Administration-dependant efficacy of ferrociphenol lipid nanocapsules for the treatment of intracranial 9L rat gliosarcoma

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International Journal of Pharmaceutics

j ou rn a l h o m e pag e :w w w . e l s e v i e r . c o m / l o c a t e / i j p h a r m

Administration-dependent efficacy of ferrociphenol lipid nanocapsules for the treatment of intracranial 9L rat gliosarcoma

Ngoc Trinh Huynh

, Catherine Passirani

, Emilie Allard-Vannier

, Laurent Lemaire

, Jerome Roux

, Emmanuel Garcion

, Anne Vessieres

, Jean-Pierre Benoit

aLUNAMUniversité,IngénieriedelaVectorisationParticulaire,F-49933Angers,France

bINSERMU646,F-49933Angers,France

cServiceCommund’AnimalerieHospitalo-Universitaire(SCAHU),AngersF-49100,France

dCNRS,UMR7223,EcoleNationaleSupérieuredeChimiedeParis,F-75231,France

a r t i c l e i n f o

Articlehistory:

Received11February2011 Receivedinrevisedform8April2011 Accepted15April2011

Available online 21 April 2011

Keywords:

Braintumour DSPE-mPEG2000

Convection-enhanceddelivery(CED) Intra-carotidinjection

PEGylatednanoparticles

a b s t r a c t

Theanti-tumoureffectofferrociphenol(FcdiOH)-loadedlipidnanocapsules(LNCs),withorwithouta DSPE-mPEG2000coating,wasevaluatedonanorthotopicgliosarcomamodelafteradministrationby convection-enhanceddelivery(CED)techniqueorbyintra-carotidinjection.Notoxicitywasobserved byMRI norby MRS in healthyrats receiving a CEDinjection ofFcdiOH-LNCs (60␮L, 0.36mg of FcdiOH/rat)whenthepHandosmolarity hadbeenadjustedtophysiologicalvaluespriortoinjec- tion.Atthisdose,thetreatmentbyCEDwithFcdiOH-LNCssignificantlyincreasedthesurvivaltimeof tumour-bearingratsincomparisonwithanuntreatedgroup(28.5daysvs25days,P=0.0009)whereas DSPE-mPEG2000–FcdiOH-LNCsdidnotexhibitanyefficacywithamediansurvivaltimeof24days.

Afterintra-carotidinjection(400␮L,2.4mgofFcdiOH/rat),hyperosmolarDSPE-mPEG2000–FcdiOH- LNCsmarkedlyincreasedthemediansurvivaltime(upto30days,P=0.0008)ascomparedtothecontrol (20%).Thiswasstrengthenedbytheirevidencedaccumulationinthetumourzoneandbythemeasure ofthefluorescentbrainsurfaceobtainedonbrainslidesfortheseDiI-labelledLNCs,being3-foldhigher thanforthecontrol.Theseresultsdemonstratedthat,dependingupontheadministrationrouteused, thecharacteristicsofLNCsuspensionshadtobecarefullyadapted.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Theincidencerateofbraintumourshasincreasedremarkably and tended to be higher in developed, industrialised countries (Parkinet al.,2005).Amongthevarioustypesof braintumour, glioblastomasrepresentthemostprevalentandmalignantgliomas inadults(gradeIVclassifiedbytheWorldHealthOrganization– WHO)(Louisetal.,2007).Inspiteofnumerous,notableadvances indiagnosistechniquesandalsoinmultimodaltherapyregimes includingsurgicalresectionfollowedbyradiotherapyandadjuvant chemotherapy(Stuppetal.,2005),thesurvivalprognosisalways remains unfavourable,mostpatientsdying within2 yearsafter diagnosis(GrossmanandBatara,2004).Intermsofchemotherapy, theeffortindrugdiscoveryanddevelopmenthasbeenlimiteddue totheobstacleoftheblood–brainbarrier(BBB)whichpreventsthe deliveryofdrugstothecentralnervoussystem(CNS)bymeansof asystemicinjection(Pardridge,2005;Segal,2000).

∗Correspondingauthorat:LUNAMUniversité,IngénieriedelaVectorisationPar- ticulaire,F-49933Angers,France.Tel.:+33244688534;fax:+33244688546.

E-mailaddress:catherine.passirani@univ-angers.fr(C.Passirani).

Overthelastdecades,thetechniqueofdrugdeliverytothebrain byconvection-enhanceddelivery(CED)hasprovidedaninterest- ingtool ofadministrationforbraincancerchemotherapy(Bobo etal.,1994).Thistechniqueconsistsinthecontinuousinjection ofatherapeuticfluidunderpositivepressure.CEDinjectionallows alocaladministrationoflargedosesofinfusatewhichismainly distributedwithintheinterstitialspacesoftissuesbyagradient ofpressurethatenhancesconstitutiveintracellularmediumcon- vection(Allardetal.,2009b).Indeed,byusinglipidnanocapsules (LNCs)whicharecharacterisedbyahybridstructurebetweenpoly- mernanocapsulesandliposomes(Huynhetal.,2009),aprevious study demonstrated the wide distribution areathroughout the injectedhemisphere(Vinchon-Petitetal.,2010).Homogeneityof NileRed-labelledLNCsaroundtheinjectionsitewasobservedfor 24hfollowingaCEDinjectionintothebrainofhealthyrats.More- over,on6-day-old9Ltumour-bearingrats,NileRed-labelledLNCs fullycoveredthetumourzoneandwerefoundinthecytoplasmof tumourcells.

