygrantsCP13/00252andPI16/02064 allergicreactionsortumours[6–12].Furthermore,lymphocyteshaveshowntobealteredin
fromCarlosIIIHealthInstitute,andbythe lungdiseases,inflammatoryprocessesleadingtoallergicdiseases,duringatheroscleroticpla-
EuropeanRegionalDevelopmentFund(ERDF).In quedevelopment,incardiovasculardiseasesandduringtumourprogression[13–20].
addition,JRHisrecipientofacontractfromthe
Metabolomicprofilingisacomprehensivemethodthatallowsthequantificationofalarge
MinistryofHealthoftheCarlosIIIHealthInstitute.
numberofdifferentmetabolitesinasingleanalysisinanon-targetedwaythatcanprovideuse-
Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,or fulinformationtostudydiseaseandtheeffectoftreatments.[21–24]Protonnuclearmagnetic
preparationofthemanuscript. resonance(1H-NMR)spectroscopyhasprovenfastandreproducibleforobtaininggoodqual-
itystructuralandsemi-quantitativeinformationaboutthemetabolomeofcells[21,25].Meta-
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. bolicprofilingofcellshasbeenpreviouslyappliedtoawiderangeofinvitromodelstohelp
gaininsightintobasicanddiseasemetabolisms,especiallyincombinationwithgenomicsand/
orproteomicsdata[26].Althoughsomestudiesaboutthemetabolicprofileofbloodcellscan
befound,toourknowledge,verylimiteddataaboutbloodcellanalysisbyNMRspectroscopy
frompatientsavailable[27–35].Theanalysisofthemetabolicprofileofbloodcellscouldnot
onlyprovideamethodforidentifyingnewbiomarkersfordiseasediagnosis,butalsoforin
vivoevaluatingtheeffectsofnewtherapeutictreatments(e.g.,nanomedicines)atapatient
level[22–24].
Nanomedicineistheapplicationofnanotechnologicalsystemstomedicine.Theimpactof
thistechnologyhasaugmenteddramaticallyoverthelastfewyearsduetoitsapplications
(drugdelivery,preventionofdrugmetabolisation,diagnosticagent,etc.)[36,37].Thankstoits
advantages,todateseveralnanometricsystemshavebeenapprovedforhumanuse,andmore
than240areindifferentclinicaltrialphases.Thissituationcreatestheneedtoimplementa
widerrangeofmethodologicaltoolstooptimizethedesignofnewnanomaterialsinearly
stagesoftheirdevelopmentandtoassesstheireffectduringclinicaltrials[38].Bloodisoneof
thefirstenvironmentsthatcomesintocontactwithananomedicinewhenitisinjectedor
whenitentersthebloodstreamviaotheradministrationtypes,whichmakesthestudyofthe
interactionofnanoparticleswithdifferentbloodcomponentshighlyrelevant.Comprehensive
studieshavebeenreportedontheeffectofnanomaterialsonboththeimmuneandcoagulating
systems.Theyincludetheanalysisoftheimpactofthesecompoundsonthemorphology,cell
cycleandproliferationofdifferenttypesofbloodcells[39–44].Indeed,newnanomaterialsare
designedtomakethisinteractionascontrolledandadvantageousaspossible,andbloodcells
haveevenbeenemployedascarriercellsfornanoparticlestoreachtheirdestinymoreeffi-
ciently[45,46].Inthiscontext,thefocusofourstudywastoevaluatethepotentialofmetabolo-
micsbyNMRtocharacterizethemetabolicprofileofperipheralbloodcellsbeforeandafter
treatmentwithnanoparticles.Totestourapproach,wehavechosengoldnanoparticlesas
modelsystems,becausetheyareoneofthemostpromisingnanomedicines,thathavebeen
suggestedforawiderangeofdifferentapplications;e.g.,medicalimagingandtherapiesincan-
cer,neurodegenerativediseasesordiabetes[47–58].Oneofitsmostpromisingpropertiesisits
capacitytoeliminateanexcessofoxidantspeciesgeneratedinstresssituations(antioxidant
behavior),whichisbeneficialformanybiomedicalapplications.Mostoftheseapplications
involveadirectcontactwithperipheralblood,whoseimpactcanbeevaluatedbyourmethod.
