SPIO labeling of endothelial cells using ultrasound and targeted microbubbles at diagnostic

17 Pages·2017·7.74 MB·English

RESEARCHARTICLE
SPIO labeling of endothelial cells using
ultrasound and targeted microbubbles at
diagnostic pressures
IlyaSkachkov1,YingLuan1,SandraT.vanTiel2,AntoniusF.W.vanderSteen1,3,Nicode
Jong1,3,MoniqueR.Bernsen2☯,KlazinaKooimanID1☯*
1 DepartmentofBiomedicalEngineering,Thoraxcenter,ErasmusMC,Rotterdam,theNetherlands,
2 DepartmentofRadiology&NucleairMedicine,ErasmusMC,Rotterdam,theNetherlands,3 Laboratoryof
AcousticalWavefieldImaging,FacultyofAppliedSciences,DelftUniversityofTechnology,Delft,the
Netherlands
☯Theseauthorscontributedequallytothiswork.
a1111111111 *[email protected]
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a1111111111 Abstract
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Invivocelltrackingoftherapeutic,tumor,andendothelialcellsisanemergingfieldanda
promisingtechniqueforimagingcardiovasculardiseaseandcancerdevelopment.Site-spe-
cificlabelingofendothelialcellswiththeMRIcontrastagentsuperparamagneticironoxide
(SPIO)intheabsenceoftoxicagentsischallenging.Therefore,theaimofthisinvitrostudy
OPENACCESS
wastofindoptimalparametersforefficientandsafeSPIO-labelingofendothelialcellsusing
Citation:SkachkovI,LuanY,vanTielST,vander
ultrasound-activatedCD31-targetedmicrobubblesforfutureMRItracking.Ultrasoundata
SteenAFW,deJongN,BernsenMR,etal.(2018)
SPIOlabelingofendothelialcellsusingultrasound frequencyof1MHz(10,000cycles,repetitionrateof20Hz)wasusedforvaryingapplied
andtargetedmicrobubblesatdiagnosticpressures. peaknegativepressures(10–160kPa,i.e.lowmechanicalindex(MI)of0.01–0.16),treat-
PLoSONE13(9):e0204354.https://doi.org/
mentdurations(0–30s),timeofSPIOaddition(-5min–15minwithrespecttothestartof
10.1371/journal.pone.0204354
theultrasound),andincubationtimeafterSPIOaddition(5min–3h).IronspecificPrussian
Editor:ChristophEHagemeyer,Monash
Bluestainingincombinationwithcalcein-AMbasedcellviabilityassayswereappliedto
University,AUSTRALIA
definethemostefficientandsafeconditionsforSPIO-labeling.OptimalSPIOlabelingwas
Received:April27,2018
observedwhentheultrasoundparameterswere40kPapeaknegativepressure(MI0.04),
Accepted:September6,2018 appliedfor30sjustbeforeSPIOaddition(0min).Comparedtothecontrol,thisresultedin
Published:September20,2018 anapproximate12timesincreaseofSPIOuptakeinendothelialcellsinvitrowith85%cell
viability.Therefore,ultrasound-activatedtargetedultrasoundcontrastagentsshowgreat
Copyright:©2018Skachkovetal.Thisisanopen
accessarticledistributedunderthetermsofthe potentialforeffectiveandsafelabelingofendothelialcellswithSPIO.
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
Introduction
withinthepaperanditsSupportingInformation
files. Invivocelltrackingisaverypromisingtechniquetovisualizecellsofinterestinsidethebody.
Funding:Theauthor(s)receivednospecific Itallowstrackingofmotiletherapeuticcellslikeimmunecells,stemcells,andendothelialpro-
fundingforthiswork. genitorcellstositesofinflammation,cancer,orischemia[1–5].Additionally,thistechnique
canbeusedtotracktumorcells[6],tumorvasculature[7,8],orendothelialcellsintissueengi-
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. neeredvalves[9]andvasculargrafts[10].