Recently,anewseriesoforganometallictamoxifenderivatives havebeendevelopedbyaddingapotentiallycytostaticferrocene moietytothetamoxifenskeleton(Jaouenetal.,2000a;Topetal., 2003;Vessieresetal.,2005).Themechanismoftheseferrocene 0378-5173/$–seefrontmatter© 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijpharm.2011.04.037

derivativesisbelievedtoactasanintramolecularcatalystforthe oxidationofthephenolgroupinmildconditions,leadingtothe productionofquinonemethideswhicharestronglytoxic(Hillard et al., 2005). These last compounds may interact with macro- moleculessuchasglutathione,DNAandcertainproteinsleading tothetumour cell’sdeathbyaccelerated senescence(Vessieres etal.,2005).Amongthesederivatives,thecompound2-ferrocenyl- 1,1-bis(4-hydroxyphenyl)-but-1-ene,orferrociphenolcompound (FcdiOH)hasprovedinvitrotobeaneffectivecytostaticcompound (IC₅₀=0.7␮MonMCF-7hormone-dependentand IC₅₀=0.44␮M onMDA-MB231hormone-independent breast cancercelllines) (Vessieresetal.,2005).FcdiOHloadedintotheoilycoreofLNCs (FcdiOH-LNCs)athighconcentrations(6.5mg/g,2%driedweight) showed remarkable antitumour effects in a subcutaneous, 9L gliosarcoma model after an intra-tumoural injection (Allard et al.,2008b).FcdiOH-LNCsimprovedthesurvivaltimeofratsinan intracraniallyimplanted9Lgliosarcomamodelwhenadministered byCEDinadose-dependentmanner(Allardetal.,2009a).More- over,asynergisticeffectwasalsoobservedbetweenchemotherapy withFcdiOH-LNCs and external irradiationrevealing theradio- sensitive properties of FcdiOH (Allard et al., 2010). However, someneuronalsideeffects intheinjectedhemisphereobserved in long-term survivors receiving the highest LNC suspension concentrationhavebeenreportedpreviously(Allardetal.,2008a).

ThisstudyaimedatevaluatingtheimpactofFcdiOH-LNCson the rat brain after CED administration by magnetic resonance imagingbeforeandafter someadjustments assessedasneeded foreffectiveandsafeadministration.Furthermore,theantitumour efficacyof thesemodified FcdiOH-LNCsdelivered byCED tech- niqueonan intracranial gliosarcoma rat model wasevaluated.

Asanalternativerouteofdrugadministrationtothebrain(Alam etal.,2010), intra-carotidinjectionofFcdiOH-LNCsthroughthe carotidarterywasinvestigatedinordertoincreasethevolumeof drugadministered.Moreover,theefficacyofFcdiOH-LNCscoated withlongerchainsofpolyethyleneglycol(PEG)wasalsoassessed inordertotesttheeffectoflong-circulatingpropertiesbroughtby thispolymer(Morilleetal.,2010).

2. Materialsandmethods

2.1. Materials

Ferrociphenol, abbreviated as FcdiOH, was prepared by a McMurrycouplingreaction(Jaouenetal.,2000b).Thelipophilic Labrafac^® CC (caprylic-capric acid triglycerides) was pur- chased from Gattefosse S.A. (Saint-Priest, France). Lipoïd^® S75-3 (soybean lecithin at 69% of phosphatidylcholine) came from Lipoïd Gmbh (Ludwigshafen, Germany); Solutol^® HS15 (a mixture of free polyethylene glycol 660 and polyethy- lene glycol 660 hydroxystearate) from BASF (Ludwigshafen, Germany) and NaCl fromProlabo (Fontenay-sous-bois, France).

Deionisedwaterwasacquiredfroma Milli-Qplussystem(Mil- lipore, Paris, France) and sterile water from Cooper (Melun, France). 1,2-Distearoyl-sn-glycero-3-phospho-ethanolamine-N- [methoxy-(polyethyleneglycol)-2000] (DSPE-mPEG2000)(mean molecularweight(MMW)=2805g/mol)waskindlyprovided by AvantiPolarLipids (Alabaster,USA).1,10-Dioctadecyl-3,3,30,30- tetramethylindocarbocyanineperchlorate(DiI)wasobtainedfrom Introgen(Cergy,Pontoise,France).

2.2. PreparationoftheLNCs 2.2.1. Anticanceragent-loadedLNCs

In this study, fifty-nanometer diameter LNCs, whose for- mulation is based on the phase-inversion phenomenon of a

microemulsion,werepreparedinconformitywiththedescribed procedure(Heurtaultetal.,2002).Briefly,thepreparationprocess involved2 steps.StepIconsistedof mixingallthecomponents (Solutol^® HS15 (17% w/w),Lipoid^® (1.5%w/w), Labrafac^® (20%

w/w),NaCl(1.75%w/w)andwater(59.75%w/w))undermagnetic stirringandheatingfromroomtemperatureto85^◦C.Threecycles ofprogressivecoolingandheatingbetween85and60^◦Cwerethen carriedout.StepIIwasanirreversibleshockinducedbysudden dilutionwithcoldwater(70%v/v)tothemixtureat70–72^◦C.Slow magneticstirringwasthenappliedtothesuspensionfor5min.

