Engineering NIR-IIb fluorescence of Er-based lanthanide nanoparticles for through-skull targeted imaging and imaging-guided surgery of orthotopic glioma

HAL Id: hal-03108241

https://hal.archives-ouvertes.fr/hal-03108241

Submitted on 13 Jan 2021

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Engineering NIR-IIb fluorescence of Er-based lanthanide nanoparticles for through-skull targeted imaging and

imaging-guided surgery of orthotopic glioma

Feng Ren, Hanghang Liu, Hao Zhang, Zhilin Jiang, Bing Xia, Cécile Genevois, Tao He, Mathieu Allix, Qiao Sun, Zhen Li, et al.

To cite this version:

Feng Ren, Hanghang Liu, Hao Zhang, Zhilin Jiang, Bing Xia, et al.. Engineering NIR-IIb fluorescence of Er-based lanthanide nanoparticles for through-skull targeted imaging and imaging-guided surgery of orthotopic glioma. Nano Today, Elsevier, 2020, 34, pp.100905. �10.1016/j.nantod.2020.100905�.

�hal-03108241�

Engineering NIR-IIb fluorescence of Er-based lanthanide nanoparticles for through-skull targeted imaging and imaging-guided surgery of orthotopic glioma

Feng Ren

, Hanghang Liu

, Hao Zhang

, Zhilin Jiang

, Bing Xia

, Cécile Genevois

, Tao He

, Mathieu Allix

, Qiao Sun

, Zhen Li

, Mingyuan Gao

aCenterforMolecularImagingandNuclearMedicine,StateKeyLaboratoryofRadiationMedicineandProtection,SchoolforRadiologicaland InterdisciplinarySciences(RAD-X),SoochowUniversity,CollaborativeInnovationCenterofRadiationMedicineofJiangsuHigherEducationInstitutions, Suzhou,215123,PRChina

bSchoolofChemistryandChemicalEngineering,AnhuiProvinceKeyLaboratoryofAdvancedCatalyticMaterialsandReactionEngineering,Hefei UniversityofTechnology,Hefei,Anhui,230009,PRChina

cCNRS,CEMHTIUPR3079,UniversitéOrléans,F-45071,Orléans,France

Keywords:

Er-basedlanthanidenanoparticles NIRIIbfluorescenceimaging Imaging-guidedsurgery Focusedultrasoundsonication Orthotopicglioma

a b s t r a c t

Highlysensitiveandspecificdiscriminationofbraintumormarginsfromthesurroundingparenchyma remainsaformidablechallenge.Limitedbytheshortofphotostableprobeswithdeeptissuepenetration andhighefficiencyofcrossingtheblood-brain-barrier(BBB),thedevelopmentoffluorescence-guided surgery(FGS)ofbraintumorswasmarkedlyconstrained.Herein,wereportthecapabilityofthestrong secondnear-infrared-IIb(NIRIIb,1500−1700nm)fluorescencefromEr-basedlanthanidenanoparti- clesinimaging-guidedsurgeryoforthotopicglioma.Wedesignedanenergy-cascadedEr³⁺-Ce³⁺-A³⁺(A

=Yb,Ho,Tm)systemandpreparedaseriesofNaErF4:Ce@NaAF4@NaLuF4down-conversionnanopar- ticles(DCNPs)foroptimizingtheinfluenceofNaAF4 interlayerandCe³⁺dopants.Wemodified the optimalNaErF4:2.5%Ce@NaYbF4(0.9nm)@NaLuF4DCNPswithDye-brushpolymer(Dye-BP)tofacilitate

4I13/2→⁴I15/2transition,whichleadstoanimpressive675-foldenhancementof1525nmfluorescencein aqueoussolutionunder808nmexcitationduetotheexcellentenergy-cascadeddownconversion(ECD), incomparisonwiththatofNaErF4nanoparticles.Wefurthermodifiedthesehighlybrightnanoparti- cleswithtumor-targetingangiopep-2peptide,andefficientlydeliveredthemtothegliomabyusing thefocusedultrasoundsonication(FUS)totemporarilyopentheBBB.Weobtainedthehighesttumor- to-backgroundratio(TBR=12.5)everreportedinthetargetedNIRIIbfluorescenceimagingofsmall orthotopicglioma(size<3mm,depth>3mm)throughintactskullandscalp,whichwasdrastically improvedto∼150aftercardiacperfusionandcraniotomytoensurethepreciseresectionoftumor.More importantly,thesizeofgliomameasuredfromthewidthoffluorescenceprofileisveryclosetothat fromT2-weightedMRIimages.OurworkprovidestheinsightsintoengineeringNIRIIbfluorescenceof lanthanidenanoparticles,anddemonstratesthegreatpotentialofNIRIIbfluorescenceimaging-guided surgeryoftumor.

©2020ElsevierLtd.Allrightsreserved.

Introduction

Finely and clearly visualizing the margins of brain tumors, especially for glioblastoma(GBM), which is the most common malignantbraintumor,fromthesurroundingparenchymaisthe lynchpinfortheirprecisediagnosisandsurgery[1–3].Although thetypicaluseofvisualinspectionandimagingguidancecanpro-

videvaluable informationforclinicians,theintrinsiclimitations ofcurrentlyavailableimaging methods,suchaslowsensitivity, non-dynamicalinspection,andhazardousionizingradiation,can causeintraoperativefailureincompletelyresectingtumortissues [1,4–6].Inaddition,forfluorescence-guidedsurgery(FGS)ofbrain tumors,photostableprobeswithstrongcapabilityofcrossingthe blood-brain-barrier(BBB)arecrucialforprecisedelineationglioma marginandthesubsequentcurativeresection.Theintrinsicdraw- backsofclinicallyusedprobes(5-aminolevulinicacid(5-ALA)[7,8]

andindocyaninegreen(ICG)[9,10]),suchasinsufficientphotosta- bility[11]andshortexcitation/emissionwavelength-inducedlow

tissuepenetrationdepth,aredrivingthedevelopmentofalterna- tiveprobesforFGSofbraintumors.

Recently,muchefforthasbeendevotedtodevelopingthesec- ondnear-infrared(NIRII,1000−1700nm)fluorescenceimaging favoringforreducedtissueabsorption,scatteringandminimized auto-fluorescence[12,13]. In particular, the fluorescencein the rangeof1500−1700nm(NIRIIb)hasattractedconsiderableinter- ests,becauseitoffersthelowestphotonscatteringeffect,according totheMietheory(␮s’∝␭^−␣,␮s’representsthereducedscattering coefficient,␭isthewavelengthofincidentlight,␣=0.2–4fordif- ferenttissues)[14],andthenegligibleautofluorescenceunderlaser irradiationinthiswindow[15,16].Currently,theNIRIIbfluores- centprobes,suchassingle-walledcarbonnanotubes[16],PbS@CdS quantumdots[17],organicdyes[18],havebeenappliedforimag- ingmousebrainvasculaturethroughintactskullandscalp.Despite of theiradvances,someother limitations drivetheresearch of complementarynanoprobes[19,20].Alternatively,Er-basedrare- earthnanoparticlesarewell-known forup-conversion(UC)and down-conversion(DC)fluorescencefromvisibletoNIRIIregion withcontrollablesize, shape and functionality [21–24]. Typical Yb³⁺-Er³⁺co-dopedlanthanidenanoparticlescouldemitNIRIIbflu- orescenceunder980nmlightexcitation,butthewaterintissues wouldinevitablyattenuate980nmlighttoproducelocalheatand reducepenetrationdepth[25].AlthoughintroducingNd³⁺sensi- tizersintoYb³⁺-Er³⁺co-dopednanoparticlesmakesthefeasibility ofexcitationwith∼800nmlight[26],precisecontrolofcoreand shellisdemandedforoptimizingNIRIIbemission.