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
Afterlabelingthecellsofinterestwithanimagingprobe,theycanbetrackedbyanimaging
modality.Magneticresonanceimaging(MRI)isinterestingforcelltrackingbecauseitispre-
cise,harmless,andthuswellsuitedforlongitudinalstudies.Moreover,singlecelltrackingis
possiblebyMRI.However,invivocelllabelingwithanMRIcontrastagentischallenging[6,
11–16].Forcelllabeling,theT2andT2(cid:3)-shorteningMRIcontrastagentsuperparamagnetic
ironoxidenanoparticles(SPIO)of80–180nminsize[17]areoftenused[18,19].Theyarerel-
ativelysafecompounds[19–22],butmostofinvitrocelllabelingtechniquesforSPIOarenot
applicableinvivo,becauseofthehightoxicityandbroadsystemiceffectsoftransfectionagents
[23].Therefore,therehasbeengrowinginterestinsafe,site-specificcelllabelingtechniques.
Onepotentialmethodinvolvesusingultrasoundcontrastagent,whicharecomprisedof
microbubbles.Themicrobubbleshavealowdiffusiblegascore(forexampleC F ),varyin
4 10
diameterfrom1–10μm,andareencapsulatedbyacoatingmaterial(forexamplephospholip-
ids).Whenultrasoundisapplied,themicrobubblesoscillateduetosequentialcompression
andexpansioncausedbypressurevariationsinthesurroundingmedium.Theoscillationof
microbubbleshasbeenshowntodelivertherapeuticmaterialsintocellsandinterstitialtissue
[24–27].Uptillnowthereisnoconsensusonthemechanismoftheenhanceduptake.Oneof
theuptakeroutesisaphenomenoncalledsonoporation,whenreversibleornon-reversiblecell
membraneporesaregenerateduponmicrobubbleoscillationsorviolentcollapse.Other
uptakeroutesincludeenhancedendocytosisandopeningofcell-cellcontacts[25,26,28].It
hasbeenreportedthattheefficacyofcellularuptakeoftherapeuticagentscanbeimprovedup
to7.7-foldinvitro[29]anduptofivefoldinvivo[30]byusingtargetedmicrobubbles(tMB)
insteadofnon-targetedmicrobubbles(non-tMB).ThetMBhavealigandaddedintheircoat-
ingbywhichthetMBcanadheretodisease-specificcellmembranebiomarkers[31,32].
Itwaspreviouslyshownthat45–60nmSPIO(Resovist)couldbedeliveredinvivointothe
swinebrainusingSonoVuelipid-coatednon-tMBandultrasound(28-kHzultrasoundwith
100-msburstlengthandrepetitionrateof1Hzat0.6–1MPa(mechanicalindex(MI)4.8–6.0)
appliedfor5min;MRIperformed3haftertreatment)[33]).BraintumordeliveryofSPIO
(meandiameter6–10nm[34]or35.7±9.2nm[35])loadedinthelipid-coatingofin-house
producednon-tMBwasshowninvivoinratsusingultrasound(0.4MHzwith1,000cyclesand
repetitionrateof1Hzat325kPa(MI0.5)appliedfor90s;MRIperformed40minaftertreat-
ment[34]or1MHzwith5,000cyclesandrepetitionrateof1Hzat300kPa(MI0.3)applied
for4min;MRIperformed1and3haftertreatment[35]).Deliveryof120–180nmSPIO(Feri-
dex)wasalsoshownintheaorticarchbySonoVueandultrasoundtreatment(8.5MHzultra-
soundatanMIof1.2appliedfor20min;MRIperformed1haftertreatment)[36].These
studiesdemonstratethepossibilityofSPIO-loadedMBorco-administratedSPIOwithMBfor
labelingextravasculartissuesandsubsequentMRIimagingoftheSPIO,butdonotcovercell
labeling.SuccessfulSPIO(Revovist)mesenchymalstemcelllabelingusingSonoVueandultra-
sound(1MHz,50%dutycycle,1.0W/cm2acousticpowerappliedfor60s)hasbeenreported
invitro[37].SPIO(12nmmeandiameter)loadedinthepolymercoatingofin-housepro-
ducednon-tMBwereusedtosuccessfullylabeltumorcellsinvitrousingultrasound(1MHz,
20cyclesperburst,repetitionrateof10kHz,0.1–0.75W/cm2acousticpowerappliedfor40s)
[38].However,MBarebloodpoolagents.Endothelialcells,whichformtheinnerliningofves-
sels,arethereforethemaintargetofintravascularadministeredMB[39,40].Exceptionsare
tumorsthatinvadeintothevasculature,asreportedforhepatocellularcancer(i.e.aprimary
livertumor)[41]andcolorectalcancer[42].Ontheotherhand,tMBwereshowntotarget
ovariancancercellspreclinicallybyanalternativeadministrationroute,namelyintraperito-
nealinjection[43].Additionally,tMBarepreferableinsteadofnon-tMBsincetheycanbespe-
cificallytargetedtothecellsofinterestanduponbindingareclosetotheendothelium,which
isaperquisitefortheMB-mediateddrugdeliveryeffectiveness[26].TheinvivostudybyGao
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
etal.demonstratedarterialwalluptakeofSPIOparticlesusingnon-tMBandultrasound[36],
butonlyunderoneacousticsetting(8.5MHzultrasoundat1.2MI,i.e.3.5MPaacousticpres-
sure),whichinducedconsiderablearterialwalldamage.Tothebestofourknowledge,noin-
depthstudieshavebeenperformedtocharacterizetheparameters(e.g.,theacousticsettings,
theSPIOadditiontimeandincubationtime)thatstronglyinfluencetheefficacyandsafetyof
SPIO-labelingofendothelialcellsusingtMBatlowMI(<0.2).