Severalapproacheshavebeenproposedtomaintainthestructureofgoldasnanoparticles
andtopreventagglomerationbyadjustingtheirpropertiesforbiomedicalapplications.For
instance,toincreasetheirbiocompatibilityandactivityagainstoxidativestress,goldnanopar-
ticleshavebeensupportedonceriananoparticlesorassembledinchitosan[59–63].Awide
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rangeofdifferentmedicalapplications,includingglucosesensors,antifilarialandantibacterial
agents,havebeenproposedforthesemodifiedgoldnanoparticles,howevernoneofthemhas
enteredclinicaltrialsyet.Bothmaterialshavetheiradvantagesanddisadvantages,forinstance,
ceriaparticlesaresolubleatphysiologicalpH,whichisnotalwaysthecaseformaterialscoated
withchitosan,thatsometimesneedanacidpHathigherconcentrations.Ontheotherhand,
ceriamaterialscanagglomerateorformcrownswithproteins,compromisingtheirbio-distri-
bution,withisminimizedinchitosanderivedmaterials.Toourknowledge,noapoptotictoxic-
ityhasbeendescribedforeitherofthesegoldnanoparticles.However,nocoherentinformation
canbefoundabouttheireffectoncellviabilityandproliferation.Whilesomeworkshavefound
noimpactinthisregard,otherstudieshavedescribedasignificantreductionincellviability
andproliferationforbothmaterials[59,64–68].Thereasonforthesedivergentresultsmaybe
variationinthesize,shapeornetchargeofnanoparticles,importantfeaturesthanhavebeen
welldescribedtocausedifferenteffectsoncells[69].Inthiscontext,thedevelopmentofnew
biomedicalmethodologiestomonitoreffectsofnanoparticles,whichcanprovideabetter
understandingofthemechanismrelatedtocertaintherapeuticand/ortoxiceffects,maycon-
tributetotheoptimizeddesignofthesematerials.Specifically,inthecaseoffunctionalizedgold
nanoparticles,forwhichawiderangeclinicalapplicationshavebeenproposedthatarewaiting
tobetransferredtoclinics,apreviousevaluationstepoftheireffectonperipheralbloodcells
couldprovideafastandeffectivefilterbeforestartinganyclinicaltrial.
Inourwork,wefirstoptimizedaglobalprotocoltoanalyzethemetabolicprofileofthe
threemaintypesofbloodcells(erythrocytes,polymorphonuclearleukocytes(PMNs)and
mononuclearleukocytes(PBMCs))thatcanbeisolatedinparallelfromonepatientsample.
Thecellfractionsfromdifferenthealthyvolunteers(n=4)werethenseparatelyexposedto
nanoparticlesbasedongold,andstabilisedonceriananoparticlesorchitosan,withantioxidant
properties.Themetabolomicprofileofthetreatedcellswasthereuponcomparedwiththepro-
fileoftheuntreatedcontrolcells.Withthisproofofconcept,weintendedtoshowthatsystem-
aticmetabolicchangescanbedetectedinperipheralhumanbloodcellsaftertreatmentwith
nanoparticles.Thisinformationcancontribute,togetherwithothertoxicologicalstudiesor
therapeuticdata,toevaluatenewnanomedicinesinpreclinicalphasesfromatranslational
pointofview,establishingaprecedentinthisfield.
Materialsandmethods
Chemicalsandmaterials
Solventsandreagentswerepurchasedfrom:Sigma-Aldrich(Ficoll-PaquePlus,Ficoll-Paque
Plus,PBS,fetalbovineserum,penicillin,streptomycin,amphotericinB,L-glutamine,chitosan,
HAuCl ,sodiumcitrate,AgNO ,sodiumphosphatedibasicdihydrate),Scharlab(methanol,
4 3
chloroform,acetone,sodiumhydroxide),Gibco(RPMI1640medium),Rhodia(CeO ),and
2
Eurisotop(deuteratedwater,deuteratedchloroform,deuteratedtrimethylsilylpropanoicacid,
trimethylsilane).MaterialswerepurchasedfromScharlab,LifeTechnologies,andFalconBD.