ToloadtheanticanceragenttotheoilycoreofLNCs,FcdiOHwas firstlydissolvedinLabrafac^®underultrasoundat4%(w/w)for1.5h andtheresultinglipophilicphasewasafterwardsmixedwithother componentsasdescribedabove.Moreover,inthisstudy,inorderto increasetheconcentrationofthedruginthefinalLNCsuspension, thecoldwateraddedtothemixtureatthestepIIwasdecreased from70%(v/v)forconventionalformulations(111mgofparticles pergramofsuspension)to28.5%(v/v)fortheconcentratedones (280mg/g).FinalLNCsuspensionswerefilteredthroughaMinisart 0.2␮mfilter(Sartorius)inordertoeliminatesomeunincorporated products.

2.2.2. FluorescentLNCs

To visualise the LNCs on the brain slides, LNCs were labelledwithDiI(emissionwavelength=549nm;excitationwave- length=565nm).TheformulationoffluorescentLNCswassimilar tothatdescribedpreviously(Garcionetal.,2006).Briefly,DiIwas dissolvedin acetoneat3mg/mland theresultingsolution was incorporatedintheLabrafac^®atproportionof1:30(v/v).Acetone solventwasthenevaporatedbeforemixingwithothercomponents toformulatefluorescentLNCs.

2.2.3. Surface-modifiedLNCs

DSPE-mPEG2000wasincorporatedtothesurfaceofLNCsatthe concentrationof10mMbythepost-insertiontechniquepreviously described(Morilleetal.,2010).Briefly,preformedLNCsuspensions andDSPE-mPEG2000micelleswereco-incubatedfor2hat60^◦C.

Themixturewasvortexedevery15minandthenquenchedinan icebathfor1min.

2.2.4. AdjustmentofthepHandosmolarityoftheLNC suspensions

IncertainexperimentalproceduresofCEDinjection,thecon- centratedLNCsuspensions(referredtoascrudesuspensions)were passedthroughaPD-10sephadexcolumn(AmershamBiosciences Europe,Orsay,France) andthen concentratedbyultra-filtration withMilliporeAmicon100kDacentrifugalfilterdevice(Millipore, St.Quentin-Yvelines,France).Theosmolarityoftheresultingsus- pensionswasthenadjustedbyaddinga5Msalinesolution.Finally, theaciditywasneutralisedbya0.1NNaOHsolutioninorderto obtainadjustedLNCs.

2.3. CharacterisationofLNCs

2.3.1. Meanparticlesizeandzetapotential

TheLNCswerediluted1:100(v/v)indeionisedwaterandthe measurementswereperformedat25^◦C.TheLNCswereanalysed intriplicatefortheirmeanparticlediameter,polydispersityindex (PdI)andsurfacechargeusingaMalvernZetasizer^®(NanoSerie DTS1060,MalvernInstrumentsS.A.,Worcestershire,UK).

2.3.2. OsmolarityandpHmeasurement

Themeasurement ofthepHand osmolarityoftheLNC sus- pensions was performed by using a Consort C561 pH meter

fromAvantec(FisherBioblock,Scientific)and5520Vaprovapour osmometerfromWescor(Logan,Utah,USA),respectively.

2.4. Invivostudies 2.4.1. Animals

SyngeneicFischerF344femalerats,weighing160–180gwere obtained from Charles River Laboratories France (L’Arbresle, France).Allexperimentswereperformedon10-to11-week-old femaleFisherrats.Animalcarewascarriedoutinstrictaccordance withFrenchMinistryofAgricultureregulations.

2.4.2. Tumourcellculture

Rat9LgliosarcomacellswereobtainedfromtheEuropeanCol- lection of Cell Culture (Salisbury, UK, No. 94110705). The cells wereculturedat37^◦Cinahumidifiedatmospherecontaining5%

CO₂inDulbecco’smodifiedEagle’smedium(DMEM)withglucose andl-glutamine(BioWhittaker,Verviers,Belgium)suppliedwith 10%foetalcalfserum(FCS)(BioWhittaker)and1%antibioticand antimycoticsolution(Sigma,Saint-QuentinFallavier,France).

2.4.3. Intra-cranialgliosarcomamodel

On the day of implantation, a cultured tumour monolayer was detached by using a mixture of trypsin–ethylene diamine tetraaceticacid,then,washedtwicewithEagle’sminimalessen- tial medium (EMEM) without FCS and antibiotics. Cells were counted,andre-suspendedinEMEMtothefinalconcentrationof 10⁵9Lcells/mlforimplantation.

Theanimalswereanaesthetisedbyintra-peritonealinjectionof 1.0ml/kgofa1:1mixtureofketamine(100mg/ml)(Clorketam^®, Vétoquinol, Lure, France) and xylazine (20mg/ml) (Rompun^®, Bayer,Puteaux,France).Theincisionsitewasshavedandthehead wasimmobilisedinastereotaxicframe(LabStandardStereotaxic;

Stoelting,Chicago,IL).A middlescalp incisionwasmadeand a burrholewasdrilledintotheskullusingasmalldentaldrill.The cannulacoordinateswere1mmposteriorfromthebregma,3mm lateralfromthesagittalsuture,and5mmbelowthedura(with theincisorbarsetat0mm).The32Gneedle(Hamilton^®),fittedto a10␮lsyringe(Hamilton^®glasssyringe700seriesRN),wasleft inplacefor5minandthen10␮lof10³ 9Lcellsuspensionwere injectedstereotaxicallyintotherightratstriatumataflowrateof 2␮l/min.Theneedlewasleftinplacefor5additionalminutesto avoidexpulsionofthesuspensionfromthebrainduringremovalof thesyringe,whichwaswithdrawnveryslowly(0.5mm/min).The scalpincisionwasthenclosedbyusingasilksuture(Perma-Hand Seide3-0,Ethicon).