Theaimofourinvitrostudywastofindoptimalparametersfornon-invasive,tMB-medi-
ated,SPIO-labelingofendothelialcellsforthefutureapplicationofMRItrackingoftumorvas-
culatureandtissueengineeredvasculaturestructures.Weusedlipid-coatedtMBtargeted
againstCD31(i.e.platelet/endothelialcelladhesionmolecule-1(PECAM1)),abiomarkercon-
stitutivelyexpressedonendothelialcellmembranes[44],asproofofconcept.Ironspecific
PrussianBluestainingincombinationwithcalcein-AMbasedcellviabilityassayswereapplied
todefinethemostefficientandsafeconditionsforSPIO-labelingofendothelialcellsinvitro.
Weinvestigatedafixedultrasounddrivingfrequencyof1MHzandaseriesoflowdiagnostic
acousticalpressures(<200kPa;MI<0.2)andtreatmentdurationtimes(0–30s).Inourstudy
weused1MHzastheultrasoundfrequencybecauseitiscommonlyusedformicrobubble-
mediateddrugdeliverystudiesandisclosetotheresonancefrequencyofmicrobubbles[26].
Althoughtheexactlinkbetweenthetypeofmicrobubblebehavioranddruguptakeisnotyet
known[26],itwasreportedthatendocytosiswasstimulatedatlonger(2,000–10,000cycles)
acousticcycles[45–47].SPIOaretypically80–150nm[17]nanoparticleswhichmayrequire
uptakebyendocytosis,asthishasbeenshowntobethemainuptakemechanismfortherapeu-
ticslargerthan~17nminradius[46].Thisisthereasonwhywechosetostudy10,000acoustic
cycles.
Materialsandmethods
Endothelialcells
Humanumbilicalveinendothelialcells(HUVECs)(Lonza,Verviers,Belgium)werecultured
inEGM-2(Lonza)mediuminT75flasks(BD,Breda,theNetherlands)inahumidifiedincuba-
torat37˚Cwith5%CO .Cellsweredetachedwith0.25%TrypsininEDTA(Lonza)and
2
replatedononesideoftheacousticallytransparentOptiCell™(NUNC,Wiesbaden,Germany)
chambers.HUVECswereculturedasdescribedbefore[48],fortwodaysuntil70%confluence
toresembleneovasculatureendothelialcells.
Targetedmicrobubbles
Biotinylatedlipid-coatedmicrobubbles(meandiameter2.5μm)consistingofacoatingof
DSPC(59.4mol%;P6517;Sigma-Aldrich,Zwijndrecht,theNetherlands),PEG-40stearate
(35.7mol%;P3440;Sigma-Aldrich),DSPE-PEG(2000)(4.1mol%;880125P;AvantiPolar
Lipids,Alabaster,AL,USA),andDSPE-PEG(2000)-biotin(0.8mol%;880129C;AvantiPolar
Lipids)withaperfluorobutane(C F )gascore(F2Chemicals,Preson,UK)weremadeby
4 10
sonicationaspreviouslydescribed[49,50].Biotinylatedanti-humanCD31-antibody
(BAM3567;R&DSystems,Europe,Abingdon,UnitedKingdom)wasconjugatedtothemicro-
bubblesviaavidin-biotinbridgingaspreviouslydescribed[50,51].Specificityofbindingof
theseCD31-targtedmicrobubbleswaspreviouslyreportedbyus[48].