GasesweresuppliedbyAir-Liquide.
Humansubjects
Theinclusioncriteriawereasfollows:Caucasianhealthymales(2)andfemales(2),35–40
years,noalcoholics,nosmoker,andnofamiliarwithpreviouschronicdiseases.Allpartici-
pantswererecruitedattheOutpatient’sDepartmentoftheEndocrinologyServiceatVall
d’HebronUniversityHospital.Thestudywasconductedaccordingtotheguidelineslaiddown
intheDeclarationofHelsinki,andallprocedureswereapprovedbytheEthicsCommitteeof
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Valld’HebronUniversityHospital.Subjectshavebeenproperlyinstructedandhaveindicated
thattheyconsenttoparticipatebysigningtheappropriateinformedconsentpaperwork.
Humanperipheralbloodcellsisolation
Theisolationoferythrocytes,PMNsandPBMCsleukocyteswascarriedoutusingaFicoll-
Paquegradientmethod[70].20mLofperipheralbloodfreshlyextractedfromhealthyvolun-
teerswascarefullypouredintoatubewith40mLofFicollandletstandforapproximately20
min,obtaining3phases.Theupperringconsistedofleukocyteswhileerythrocytesconcen-
tratedasapelletatthebottom.Theleukocyteringwascarefullytransferredinatubewiththe
samevolumeofFicoll,avoidingmixingand,subsequently,centrifugedat300gfor25minat
20˚C.ApelletofPMNscellsandanintermediateringwithPBMCscellswereisolatedsepa-
ratelythroughthefollowingmethodology.FortheisolationofPBMCs,thePBMCsringwas
transferredtoatubewiththesamevolumeofPBSandcentrifugedat300gfor5minat20˚C
during5min.ThesupernatantwasdiscardedandthepelletcontainingthePBMCswaskept
onice.FortheisolationofPMNs,thepelletofPMNs,containingremaindersoferythrocytes,
wastreatedfor5minwith1mLoferythrocytelysisbuffer.Subsequently,themixturewascen-
trifugedat300gfor5minat20˚Candthesupernatantwasdiscarded.Theresultingpelletwas
resuspendedinthesamevolumeofPBSandcentrifugedat300gfor5minat20˚C.Thesuper-
natantwasdiscardedandthepelletcontainingthePMNswaskeptonice.Fortheisolationof
erythrocytes,theerythrocytepelletwastransferredtoatubewiththesamevolumeofPBSand
centrifugedat200gfor5minat4˚Cwithoutaccelerationandbrake.Thesupernatantwasdis-
cardedandtheerythrocytepelletwaswashedwithPBSagain.Finally,afterdiscardingthe
supernatant,thepelletcontainingtheerythrocyteswaskeptonice.
Theresultantpelletofalltheperipheralbloodcellswere:i)fordirectmetabolomicanalysis,
washedwithPBSagainandfinallystoredat-80˚Cafteradding0.5mLofice-coldmethanol
(for20millioncells)orii)fortreatmentwithnanoparticles,resuspendedin1mLofcomplete
RPMI1640mediumforcellcountinganddilutedinmoreRPMI1640untilobtainingasolu-
tionof5millioncells/mL(M/mL)
Treatmentofbloodcellswithnanoparticles
TwentymillionPMNsandPBMCs,and40millionerythrocytesweretransferredtocellculture
flasksataconcentrationof5M/mLinaTelstarBIOLaminarflowcabinet.Cellswerecultured
inaRPMI1640mediumconsistingof10%fetalbovineserum(FBS),1%ofantibioticmixture
(50μg/mLPenicillin,50μg/mLStreptomycin),1%(2.5μg/mL)AmphotericinBand2.05%
L-Glutamine.Toaddthenanomaterials,thefollowingprocedureswerecarriedout:a)AuCeO :
2
a1mg/mLdispersionofAuCeO inwaterwasprepared,andthendilutedto20μg/mLwith
2
medium.ThepHwasreadjustedto6.5with0.1Mofaceticacid.b)AuChi:a1mg/mLsolution
ofAuChiin0.1%aceticacidwasprepared,andthendilutedto20μg/mLwithmedium.ThepH
wasreadjustedto6.5with0.1MofNaOH.Forcontrolsamples,thepHofthemediumwas
directlyadjustedto6.5with0.1Mofaceticacid.FlaskswerethenincubatedinanIGO150
(Jouan,Saint-Herblain,France)incubatorduring24hwithoutstirringat37˚Cand5%ofCO .