OnDay6after9Lcellimplantation,ratswereinjectedbymeans ofCEDorintra-carotidinjectionofdifferentLNCformulations.

2.4.4. CEDprocedure

Tumour-bearing-ratswereanaesthetisedbyanintra-peritoneal injectionof1.5ml/kgofa2:1mixtureofketamineandxylazine.

Thesameprocedureofinjectionasdescribedfor cellimplanta- tionwasapplied.The 32Gneedle (Hamilton^®)fitted toa 10␮l Hamilton^®syringewasplacedinthesamecoordinates.Thissyringe wasconnectedtoa100␮lHamiltonsyringe22G(HarvardAppa- ratus,LesUlis,France)containingtheproductthroughacannula (CoExTMPE/PVCtubing,Harvardapparatus,LesUlis,France).Sixty microlitres(60␮l)ofLNCsuspensionwereinjectedbyCEDwhich wasperformedwithapumpPHD2000infusion(HarvardAppara- tus,LesUlis,France)bycontrollinga0.5␮l/minratefor2h.The needlewasleftinplacefor5additionalminutestoavoidexpulsion ofthesuspensionfromthebrainduringremovalofthesyringe, whichwaswithdrawnveryslowly(0.5mm/min).

2.4.5. Intra-carotidinjection

Tumour-bearing-ratswereanaesthetisedbyanintra-peritoneal injectionof1.0–1.5ml/kgofa1:1solutionofketamine(100mg/ml) (Clorketam^®, Vétoquinol,Lure, France) and xylazine(20mg/ml) (Rompun^®,Bayer, Puteaux, France). The middle neck skin was shavedandincised.Therightcommoncarotid(thesamesideof9L cellimplantation)wasexposedandligated.APE10polyethylene catheter(BDIntramedic^TMPolyethyleneTubing,BectonDickinson, USA)wasinsertedretrogradelythroughasmallarteriotomy.Four hundredmicrolitresofLNCsuspensionboluswereinjectedininto eachrat.Thecatheterwasthenremovedandthecarotidarterywas ligated.

2.4.6. Sideeffectmonitoringbymagneticresonanceimagingand

1H-magneticresonancespectroscopy

SixtymicrolitresofFcdiOH-LNCswereadministeredbyCED(as describedinSection2.4.4)tohealthyratsatthesamecoordinates oftumourimplantation.Magneticresonanceimaging(MRI)and¹H magneticresonancespectroscopy(MRS)weretakenonDays14,42, 70andDay100afterCEDinjection,tomonitorsideeffectsofcrude suspensionandadjustedsuspensionofFcdiOH-LNCswithphysio- logicalosmolarityandpHvalues.MRIwasperformedonaBruker AvanceDRX300equippedwitha verticalsuperwide-bore mag- netoperatingat7T.RapidqualitativeT2-weightedimageswere obtained using rapid acquisition with relaxation enhancement (RARE) sequence (TR=2000ms; meanecho time (TE)=31.7ms;

RAREfactor=8;FOV=3cm×3cm;matrix128×128;ninecontigu- ous slicesof 1mm, eightacquisitions). ¹H MRSwas performed usingaPRESSsequencewithwatersuppressionunderthefollow- ingparameters:TR/TE=1500/11ms; NEX=128;voxel size27␮l (3mm×3mm×3mm), as already determined (Lemaire et al., 1999).

2.4.7. VisualisationoffluorescentLNCsinratbrain

ForthepurposeoftrackingtheinjectedLNCs,theywereloaded with a fluorescent agent (DiI) instead of an anticancer agent (FcdiOH). DiI-LNCs or DSPE-mPEG2000–DiI-LNCs (400␮l) were administeredintotumour-bearingratsthroughtherightcommon carotidartery.Untreatedtumour-bearingratsservedasthecon- trolgroup.Theratswereeuthanised24hafterinjectioninaCO₂ chamber.Thebrainwasremovedandthensnap-frozeninliquid nitrogen-chilledisopentaneandstoredat−80^◦C.Coronalcryosec- tions(14␮m)throughoutthetumourzonewereperformedand recoveredonslides.Theslideswerekeptatleast24hat−20^◦C beforebeingprocessed.

Frozensectionsweredefrostedatroomtemperatureandrehy- dratedinDulbecco’sphosphate-bufferedsaline(DPBS).Sections werefixedin4%paraformaldehydeandwashedwithDPBS.Slides wereobservedunderanAxioskop-2Zeissfluorescentmicroscope (LePeck,France)usinga20×objective.Theacquiredimageswere analysedbymeasuringthefluorescentsurfaceareaonthebrain tissueslidesviaintegratedmorphometryanalysisofaninclusive thresholdimagebyuseoftheMetamorph^®software(RoperSci- entific,Evry,France). Inorder tobettervisualise thenucleusof thetumourcells,somesectionswerefurtherstainedwith1/1000 4,6-diamidino-2-phenylindole(DAPI)inDPBSfor10minandthen washedwithDPBSbeforemounting.

2.4.8. Efficacystudy

The treatment was assessed by CED administration (60␮l, 0.36mgofFcdiOH/rat),orintra-carotidinjection(400␮l,2.4mgof FcdiOH/rat)withFcdiOH-LNCsorDSPE-mPEG2000–FcdiOH-LNCs.