Celltreatment
TheconcentrationoftMBwasevaluatedbyCoulterCounter(Multisizer3,BeckmanCoulter,
Mijdrecht,theNetherlands)measurements(n=3)usinga20-μmaperturetubeallowing
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
quantificationofparticlediametersbetween0.4and12μmusingalinearspacingbetweenthe
256channels.TenmilliontMBwereaddedtoanOptiCell™chamberwithcellsplatedonthe
bottom(celltobubbleratioof1:3),whichwasturnedupsidedowntoletmicrobubblesadhere
tothecellsbyflotation.After5minincubationat37˚C,thechamberwasrevertedforthe
experimentsotheboundtMBwereontopoftheendothelialcellsasshowninFig1A.SPIO
nanoparticles(Endorem™,GerberS.A.,Paris,France)wereaddedatfourtime-points:5min
before,immediatelybefore(0min),5minafter,and15minafterinsonificationasillustrated
inFig1B,atafinalconcentrationof22.4μgFe/ml.EachOptiCell™chamberwasdividedinto
sixacousticallynon-overlappingareas(25×30mmeach;seeFig1C),whichcoveredthebeam
area(6.5mmfor-6dBbeamwidth)atthefocusofthe1.0MHztransducer(V303;Pana-
metrics-NDTTM,OlympusNDT,Waltham,MA,USA),asverifiedinadvancewithacali-
brated0.2mmPVDFneedlehydrophone(PrecisionAcousticsLtd,Dorchester,UK).The
OptiCellchamberwasplacedintoa37˚Cwaterbathandconnectedtoa2Dmicropositioner
(Fig1D).The1MHzfocusedtransducerwasconfiguredata45˚anglebelowthesampleand
theacousticfocuswasalignedwiththecenterofeachsubsection.
Duringtheexperiment,thepositionoftheOptiCellchamberwasmanipulatedsothatthe
centerofeachsubsectionwasinsonifiedinsequenceatapredefinedpressure(10to160kPa
peaknegativepressure(PNP),Fig1C).Aprolongedburstof10,000cycleswitharepetition
rateof20Hzwasappliedgeneratedbyanarbitrarywaveformgenerator(33220A,Agilent,
PaloAlto,CA,USA)andamplifiedusingabroadbandamplifier(ENIA-500,Electronics&
Innovation,Rochester,NY,USA).Thefirstsubsection,withouttheapplicationofultrasound,
wasusedasthecontrol.Theeffectofthedifferenttotalinsonificationtimewasdetermined(1
s,10s,and30s)at40,80,and160kPaPNPwhenSPIOwereadded5minpriortoinsonfica-
tion.ToinvestigatetheeffectoftheincubationtimewithSPIO,theOptiCellswereincubated
at37˚Cfor5min,1h,and3hafterinsonificationwhenSPIOwereadded5minpriorto
Fig1.Experimentalsetup.(A)SchematicrepresentationofthetMBadheringtoHUVECsduringtreatment.(B)
Timingdiagramoftheexperiment.Thetimeofinsonification(0min)wasusedasthereferencetime.Targeted
microbubbles(tMB)wereadded5minbeforetheultrasoundwasapplied;SPIOwasadded5minbefore(i.e.-5min),
justbefore(i.e.0min),5minafter,and15minafterinsonification.Cellswerefixated60minafterSPIOaddition.(C)
SchemeofinsonificationofsubsectionsoftheOptiCell™chamber(toscale).TheacousticpressureisgiveninPNP.(D)
Thetreatmentsetup.
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
insonification.TheeffectofSPIOadditiontime(-5,0,5,and15minwithrespecttothestart
ofinsonification)wasdeterminedat10,20,40,80,and160kPaPNP.Tochecktheeffectof
insonificationofHUVECsonSPIOuptakeintheabsenceoftMBs,theOptiCellswereinsoni-
fiedat40,80,and160kPaPNPfor30s,whileSPIOwasadded5minpriortoinsonification
(n=2).Allotherexperimentswererepeatedthreetimes.Fromthesethreedatasetstheaverage
andstandarddeviationareplotted.