2
Afterincubationwiththedifferenttreatments,thecontentofeachflaskofcellswastransferred
toafalcontubeandtheremainingcellswerescrapedoffinPBSandaddedtothetube.Leuko-
cytecellswerecentrifugedat300gand20˚Cduring5minanderythrocytesat200gand4˚C
during10minwithoutaccelerationandbrake.Supernatantswerethendiscarded,thepellets
resuspendedinthesamevolumeofPBSandcentrifugedagainunderthesameconditions.
Then,leukocytescellswerecentrifugedat11000gand20˚Cduring5minanderythrocytesat
200gand4˚Cduring10min.Supernatantswerediscarded,0.5mLofice-coldmethanolwas
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addedtothepellets,thatwerestoredat-80˚Cuntilperformingtheextractionfor1H-NMR
analysis.
Extractionofpolarandnonpolarmetabolitesfor1H-NMRexperiments
Frozensampleswereplacedonice,allowedtothatfor5min,andthensubjectedtoanextrac-
tionprocedure.Then,250μlofchloroformat4˚Cper20millioncellswereaddedtothecorre-
spondingpelletsandletstandfor30min.Sampleswerethenhomogenizedwithavortex,cells
resuspendedwithapipetteandtransferredtoa1.5mltube.Foruniformcellbreakage,samples
weresubmittedtothreefreeze-thawcycleswithliquidnitrogen.Then,400μLofdistilledwater
and400μLofchloroformwereaddedtoeachsamplewhichthenwasvortexed.Sampleswere
thencentrifugedat13000gfor20minat4˚Ctoseparatephases.Thesolutionwasseparated
intoanupperwater/methanolphase(withpolarmetabolites,aqueousphase),aninterphase
containingmainlyproteins,DNA/RNAandcellmembranes,andalowerchloroform/metha-
nolphase(withlipophiliccompounds,organicphase).Toobtaindryextracts,theaqueous
phasewaslyophilizedovernightandtheorganicphaseremovedusingaspeedvacuumconcen-
trator.Extractswerestoredat-80˚Cuntilsamplepreparationforthe1H-NMRexperiments.
1H-NMRexperiments
Frozencellpelletswereplacedoniceandallowedtothawfor5min.Totheaqueousphasewas
solubilisedin550μLofphosphatebuffer(100mMNa HPO pH7.4,inD O)containing0.1
2 4 2
mMofdeuteratedtrimethylsilylpropanoicacid(TSP-D4).Theorganicextractwasdissolved
in550μLofcolddeuteratedchloroform(CDCl with0.03%trimethylsilylpropanoicacid,
3
TMS).Sampleswerestoredat4˚C,equilibratedatRTfor15minbeforeanalysisandanalysed
thesameday.1H-NMRspectraofextractswererecordedat27˚ConaBrukerAVII600MHz
spectrometerusinga5mmTCIcryoprobeandprocessedusingTopspin3.2software(Bruker
GmbH,Karlsruhe,Germany).1H1DnoesyNMRspectrawereacquiredwith256freeinduc-
tiondecays(FIDs),64kdatapoints,aspectralwidthof30ppmandarelaxationdelayof4s.