Unlikeotherlanthanideionsservingaseithersensitizersoracti- vators,Er³⁺ionscanactasboththesensitizersandactivatorsto rendereffectiveUCandDCprocesses[22].Toincreasenear-infrared absorptionasmuchaspossible,highconcentrationofsensitizers isusuallyrequired[27],whichcaninevitablycausenon-radiative cross-relaxationandenergymigrationlossofEr³⁺-heavilydoped nanoparticles.Toreducethenegativeeffectscausedbyactivators (Er³⁺ions),variousenergytrappingcenters(Yb³⁺,Tm³⁺,Ho³⁺,etc) wereintroducedtoconfinetheexcitationenergytoenhancethe UCprocess[28,29].However,thereisrarereportonboostingDC processofEr³⁺-heavilydopedlanthanidenanoparticles,whichare facingthechallengeofweakNIRIIbfluorescence.

TosignificantlyimproveNIRIIbemissionofEr³⁺-heavilydoped nanoparticles under 808 nm excitation, maximally increasing absorptionand optimizingtheenergytransferto⁴I_13/2 arecru- cial.Herein,wereportanenergy-cascadedstrategytoboostNIRIIb emissionofEr-basedcore-shell-shelldown-conversionnanoparti- cles(DCNPs),andacooperativetactictodeliverthemtoorthotopic glioma for targeted imaging and imaging-guided surgery. The resultingnanoparticlesexhibit675-foldenhancementofemission at1525nminaqueoussolutionincomparisonwithcorenanopar- ticles.TheirstrongNIRIIbfluorescenceandlowbackgroundmake themveryusefulinimagingandimaging-guidedsurgeryofdeep- seatedtumor.

Resultsanddiscussion

ModulatingNIRIIbemissionofEr-basedDCNPsvia energy-cascadedprocess

We designed core-shell-shell NaErF₄:Ce@NaYbF₄@NaLuF₄ DCNPs by using excitation energy trapping and non-radiative cross-relaxation approaches (Fig.1a)[20,29,30]. In this energy- cascadedEr³⁺-Ce³⁺-Yb³⁺ system, NaYbF₄ interlayer can bounce backthe radiative energy from ⁴I_11/2 of Er³⁺, and sequentially drivecross-relaxationto⁴I_13/2ofEr³⁺viaCe³⁺ionstomaximize

4I_13/2→⁴I_15/2 transition (Fig.1b). Although other self-sensitized activators,suchasEr³⁺,Ho³⁺,andTm³⁺ions,couldpromotethe

energy transfer from ⁴I_11/2 to ⁴I_13/2 of Er³⁺ withtwo involved energylevels(Fig.1c),wehypothesizethatEr³⁺-Ce³⁺-Yb³⁺system ismorefavorablefor⁴I_13/2→⁴I_15/2 transitiontogenerateNIRIIb fluorescence,due tothepresenceofradiativephotonrelaxation inducedbytheintermediateenergylevel(dashlineinFig.1c).

WepreparedaseriesofNaErF4@NaAF4(A=Yb,Ho,Tm)@NaLuF4

nanoparticleswithvariedinterlayerthicknessbysimplychang- ingtheamountofclusterprecursors.TheinertNaLuF₄shellwas usedtoinhibitsurfacequenchingeffectofEr-basednanoparticles [22,31].Transmissionelectronmicroscopy(TEM)imagesofthese nanoparticlesareshowninFig.S1−3,theeffectsoftheirinterlayer thicknessonUC(655nmemission)andDC(1525nmemission) processesarealsodistinctivelyillustratedinFig.1d.WhenNaYbF₄ wasusedastheinterlayer,theoptimizedNaYbF₄(0.9nminthick- ness)endowed1.8-foldand5.8-foldenhancementofUC(655nm) andDC(1525nm)emissionscomparedtothoseofNaErF4@NaLuF4

nanoparticles(Figs.1dandS4a).ForNaHoF₄interlayer,theopti- mizedNaHoF₄(0.2nminthickness)promotedtheUCprocessto resultin4.8-foldenhancementof655nmemission,whichisnearly twiceof1525nmemission(Figs.1dandS4b).ThedominantUCpro- cesscouldbeattributedtothatHo³⁺ionswith⁵I₆levelconfined excitationenergyto⁴I_13/2ofEr³⁺andimmediatelyintegrated808 nmphotonstogenerateup-conversionemission(Fig.1c). Since Tm³⁺ ionshave seriousproblemwithcross-relaxation [32],the NaTmF4interlayerhasnocontributiontoUCandDCprocesseswith thevariationofthicknessfrom0.4to2.1nm(Figs.1dandS4c).

TheaboveresultsdemonstratethatYb³⁺andHo³⁺ionscaneffi- cientlytrapexcitationenergytosimultaneouslypromoteUCand DCprocesses,andshowthelimitedimprovementof⁴I_13/2→⁴I_15/2 transitiontogenerateNIRIIbemission.

RecentprogresshasexemplifiedthatCe³⁺ionscanefficiently shorten the ⁴I_11/2 of Er³⁺ lifetime to enhance the DC process andimproveemissionbeyond1500nmunderirradiationby980 nmlight[19,20,33].ButfortheEr³⁺-heavilydopednanoparticles excitedwith808nmlight,theeffectandtheroleofCe³⁺ionsare stillunknown.WefirstlytestedtheeffectofCe³⁺dopantonthe

4I_13/2→⁴I_15/2 transition.TEMimagesofpreparedNaErF₄:y%Ce(y

=0,1,2.5,5)@NaLuF4nanoparticlesinFig.S5a-dshowanaverage sizeof12.5nmwith1.1nmdeviation(Fig.S5e).Theoptimizeddop- ingratioofCe³⁺ions(2.5%)leadsto1.4-foldenhancementof1525 nmfluorescenceatthecostof2.9-folddecreasesof655nmfluo- rescence(Fig.S5f),whichprovestheeffectivenessofCe³⁺dopants.

Basedontheaboveresults,weintroducedCe³⁺dopantsinto NaErF4@NaYbF4(0.9nm)@NaLuF4nanoparticlesbecausetheYb³⁺

ionscantrapenergyto⁴I_11/2 ofEr³⁺moreefficientlythanother ions (Er³⁺, Ho³⁺, Tm³⁺), and cooperate with Ce³⁺ dopants to boost ⁴I_13/2→⁴I_15/2 transition by populating the ⁴I_13/2 of Er³⁺. Uniform size and spherical morphology of NaErF4:y%Ce(y = 1, 2.5, 5, 10)@NaYbF₄(0.9 nm)@NaLuF₄ nanoparticles were pre- pared and characterized by TEM(Fig. S6). Theircorresponding fluorescence spectra present that the optimized doping con- centration (2.5 %) leads to 3.5-fold enhancement of 1525 nm fluorescence(Fig.S7a).Moreimportantly,theoptimizedNaErF₄:2.5

%Ce@NaYbF4(0.9nm)@NaLuF4 nanoparticlesshowanimpressive enhancementof1525nmfluorescence(13.4-folds),comparedto thatofNaErF₄@NaLuF₄nanoparticlesundertheexcitationof808 nmlight (Fig.1d). In addition,theoptimized Ce³⁺ dopantscan alsodrasticallyincreasethelifetimeofdown-conversionlumines- cenceat1525nmto2.88ms,whichismuchhigherthanthatof NaErF₄@NaLuF₄ nanoparticles(0.28ms)andNaErF₄@NaYbF₄(0.9 nm)@NaLuF4 nanoparticles (0.4 ms) (Fig. S7b-c). The results demonstratethatCe³⁺dopantsinducedpopulationof⁴I_13/2energy levelofEr³⁺ions,whichcanenhancetheNIRIIbfluorescenceand prolongitslifetimesimultaneously.