SPIOlabeling
Afterthetreatmentdescribedabove(seealsoFig1B),cellswererinsedthreetimeswithphos-
phate-buffersaline(PBS;Invitrogen,Groningen,theNetherlands)toremovenon-internalized
SPIO.Then,cellswerefixatedwith4%formaldehyde(Sigma-Aldrich,Zwijndrecht,theNeth-
erlands)for10min.Afterfixation,thecellswerewashedthreetimeswithPBSandthenincu-
batedwithPrussianBluesolutionfor30min(aqueoussolutionof10%hydrochloricacid
(Sigma-Aldrich)and5%potassiumferrocyanide(Sigma-Aldrich))toassesstheSPIO-labeling
[52].Next,thecellswerewashedthreetimeswithPBSandthenucleiwerestainedwith0.1%
nuclearfastredsolution(Sigma-Aldrich).ThereaftertheOptiCellsweredriedfor48hand
microscopicallyexaminedusingamicroscope(Olympus,Zoeterwoude,theNetherlands)
equippedwith20×Plan(NA0.4)objective(Olympus)andacolorcamera(AxiocamMRc,
CarlZeiss,Germany).SPIOuptakewasassessedbymanuallycountingPrussianBluepositive
cellsamong~500cells(acquiredin5fieldsofview)locatedwithinacircleof6mmdiameter
aroundthecenterpointofeachinsonifiedarea.AcellwascountedasSPIOpositivewhenit
containedoneormorePrussianBluestainedironparticles.
Cellviabilityassay
ForeachSPIOuptakemeasurement,cellviabilitywasdeterminedintriplicatebycalcein-AM
andpropidiumiodide(PI)assaysinparallel.CellsweretreatedwithSPIO,tMBandultra-
soundasdescribedbefore(seealsoFig1).Within3to4minaftertheUStreatmentofallsub-
sectionsoftheOpticells,HUVECswereincubatedat37˚C,5%CO .Thirtyminbefore
2
assessingthecellviability,calcein-AMwasaddedtotheOptiCellchamber(C3100MP;Invitro-
gen;0.25μMfinalconcentrationfroma1mMstockpreparedinDMSO(Sigma-Aldrich))and
incubatedfor30minunderthesameconditions.ThereafterPI(P4864,Sigma-Aldrich,25μg/
mlfinalconcentration)andHoechst33342(Invitrogen;5μg/mlfinalconcentration)were
addedtotheOpticellchamber.MicroscopicexaminationwasperformeddirectlyafterthePI
andHoechstadditionwithafluorescentmicroscope(Olympus)equippedwiththesamesetup
asappliedforSPIOlabelingmeasurements,onlythata5×LMPlanFl(NA0.13)objective
(Olympus)wasusedhere.Foreachconditionfivedifferentfieldsofviewwereacquired
(~2900cells)withinthe6mmcirclearoundthecenteroftheinsonifiedarea.Differentfilter
sets(U-MWU2,330–385/420nm;U-MWIB2,460–490/510nm;U-MWG2,510–550/570nm,
Olympus)wereappliedfordetectingallcells(stainedwithHoechst),viablecells(stainedwith
calcein-AM),anddeadcells(stainedwithPI)respectively.Allimageswereautomaticallyana-
lyzedinImageJ[53].TheFindMaximafunctioninImageJwasusedtodefinetheexactnum-
berofcells.TofindanappropriatetolerancefortheFindMaximafunctionineveryimage,the
numberoflocalmaximawasdefinedfortoleranceparametersof0to200instepsoftwo.We
analyzedthedifferencesinnumberofmaximabetweenthesteps.Whenthedifferencebecame
smallerthan20,thispointwasconsideredasthecorrecttoleranceandthecorresponding
numberofcellsascorrectnumberofcells.Thisapproachwasvalidatedbyselectivemanual
countingofnumberofcells(n=10).Thedifferencebetweenmanualandautomaticcounting
was2.1±0.4%.AsshowninS1Fig,the%ofviablecellsdeterminedfromthecalcein-AM
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
staining(livecells)wasthesameasthecellviabilitydeterminedfromthePIstaining(dead
cells).Thecellviabilitydatawasthereforepresentedasthe%ofviablecellsdeterminedfrom
thecalcein-AMstaining.
Results
Microbubbledynamics
Duringallstudiedultrasoundbursts,weobserveddisplacementanddisappearanceoftMB.