Waterpresaturationwasappliedforaqueoussamples.TheFIDvaluesweremultipliedbyan
exponentialfunctionwitha0.5Hzlinebroadeningfactor.TotalCorrelationSpectroscopy
(TOCSY)andmultiplicityHeteronuclearSingleQuantumCorrelation(HSQC)wereper-
formedonrepresentativesampleswith256–512t1increments,32–96transientsandarelaxa-
tiondelayof1.5s.TOCSYspectrawererecordedusingastandardMLEV-17pulsesequence
withmixingtimes(spin-lock)of65ms.
Synthesisandcharacterizationofgold-chitosannanoparticles(AuChi)
AuChinanoparticlesweresynthesizedaspreviouslyreported[60].Briefly,200mgoflow
molecularweightchitosanwereaddedto100mLofmiliQwatercontaining1%ofaceticacid.
Then,themixturewasheatedat90˚Cwithvigorousstirringuntilcompletedissolutionofchit-
osan.Subsequently,1.3mLofa9.6mMHAuCl x3H Oaqueoussolutionwasaddedslowly
4 2
tothechitosansolutionandthemixturewasstirredfor5min.Afterward,250μLofa0.1M
sodiumcitratesolutionwasaddedandthemixturewasstirredforanother5min.Later,the
mixturewasquicklycooledinawater-icebath.Then,thesolutionwasfilteredthrougha
0.22μmcellulosefilterandcharacterized.Thehydrodynamicsizeandthezetapotentialwere
determinedbydynamiclightscattering(DLS)(ZetasizerNanoZS(MalvernInstrument,
UK)).Thegoldnanoparticlesizewasdeterminedbyhighresolutiontransmissionelectron
microscopy(HR-TEM)(PhilipsCM300FEG100kV).Finally,thecontentofgoldwasdeter-
minedbyinductivelycoupledplasma(ICP)(Varian715-ESICP-Plasma).
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Synthesisandcharacterizationofgold-ceriananoparticles(AuCeO )
2
AuCeO nanoparticlesweresynthesizedfollowingaprotocolpreviouslydescribedbyour
2
group[59].Briefly,200mgofHAuCl x3H Oweredissolvedin40mLofdistilledwaterat
4 2
RT.Then,asolutionof0.2MNaOHwasaddeduntilpH=10whilethemixturewasstirred
vigorously.OncethepHwasstable,asuspensionof1.0gCeO in13mLofdistilledwaterwas
2
addedslowly,adjustingthepHofthemixtureto10with0.2MNaOH.WhenthepHwassta-
ble,themixturewasstirredvigorouslyovernight.Afterthat,thedispersionwasfilteredand
washedwithseverallitresofdistilledwateruntilnotracesofchloridesweredetectedbythe
AgNO test.Then,thesolidwaswashedwith2x100mLofacetone,driedandplacedinafur-
3
nace.ThesamplewasthenheatedfromRTto300˚Catarateof8˚C/minduring4.5h,inpres-
enceofH .Afterthat,thefurnacewasshutdownuntilreachingroomtemperature.Then,
2
nanoparticlesweredissolvedonPBSandtheresultantsolutionwasfilteredthrougha0.22μm
cellulosefilter.ThegoldnanoparticlesizewasdeterminedbyHR-TEM(PhilipsCM300FEG
100kV).ThehydrodynamicsizeandzetapotentialofthesolidwereanalyzedbyDLS(Zetasi-
zerNanoZS(MalvernInstrument,UK)).Finally,thecontentofgoldwasdeterminedbyICP
(Varian715-ESICP-Plasma).
Dataanalysisandstatistics
1H-NMRspectraweretransformedwitha0.5line-broadening,andmanuallybaselineand
phasecorrectedwithTopspin3.2.NMRsignalsofTSP-D (polarspectra)andTMS(non-polar
4
spectra)werereferencedto0ppm.Formetaboliteidentification,the1Hand13Cchemicalshift
valuesandmultiplicityofthesignalswerecomparedwithreferencedatafromthespectraldata-
basesHumanMetabolomeDatabaseandtheBiologicalMagneticResonanceBankandseveral
literaturereports[29,71,72].TheassignmentNAD,NADH,NADP,NADPH,ATP,ADP,acet-
oacetateandsarcosine,wasconfirmedbyspikingthesamplewithreferencecompounds.Spec-
trawerenormalizedtototalintensitytominimizethedifferencesinconcentrationand
experimentalerrorduringtheextractionprocess.Optimalintegrationregionsweredefinedfor
eachmetabolite,tryingtoselectsignalswithoutoverlapping.Integrationwasperformedwith
MestreNova8.1utilizationtheGSDdeconvolutionoption.Inthestudywithgold-nanoparticle
treatments,p-valueswerecalculatedwiththenon-parametricMann-WhitneyUtestwithIBN
SPSSstatistics21.PathwayanalysiswasperformedwithMetaboanalyst[73].