A representative TEM image of the optimized NaErF₄:2.5

%Ce@NaYbF₄(0.9 nm)@NaLuF₄ nanoparticles (Er-DCNPs) shows

Fig.1. a)schematicillustrationofpreparedNaErF4:2.5%Ce@NaYbF4(0.9nm)@NaLuF4nanoparticleandb)correspondinglyproposedenergydiagramshowingtheUC(655 nmemission)andDC(1525nmemission)processesunder808nmlightexcitation;c)proposedenergydiagramshowingexcitationenergytrappingmechanismsofYb³⁺

ionsbetweenEr³⁺ions,andofA³⁺(A=Er,Ho,Tm)ionsbetweenEr³⁺ions;d)normalizedemissionintensitiesof655nmand1525nmofNaErF4core,NaErF4@NaLuF4, NaErF4@NaAF4@NaLuF4(A=Tm-0.4nm,Ho-0.2nm,Yb-0.9nm),NaErF4:2.5%Ce@NaLuF4,andNaErF4:2.5%Ce@NaYbF4(0.9nm)@NaLuF4nanoparticlesunder808nmlight excitation;e)TEMimage,f)High-resolutionTEMimage,g)HAADF-STEMimage,andcorrespondingelementalmapsofCe,Er,Yb,andLuoftheas-preparedNaErF4:2.5

%Ce@NaYbF4(0.9nm)@NaLuF4nanoparticles.

highlyuniformandmonodispersedsphericalparticleswithadiam- eter of 17.9 ± 0.9 nm (Fig. 1e). Their core-shell structure and highcrystallinityaredemonstratedbytheircorrespondinghigh- resolutionTEMimage(HRTEM,Fig.1f).Thelatticespacingsforthe coreandshellaremeasuredtobe∼2.97Åand∼3.94Å,respectively, matchingwellwiththe(110)planesofhexagonal-phaseNaErF₄ (PDF#27-0689)andthe(300)planesofhexagonal-phaseNaLuF₄ (PDF#48-0830).Thehigh-angleannulardarkfield-scanningTEM (HAADF-STEM)and correspondingelementalmapsof Er-DCNPs exhibithomogeneousdistributionofEr³⁺inthecoreoftheNPs, alongwithsuccessfuldopingofCe³⁺ionsandYb³⁺/Lu³⁺ionslocal- izedintheshells(Fig.1g).

SurfacemodificationofEr-DCNPsforfurtherenhancementoftheir NIRIIbfluorescenceandbiocompatibility

For invivo bio-imaging, a hydrophilicsurface is requiredto endowbiocompatibilitytoEr-DCNPs.Duetothequenchingeffect ofhydroxylgroupsontheDCemission(∼1500nm)andthenar-

row(∼10nm)andweakabsorption(cross-sectionof10^-20cm^-2)of Er³⁺ions[20,27], amphiphilicpolymerssuchasDSPE-PEG were usually usedtowrapdyes (e.g.IR-808, IR-806)as organicsen- sitizer, which improvedthesolubility of nanoparticlesand the NIR IIfluorescence [34–36]. However,the wrapped dyes could beleakedandinstable,whichdirectlyinfluencedthesensitizing efficiencyinvitroandinvivo.Toresolvethisissue,wedesigned and synthesized a novel dye-brush polymer (Dye-BP) through reversibleaddition-fragmentationchaintransfer(RAFT)polymer- ization tointegratesurfaceligands withdyesensitizer(see the detailsonthesynthesisandcharacterizationofDye-BPintheSup- portingInformation, SchemesS1−2,andFigs.S8–14).Following thesamesyntheticroute,wealsopreparedasimilarbrushpoly- mer(BP)withoutdye.ByusingdifferentratiosofBPandDye-BP, wecantunetheamountofdyeonthesurfaceofnanoparticlesto achievetheoptimizedsensitizationeffect,andsimultaneouslypro- videbiocompatibilityandfunctionalgroupsforconjugatingwith targetingmolecules(Fig.2a).Fig.2bshowstheproposedenergy- cascadeddown-conversion(ECD)process,wherethedyeasthe

Fig.2. a)schematicillustrationofthesynthesisofEr-DCNPs-Dye-BP-ANGthroughligandexchangewithoptimizedDye-BPandcovalentconjugationwithangiopep-2 peptide;b)proposedenergydiagramshowingenergytransferfromDye-brushpolymertoEr³⁺ions,involvingphotontransitionamongEr³⁺-Ce³⁺-Yb³⁺ions,leadingto1525 nmemissionenhancement;c)hydrodynamicsizeofEr-DCNPsmodifiedwithvariousamountsofbrushpolymer(BP)andDye-BP;d)theabsorptionspectraofEr-DCNPsand Dye-BP,andtheNIRIIbemissionspectraofEr-DCNPsmodifiedwithdifferentmolarratiosofDye-BPrelativetothemolaramountofEr³⁺ions,under808nmexcitation;

e)thedependenceofNIRIIbintensityonthemolarratiosofDyemoleculesandEr³⁺ions.f)NIRIIbemissionspectraofEr-BP,Er@Lu-BPnanoparticles,Er-DCNPs-BP,and Er-DCNPs-Dye-BP;g)changeofthehydrodynamicsizeofEr-DCNPs-Dye-BPbeforeandafterfunctionalizationwithANG;h)therelativeviabilityofU87cellsafterincubation withEr-DCNPs-Dye-BPandEr-DCNPs-Dye-BP-ANGfor24h.

excitation-energyco-harvesterabsorb808nmphotonsandtrans- ferto⁴I_9/2ofEr³⁺,andthenactivatetheenergy-trappingYb³⁺ions andcross-relaxationCe³⁺ionstoboost⁴I_13/2→⁴I_15/2transitionin theEr-DCNPs.

The Er-DCNPs modified with varied mole ratios of BP to Dye-BPexhibit a similaraverage hydrodynamic size(39.9 nm) with a very small deviation (2.8 nm) (Fig. 2c), which indi- cates superior water-dispersity of polymer-modified Er-DCNPs.

The highly overlapping absorption of Dye-BP and Er-DCNPs at 808 nm (Fig. 2d-left) laid a solid ground for maximiz- ing the ECD process. The NIR IIb fluorescence (1525 nm) of resultant Er-DCNPs-Dye-BP was enhanced by 8.5-fold in com- parisonwiththat of Er-DCNPs-BP(Fig.2d-right),and themole ratio of dye to Er³⁺ ions was calculated to be 4.48 × 10⁻⁶ based on UV absorption quantitation (Figs. 2e and S15). In addition,theNIRIIbemissionofoptimizedDye-BPmodifiedEr- DCNPs(Er-DCNPs-Dye-BP)inaqueoussolutionwasimpressively improvedby43.1-foldand675-foldcomparedtothatofBP-coated NaErF₄@NaLuF₄ (Er@Lu-BP) and NaErF₄ (Er-BP) nanoparticles, respectively(Fig.2f).

Theirfouriertransforminfrared(FTIR)spectraconfirmthesuc- cessfulsurfacemodificationofEr-DCNPswithDye-BP(Fig.S16a).

TEMimage of Er-DCNPs-Dye-BP shows excellent dispersibility, witha similar size to that of oleic acid capped Er-DCNPs (i.e.