ForCEDinjection,theosmolarityandpHoftheLNCsuspensions wereadjustedtophysiologicalvalueswhereasthecrudesuspen- sionswereusedforintra-carotidinjection.Theuntreatedcontrol groupdidnotreceiveanytreatment.Animalswereweighedevery

6days.TheanimalsweresacrificedinaCO₂chamberwhenthey lost20%ofbodyweightand/orassoonastheypresentedseizure, ahunchedposture,orhaemorrhagingaroundtheeyes,mouthand nose.Thedeathwasrecordedasifithadoccurredonthenextday ofsacrificeandwasrepresentedasthesurvivaltimeofanimalson theKaplan–Meiercurves.

Thestatisticalanalysiswasestimatedfromthelog-ranktest (Mantel-CoxTest)byusing StatView software,version5.0 (SAS instituteInc.).ThelevelofsignificancewassetatP<0.05.Thediffer- enttreatmentgroupswerecomparedintermsofmedianandmean survivaltime(days).Thepercentageofincreaseinsurvival time (%IST)wasdeterminedrelativetothemedianandmeansurvival timesoftheuntreatedcontrolgroupaspresentedinthefollowing equation:

% IST= [Median_T(Mean_T)−Median_C(Mean_C)]

Median_C(Mean_C)

whereMedianT/MeanTwasthemedian/meanofsurvivaltimeof treatedgroupwhileMedian_C/Mean_Cwasthemedian/meanofsur- vivaltimeofcontrolgroup.

3. Resultsanddiscussion

3.1. PhysicochemicalpropertiesofLNCsuspensions

ThephysicochemicalpropertiesofdifferenttypesofLNCsare presented in Table 1. All types of LNCs had theiraverage size ranging44–53nmwithvery narrowsizedispersion(PdI<0.08).

LNCswerecharacterisedbyaslightlynegativezetapotential(from

−3to−5mV),exceptforDSPE-mPEG2000–FcdiOH-LNCsthatpre- senteda zetapotentialofbout -22mVduetotheformation of dipolesbetweenPEGmoleculesandwater,asalready described (Vonarbourgetal.,2005).TheencapsulationofFcdiOHdidnotaffect theaverageparticlesizeandzetapotentialaspreviouslyreported (Allardetal.,2008b).ThisdenotedefficientencapsulationofFcdiOH intheoilycoreofLNCs.

Thereduction of cold water added tothe final temperature cycleallowedformulating FcdiOH-LNCsat a highdrugconcen- tration(6.5mgofFcdiOHpergofLNCsuspension,corresponding to2%w/wdryweight).Thismodificationdidnotaltertheparti- clesizenorthezetapotentialofresultingLNCscomparedtothe conventionalones(Table1).However,concentrated blankLNCs as wellas FcdiOH-LNCs werehyperosmolar (780.25±7.80 and 773.50±7.05mmol/kg,respectively)whereasconventionalblank LNCswerehypoosmolar(227.50±2.12mmol/kg).Withrespectto thesuspensionacidity,allLNCsuspensionspresentedanacidpH level (pH=5.3–5.7) and there was no differencebetween con- ventionalformulationsandconcentratedones.Afteradjustments concerningosmolarityandpH,measurementofparticlesizeand zetapotentialshowednochangesintheseparameters(datanot shown).

3.2. DrugdeliverybymeansoftheCEDtechnique

3.2.1. Monitoringofsideeffectsbymagneticresonanceimaging Inapreviousstudyreportingthetreatmentof9Lgliosarcomain ratsbyCEDinjectionofaconcentratedsuspensionof¹⁸⁸Re-loaded LNCs, the T2-weighted images taken from long-term survivors showed a hyper-intense signal, which wasmainly localised in thestriatumandpersisteduntilDay140ofexperiments(Allard etal.,2008a).Moreover,¹H MRSshoweda decreaseinthesig- nalintensityforN-acetylaspartate(NAA)andcreatine(Cr).NAAis consideredasavaluablemarkerofbraininjury(Demougeotetal., 2001).AdecreaseinNAAlevelsdetectedfromMRSsuggestsneu- ronal/axonalloss,orcompromisedneuronalmetabolism(Moffett etal.,2007;Schuffetal.,2006).Theauthorspostulatedthatthis

lesionwasrelatedtointernalradiationinjury.Unfortunately,these sideeffectswerealsoobservedinalong-termsurvivorratthatwas treatedwithconcentratedsuspensionsofFcdiOH-LNCsinaprevi- ousstudyconcerningtumour-bearingrats(unpublisheddata).

Therefore,thefirstsetofexperimentsinthepresentstudywas toinvestigatewhethercrude FcdiOH-LNCsaltered theneuronal metabolismandcausedthesideeffectsonhealthyratsafteraCED administration.T2-weightedimagesshowedthattheinjectionof suchasuspensioncausedalesion,highlightedbyahyper-intense signalregionontheinjectedhemisphere untilDay70 (Fig.1A) andcharacterisedbyasignificantdecreaseinNAAandCrinten- sityoftheinjectedhemisphereascomparedtothecontralateral hemisphereonprotonspectra(Fig.1B).Ontheotherhand,two highpeaksaround0.9–1.5ppmwereobservedfromthespectrum takenonDay14post-injection,whichprobablycorrespondedto thepeaksofLNClipids.However,thesepeaksnearlydisappeared bythefollowingreading(Day42).Thisunderlinedthemetabolism ofLNCsduring6weeksafterinjectionintothehealthyratstria- tum.Moreover,theseneuronallesionswerealsoobservedinrats receivingblankLNCswithsimilarhyper-intensesignalregionon theinjectedhemisphereonMRIand aconsiderabledecreasein NAAandCrlevelson¹HMRS(figuresnotshown).Therefore,such sideeffectscouldberelatedtotheintrinsicpropertiesofLNCsus- pensions.