Resultsanddiscussion
Optimisationofbloodcellisolation,treatmentandmetaboliteextraction
Aprotocolfortheisolationofthedifferenthumanbloodcelltypesandtheirmetaboliteswas
initiallyoptimized.Tothisend,peripheralbloodwasextractedfromhealthyindividualsand
thethreemayorbloodcellfractions(RBCs,PMNsandPBMCs)wereseparated.Twodifferent
methodsweretestedforevaluatingtheseparationofthedifferentfractions:theFicoll-Paque
gradientmethod,anddextranfollowedbytheFicoll-Paquegradientmethod.Nosignificant
differencesinthequalityofthemetabolomicprofileobtainedusingbothprocedureswere
found.Therefore,theFicoll-Paquegradientmethodwasselectedforitssimplicityandminimal
samplehandling.Anotherimportantfactortoconsideristhaterythrocytesareparticularly
fragile,sotheymustbecentrifugedatalowerspeedwithoutaccelerationorbraking.
Afterachievingphaseseparation,cellsweresubmittedtotreatmentwithnanoparticles(see
detailsintheSupplementaryInformation).Threedifferentconcentrationsofcellsweretested:
30,15and5M/mL.Agglomerationandcelldeathwasobservedatthetwohigherconcentra-
tions(30and15M/mL),butnotatthelowestconcentration(5M/mL).Finally,cellswere
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MetabolomicsofgoldnanoparticlesonhumanbloodcellsbyNMRastranslationalresearchapproach
harvestedandsubjectedtoanextractionprocesswithmethanol,chloroformandwater,a
methodthathadbeensuccessfullyappliedinpreviousmetabolomicsstudies[74].Thisprocess
isextremelysensitivetotheeliminationofmetabolitesfromthemediumthatcouldinterfere
withtheanalysis,aprocessthatwasachievedbywashingwithPBS.Furthermore,coldmetha-
nol(-20˚C)wasaddedtoeffectivelyquenchthemetabolismofcells.Solventamountswere
optimisedto500μLmethanol(addeddirectlyaftercellharvesting),650μLofchloroform
(addedintwotimes,beforeandaftercellbreakage)and400μLofwater(addedaftercell
breakage,toavoidice-buildingduringthefreeze-thawcycles)per20millioncells.Smallersol-
ventvolumesproducedpoorerextractionsyieldsandalessefficientphaseseparation,while
biggervolumesshowednoimprovement.Asaresultoftheextraction,twodifferent,aqueous
andorganic,fractionswereobtainedwithpolarandnon-polarmetabolites,respectively.For
theNMRanalysis,cellextractsweredriedandlaterondissolvedindeuteratedsolventsto
improvespectrumquality,whichcontainedinternalstandards(forquantification),andwere
bufferedintheaqueousphase(forpHcontrol).ThewholeprocessissummarisedinFig1.
MetabolicprofileofRBCs,PMNs,andPBMCsbloodcells
1H-NMRspectracorrespondingtotheaqueousandorganicextractsofthethreeperipheral
bloodcelltypesareshowninFig2.Adetailedassignmentofthedifferentspectrawasper-
formedbasedonthe1Dand2D-NMRspectraacquiredinthisstudy,aswellasthegeneral
informationavailablefrompublicdatabases,sincenoexhaustiveinformationregardingthe
metaboliccontentofthesebloodcellscouldbefoundintheliterature.Thus,itwaspossibleto
identifymorethan80differentpolarandnon-polarmetabolites,orfunctionalgroups(Fig2).