18nm,Fig.S16b-c).Thestrongcoordinationbetweenthephos- phategroups ofbrushpolymer andthesurfacelanthanideions

ofnanoparticlesguaranteestheexcellentcolloidalstabilityofthe nanoparticlesin0.9wt%NaClsolution[37],asevidencedbythe similarhydrodynamicsizearound34nmretainedoveroneweek ofstorage(Fig.S16d-e).

TargetingcapabilityofEr-DCNPs-Dye-BP-ANG

To enhance the uptake by glioma cells, we functional- ized Er-DCNPs-Dye-BP with angiopep-2 peptide (ANG), which can specifically bind to overexpressed low density lipopro- teinreceptor-relatedprotein(LRP) onglioma cells[38–40].The increment in the hydrodynamic size of the nanoparticlesafter modificationwasmeasuredtobe6.2nm (Fig.2g).Thein vitro cytotoxicityofthenanoparticlesagainstgliomacellswastestedby atypicalMTTassay(Fig.2h).Theviabilityofgliomacellsisover 75%,even thoughtheywereincubatedwitha highconcentra- tionofnanoparticles(800␮g/mL)for24h,whichdemonstrates theexcellentbiocompatibilityofEr-DCNPs-Dye-BPmodifiedwith orwithoutfunctionalangiopep-2peptide.Sinceournanoparticles have both UC and DC luminescence, we usedthe UC lumines- cencetocharacterizetheiruptakeby gliomacells (Figs. 3a and S16f).Thein vitro confocalmicroscope imagesinFig. 3bshow that strong UC fluorescence under excitation by the 980 nm laser was observed around thenuclei of the glioma cells after incubationwithEr-DCNPs-Dye-BP-ANG,implyingthattheywere efficientlyendocytosedbygliomacellsintocytoplasm.Incontrast,

Fig.3. a)schematicillustrationoftheuptakeofEr-DCNPs-Dye-BP-ANGbygliomacells,monitoredthroughUCluminescence(540nm)andDCluminescence(1525nm);b) representativeconfocalUCfluorescenceimagesofU87cellsincubatedwithEr-DCNPs-Dye-BPandEr-DCNPs-Dye-BP-ANG,andirradiatedwitha980nmlaser(scalebar:100

␮m);c-d)TEMimagesofEr-DCNPs-Dye-BPandEr-DCNPs-Dye-BP-ANGinU87cells(scalebar=500nm);e)bright-field(scalebar=1cm)andNIRIIbfluorescenceimagesof subcutaneousgliomafrommiceintravenouslyinjectedwithEr-DCNPs-Dye-BPandEr-DCNPs-Dye-BP-ANG(12mg/kg),andirradiatedwitha808nmlaser(100mW/cm²).

TheNIRIIimageswerecollectedusinganLP1400nmopticalfilter;f)thewidthofvesselsindicatedbythewhitedashedlineintheNIR-IIbfluorescenceimageshownin(e);g) thecorrespondingvariationofthetumor-to-liversignalratiocalculatedfromtheNIRIIbfluorescenceimagesin(e);h)invivovisualizationofangiogenesisinsubcutaneous glioma,NIRIIbfluorescenceimageswereacquiredat1minpostintravenousinjectionofEr-DCNPs-Dye-BP(12mg/kg,scalebar:5mm)andi)correspondingvariationofthe vascularwidth(redarrowpointed);j)NIRIIbfluorescenceimagingofbrainvasculatureinahealthynudemouse.

thecellsincubatedwithEr-DCNPs-Dye-BPonlyshowweakfluo- rescence.Additionally,therepresentativeTEMimagesshowmore nanoparticlesdistributedinthecytoplasmofcellsincubatedwith Er-DCNPs-Dye-BP-ANG,whichfurtherprovesthehighspecificity ofcovalentpeptidetoglioma(Fig.3c–d).

ToassessthespecificityofEr-DCNPs-Dye-BP-ANGinvivo,NIR IIbfluorescenceimagingofsubcutaneouslygraftedgliomatumors wascarried out withintravenous injectionof passive-targeting (ANG(-) group) and active targeting (ANG(+)group) nanoparti- cles(12mg/kg)(Fig.3e).The808nmlaserwithapowerdensity of100mW/cm² wasusedfortheNIRIIbimaging.Thewidthof bloodvesselscouldbemeasuredbythefluorescencesignal,e.g.

two blood vessels (whitedashedline inFig. 3e) in theNIRIIb fluorescenceimageofa mouseacquiredat1minpost-injection ofEr-DCNPs-Dye-BP-ANGweremeasuredtobe6mmand7mm (Fig.3f),respectively.Moreimportantly,thetumorfromtheANG(+) groupgraduallybecamebrightandachieveditsmaximalbright- nessafter30minpost-injection,whichismuchbrighterthanfor theANG(-)group.Thecalculatedtumor-to-liverfluorescenceratio fromtheANG(+)groupcollectedat30minpost-injectionis2.2-fold thatfromtheANG(-)group(Fig.3g),duetotheexcellenttargeting capabilityofANGpeptidetotheLRPreceptor[38].Moreover,the superiorNIRIIbfluorescenceofwell-designedEr-DCNPs-Dye-BP canbeappliedtomonitortheevolutionoftumorrelatedvessels (labeledwithredarrowinFig.3h),whichclearlydemonstratesthe angiogenesisprocessinthesubcutaneousglioma,evidencedbythe variationofatypicalvesselincreasedfrom0to1.26mminthefirst week,thento1.79mmtillday14,andmaintainedat1.79mmon day21(Fig.3i).

As the basis for the targeted imaging of orthotopic glioma, NIR IIb fluorescence imaging of brain vasculature and circula- tion, distributionand toxicity of ourwell-designed nanoprobes (Er-DCNPs-Dye-BP)inhealthymicewereconducted.TheNIRIIb fluorescenceimageofa mousebrainwasimmediatelycollected underexcitationbythe808nmlaser(120mW/cm²)aftertailvein injectionofnanoprobes(20mg/kg)(Fig.3j).Thebrainvascularnet- workwasclearlyobservedduetothestrongluminescenceofour nanoprobesandthedeeppenetrationoftheNIRIIbfluorescence throughtheintactmouse’sscalpandskull.

Bio-circulation,distributionandtoxicityofEr-DCNPs-Dye-BP Highlysensitivesingle-photonemissioncomputedtomography (SPECT)imagingwithunlimitedpenetrationdepthwasemployed toevaluatebio-circulationanddistributionofEr-DCNPs-Dye-BPin healthymice[41].Weutilizedthephosphategroupsonthesur- faceofnanoparticlestolabel^99mTcfortracingthecirculationand distributionofnanoparticles(Fig.4a).Time-dependentSPECT-CT imagesandthecorrespondingstatisticalanalysisofmajororgans demonstratethattheaccumulationofournanoprobesinliveris over20%ID/gandhalf-lifetimeofbloodcirculationis5.1±0.3h (Figs.4b-dandS17).Tofurtherdemonstratethebiosafetyofour nanoprobes,we performedhematoxylin-eosin(H&E)stainingof majororgans(heart,liver,spleen,lung,kidney,brain)ofmice.No clearpathologicaltissueisobservedfrommiceintheexperimental groupsafterdifferentdayspost-injectionofnanoprobes,incom- parisonwithmicefromthecontrolgroup(Fig.4e),whichindicates anexcellentbiocompatibilityofournanoprobes.