Thiswasmostpronouncedforthe30sinsonificationperiod,asshowninFig2.Duringinsoni-
fication,tMBalsoclustered(Fig2B–2D).Afterthe30sinsonificationperiod,notMBwere
observedinthefieldofview(Fig2E),suggestingtheyhadbeendestroyed.
Insonificationduration
Intheabsenceofultrasound,lessthan2%ofcellsintracellularlyincorporatedSPIOnaturally
(Fig3A).TheefficacyofSPIOuptakebyHUVECsandthecorrespondingcellviabilityasa
functionoftheacousticPNPandthetotalinsonificationduration(1,10,or30s)atlowMI
(<0.16)areshowninFig3.ThetotalultrasoundexposuretimewasakeyfactorforSPIO
uptakeefficacy.ThiswasdemonstratedbytheamountofSPIOpositivecellsnotexceeding4%
for1sand6%for10sofinsonification,butwith30sofinsonificationtheamountofSPIO
positivecellsincreasedtomorethan10%.Additionally,thePNPalsoinfluencedSPIOuptake
significantly.With30sofinsonification,theproportionofSPIOpositivecellssignificantly
increasedwiththePNP(i.e.,from~10%at40kPato~16%at160kPa).Atthesametime,cell
viability(Fig3B)decreasedwithboththeincreasingacousticalpressureandtheinsonification
time.Forexampleat80kPaPNP,thecellviabilitydecreasedfrom~70%for10sofinsonifica-
tionto~60%for30sofinsonification.Foratreatmenttimeof30s,thecellviabilitydropped
bynearlytwo-foldfrom40kPato160kPaPNP.Ingeneral,thecellviabilityremainedhighfor
upto40kPaPNP.Specifically,insonificationfor30sdemonstratedthebestSPIOuptakeand
wasselectedforfurtherexperiments.Wedidnotinvestigatealongerinsonificationtime
becauseafter30salltMBweredestroyed(seeFig2E).
SPIOincubationtime
TheinfluenceoftheSPIOincubationtime(5min,1hor3h)afterthetreatmentwithultra-
soundandtMBonSPIOuptakeandcellviabilityisillustratedinFig4.Ingeneral,SPIOuptake
increasedprominentlywiththeincubationtime,forexamplefor180kPaPNPSPIOuptake
increasedfrombelow~4%to~22%.Thelargestratiobetweencontrolandtreateduptakewas
at1hofincubationforallPNPs.Cellviability(Fig4B)remainedhigh(>75%)at40kPaPNP
forallincubationtimes.Itdecreasedwiththepressure(80–160kPaPNP)forboth1hand3h
ofincubationtime.Alongerincubationtimedidnotlowercellviability,ascellviabilitywas
slightlyhigherafter3hofincubationthanafter1hofincubation.Basedontheresultsfrom
thisexperiment,1hofincubationwithSPIOwasselectedforfurtherinvestigations.
SPIOadditiontime
InFig5thepercentageofSPIOpositivecellsandcellviabilityareplottedforfourdifferent
timesbetweeninsonificationandSPIOadditionfordifferentacousticPNP.Similartothepre-
viousobservations,noultrasoundapplicationresultedinlessthan2%ofSPIOpositivecells.In
addition,ultrasoundapplicationwithouttMBspresentshowednosignificantdifferencein
SPIOuptakeincomparisontothecontrolwithoutultrasoundapplicationforallstudiedPNP
whentheSPIOwereadded5minpriortoinsonification.ForbothadditionsofSPIOat5min
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
Fig2.OpticalrecordingoftMBonHUVECsduringultrasoundtreatment.(A)beforetreatment.(B-E)tMBdisplacement,
clustering,anddestroymentduring30sinsonification(1MHz,80kPaPNP,10,000cycles,repetitionrateof20Hz,30s
insonificationtreatment,SPIOaddedat-5minwithrespecttothestartofinsonification).
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prior(-5min)andjustbefore(0min)theultrasoundapplication,weobtainedarelatively
largepercentageofSPIOpositivecells(~>10%)foracousticpressuresabove20kPaPNP.
Moreover,thepercentageofSPIOpositivecellsincreasedupto~12–15%withhigherPNPfor
SPIOadditionbeforetheinsonification.Incontrast,SPIOadditionat5and15minafterultra-
soundapplicationresultedinmuchlowerSPIOuptake(<8%).Similarly,thecellviability
remainedabove50%forallsettings.