Themainmetabolitesidentifiedintheaqueousphasewereaminoacids,sugars,organicacids
andnucleotides,whereasintheorganicfractionitwaspossibletoidentifydifferenttypesof
lipids,suchasmonoandpolyunsaturatedlipids,di-andtriglycerols,phospholipids,aswellas
cholesterol.Thisisthefirsttimethatthemetabolicprofileofdifferentkindofbloodcellshas
beensystematicallydeterminedbyNMR,andtheresultsarecoherentwithdataobtainedin
previousstudiesusingGC-MSandLC-MS[75,76].
Itshouldbestressedthateachcelltypehadaspecificmetabolomicprofile.RBCs,for
instance,exhibitedlargeamountsoflactate,glucose,glutathione,ATP/ADP,ascorbicacid,
creatineandphospholipids.Thepresenceofseveralofthesemetaboliteshasbeenpreviously
describedinRBCsbyothertechniques,andtheirlevelalterationsareassociatedwithdiseases
suchassicklecell,Alzheimer’sdisease,Wilson’sdiseaseoranaemia[77–81].BothPMNsand
PBMCsleukocytescontainedverylargeamountsofglycerol,acompoundthat,amongother
functions,isavitalosmoprotectiveagentforcellsinsuspension.Thelevelsofmetabolitessuch
asglucose,pyruvateandsuccinatewerehigherinPBMCsthaninPMNs,perhapsreflecting
theimportanceofoxidativebioenergeticmetabolismoflymphocytes(mainfractionof
PBMCs)underbasalconditions[82].Moreover,ithasbeendemonstratedthatthebioener-
geticmetabolismofneutrophils(mainfractionofPMNs)ismainlyglycolytic[82].Accord-
ingly,lactateconcentrationsweresignificantlyhigherinthisbloodcelltype.
Insummary,metabolomicsprofilesobtainedby1H-NMRproviderelevantinformationof
thespecificmetabolismassociatedwitheachcelltype,thatcouldbeextremelyusefulfroma
clinicalpointofview.
EffectofnanoparticletreatmentonthemetabolicprofileofRBCs,PMNs,
andPBMCs
Onceareliableprotocolforevaluatingthemetabolomicprofileofeachcelltypewasestab-
lished,weevaluateditspossibleapplicationinnanomedicine.Tothisend,theeffectscaused
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MetabolomicsofgoldnanoparticlesonhumanbloodcellsbyNMRastranslationalresearchapproach
Fig1.Strategyforperipheralbloodcellisolation(PBMCs,PMNsanderythrocytes),nanoparticletreatment,polarandno-polar
metaboliteextraction,and1H-NMRanalysis.PBMCs,PMNsanderythrocytessampleswereisolatedfromperipheralbloodof
healthyhumanindividuals.Samplesofeachcelltypeweresplitanaliquot(20millioncells)forcharacterization(a),andanotheraliquot
(40millioncells)fornanoparticletreatments(b).Finally,polarandnonpolarwereextractedandthe1H-NMRmetabolicprofiles
determined(c).
https://doi.org/10.1371/journal.pone.0182985.g001
bychitosan-cappedgoldnanoparticles(AuChi)andgoldnanoparticlesstabilisedoncerianano-
particles(AuCeO ),twopromisingtypeofgoldnanoparticlesforbiomedicine,wereassessed.A
2
detailedphysicalcharacterizationofbothnanoparticlesrevealedmeansizes,determinedby
HR-TEM,of5.65nm(AuChi)and5.90nm(AuCeO )(S1File,FigAandD,respectively).
2
Moreover,zetapotentialsof+17mV(AuChi)and-19mV(AuCeO ),aswellashydrodynamic
2
sizesof205nm(AuChi)and133nm(AuCeO ),thatareoptimalvaluesforbiomedicalapplica-
2
tions,weredeterminedbyDLS(S1File,FigBandC,respectively).Finally,thegoldcontentof
bothnanoparticles,characterizedbyICP,was1.2%(AuChi)and0.8(AuCeO ).Pictorialrepre-
2
sentationsofthetwo-particlesystemscanbefoundinFig3.Thenon-toxicityofthenanomater-
ialswasconfirmedwithanMTT-assay(S2File).