Indeed,administratingahyperosmoticinfusateintotheextra- cellular space produces the movement of water from the intracellularspacetotheextracellularspace,leading tocellular dehydration.Thiseffectpotentiallyinducesbraintissuedamage, resultinginneurologicaldysfunction(Petitetal.,2006;Verbalis, 2010).Amarkedcellvolumedecreasewasobservedwhenincu- bating cultured human astrocytes with hyperosmotic medium (Anderson et al., 2000). Luh et al. demonstrated that thepro- longedexposureofendothelialcells toahyperosmolarmedium (>460mOsm/L)significantlyreduceditsviabilityandfunction(Luh etal.,1996).Therefore,thesepreviouslyobservedsideeffectscould belinkedtothehyperosmolarityofcrudeLNCsuspensionwhich was2.5-foldhigherthanphysiologicalosmolarity.

Asaconsequence,forasafelocaladministration,weadjusted theirosmolarityandpHtophysiologicalconditionsasdescribed inSection2.2.4.Effectively,thisformulationdidnotdisturbthe normal condition of the brain environment since MRI showed alimited hyper-intensesignalzone ontheinjectedhemisphere (Fig.1C)associatedwithnodifferenceinNAAandCrsignalintensity between2hemisphereson¹HMRS(Fig.1D).

3.2.2. EfficacyofCEDtreatment

Thesurvivaldatafromtreatedtumour-bearingratsaresum- marised inTable 2 andKaplan–Meier survival plots are shown inFig.2.Byapplyingthecriteriaforeuthanasiaofexperimental animals,ratsweresacrificedwhentheylost20%ofbodyweight associatedwithahighdegreeofdepression.Alluntreatedcontrol ratsweresacrificedfromDay22toDay26after9Lcellimplantation resultinginamedianandmeansurvivaltimeof25days.

ThetreatmentwithadjustedFcdiOH-LNCs(60␮l,0.36mg/rat) bymeansofCEDsignificantlyincreasedthemedianandthemean survivaltimeofanimalsascomparedtothecontrolgroup(28.5 and30days,respectively).Onthecontrary,treatmentwithDSPE- mPEG2000–FcdiOH-LNCsin thesame dosedidnotimprovethe survivalofanimalsastheirmedianremainedat24days.

ThefailureofDSPE-mPEG2000–FcdiOH-LNCstreatmentcould beexplained bytheabundantpresence of PEG,especially with thelongchainsofPEGatthenanoparticulatesurface.Indeed,PEG coatingpotentiallyinhibitstheinternalisationofdrug-loadedNPs bytumourcellsbecauseitisknowntopreventtheinteractions betweentheNPsandthetumourcellsurface(Hongetal.,1999;

Morilleetal.,2009).Moreover,themoderatedefficacyofFcdiOH

Table1

PhysicochemicalpropertiesofLNCsuspensions.

Meanparticlesize(nm) Poly-dispersityPdI Zetapotential(mV)

Conventionalformulation BlankLNCs 49.13±2.18 0.026±0.012 −3.38±0.42

Concentratedformulation BlankLNCs 49.01±2.18 0.029±0.009 −3.82±0.21

FcdiOH-LNCs 44.10±0.91 0.025±0.007 −5.05±1.61

DSPE-mPEG2000–FcdiOH-LNCs 49.08±0.15 0.080±0.006 −21.67±1.10

DiI-LNCs 49.94±0.22 0.040±0.008 −3.28±0.10

DSPE-mPEG2000–DiI-LNCs 53.22±1.40 0.041±0.009 −22.50±1.11

byCEDadministrationmightbelinkedtothedoseofdrugusedin thisstudybeingnotenoughhigh.Indeed,aspreviouslyobserved, FcdiOHexhibiteda cytostaticactivityinvivoontheintracranial 9Lgliosarcomamodelinratsinadose-dependentmanner(Allard

etal.,2009a).Inthispreviouswork,thetreatmentbyCEDofFcdiOH dissolvedinLabrafac(2.4mg/rat)atitslimitedsolubility(40mg/g) gained the median survival time up to31 days. Unfortunately, some toxic signs were observed in rats treated withdrug-free

Fig.1.RepresentativeMRIand¹HMRStakenfromhealthyratsthatreceivedcrudeFcdiOH-LNCs(A,B)andadjustedFcdiOH-LNCs(C,D).CEDinjectionofcrudeFcdiOH-LNCs causedalesionontheinjectedhemisphereasseenonT2-weightedimages(A).ThelesionswerecharacterisedbyadecreaseinNAAandCrintensity(i)ascomparedto thecontralateralhemisphere(ii)thatwaspresentedbythedifferenceinsignalintensitybetween2hemipheres(iii)onthe¹HMRS(B).Byadjustingtheosmolarityand pHofFcdiOH-LNCstothephysiologicalvalues,CEDinjectionofadjustedFcdiOH-LNCsshowedalimitedhyper-intensesignalontheinjectedhemisphereonT2-weighted images(C)andnodifferenceinNAAandCrintensitywasfoundin¹HMRSbetweenthe2hemispheres(D).SpectratakenonDay14revealed2peaksofLNCs(circle)which disappearedonDay42.

Table2

EfficacyofFcdiOH-LNCsandDSPE-mPEG2000–FcdiOH-LNCsinanintracranial9LgliosarcomamodelinFisherrats(IST:increasedsurvivaltime).