Fig3.TheinfluenceofultrasoundinsonificationtimeonintracellularSPIOuptakeefficiency.(A)SPIOpositivecells.(B)Cellviability.
HUVECsweretreatedwithtMBandnoultrasound(-US)orultrasoundatvaryingPNP(40,80,or160kPa)for1,10,or30s;SPIOadded5min
beforeinsonification(-5min);1hofincubationafterSPIOaddition.
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
Fig4.TheinfluenceofSPIOincubationtimeonintracellularSPIOuptakeefficiency.(A)SPIOpositivecells.(B)Cellviability.HUVECs
weretreatedwithtMBandnoultrasound(-US)orultrasoundatvaryingPNP(40,80,or160kPa)for30s.SPIOwereadded5minbefore
insonification(-5min).IncubationtimeafterSPIOadditionwasvariedfrom5minto1hand3h.
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SPIOcelllabeling
Intheabsenceofultrasound,intracellularlyincorporatedSPIOweredetectedassmalliron
particleaggregatesdistributedinthecytoplasm(Fig6Aand6B).UltrasoundandtMBtreated
cellsdemonstratedmuchhigherSPIOuptakeasshowninFig6C–6G.Ironparticleswere
detectedasaggregatesofdifferentsizesinthecytoplasm(Fig6Cand6D).Othertypicalindi-
vidualexamplesofintracellularSPIOdistributionpatternsafterultrasoundandtMBtreatment
areshowninFig6Eand6F.Thesestainingpatternsincludeddistributionofaggregatesvary-
inginsizeandblueintensitythroughoutthecytoplasm(Fig6E)andonelargeaggregate
mainlylocatednearthenucleus(Fig6F)havingahigherblueintensityincomparisontothe
aggregatesinFig6E.Theintensitydifferencesofthebluestainsuggestdifferentconcentrations
ofSPIOparticles.
Fig5.TheinfluenceofSPIOadditiontimeonintracellularSPIOuptakeefficiency.(A)SPIOpositivecells.(B)Cellviability.HUVECswere
treatedwithtMBandnoultrasound(-US)orultrasoundatvaryingPNP(10,20,40,80,or160kPa)for30s;SPIOwereadded5minbefore
insonification(-5min),justbeforeinsonification(0min),5minafteror15minafterinsonification;1hofincubationafterSPIOaddition.
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
Fig6.SPIOcelllabeling.PrussianBluestainingofSPIOuptakein(A,B)thecontrol(i.e.noultrasound,1hafter
incubation)andin(C—F)theultrasoundandtMBtreatedHUVECsatdifferenttreatmentconditions.C,D,E:80kPa
PNP,SPIOaddedat-5min,30sinsonification,1hincubationafterSPIOaddition;F:160kPaPNP,SPIOaddedat15
min,30sinsonification,1hincubationafterSPIOaddition.BandDarezoomedinfromAandCasillustratedbythe
dashedrectangles.InBandD,oneexampleofaSPIOaggregateisindicatedbyanarrow.
https://doi.org/10.1371/journal.pone.0204354.g006
Discussion
Trackingofendothelialcellsisimportantforcancerandcardiovasculardisease.Therearesev-
eralwaysofSPIOcelllabelinginvitro[22,54].Mostofthesetechniquesrequiredifferent
transfectionagents,whichcannotbeusedinvivoduetotheassociatedhightoxicityandsys-
temiceffects.Wethereforestudiedatechnique,basedonultrasound-activatedultrasoundcon-
trastagentsthatwillbecompatibleforinvivouse.TheSPIOuptakewasdependenton
multiplefactors,includingtheultrasoundsettings,thetimeofSPIOaddition,andtheincuba-
tiontimeofSPIOwithcellsaftertheultrasoundtreatment.Optimallabelingat1MHzultra-
soundfrequencywasobservedwhentheultrasoundparameterswere40kPapeaknegative
pressure(MI0.04),10,000cyclesandrepetitionrateof20Hz,appliedfor30swhenSPIOwere
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SPIOlabelingofendothelialcellsusingultrasoundandtargetedmicrobubblesatdiagnosticpressures
addedat0min.Comparedtothecontrol,thisresultedinanapproximate12timesincreaseof
SPIOuptakewith85%cellviability.