Fig.4. a)schematicillustrationofradiolabelingofEr-DCNPs-Dye-BP;b-d)SPECT-CTimagesandbiodistributionoflabelednanoparticlesinheartandliverofahealthymouse, collectedatdifferenttimepointspost-injectionof^99mTclabeledEr-DCNPs-Dye-BP;e)H&EstainingimagesofmajororgansofmicetreatedwithEr-DCNPs-Dye-BP(20mg/kg) andsacrificedatdifferentdaysincomparisonwithnon-treatedones(control)(scalebar:50␮m).

NIRIIbfluorescenceimagingoforthotopicgliomawith nanoprobes

Encouragedbytheaboveexperimentalresults,weevaluated thecapabilityofournanoprobesforactivelytargetedimagingof orthotopicgliomas.WefirstemployedT2-weightedMRItolocate deep-seatedandsmall-sizedorthotopicgliomas(∼3mmindepth,

<3mmindiameter)(Fig.S18a-left).Then,weperformedNIRIIb fluorescenceimagingoftheorthotopicgliomasafterinjectionof Er-DCNPs-Dye-BPandEr-DCNPs-Dye-BP-ANGnanoprobes,respec- tively.Thebrainvasculatureisclearlyobservedinbothcases,but theintratumoralenrichmentofnanoprobesthroughtheenhanced permeabilityandretentioneffect(EPR)aloneislowduetothepro- tectionofBBBandnon-specificity totumor(Fig. S18a-right).In contrast,ANGfunctionalizednanoprobescanbeefficientlyaccu- mulatedinthetumor,leadingtoa highertumor-to-background ratio(TBR)of7.0at1minposttail-veininjection,which is1.8- foldthatobtainedfrominjectionofEr-DCNPs-Dye-BPprobes(Fig.

S18b).

To further improve the intratumoral accumulation of nanoprobes for the NIR IIb fluorescence imaging, we used focused ultrasound sonication (FUS) to assist the delivery of nanoprobes. Previous reports have demonstrated the advances oftheFUStechniqueincontrollableopeningofBBBforpassively deliveringnanoprobestoglioma[34,42,43].Sinceactivetargeting enablesmoreaccumulationofnanoprobesinthetumor[44],we believethattemporaryopeningof BBBby FUScouldefficiently facilitatetheintratumoral accumulationof nanoprobes for pre- cisely delineating glioma marginsand imaging-guided surgery.

Undertheguidanceofpre-positionedT₂-weightedMRIandNIR IIbfluorescenceimagingofcerebrovascularnetwork(Fig.5a),we couldprecisely pinpointthepositionof thetumorand usethe FUStoopenthelocalBBBinconjugationwithmicrobubbles.MRI imagesshowthattheestablishedorthotopicgliomaswereseated belowtheskullofmicebyaround3.7mmwithadiametersmaller

than3mm(Fig.5b-left,whitedashedline).Fluorescenceimaging wasthen carried out atdifferent time pointsafter intravenous injectionofNIRIIbnanoprobes(Fig.5b-right).Undertheassistance ofFUS,muchbrightertumorsareobservedthanwithoutFUStreat- ment.TheTBRforthemiceadministeredEr-DCNPs-Dye-BP-ANG couldbeupto12.5at25minpost-FUStreatment(Fig.5c),which is higher than that (10.2) of mice intravenously injected with Er-DCNPs-Dye-BP,duetothesynergeticeffectsofFUSandactive targeting.Inaddition,theTBRdeclinehalf-timeforthegroupof mice administered Er-DCNPs-Dye-BP-ANG is around 150 min, whichislongerthanthatformiceinjectedwithEr-DCNPs-Dye-BP (only∼90min).Theresultsclearlyshowthattheabsenceoftumor- targetingpeptideresultedinarelativelylowTBRandreducedthe retentiontimeofnanoparticles.Previously,weshowedthatactive targetingstrategy alone couldnot ensureand controlsufficient nanoprobestocrossBBBandaccumulatewithinthetumor.Since gliomahashighervascular densitythan normalbraintissue by over3-fold(Fig.S19),FUStreatmentcouldalsoproduceruptures in the vasculature to increase its permeability for nanoprobes diffusingintothetumor[45],whichwouldfurtherleadtostrong NIRIIfluorescenceandahighTBR.

Similarly,thefullwidthathalfmaximum(FWHM)oftheinten- sityprofilecanbeusedtocharacterizethediameterofvesselsor thesizeoftumors.TheGaussAMPfittedFWHMofthevesselatthe tumorspotismeasuredtobe0.45mmintheimageacquiredat1 minpost-injection(Fig.5d).Moreimportantly,thesizesoftumors measuredfromtheFWHMofthefluorescenceintensityacquired at25minpost-FUStreatment(Fig.5e–f)are2mmand3mm(from multiplyingtheFWHMby2)forthemiceinjectedwithEr-DCNPs- Dye-BPandEr-DCNPs-Dye-BP-ANG,respectively,whicharevery closetothose (i.e.,1.7 mm and 2.7 mmrespectively) obtained fromT2-weightedMRIimagesduetothelowbackgroundofNIR IIbfluorescence.Thesubsequentbloodvesselandrare-earthstain- ingoftumortissuesconfirmthatlanthanidenanoprobeswerestill retainedinthevasculature-abundanttumortissue(blackarrow),

Fig.5. a)SchematicillustrationofT2-weightedmagneticresonanceimaging(MRI),andnon-invasiveNIRIIbfluorescenceimagingofadeep-seatedandsmall-sizedorthotopic gliomaundertheassistanceoffocusedultrasound(FUS);b)thepre-contrastT2-weightedMRIimagesandtheNIRIIbfluorescenceimagesofsimilarsizedorthotopicgliomas collectedatdifferenttimepointspostintravenousinjectionwithEr-DCNPs-Dye-BPandEr-DCNPs-Dye-BP-ANG(20mg/kg),respectively,beforeandafterFUStreatment;

c)thetumor-to-backgroundratiooftheNIRIIbfluorescenceimagesshownin(b);d)thevesselwidthmeasuredfromtheNIR-IIbfluorescenceimageoforthotopicglioma collectedat1minpostinjectionwithEr-DCNPs-Dye-BPandshownin(b)(whitedashedline);e-f)thesizesoftumorsmeasuredfromtheNIRIIbfluorescenceintensityof imagesacquiredat25minwithoutandafterFUStreatment;g)immunohistochemicalassayforbloodvesselsandArsenazoIIIstainingofthegliomatissues,withtheblack andredarrowsindicatingthecapillariesandrare-earthionsinthetumortissues(scalebar:50␮m).

even180minafterNIRIIbfluorescenceimaging,andtherewas longerretention of Er-DCNPs-Dye-BP-ANGthan Er-DCNPs-Dye- BPinthetumor,asshowninFig.5g-right(redarrow).Basedon theseresults,wecanclaimthatsequentialoperationswithpre- positionedT₂-weightedMRIandpreoperativeNIRIIbfluorescence imaging enable usto pinpoint thelocation of deep-seated and small-sizedorthotopicgliomasforpreciselydepictingthemargins oftumorswithahighTBRunderthecooperationofactivetargeting andFUStreatment.

SurgicalresectionoforthotopicgliomaguidedbyNIRIIb fluorescenceimaging

ThecloselymatchingtumorsizefromNIRIIbfluorescenceimag- ingand MRIsuggeststhat portableNIRIIfluorescence imaging couldbeusedforimaging-guidedsurgeryduetothesignificanceof real-timevisualizationoftumormarginswithprecision.Toclearly showthemarginoftumor,wefirstusedT₂-weightedMRIandNIR IIbfluorescenceimagingtolocatetheglioma,appliedtheFUStreat- ment,andthendidcardiacperfusiontoeradicatetheinterference

ofnanoprobesintheblood.Afterthat,weresectedtheorthotopic gliomaundertheguidanceofNIRIIbfluorescenceimaging(Fig.6a).