Administrationroute(injecteddoseofFcdiOH) Treatment n Survivaltime(days) %IST

Range Median Mean±SD Median Mean

Convection-enhanceddelivery(0.36mg/rat) FcdiOH-LNCs 8 25–36 28.5 30.13±3.98 14.00 19.90

DSPE-mPEG2000–FcdiOH-LNCs 8 20–29 24 23.75±3.01 – –

Intra-carotid(2.4mg/rat) FcdiOH-LNCs 7 25–30 27 27.57±2.07 8.00 10.29

DSPE-mPEG2000–FcdiOH-LNCs 8 26–33 30 29.38±2.50 20.00 17.50

Untreatedcontrol 8 23–27 25 25.13±1.25 –

Fig.2.Kaplan–Meiersurvivalplotsfor9Lglioma-bearingratsreceivingCEDinjec- tionofDSPE-mPEG2000–FcdiOH-LNCs,FcdiOH-LNCs (60␮lofLNCsuspension, correspondingto0.36mgofFcdiOHperrat)atDay6aftercellimplantationand untreatedanimals.

LabrafacshowingthelimitationofclinicaltolerancetoLabrafac wheninfused alone.Thus, takingintoconsideration thebenefit ofLNCsinimprovingintracellularbioavailability(Garcionetal., 2006),theseresultsrevealthenecessityofloadingFcdiOHintothe oilycoreofLNCsandincreasingthevolumeoftheinjectionaswell.

3.3. Drugdeliverybyintra-carotidinjection

Intra-carotidadministration can beconsidered asa regional deliveryofdrugtothebrainthatleadingtoasignificantenhance- mentindrugexposureofthetumouralvasculature(Joshietal., 2008).Inthiscase,thedrugisinjecteddirectlyintothebloodartery anddirectedtowardsthebrainvasculaturebeforeenteringperiph- eraltissuesbyavoidingthefirstpassmetabolism(Alametal.,2010).

Extensivestudiescarriedoutinbothexperimentalanimalsaswell asinhumans,havedemonstratedbenefitsofintra-arterialadmin- istrationinthetreatmentofbraintumour(Chertoketal.,2010;

Figueiredoetal.,2010;Fortin etal.,2005;Fujiwaraetal.,1995;

Newton,2006;Newtonetal.,2003;SchemandKrossnes,1995).

Inordertotestthebenefitofthisadministrationroute,LNCs werelabelledwithDiIforthepurposeoftrackingtheinjectedLNCs.

Moreover,thisadministrationrouteallowedincreasingthevolume ofinjectionofLNCsfrom60␮lforCEDinjectionto400␮l.

3.4. LNCvisualisationinratbrainslices

Fluorescentsectionimagesofcontrolratsthatdidnotreceive DiI-LNCsshowedtheauto-fluorescentspotsofbraincellswhich wereconsideredasbackgroundnoise, and nofluorescence was detectedinthebraincapillarylumen(Fig.3A).24hafteradminis- trationoflabelledLNCs,includingDiI-LNCsorDSPE-PEG-DiI-LNCs bycarotidinjection,redfluorescenceofDiIwasobservedandcould beassignedwithinthebraintissue(Fig.3BandC).Especially,in thebrainsectionsofratsreceivingDSPE-mPEG2000–DiI-LNCs,the fluorescentspots werelargelyenhanced throughoutthetissues associatedwithobviousfluorescenceofcapillarylumen.Themea-

Fig.3. Sectionrepresentativeimageunderfluorescentmicroscopetakenfromthecontroltumour-bearingrat(A)andratsreceivinganintra-carotidadministrationofDiI- LNCs(B)orDSPE-mPEG2000–DiI-LNCs(C).Theblue-fluorescentDAPI(D)denotedthetumourzonewhereastheredfluorescenceofDiI(E)showedthedistributionofLNCs aroundthetumourillustratedbythemergedimage(F).

Fig.4. Semi-quantifiedpresenceofLNCs24hafteradministrationinthebrain slidesbymeasurementofDiI-fluorescentsurfaces.*indicatesthestatisticallysignif- icantdifferenceinfluorescentsurfaceuponDiI-LNCandDSPE-mPEG2000–DiI-LNC administrationfromcontrolrats.=/ indicatesthestatisticallysignificantdifference betweenDSPE-mPEG2000–DiI-LNCadministrationandDiI-LNCone.

surementsofthefluorescentsurfaceinpixelsofinclusivethreshold imagesallowedtosemi-quantifythepresenceofLNCsinthebrain slides.AspresentedinFig.4,incomparisonwiththecontrol,the fluorescentsurfacesignificantlyincreasedwithDiI-LNCadminis- trationandachievedthehighestvalueinthebrainslideforrats receivingDSPE-mPEG2000–DiI-LNCs.Indeed,thefluorescentsur- facemeasuredfromtheseslideswasabout1.75-foldhigherthan thatofDiI-LNCsandabout3-foldthanthatofcontrol.

BylabellingcellnucleiwithDAPI,thetumourwasrecognised bybluefluorescence (Fig.3D)whereas ofDiI-labelled nanopar- ticles was detected by red fluorescence (Fig. 3E). The merged imageshowedtheaccumulationofnanoparticlessurroundingthe tumourzone(Fig.3F).Consequently,thesepromisingresultscould potentiallyleadtoanimprovementinthetherapeuticefficacyof anticancerdrug-loadedLNCsontumour-bearingrats.