Apilotstudywasthenconductedtoevaluatetheimpactofbothnanoparticlesineachcell
type.Thus,bloodwasextractedfromhealthyvolunteersandsubjectedtothepreviously
describedprotocol(Fig1).Toevaluatetheeffectthatthecoatinghadonthebloodcells,con-
trolexperimentswithcommercialchitosanandCeO werealsocarriedout.Therepresentative
2
1H-NMRspectraofRBCs,PMNsandPBMCsfromfreshlyperipheralhumanbloodafter
beingexposedfor24htoAuChi,AuCeO andthevehicleareshowninFig3.Significantdif-
2
ferencesinthemetabolomicprofilesofbloodcellsaftertreatmentwithbothnanomaterials
weredetected,asshowninTables1–3andTablesA–CinS3File.Togetanoverviewofthe
affectedpathways,pathwayanalysiswasperformedwiththeprogrammetaboanalyst,using
metabolitesthatchangesignificantlyasinputdata(FigAtoFinS4File).Ingeneral,itwas
foundthatthemetabolicchangesinducedbyAuChiwerelesspronouncedthanthoseassoci-
atedwithAuCeO .ThisresultisinagreementwithpreviousstudiesindicatingalowerAuChi
2
toxicitycomparedwithAuCeO [59,67,68].Interestingly,itwasalsofoundthatthemetabolic
2
impactofthenanoparticleswascell-typespecific,anindicationoftheexistenceofdifferent
mechanismofactionsofthenanoparticlesineachbloodcelltype.
Theantioxidanteffectofnanoparticles,asindicatedbyanincreaseintheGSH/GSSGratio
aftertreatmentwithAuChinanoparticles,wasbetterreflectedinPMNcellsastheycontaina
largenumberofmitochondriaasothercelltypes.Simultaneously,adropinlactatewas
observed,probablyreflectingareducedglucoseconversion(mainenergysourceinneutro-
phils)intolactate,inparallelwithanincreasedinsertionofpyruvateintotheoxidativecitric
acidcycle.Thesecellshavebeenreportedtouseglycerol-phosphateforenergyproductionin
mitochondria[83].Thus,antioxidantmaterialscouldhaveactivatedglycerol-phosphateoxida-
tioninmitochondria,aprocessthatwouldexplaintheobservedreductionofglycerollevels.In
thissituation,theobservedincreaseofbetainelevels,aknownosmolyte,couldcompensatethe
decreaseofgycerollevels[84].Anotherimportantobservationwasthedecreaseinglutamine
levels,revealinganalterationinglutaminolysis,aprocessthatoccursathighratesinimmune
systemcells[83].
Ingeneral,theantioxidantimpactofthenanoparticleswaslesspronouncedinPBMCcells
thaninPMNcells,anindicationperhapsofthefactthatlymphocytesalreadyrelyonanoxida-
tivemetabolismforenergyproduction.Alsointhiscase,adropinglycerolandanincreasein
betaineuponAuChitreatmentwereobserved,buttheseeffectsdidnottakeplaceforAuCeO .
2
Thedecreaseinphosphocholinederivatives,glycerides(AuChi)andMUFA,PUFAand
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Fig2.1H-NMRspectraoferythrocytes,PMNsandPBMCs.Polar(a)andnonpolar(b)1H-NMRmetabolomic
profilesofextractsofthemaintypesofperipheralbloodcells.Metaboliteassignmentsareindicatedwiththefollowing
numbers:1)2-hydroxybutyrate,2)leucine,3)valine,4)ethanol,5)lactate,6)2-aminoisobutyrate,7)alanine,8)lysine,9)
acetate,10)glutamate,11)reducedglutathione(GSH),12)oxidizedglutathione(GSSG),13)pyroglutamate,14)
pyruvate,15)succinate,16)glutamine,17)creatine,18)phosphocreatine,19)malonate,20)spermidine/spermine,21)
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