Large-sizedgliomaswereusedtotestthefeasibilityofthisdesigned operationalprocedure.TheT2-weightedMRIimagesofbrainshow thatthesizesof thetumors are7.9mmand6.8mmin diame- terforthemicefromtheANG(+)+FUSandANG(-)+FUSgroups, respectively,whichareclosetothesizeofthegliomasmeasured fromtheNIRIIbfluorescenceimages(e.g.8.1mmand8.5mm, respectively,inFig.6b-d).Thesmallerdifferenceinthetumorsize betweenMRI and NIRIIb fluorescenceimages(0.2 mm)in the ANG(+)+FUSgroupfurthersupportsthemoreaccumulationof nanoprobeswithinthetumorandstrongNIRIIbfluorescence,due tothetumor-targetingcapacityoftheANGpeptide.In contrast, non-specificbindingcausedonlyalowaccumulationofnanoprobes andweakerluminescence,resultinginanotablesizedifference(1.7 mm).

TofurtheraddressthegreatpotentialofNIRIIbfluorescence inimaging-guidedsurgery oforthotopicglioma,we utilizedEr- DCNPs-Dye-BP-ANGas probestoperformthesurgicalresection ofsmallgliomas.Smallgliomas(3.8mmin depthand2mmin

Fig.6.a)SchematicillustrationofNIRIIbfluorescenceimagingguidedresectionofdeep-seatedandsmall-sizedorthotopicglioma;b)T2-weightedMRIimagesoflarge- sizedglioma(left),andNIRIIbfluorescenceimagesoftheresectedbrainaftercardiacperfusion(right);c-d)thecorrespondingtumordiametermeasuredfromtheNIR IIbfluorescenceintensityofimagesacquiredat25minpostinjectionofnanoprobesandFUStreatment;e)preoperativeT2-weightedMRIdiagnosisofsmalldeep-seated orthotopicglioma2mmindiameter;f)NIRIIbfluorescenceimagesofresectedbrain,afterthemicewereintravenouslyinjectedwithEr-DCNPs-Dye-BP-ANG,treatedwith focusedultrasound,andsubjectedtocardiacperfusion(scalebar:5mm);g-h)thecorrespondingNIRIIbfluorescenceintensityofthegliomaareabeforeandafterresection;

i)immunohistochemicalassayofbloodvesselsandArsenazoIIIstainingofrareearthionsintheresectedtumortissue,withtheblackandredarrowsindicatingthecapillaries andrare-earthions(scalebar:50␮m).

diameter)weremeasuredwithT2-weightedMRI(Fig.6e).Afterthe NIRIIbfluorescenceimagingproceededat25minpostFUStreat- ment,similarcardiacperfusionandcraniotomywerecarriedoutto reducetheinterferencefromblood.TheTBRofthelocatedglioma isupto150incomparisonwithnormalbraintissue(blank),and thecorrespondingtumorsizemeasuredfromNIRIIbfluorescence intensityis2mmindiameter(Figs.6f-left,6g),whichisexactly sameasfortheT2-weightedMRIimages.Aftertheresectionofa gliomaundertheguidanceofNIRIIbfluorescenceimaging,noflu- orescencesignalwasleftinthesurroundingtissueoftheresected tumorcomparedtothenormalbraintissue(Fig.6f-right,6h).The highTBR is mainly attributedto the high accumulationof Er- DCNPs-Dye-BP-ANG(redarrow)inthetumorareawithabundant vasculature(blackarrow),whichisconfirmedthroughbloodves- selandrare-earthstaininganalysesoftheresectedsection(Fig.6i).

TheseresultsdemonstratethatintraoperativeNIRIIbfluorescence imaging is promising for finely delineating tumormargins and imaging-guidedsurgery.

Conclusions

Insummary, we proposedan energy-cascadeddownconver- sion strategy for drastically enhancing 1525 nm emission of Er-basedDCNPs,andanefficientdeliveryofresultanthighlybright

nanoparticlesfor NIRIIb fluorescence imaging of brain tumors and imaging-guided surgery. Specifically, we designed energy- cascadedNaErF₄@NaAF₄@NaLuF₄(A=Yb,Ho,Tm)DCNPs,inwhich threeinterlayerswithdifferentcomponentsand thicknesswere firstlyinvestigatedtheirenergytappingeffectsonDCprocess,sec- ondly,Ce³⁺ionsweredopedintoNaErF₄ coretomaximizeECD processforimproving⁴I_13/2→⁴I_15/2transition,andfinallyDye-BP containingIR-806asamajorenergyharvesterwasusedtomodify nanoparticlesurface.Theefficientenergycascadeintheoptimized nanoparticles(Er-DCNPs-Dye-BP)synergisticallyboosted1525nm fluorescenceby675timesinaqueoussolution,incomparisonwith thatofNaErF₄nanoparticles.WemodifiedtheEr-DCNPs-Dye-BP withtumor-targetingpeptideandusedtheFUStechniquetotem- porarilyopentheBBBtocooperativelydeliverthemintoorthotopic glioma.Owingtothestrongfluorescenceofournanoprobes,wecan preciselydelineatethetumormargin,andresectthemunderthe guidanceofNIRIIbfluorescenceimagingwithahighTBR.Thesizes ofthetumorsmeasuredfromtheintensityofNIRIIfluorescenceare veryclosetothosemeasuredfrompreoperativeT₂-weightedMRI images,duetothelowbackgroundofNIRIIbfluorescencefromtis- sue.Theseresultsdemonstratethegreatpotentialofourstrategies inengineeringNIRIIbfluorescenceoflanthanidenanoparticlesand inimaging-guidedsurgeryoftumor.Theresultsalsodemonstrate thepromiseofefficientdeliveryoftheranosticnanoparticlesfor

diagnosisandtreatmentoftumorbycombiningactivetargeting strategywithfocusedultrasound(FUS)technique.

Materialsandmethods

Materials

ErCl₃·6H₂O,CeCl₃·7H₂O,YbCl₃·6H₂O,TmCl₃·6H₂O,HoCl₃·6H₂O, LuCl₃·6H₂O, sodium fluoride (NaF, 99.99 %), oleic acid (OA, 85 %), 1-octadecene (ODE, 90 %), 4-mercaptobenzoic acid (90 %), 2-aminoethyl methacrylate hydrochloride (AMA, 90

%), N,N-dicyclohexylcarbodiimide (DCC, 99.0 %), 4-cyano- 4-(phenylcarbonothioylthio)pentanoic acid (CTA, >97 %), 2,2-azobis(2-methylpropionitrile) (AIBN, 99 %), and tris(2- carboxyethyl)phosphine (TCEP, 98 %) were purchased from AladdinCo.Ltd.Sodiumhydroxide(NaOH,96%)waspurchased fromBeijingChemicalReagentsCo.Ltd.Trisbuffersolution(pH= 8.0)andphysiologicalsalinewereobtainedfromShanghaiYuanye Biotech, Inc. Thiolated-Angiopep-2 (TFFYGGSRGKRNNFKTEEYC, ANG-SH) was purchased from Xi’an Ruixi Biological Technol- ogy Co. Ltd. Poly(ethylene glycol)methyl ether methacrylate (number average molecular weight, Mn = 300, containing 100 ppm hydroquinone monomethyl ether (MEHQ) and 300 ppm butylatedhydroxytoluene(BHT)asinhibitor),andPoly(ethylene glycol)methacrylate(averageMn=360,containing500−800ppm MEHQ as inhibitor)were purchased fromSigma-Aldrich. N-(2- aminoethyl)maleimidehydrochloride(HPLC,>93.0%)wasbought fromTCICo.,Ltd.Phosphorusoxychloride(99%)waspurchased fromXiyaReagent.Allreagentswereusedasreceivedwithoutany purification.