Microbubbledynamics
WefoundanincreasingtrendofbothSPIOpositivecellsandcelldeathwiththeacousticPNP
increase.Notethatwestudiedacousticpressuresupto160kPaPNP,aregimeinwhichthe
amplitudeofradiallipid-coatedmicrobubbleoscillationsincreaseswithpressure[55,56].A
previousstudybyVosetal.[57]hasreportedthathighlynon-sphericalmicrobubblevibrations
canbeinducedatpressuresaslowas140kPaPNPforlipid-coatedmicrobubblesatresonance.
Inthisregime,theacousticstreaminggeneratedbyoscillatingmicrobubblesandtheproduced
shearstresses[58–61]canbeoneofthemechanismsforenhancedpermeabilityofthecell
membrane[26].Aswasexpected,thetotaldurationofinsonificationalsoshowedaneffecton
theSPIOuptake(Fig3A).Ithasbeenreportedthatat1MHzfrequencyatalowmechanical
index(MI<0.1),alipid-coatedmicrobubblecanrepeatedlyoscillateforthousandsofcycles;
whileathigherMImicrobubblesaredestroyedwithinabout100μs(i.e.100cycles)irrespective
ofpulselength[62].Weindeedobservedmicrobubblesstillpresentupto30sat80kPaPNP
(MI=0.08)(Fig2).Theimproveduptakewithprolongedinsonificationmayberelatedwith
thepersistenteffectproducedbymicrostreaminggeneratedbymicrobubblevibrationsasfor-
mulatedearlier.Moreover,wenoticeddisplacementoftMBwithsubsequentmicrobubble
clusteringandmergingdrivenbysecondaryradiationforceovertheprolongedburst,asillus-
tratedbyFig2.ThesefindingsareinlinewithourpreviousstudywheretMBboundtoendo-
thelialcellsalsodisplaced,clustered,andmergedbyinsonificationat1MHz,albeitforasingle
burstofupto50,000cycles[63].Detachmentofboundlipid-coatedtMBhasbeenreportedto
beduetotheattractivesecondaryBjerknesforcebetweentwotMB[64,65].Aggregationof
detachedtMBformingbiggermicrobubbleclustersmayhaveinfluencedtheiroscillation
dynamicsaslargermicrobubbleshavealowerresonancefrequencythanindividualsmall
microbubbles[66].Atalowdrivingfrequency(forexample1MHzasappliedinthisstudy),
microbubbleclustersareexpectedtohaveahigheramplitudeofoscillationastheywillbe
closertoresonance,whichcouldhavecontributedtotheenhancedSPIOuptake.
SPIOuptake
Inourstudy,HUVECsshowed~1%naturaluptakeafter1hofincubation,andthisvalue
increasedto~5%after3h(Fig4A).AlthoughthenaturaluptakeofSPIObyHUVECswaspre-
viouslyreportedbyvanTieletal.[67],thispercentageoflabeledcellsisnotsufficientforcell
tracking.TreatmentofHUVECswithultrasoundandtMBledtoadramaticincreaseof
~10-foldinSPIOuptakeafter1hincubation(Fig4A).Ontheotherhand,cellviability
decreasedbetween5minand1hincubationtime(Fig4B),suggestingthatinstantaneouscell
death(i.e.irreversiblesonoporationduetolargepores)islessprominentthaninducedcell
death.Inducedcelldeathcouldoccurviatheapoptoticpathway,aprocessthattakestime[68,
69],thatcanbeactivatedbyultrasoundandmicrobubblesaspreviouslyreportedbyothers
[70–72].WealsoobservedthattheSPIOlabelingefficiencywasinfluencedbytheSPIOaddi-
tiontimeinrespecttothetimeoftreatmentwithtMBandultrasound(Fig5).Weobserved
thehighestefficacywhenSPIOwereaddedwiththetMB(0min)foracousticPNPsupto80
kPa.WhenSPIOwereadded5or15minaftertreatment,SPIOuptakewaslower,butstillsig-
nificantlyhigher(morethanfivefoldat5min)thannaturaluptake.Thismaysuggeststimu-
latedendocytosisasuptakemechanismratherthansonoporation,sinceresealingofpores
createdbyultrasoundactivatedmicrobubbleshasbeenreportedonarelativelyshorttime
scaleofuptoaminute[28,73].Ontheotherhand,ourresultsmayalsosuggestthatboth
PLOSONE|https://doi.org/10.1371/journal.pone.0204354 September20,2018 10/17