3.5. Efficacyofintra-carotidtreatment

Survival of experimental animals was plotted on the Kaplan–MeiercurveasshowninFig.5.Resultsshowedthatintra- carotidtreatmentwithFcdiOH-LNCsslightlyincreasedthesurvival

Fig.5.Kaplan–Meiersurvival plotsfor9Lglioma-bearingratsreceivingintra- carotidinjectionoftreatmentatDay6aftercellimplantation.Survivaltimeswere plottedforuntreatedanimalsandtreatedanimalswithFcdiOH-LNCsorDSPE- mPEG2000–FcdiOH-LNCs(400␮lofLNCsuspension,correspondingto2.4mgof FcdiOHperrat).

time oftreatedanimals(median=27days)andwasstatistically significantcomparedtotheuntreatedcontrolgroup(P=0.0177), and wascomparable totreatmentbyCED injection(P=0.3172) (Fig.5).TreatmentwithDSPE-mPEG2000–FcdiOH-LNCsincreased the median survival time of rats to 30 days with an IST per- centage of 20% (P=0.0008). This result wasin agreement with thepreviously obtainedresultsabouttheenhanced fluorescent intensitymeasuredfromtheimagesofcoronalbrainslidesofrats receivinganintra-carotidinjectionofDSPE-mPEG2000–DiI-LNCs.

Thissurvivaltimegainissignificantandindicatesarealtreatment benefitas,inclinicaltrialsonhuman(EORTC/NCIC-protocol),the combination ofTemozolomide withfocalradiotherapy presents alsoa20%ISTincomparisontoradiotherapyalone(from12.1to 14.6months)(Stuppetal.,2005).

Asawell-knowncharacteristic,thecoatingwithlongchainsof PEGrendersnanoparticles‘invisible’totheimmunesystemafter injectioninthebloodthatconfersstealthpropertiestoPEGylated nanoparticles(Huynhetal.,2010).Thus,PEGylatednanoparticles havemorechancetoreachtumourtissuesandexhibittheintrinsic activityoftheentrappeddrug.Previously,Morille etal.demon- stratedthelong-circulatingpropertiesofDSPE-mPEG2000–LNCs owingtotheirweakcomplementactivationandlowmacrophage uptakeassociatedtoanextendedhalf-lifeinmiceascomparedto conventionalLNCs(Morilleetal.,2010).

Inaddition,for thisadministrationroute,crudeLNCsuspen- sionswereusedwithouttheadjustmentthatwasnecessaryfor CED injection.Toxicitywasnot evaluatedinthiscase asallthe componentswerescatteredinthebloodflow,whichisadifferent situationfromwhathappensinCEDinjection.Themainpurpose fortheutilityofcrudeLNCswastotakeadvantageofthehyperos- molarpropertiesofthesesuspensions.Indeed,theadministration of hypertonic substratescan leadtothe openingof tight junc- tionsoftheBBBduetotheshrinkageofendothelialcells,bywhich extracellularproteinsaredisorganised(Alametal.,2010).Conse- quently,drugentrytakesplaceinaparacellularfashionandduring thisstate,bothdiffusionandconvectionflowacrosstheBBBcan beconsiderablyenhanced (Rapoport,2000).Thus, nanoparticles couldpotentiallyreachthebraintumourtissues.Themechanism ofbrainentryaswellasthereversibilityandtimeofjunctionopen- ingwouldbeessentialtodetermineandwillbestudiedinfurther experiments.

Theremainingmatterwillbetoimprovetheinternalisationof drugintothetumourcells afterextravasation.To thisend, fur- theroptimisationoftheLNCsurfacebyattachingactiveligands whichcanspecificallyrecognisetumourcellsshouldbetakeninto consideration.Forexample,thecovalentcouplingofOX26mono- clonalantibodiestoLNCsattheendofthePEGmoleculesleadsto theformationofimmunonanocapsuleswhichspecificallyassociate invitrotocellsover-expressingthetransferrinreceptor(Beduneau etal.,2008).Indeed,OX26targetsthetransferrinreceptorswhich arehighlyexpressedonthecerebralendotheliumandalsoover- expressedonthesurfaceofproliferatingcellssuchasgliomacells (Hall,1991).ThisshouldfacilitatethepassageoftargetingLNCs throughtheBBB,followedbytheirinternalisationintothetumour cells.

4. Conclusion

To our knowledge, this is the first report involving a neu- ronal,side-effectstudyafterCEDadministrationofahyperosmotic infusate. At the same time, intra-arterial injection through the carotid artery was shown to represent a promising route of administration fordrug deliverytothebrain tumour bygiving nanoparticles more chance to cross the BBB. Results revealed theimportanceofthephysicochemicalpropertiesoftheinfusate

versusthebrainenvironment.Ineachadaptedcase,thebenefitof regionaldeliveryofadrugforbraincancerchemotherapywasevi- dencedandalsoconfirmedtheantitumourefficacyofFcdiOHinan orthotopic9Lgliosarcomamodelinrats.

Acknowledgments

TheauthorswouldliketothankPierreLegras(ServiceCommun d’AnimalerieHospitalo-Universitaire(SCAHU),Angers,France)for hisskilfultechnicalsupportinanimalexperiments,PascalPigeon for thesynthesis of FcdiOH and Gerard Jaouen for fruitful dis- cussions.We arealsogratefultoJoseHureaux,LaurenceSindji, FredericLagarce(InsermU646,Angers,France)andFlorenceFran- coni(Plateformed’Ingénierieetd’AnalysesMoléculaires(PIAM), Angers,France)fortheirhelpfultechnicalassistance.Thisworkis supportedbygrantsfrom“LaLigueNationaleContreleCancer”.

NgocTrinhHuynhwouldliketothanktheEmbassyofFrancein VietnamforitsEvaristGaloirfellowship.

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