SynthesisofEr-basedcore-shellnanoparticles

SynthesisofNaErF₄@NaAF₄(xnm,A=Yb,Ho,andTm)@NaLuF₄ core-shell-shellnanoparticles

TheNaLnF₄ (Ln=Er,Ce,Yb,Lu)precursorswerepreparedas described elsewhere [30,46,47], and the resultant NaLnF₄ nan- oclustersweredispersedin cyclohexaneforfurtheruse.NaErF4

nanoclusterssolution(2mL,0.5mmol)wasmixedwithOA(4mL) andODE(10mL) ina three-neckflask(100mL). Theflaskwas purgedwithnitrogen(N2)at70^◦Cfor30mintofullyremovethe cyclohexane,andthenheatedupto280^◦Catarateof∼10^◦C/min.

Afterreactedat280^◦Cfor30min,thesolutionwascooledto70

◦C,followedbytheadditionofthemixtureofNaYbF₄nanoclusters solution(0.2,0.4,1mL,0.25mmol/mL),OA(2mL)andODE(5mL).

Theresultantsolutionwasmaintainedat70^◦Cfor20min,heated upto280^◦Cattherateof∼10^◦C/min,andthenkeptat280^◦Cfor 30mintoformNaErF4@NaYbF4nanoparticleswithdifferentthick- nessesoftheirNaYbF₄shells.ThegrowthofNaLuF₄inertshellsis similartothegrowthofNaYbF₄shells,exceptthattheamount(2 mL,0.25mmol/mL)ofprecursorandthereactiontimeat280^◦C(60 min)werefixed.Aftercoolingtoroomtemperature,thereaction mixturewascentrifugedat11,000rpmfor5mintocollectedcore- shell-shellnanoparticles,whichwerewashedtwicewithabsolute ethanolandfinallydispersedintetrahydrofuran(THF).

ThesynthesisandpurificationofNaErF₄@NaHoF₄(xnm,x=0.2, 0.8,1.1,1.8)@NaLuF₄,andNaErF₄@NaTmF₄(xnm,x=0.4,0.8,1.6, 2.1)@NaLuF4core-shell-shellnanoparticlesweresametotheabove methods,excepttheNaHoF₄ andNaTmF₄nanoclustersandtheir amounts.

SynthesisofNaErF₄:y%Ce(y=1,2.5,5,10)@NaYbF₄(0.9 nm)@NaLuF4core-shell-shellnanoparticles

Doping different concentrations of Ce³⁺ ions into the host matrix of NaErF₄ was carried out by using the Liquid-Solid- Solution(LSS)strategyatroomtemperature.Thegrowthmethod

forNaErF₄:y%Ce(y=1,2.5,5,10)cores,andNaYbF₄ andNaLuF₄ shellswasexactlysameasthatusedabove.

ModificationofEr-DCNPswithDye-BPandANG-SH(simplifiedto Er-DCNPs-Dye-BP-ANG)

Tooptimizethedyesensitization,theoleicacidonthesurface ofEr-DCNPswasreplacedwithdifferentratiosofDye-BPthrough ligandexchange,asreportedpreviously[37].Typically,10mgEr- DCNPswererespectivelymixedwithBPandDye-BPinamassratio of50:0;50:10;50:30;50:50mg),ormixedwithDye-BPalone(50, 100,200mg)inTHF(5mL).Thesolutionwasstirredovernight.

Then,thepolymercoatedEr-DCNPswereprecipitatedbyadding cyclohexaneanddriedunderavacuumatroomtemperaturefor 2h.Thedriednanoparticleswereredispersedindeionizedwater (5mL),andtheresultantsolutionwaspurifiedbyultrafiltration threetimeswiththecentrifugalforceof 1500gtoremovefree polymer.

ThesynthesisofANG-SHpeptidefunctionalizedEr-DCNPs-Dye- BPwasconductedreferringtothetypical“clickreaction”.ANG-SH peptide(2mg)andTCEP(1mg)werefirstdissolvedin3.5mLofTris bufferedsalinesolution,followedbytheadditionofEr-DCNPs-Dye- BP(2mg).Afterstirringfor1hatroomtemperature,thereaction solutionwaspurifiedthreetimesbyultrafiltrationwiththecen- trifugalforceof1000gtimestoremovetheTCEPandfreeANG-SH, andthenthefinalEr-DCNPs-Dye-BP-ANGwasobtained.

Characterization

TEMimageswerecollectedwithaFEITecnaiG20transmission electronmicroscopeoperatingwith200kVaccelerationvoltage.

X-rayEnergyDispersiveSpectroscopy(EDS)elementalmapping, high-resolutionTEMandhighangleannulardarkfieldscanning transmission electron microscopy (HAADF-STEM) imaging (Z- contrastimagingmode)wereperformedonaJEOL-ARM200Cold FEGtransmissionelectronmicroscopeoperatingat80kV,equipped witha double spherical aberrationcorrectorsand fitted witha JEOLSDDCENTURIOEDS system.Thefluorescencespectrawere recordedonaFLS980spectrometer(EdinburghInstruments,UK) equippedwithexcitationsources(808nmand980nmcontinuous- wavelaserdiodes).Thehydrodynamicsizewasmeasuredat25^◦C withaMalvernZetasizerNanoZS90equippedwithasolidstate He–Nelaser(␭=633nm).

Animaltumormodel

Allmicewereculturedandexperimentedoninaccordancewith guidelinesapprovedbytheethicscommitteeofSoochowUniver- sity(Soochow,China).Specificpathogenfree(SPF)gradeBALB/c nudemice(6–8weeksold)wereprovidedbythelaboratoryofthe AnimalCenterofSoochowUniversity.Thexenografttumormodels wereestablishedbysubcutaneousinjectionofU87cells(2×10⁶in 50␮LPBS)intotherightflankregiononthebacksofthemice.The tumorimagingwascarriedoutat7–10daysaftertheinoculation oftumorcells.Theorthotopictumormodelswereestablishedwith theinjectionofU87cells(5×10⁵in5␮LPBS)intothestriatumof mice.Theinjectionlocationwassetwiththeassistanceofstereo- taxicapparatus,whichwasbregma1.0mm,rightlateral2.0mm, depth2.5mm.Toobservethegrowthoftumors,anMRS3000scan- ner(3T)wasusedtodepictthesizeanddepthoforthotopicgliomas byT₂-weightedimaging.Theorthotopicgliomabearingmicewere experimentedonafterdifferentnumbersofdays(7,14)afterthe inoculationoftumorcells.

Cytotoxicityassay

Thestandardtetrazoliumdye(MTT)assaywascarriedoutto evaluatethecytotoxicityofEr-DCNPs-Dye-BPandEr-DCNPs-Dye- BP-ANGtowardsU87 cancercells. U87 cells (1× 10⁴ cells per well)were firstseeded into96-well platesand cultured for 24 hin a standard cellmedium at 37 ^◦C in 5 %CO₂ atmosphere.

Then, these cells were washed with phosphate bufferedsaline (PBS)andincubated withEr-DCNPs-Dye-BP andEr-DCNPs-Dye- BP-ANGin differentconcentrations (i.e. 6.25, 12.5, 25, 50, 100, 200,400,and800␮g/mL)for24h.Afterthat,theU87cellswere washedwithPBSsolution,followedbytheadditionofMTT[(3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)]solution (20␮L,5mg/mL).Afterthecellswerefurtherincubatedfor4h andtheculturemediumwasremoved,150␮Ldimethylsulfoxide (DMSO)wasaddedintoeachwell.Theabsorbanceofeachsolu- tionat490nmwasmeasuredby amicroplate reader(Thermo, VarioskanFlash).

