M.Rouchota1,S.Sarpaki2,E.Fragogeorgi2,N.Shraga-Heled3,N.Günday-Türeli4,E.Türeli4,R.Popovtzer5,C.Maeseneire6,N.Passon6,M.Masa7,G.Loudos1,21BioemissionTechnologySolutions,Research&Development,Athens,Greece2NationalCenterforScientificResearch(NCSR)“Demokritos”,InstituteofNuclear&RadiologicalSciences&Technology,Energy&Safety,Athens,Greece3PluristemTherapeuticsInc,Haifa,Israel4MJRPharmJetGmbH,Überherrn,Germany5FacultyofEngineeringandtheInstitutesofNanotechnologyandAdvancedMaterials,Bar-IlanUniversity,Ramat-Gan,Israel6CenterforMicroscopyandMolecularImaging(CMMI),UniversitéLibredeBruxelles(ULB),Charleroi(Gosselies),Belgium

6LEITAT,Terrassa,Spain

STRUCTURALANDFUNCTIONALTRACKINGOFSTEMCELLSONMUSCLEREGENERATIONMODELTHROUGHNON-INVASIVEMULTIMODALIMAGING

PosterNo21

BACKROUND&PROJECTPLAN

Stemcelltherapykeepsgaininggroundasapromisingapproachfor a broad range of diseases, with currently no alternativeeffectivetherapies.However,toolsprovidingreal-timetrackingoftransplantedcellson theirearlybiodistributionandviability,aremissing from the current therapeutic approaches. At the sametime gold nanoparticles (GNPs) have shown that except ofexcellentbloodpoolandheartcontrastagents,canalsoserveascan serve as imaging agents to perform cell tracking, in celltherapy schemes. Moreover their ability to be easilyradiolabelled,rendersthemavaluabletoolwhichcouldevaluateandpredict the safetyand successof cell-based treatments.arealready used as contrast agents in Computed Tomography (CT)andasdrugcarriersfortargetedtherapyTheEU-fundedprojectnTRACK,started inOctober2017aimstodevelopasafe,scalableandhighlysensitivemultimodalcellnano-imaging agent ready for first in humans. The nTRACK approachwillenablenon-invasivewhole-bodymonitoring,longitudinalandquantitative discrimination of living stem cells in humans usingCT,MRIandPET,simultaneously.

Figure1:SchematicillustrationofthenTRACK’sprojectplan.

•  Biocompatibleandfunctionalnano-basedmultimodalimagingagents were developed in order to enable non-invasivemonitoringoflivingstemcellsinsmallanimalmodelsthroughSPECTandCTimaging.

•  Theinvivoplatformandmethodologytotrackstemcellsfatewas established based on GNPs samples (gold coated -magneticcoreNPsmanufacturedbyBar-IlanUniversity).

•  TheimagingstudieswereperformedonaSPECTandonaCTimaging system either by Molecubes (γ-CUBE and x-CUBE),providing spatial resolution of 0.6 mm and 0.05 mm,respect ive ly or by Mediso (nanoSPECTPlus andnonoScanPETCT), providing spatial resolutionof 0.3mmand0.01mm,respectively

METHODS&MATERIALS:

Figure2:Imagesofγ-CUBEandx-CUBEfromMolecubes,tomographicsystemsthatforthesestudies.

RESULTS:

Figure5:SPECT/CTimagingofahealthyC57Bl/6Jmouse,

intramuscularlyinjectedwithapproximately2.6MBqof[111In]InCl3andimagedat(a)2hrsp.i.,(b)4hrsp.i.,(c)24hrsp.i.and(d)48hrsp.i.

Figure6:CTimagingofahealthyWTfemale

mouse,intramuscularlyinjectedwith50uLofNPssolutionBIU-

DTPA-Au@IONPStherightlegisinjectedwith30mgAu/mLNPsandtheleftlegwith7.5

mgAu/mLNPsat(a)0hrp.i.,(b)2hrsp.i.,(c)4hrsp.i.and(d)24hp.i

The first in vivo control tests havebeenperformed, to establishthebiodistributionofthe(i) ligand,ofthe(ii)NPsandofthe(iii)labeled NPs. These biodistributions are shown in the followingFigures(Fig.5,6,and7).Theseresultsshowthatwhenthesubstancesareadministeredontheirown,theyfollowagradualclearancefromthebody.Whenadministeredwithinthecells,thiswillallowtotestthatthecellsindeedstayinthedesiredregion.