Cellularuptake

U87cellswereseededatadensityof2×10⁴–3×10⁴cellsper cellculturedishandincubatedfor24hat37^◦Cunder5%CO₂atmo- sphere.The Er-DCNPs-Dye-BP and Er-DCNPs-Dye-BP-ANGwere dispersedintoDulbecco’sModifiedEagleMedium(DMEM)forcell- culturewithaconcentrationof400␮g/mL,andthenaddedinto theculturedishafterremovaloftheoriginalculturemedium.After co-incubationfor24h,thecellswerewashedtwotimeswithPBS toremovethenon-uptakenanoparticles,andthenfixedwith4% paraformaldehydefor30min.Thecellswerewashedtwicewith PBSsolution,followedbytheadditionof5␮g/mLHoechststainfor stainingthenucleistainingfor10min.Theconfocalfluorescence imagingexperimentswerecarriedoutafterwashingthreetimes withPBS.

SPECT-CTimaging

2.5mCi^99mTcradionuclides(providedbyShanghaiGMSPhar- maceuticalCo.,Ltd.)wereusedtolabeltheEr-DCNPs-Dye-BPfor SPECT-CTimaging.200␮L(2mg/mL)Er-DCNPs-Dye-BPsolution wasadded into a freshly prepared stannous chloride (SnCl2, 1 mg/mLin0.1MHCl)solutioncontaining2.5mCi^99mTcradionu- clidesandreactedfor30minunderambientconditions.Themixed solutionwasultra-filtratedforthreetimestoremovefree^99mTc radionuclidesusing30kDaMilliporemembrane,andfinallabelling yieldwas82.2%.TheresultingEr-DCNPs-Dye-BP-^99mTcsolution was injected into an anaesthetized BALB/c healthy mouse for SPECT-CTimaging,whichwasrecordedwiththeMILabsimaging system.

Focusedultrasoundsonication

Anultrasoundtransducer(0.5MHzinfrequencyand30mmin diameter)drivenbyafunctiongeneratorandconnectedtoapower amplifierwasusedtoopentheBBBofmicebearing orthotopic gliomas.Theacousticparametersweresetat0.5MPainacoustic pressure,0.5MHzinfrequency,1msinpulseinterval,and90sfor sonication.50␮Lofmicrobubblesolution(∼1×10⁹bubbles/mL) wasintravenouslyinjectedintothemicebeforeultrasoundsonica- tion.

In-vivoNIRIIfluorescenceimaging

In-vivoNIRIIfluorescenceimagingwasconductedwithaNIRII ImagingSystem(SeriousII900–1700,SuzhouNIR-OpticsCo.,Ltd.).

Thepowerdensityofthe808nmlaserwassetto100mW/cm²for

thesubcutaneousgliomatumormodeland120mW/cm²forthe orthotopicgliomatumorimagingand200mW/cm²forimaging- guided surgery. The mice were anaesthetized with isoflurane, placedinananimalbed,andthenintravenouslyinjectedwithEr- DCNPs-Dye-BPandEr-DCNPs-Dye-BP-ANGinphysiologicalsaline solutionwitha dose of12 mg/kg for the subcutaneousglioma tumormodel,and20mg/kgfortheorthotopicgliomatumormodel, respectively.TheNIRIIfluorescenceimageswerecollectedatdif- ferenttimepointspostinjection.ForthemicetreatedwithFUS, theirNIRIIfluorescenceimageswerecollectedat1minpostinjec- tionofnanoprobes.

Authorstatement

F.R.performedthepreparation,modification,andcharacteriza- tionofnanoprobes, anddraftedthemanuscript.H.L.,H.Z.,and Z.J.performedthecelland animalexperiments.B.X. andT.H.

synthesizedandcharacterizedtheblockpolymers.C.G.andM.A.

performedtheTEManalysisofsamples.Z.L.,Q.S.,andM.G.didthe criticalrevisionsofthemanuscript.

DeclarationofCompetingInterest

Theauthorsdeclarenoconflictofinterest.

Acknowledgements

Z.LiacknowledgessupportfromtheNationalNaturalScience FoundationofChina(81971671,81527901),NationalKeyResearch andDevelopment ProgramofChina(2018YFA0208800), Jiangsu ProvincialKeyResearchandDevelopmentProgram(BE2019660).

TheauthorsalsoaregratefulforsupportfromtheJiangsuProvincial KeyLaboratoryofRadiationMedicineandProtection,thePriority AcademicDevelopmentProgramofJiangsuHigherEducationInsti- tutions(PAPD).TheauthorswouldliketothankDr.TaniaSilver forhelpfuldiscussionofthemanuscript.Thisprojectisco-funded bytheEuropeanUnion,theRégion–CentreValdeLoireandThe Frenchministerofresearch(MESRI –DRRT).Europeiscommit- tedtotheCentre-ValdeLoireregionwiththeEuropeanregional developmentfund(ERDF).

AppendixA. Supplementarydata

Supplementarymaterial relatedto this articlecanbe found, intheonlineversion,atdoi:https://doi.org/10.1016/j.nantod.2020.

100905.

References

[1]J.Y.Lee,J.P.Thawani,J.Pierce,R.Zeh,M.Martinez-Lage,M.Chanin,O.Venegas, S.Nims,K.Learned,J.Keating,S.Singhal,Neurosurgery79(2016)856–871.

[2]C.Li,L.Cao,Y.Zhang,P.Yi,M.Wang,B.Tan,Z.Deng,D.Wu,Q.Wang,Small11 (2015)4517–4525.

[3]D.Y.Zhang,S.Singhal,J.Y.K.Lee,Neurosurgery85(2019)312–324.

[4]P.L.Kubben,K.J.terMeulen,O.E.Schijns,M.P.terLaak-Poort,J.J.van Overbeeke,H.vanSantbrink,LancetOncol.12(2011)1062–1070.

[5]M.F.Kircher,J.K.Willmann,Radiology264(2012)349–368.

[6]M.M.Kim,A.Parolia,M.P.Dunphy,S.Venneti,Nat.Rev.Clin.Oncol.13(2016) 725–739.

[7]W.Stummer,U.Pichlmeier,T.Meinel,O.D.Wiestler,F.Zanella,R.

Hans-Jurgen,LancetOncol.7(2006)392–401.

[8]P.Schucht,K.Seidel,J.Beck,M.Murek,A.Jilch,R.Wiest,C.Fung,A.Raabe, Neurosurg.Focus37(2014)E16.

[9]J.X.Jiang,J.J.Keating,E.M.Jesus,R.P.Judy,B.Madajewski,O.Venegas,O.T.

Okusanya,S.Singhal,Am.J.Nucl.Med.Mol.Imaging5(2015)390–400.

[10]R.Zeh,S.Sheikh,L.Xia,J.Pierce,A.Newton,J.Predina,S.Cho,M.Nasrallah,S.

Singhal,J.Dorsey,J.Y.K.Lee,PLoSOne12(2017),e0182034.

[11]W.Stummer,S.Stocker,S.Wagner,H.Stepp,C.Fritsch,C.Goetz,A.E.Goetz,R.

Kiefmann,H.J.Reulen,Neurosurgery42(1998)518–526.

[12]C.Li,Q.Wang,ACSNano12(2018)9654–9659.