Figure4:RadioTLCof(a)[111In]InCl3and(b,c)[111In]In-DTPA-nTRACKNPsdevelopedonC18chromatographypaperwithMeOH/NH4OAc5Mat2hpostpreparation(b)and24hpostpreparation(c)Thechromatographicpaperswerereadusingaphosphorimager.

ExposureofPLX-PADcellstonTrackNPsinsalinesolutionwasexplored and resulted in the successful staining of cells withnTRACKNPs(Fig.3).

Thelabellingprotocolforthechelationof[111In]InCl3toDTPA

functionalisednTRACKNPswasoptimised inordertoobtainthehighestpossibleradiochemicalincorporationincombinationwithagoodkineticstability.(Fig.4)

(a) (b) (c) (d)

This project has received funding from the European Union’s Horizon research and innovation programme under grand agreement No 761031. This publication reflects only the author’s views and the European Union is not liable for any use that may be made of the information contained therein.

1.  O.Betzer,R.Meir,T.Dreifuss,K.Shamalov,M.Motiei,A.Shwartz,K.Baranes,C.J.Cohen,N.Shraga-Heled,R.Ofir,G.YadidandR.Popovtzer,ScientificReports,2015,5,15400.

2. R.Meir,K.Shamalov,O.Betzer,M.Motiei,M.Horovitz-Fried,R.Yehuda,A.Popovtzer,R.PopovtzerandC.J.Cohen,ACSNano,2015,9,6363-6372.

3. M.Motiei,T.Dreifuss,O.Betzer,H.Panet,A.Popovtzer,J.Santana,G.Abourbeh,E.MishaniandR.Popovtzer,ACSNano,2016,10,3469-3477.

REFERENCES:

CONCLUSION&FUTUREWORK:Radiolabelling of nTRACK NPs has successfully been performedandthefirst imagingresultswereobtained.TheCTenhancedofthe nTRACK NPs has been proven from CT and Gold coremagneticNPs have successfully been imaged through SPECT/CTforthefirsttime.SotheabilityofnTRACKNPstoprovideamulti-purposeimagingtoolisalreadydemonstrated.Ontheotherside,the labelling protocol of the nTRACK NPs to the stem cells hasbeenestablished.The in vivo platform and methodology to study GNPs as animaging tool, for cell tracking applications have been preparedanditwillbefurtheroptimisedinthenearfuture.

•  EuropeanUnion’sHorizon2020•  Researchandinnovationprogram•  IndustrialScholarshipsofStavrosNiarchosFoundation.•  TherestofthenTRACKconsortium.•  TheCMMIissupportedbytheEuropeanRegionalDevelopment

Fund,theWalloonRegion,theFondationULB,theFondsErasmeand“AssociationVinçotteNuclear

ACKNOLEDGMENTS:

§  Simultaneous SPECT andCT imagingof [111In]In-DTPA labeledNPs

§  VeryfastclearanceofNPs,shownviaCTmonitoringtest§  Very good CT control, as if signal is detectedwhen cells areinjected,itwilldefinitelycomefromtheactuallabeledcells

§  Theligandindeedfollowsthe“carrier”NPsandisnotdetachedinvivo(furthervalidationoflabelingstabilityresults)

§  Significantly higher sensitivity with SPECT imaging – smalleramountsofNPscanbeeasilydetectedthanwiththeCTalone.

ControlSample(NonTRACKNPs) CellstainedwithnTRACKNPs

Figure3:MicroscopeImagesofNPstainedcells.

Figure7:SPECT/CTimagingresultsofahealthymale

C57Bl/6Jmouse,intramuscularlyinjectedwith50uLofNPssolutionBIU-DTPA-Au@IONPS_Feb2019

Feb2019theleftlegisinjectedwith1.5mgAu/mLNPsat(a)2hrp.i(40min

scan).and(b)4hrsp.i(1hr10minscan)and(c)24hrs(1

hr30minscan)

1.Synthesisof

biocompatibleandfunctionalnano-basedmultimodalimagingagents(nTRACKNPs)

2.Labellingof

stemcellswithnTRACKNPs

3.Characterizationandfunctionalanalysisofnanoparticlelabelled

stemcells

4.Invivo

characterizationandcelltrackingbynon-invasiveimagingtechnologiesalgorithms.

5.Injectionoflabelledstemcellsintoaninjuredmuscle

6.Invivo

characterizationandcelltrackingbynon-invasiveimagingtechnologies.

FIGURES: