Combined anti-Galectin-1 and anti-EGFR siRNA-loaded chitosan-lipid nanocapsules decrease temozolomide resistance in glioblastoma: In vivo evaluation

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Pharmaceutical nanotechnology

Combined anti-Galectin-1 and anti-EGFR siRNA-loaded chitosan-lipid nanocapsules decrease temozolomide resistance in glioblastoma: In vivo evaluation

Fabienne Danhier

^a,1

, Khaled Messaoudi

^b,1

, Laurent Lemaire

^b

, Jean-Pierre Benoit

^b,c

, Frédéric Lagarce

^b,c,

*

aUniversitécatholiquedeLouvain,LouvainDrugResearchInstitute,AdvancedDrugDeliveryandBiomaterials,AvenueMounier,73,bteB173.12, B-1200Brussels,Belgium

bL'UniversitNantesAngersLeMans,INSERMU1066,Microetnanomédecinesbiomimétiques,IBS-CHUAngers,4rueLarrey,49933Angers,Cedex9,France

cPharmacyDepartment,AngersUniversityHospital,CHUAngers,4rueLarrey,49933Angers,Cedex9,France

ARTICLE INFO

Articlehistory:

Received25November2014

Receivedinrevisedform26January2015 Accepted29January2015

Availableonline30January2015

Keywords:

Galectin-1 EGFR siRNA Temozolomide Glioblastoma Lipidnanocapsules

ABSTRACT

Glioblastomaisthemostfrequentprimarymalignantbraintumorinadults.Despitetreatmentsincluding surgery,radiotherapyandchemotherapybyoralTemozolomide(TMZ),theprognosisofpatientswith glioblastoma remainsverypoor.This is partlydue totheresistance ofmalignant cellstotherapy particularlyTMZ.Overexpressionofepidermalgrowthfactorreceptor(EGFR)andGalectin-1bytumor cellssigniﬁcantlycontributestoTMZresistance.Thepurposeofthisstudywastoevaluateinvivo,the effectoflocaladministrationbyconvectionenhanceddelivery(CED)oftheanti-EGFRandanti-Galectin- 1siRNAsadministeredseparatelyorincombinationon(i)thesurvivalofnudemice-bearingorthotopic U87MGglioblastomacellsandon(ii)theEGFRandGalectin-1expressioninexcisedU87MGtumortissue.

BothsiRNAswerecarriedbychitosanlipidnanocapsules(LNCs).Survivalofmicetreated14daysafter tumorimplantationbythecombinationofanti-EGFRandanti-Galectin-1siRNAsandTMZ(40mg/kg) wassignificantlyincreasedcomparedtoanimalstreatedbysingleanti-EGFRoranti-Galectin-1siRNAs carriedbychitosan-LNCs.ThiswasconfirmedbyadecreasedEGFRandGalectin-1expressionatthe proteinlevelinexcisedU87MGtumortissue,8dayspost-transfection,visualizedbyimmunofluores- cence.Thisstudydemonstratesthepotentialofourstrategyinglioblastomatherapy.

ã2015ElsevierB.V.Allrightsreserved.

1.Introduction

Glioma are brain tumors that arise from astrocytes or supportive tissue of the brain. These tumors can be highly malignant:multiformglioblastomais themostcommonmalig- nant tumor of the central nervous system in adults and corresponds to stade IV gliomas. These tumors show a high proliferationrate,variabilityintumorhistopathologyanddiffusely inﬁltrate adjacent brain tissue (Bernardi et al., 2009). The treatmentof glioblastomaremainsoneof themostchallenging cancerproblems, as nocurative treatment hasyet been found (Stuppetal.,2009).Conventionaltherapeuticproceduresfocuson surgicalresection combined withadjuvant radiotherapyand/or

chemotherapybyoralTemozolomide(TMZ)(Stuppetal.,2005).

However, tumor recurrences are common, median survival is 14monthsandthepercentageofpatientslivingfor5yearsormore islessthan10%(Lefrancetal.,2006).TMZisanalkylatingagent from the imidazotetrazine family (Zhang et al., 2012). The sustained efﬁcacy of TMZ for treating glioblastoma is limited.

Indeed,amongothers,overexpressionofEpidermalGrowthFactor Receptor (EGFR) and Galectin-1 by tumor cells significantly contributes to TMZ resistance (Friedman et al., 2000). EGFR overexpression or overactivaton is observed in 40–70% of the patients.Ithasbeendemonstratedthattheexpressionofaspecific mutantformofEGFR(EGFRvIII)promotestumorformationand growth (Hatanpaa et al., 2010; Verreault et al., 2013). The oncogenic properties of EGFR overexpression are suggested to bea consequence of theconstitutive activationofdownstream pathwayssuchasPI3K/AKT(Moscatelloetal.,1998).Galectin-1, whichisalectinwithspecificityfor

b

-galactosides,significantly influencesglioblastomacellmigration.Galectin-1positivepatients survive for a significantly shorter period than individuals whose glioma expresses less Galectin-1 (Camby et al., 2002).

*Corresponding author at: L'Université Nantes Angers Le Mans, Micro et Nanomédecines Biomimétiques, bâtiment IBS (Institut de Biologie en Santé) INSERMU1066,4rueLarrey,49933Angers,Cedex9,France.Tel.:+33244688568;

fax:+33244688546.

E-mailaddress:frederic.lagarce@univ-angers.fr(F.Lagarce).

1Thesetwoauthorscontributedthesameway.

http://dx.doi.org/10.1016/j.ijpharm.2015.01.051 0378-5173/ã2015ElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

International Journal of Pharmaceutics

j o u r n al h o m ep a g e: w w w . el s e v i e r . c o m / l o c at e / i j p h a r m

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Galectin-1expressionisincreasedunderhypoxicconditionsand radiotherapy stimulates Galectin-1 expression in glioblastoma cells. Elsewhere, hypoxia also confers cellular resistance to conventional chemotherapy (Le Mercier et al., 2008; Le et al., 2005).Galectin-1isnegativelyregulatedbyp53,whichtriggers apoptosis response to cellular stress including chemotherapy, while loss of p53 functionality leads to chemoresistance (Le Mercieretal.,2008;Cambyetal.,2005).

In recent years, thestrategy of ribonucleic acid interference (RNAi),whoseprincipleprocessistoreducetheexpressionoftarget messengerRNA(mRNA),wasdiscovered,openingupnewperspec- tives in different areas of medicine. RNAi is an intrinsically cellularpathwaydiscoveredin1998(Fireet al.,1998;McManus andSharp,2002;Ngoetal.,1998).Thispathwaycanbeinitiatedby introducingsmall interfering RNA (siRNA) consisting ofdouble- strandedRNAof21–23nucleotides.TheuseoftherapeuticsiRNA aloneisnotpossibleforseveralreasons,including(i)theirnegative charge,preventingthemfromcrossingtheplasmamembraneto reach their target in the cytoplasm (Aagaard and Rossi, 2007; Kim and Rossi, 2007; Reischland Zimmer, 2009); (ii) the presence of nucleases inplasma and the cytoplasm that deteriorate siRNA rapidly after their injectionintotheblood;(iii) theimmune responsethatcan be triggeredby theirpresence inthe bloodmayalso leadto their degradation(Volkovetal.,2009).Thesereasonsemphasizetheneed forasuitablevectorabletodeliverthesiRNAintothetargetedcells.

Previously,wedevelopedchitosan-graftedlipidnanocapsules (LNCs) for the delivery of both anti-EGFR and anti-Galectin-1 siRNA(Messaoudietal.,2014a,b,b).LNCswerepreparedusinga phase-inversationprocess(Heurtaultetal.,2002).Thisprocessis green: no organic solvents are used. Moreover, only generally recognized as safe (GRAS)and genetically modiﬁedorganisms (GMO)freeexcipientsareused(Hureauxetal.,2009).Chitosan wasgraftedattheLNCsurfacebyatransacylationreaction.The positive charge of chitosan allows for ﬁxing the negatively chargedsiRNA tothevector surfacebyelectrostatic attraction.

Chitosan was selected as the transacylation polymer for its interesting physicochemical properties in particular its non- toxicityandbiocompatibility(Malmoetal.,2012).Wepreviously demonstratedinvitrothattreatmentofU87MGcellswithanti- EGFRor anti-Galectin-1 siRNA/chitosan-LNCscomplexes (siRNA concentration of 40nM) resulted in a reduction of EGFR or Galectin-1expressionof52%and38%,respectively,after96hof incubation.Wealsoobservedamortalityreaching82%whenthese two siRNA were applied on U87MG cells 72h before TMZ treatmentat0.5mg/mLwhileTMZaloneinducedamortalityof 26%andanti-EGFRorGalectin-1siRNAaloneinducedamortality of67%and59%,respectively.Theseresultsclearlydemonstratedin vitrotherationale of combiningthe two anti-EGFR(Messaoudi etal.,2014a)andanti-Galectin-1(Messaoudietal.,2014b)siRNA toincreasethesensitivityofU87MGcellstoTMZ.

Intracranial administration of treatments by a convection enhancement delivery (CED) procedure was selected since this techniquewasshowntoimprovethediffusionofnanomedicinesin thebrainparenchyma.CEDisarecentapproachtodeliverdrugs intobraintissue andisdefinedasthecontinuousinjectionofa therapeutic fluid agentunder positive pressure. This technique using convection or “bulk flow” was proposed to supplement simplediffusion,whichcharacterizeslocalintracerebraldelivery, bystereotaxicinjections.Inpractice,drugsaredeliveredcontinu- ouslyviaacatheterconnectedtoasyringepump,thusenablingthe distributionof largevolumes of high drugconcentrations with minimumsystemictoxicity.DuringCED,diffusionandconvection take place simultaneously. By using convection to supplement simple diffusion, an enhanced distribution of small and large molecules can be obtained in the brain while achieving drug concentrationgreaterthansystemiclevels(Allardetal.,2009).

Hence, in this study, we aim to evaluate the preclinical relevanceof thecombination of thesetwo anti-EGFR andanti- Galectin-1siRNAtoincrease thesensitivityofglioblastoma.For thispurpose,weadministeredsiRNA/chitosan-LNCscomplexes,by intracranialinjectionusingtheCEDprocedure,threedaysbefore intraperitoneal injection of TMZ. The influence of the time schedule of administration after tumor implantation on the treatment efficacywas alsoevaluated.Invivo EGFRand Galec- tin-1 expression was evaluated by immunofluorescence. All experiments were performed on orthotopic human U87MG glioblastoma-bearingnudemice.

2.Materialandmethods

2.1.PreparationandcharacterizationofsiRNA/chitosan-LNCs complexes

2.1.1.FormulationofLNCs

LNCswerepreparedaccordingtoaphase-inversionprocessas previouslydescribed(Heurtaultetal.,2002).Brieﬂy,Solutol¹H15, Lipoïd¹S75-3,NaCl,Labrafac¹andwater(0.846g,0.075g,0.089g, 1.028g,2.962g,respectively)weremixedandwarmedupto85C undermagneticstirring.Theformulationwasthencooledto60C;

thiscyclewasrepeatedthreetimes.Duringthelastcycle,12.5mL ofwaterat4Cwasaddedtotheformulation.

2.1.2.SurfacemodiﬁcationofLNCs

AtransacylationreactionwasobtainedbetweenthePEGylated hydroxystearate and a functional amino group of a polymer (Messaoudi et al., 2014a).Previously-made suspensionof LNCs (20mL)weremixedwith1mLofNaOH10Mand50mgofchitosan oligosaccharide lactate5000Da (Sigma–Aldrich,St.Louis, USA).

Thereactionwasperformedina25Cwaterbathfor15min.The reactionwasstoppedbyadding20mLofglycinebuffer.ThepHwas adjustedto5.50.5toensureacorrectionizationofthechitosan before adding siRNA. Finally, puriﬁcation was performed by dialysiswithmembranes(SpectrumLaboratories,RanchoDom- inguez,USA)havingamolecularweightcut-offof50KDa.Chitosan nanoparticles were allowed for staying inside the dialysis membranewhilethefreechitosandiffusedinthedialysiswater.

Dialysiswasperformedfor24hwithMilli-Qwaterundermagnetic stirring.

2.1.3.Complexationofchitosan-LNCswithsiRNA(siRNA/chitosan- LNCs)

ThecomplexationoftransacylatedLNCsandanti-EGFRand/or and Galectin-1 siRNA (Eurogentec, Angers, France; sequences illustratedinTable1)wasperformedasfollowed:100

m

^L^of^LNCs

and2.32

m

^L^siRNA⁽¹⁰⁰⁰

m

^M)^were^mixed^with^RNAse-free^water

untilaﬁnalvolumeof500

m

^L.^The^solution^was^diluted^two^times

in glucose 5% toobtain a ﬁnalconcentration of LNCs of about 100

m

^g/mL^andconcentrationsofsiRNAof40nM(Messaoudietal., 2014a).

2.1.4.Physico-chemicalcharacterizationofnanoparticles

siRNA/chitosan-LNCswerecharacterized intermsofsizeand zeta (

z

⁾ ^potential. ^The ^average hydrodynamic diameter and polydispersity index of particles were determined by photon correlation spectroscopy, while

z

^potential ^was ^determined ^by

laser Doppler velocimetry, combined with phase analysislight scattering (PALS), both using a Zetasizer¹ Nano ZS (Malvern Instruments,UK).Theinstrument wascalibratedwithreference polystyrene nanoparticles (MalvernInstruments, UK). Measure- ments wereperformedafterdispersionof 50

m

^L^of ^particles ⁱⁿ

2.95mL of RNAse-free water. All assays were performed in triplicate(Messaoudietal.,2014a).

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2.2.TMZformulation

TMZ was dissolved with the dissolution-enhancing agent ^L- histidine,whichisanexcipientapprovedbytheFoodandDrug Administration(FDA,USA).TMZ(40mg)and L-histidine(40mg) weresolubilizedinphosphatebuffer(10mL,pH5.0).Thesolution wasfilteredon0.22mmpolyvinyldifluoridefilter(Millipore,MA, USA)(Wauthozetal.,2010).

2.3.Invivoanti-tumorefﬁcacy 2.3.1.Cellculture

Human glioblastoma U87MG cells were obtained from LGC (Molsheim, France). These cells were cultured in Dubelcco’s modiﬁedEagle’smedium(DMEM)with1g/Lofglucosesupple- mentedwith10% ofFetalBovine Serum(FBS)(Lonza, Verviers, Belgium)and1%ofantibiotics(Sigma–Aldrich,St.Louis,USA)and incubatedin5%CO2at37C(Messaoudietal.,2014a).

2.3.2.Tumormodel:orthotopicU87MGglioblastoma

FemaleathymicSwissnudemice,aged6weeks,wereobtained from Charles River (L’Arbresle, France). Experiments were conducted in accordance with the regulations of the French MinistryofAgricultureandtheregionalethiccommittee.Animals wereanaesthetized by anintraperitoneal injection of xylazine (13mg/kg)andketamine(100mg/kg) andwerepositionedin a stereotaxicframe(DavidKopfInstruments,Tujunga,USA).Onday zero,micereceivedanintracranialinjectionof5

m

^L^U87MG^cells

inHBSSwithCa²⁺andMg²⁺(310⁴cells)intotherightfrontal lobe (striatum) (coordinates: 2.1mm lateral from the bregma, 0.5mm anteriorand 3mm deep from the outer borderof the cranium),using a 5

m

^L ^Hamilton¹ ^syringe ^with^a ^26-G ^needle

(Clavreuletal.,2014).

2.3.3.Convectionenhancementdelivery(CED)ofsiRNA/chitosan-LNCs andTMZtreatment

Atdifferenttimesaftertumorimplantation(day6,10and14), mice were randomly assigned to groups receiving intracranial injectionoftreatments.CEDinjectionofsiRNA/chitosan-LNCswas similarto tumorimplantation, except that the5

m

^l ^Hamilton¹

syringewitha32-Gneedlewas connectedtoanosmoticpump PHD 2000 infusion (Harvard Appartus, Les Ulis, France) by controlling0.5

m

^l/min^rate(Vanpouille-Boxetal.,2011).Control groupconsistsinsiRNAnegativecontrol.Threedaysaftertumor implantation,TMZwasintraperitonealy(IP)injected,atadoseof 20 or 40mg/kg. Control group consisted in intraperitoneal injectionofHBSSwithCa²⁺andMg²⁺(250

m

^L).^The^different^time

schedulesareillustratedinFig.1.

Foreachcondition,miceweredivedinto5groups:Group1:

HBSS(CED)+HBSS(IP);Group2:siRNAnegativecontrol(CED)+ TMZ(IP);Group3:anti-Galectin-1siRNA/chitosan-LNCs(CED)+ TMZ(IP);Group4:anti-EGFRsiRNA/chitosan-LNCs(CED)+TMZ (IP); Group 5: anti-Galectin-1+ anti-EGFR siRNA/chitosan-LNCs (CED)+TMZ(IP).Animalsweresacriﬁcedwhentheyhadlostmore

than20%oftheirbodyweight,whichwasdirectlyattributedtothe tumordevelopment.

2.4.MRI

TheorthotopicU87MGtumorgrowthproﬁlewasdetermined by Magnetic Resonance Imaging (MRI). MRI was performed using a 7T scanner (Biospec 70/20 Avance III, Bruker Wissembourg, France) equipped withBGA12S gradient system (675mT/m).Animalbodytemperaturewasmaintainedthrough- outtheexperimentbyhotwatercirculationintheanimalbed.

During the MR protocol, mice wereanesthetized with 0.5% of isoﬂurane addedto0.4Lmin¹ of pureO2 and respirationwas monitored.Tumorvolumewasassessedtwiceaweekusinga¹H cryoprobeandarapidacquisitionwithrelaxationenhancement (RARE) sequence (TR=3200ms; effective echo time (TEeff)= 21.3ms; acceleration factor=4; FOV=22cm; matrix 256256; nine contiguous slices of 0.5mm,Nex=1). Volumes were calculated frommanually drawn regionof interest (ROI).

Thesetumorvolumegrowthcurvesarethenﬁttedbythemethod of least squares with an exponential function (Clavreul et al., 2014).

To checkthetumordevelopment andthelocalizationin the rightstriatum,beforetreatments,brainsofmiceofthewholestudy wereimagedatday10withMRI,usingthesameaforementioned protocol.

2.5.Immunoﬂuorescence

14 days after U87MG implantation, mice were dived into 3groups(n=3)receiving intracranialinjectionoftreatmentsby CED:Group1:HBSS(CED);Group2:siRNAnegativecontrol(CED);

Group3:anti-Galectin-1+anti-EGFRsiRNA/chitosan-LNCs(CED).

8daysaftertreatment,micewerekilled.Brainswereimmediately placed in Tissue-Tek OCT embedding medium (Sakura Finetek, Tokyo,Japan),frozeninliquidnitrogenandstoredat–80C.Brain coronalsectionswerecut(10

m

^m^thick)^and^collected^onto^slides.

Cryosectionswereair-dried,rehydratedinPBSandﬁxedfor2min inice-coldacetone,forEGFRstaining,orin4%PFA,pH7.4,at4C, for Galectin-1 staining. To block non-speciﬁc binding, sections wereincubatedinPBScontaining4%bovineserumalbumin(BSA) and10%normalgoatserum.Triton0.25%wasaddedtothemedium fortheGalectin-1staining.Sectionswereincubatedovernightat 4CwithisotypecontrolsandprimaryantibodiesagainstEGFR(1/

100)(Ratmonoclonalanti-EGFR;Abcam)andagainstGalectin-1 (1/1000)(Rabbitpolyconalanti-Galectin-1;Abcam).Primaryanti- EGFR antibodies were detected using biotinylated anti-rat secondaryantibodyandthesignalwas amplifiedusingstrepta- vidin-FITC (Dako). Primary anti-Galectin-1 antibodies were detectedusingsecondaryantibodyOregongreen488goatanti- rabbit;LifeTechnologies).NucleiwerecounterstainedwithDAPI (1/1000)(Sigma).Cryosectionsfromfivemiceofeachgroupwere analyzedunderafluorescencemicroscopewith350nm(blue)and Table1

anti-EGFRandanti-Galectin-1siRNAsequences.

siRNA Sequence

Negativecontrol(scrambled) sense:5⁰-GGAAAUCCCCCAACAGUGAdTdT-3⁰

antisense:5⁰-UCACUGUUGGGGGAUUUCCTdT-3⁰

Anti-EGFR sense:

5'CACAGUGGAGCGAAUUCCUdTdT3⁰

antisense:3⁰dTdTGUGUCACCUCGCUUAAGGA5⁰

Anti-Galectin-1 sense:5⁰GCUGCCAGAUGGAUACGAAdTdT3⁰

antisense:3⁰UUCGUAUCCAUCUGGCAGCdTdT5⁰

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470–490nm(green)excitationﬁlters(Axioscope¹2optical,Zeiss, LePecq,Germany)(Zhaoetal.,2012).

2.6.Statistics

Allresults arerepresented asthemeanstandard deviation (SD).Survivalcurveswereobtainedbya Kaplan–Meiersurvival rateandtumorvolumeswereanalyzedusingtwo-wayANOVAand Bonferronipost-hoctest,usingthesoftwareGraphPadPrism6.2 forMac.p<0.05wasconsideredtobestatisticallysigniﬁcant.

3.Resultsanddiscussion

3.1.SizeandzetapotentialofsiRNA/chitosan-LNCscomplexes Physico-chemicalcharacteristicsofsiRNA/chitosan-LNCscom- plexesaresummarizedinTable2.Afterpuriﬁcationbydialysis, size, PDI index and

z

^potential ôf ânti-EGFR ôr anti-Galectin- 1 siRNA/chitosan-LNCs complexes were about 94nm, 0.26 and 15mV respectively. As previously described (Messaoudi et al., 2014a,b,b), theco-complexation ofanti-EGFRandanti-Galectin- 1 siRNA on the chitosan-LNCs did not modify the size of nanoparticlesbutledtoaslightdecreaseof

z

^potential⁽¹²^mV).

Chitosanwasusedforthetransacylationreactionduetoitshigh transfection efﬁciency and low toxicity. The chitosan used presented a low molecularweight, allowingit toobtain nanoparticles with small size forming complexes with siRNA and dissociatingatthecellularleveltoallowtheactionofsiRNA.Low molecularweightwaspreferredbecausehighmolecularweights havebeen reportedtoformverystable complexeswithsiRNA, reducingthereleaseofsiRNAandleadingtoasmallereffectonthe targetmRNA(Messaoudietal.,2014a;Techaarpornkuletal.,2010).

Allcomplexestestedinthisstudywerestableat4Cand25Cat least for 72h, in terms of hydrodynamic diameter, PDI and

z

potential(Messaoudietal.,2014b).

3.2.EvaluationoforthotopicU87MG-tumorgrowthproﬁle

Tochoosethedifferenttimeschedulesfortheadministrationof thecombinedtherapysiRNA+TMZ,theU87MGtumorgrowthwas evaluatedbyMRI.Usingrapidanatomicalprotonimages,tumor volumes werecalculated. Fig.2A showsthe orthotopicU87MG tumorgrowthinfunctionofthetime.Thegrowthwasfoundtobe exponentialwithatime constantof2.930.77.Additionally,all miceofthestudy(n=158)wereimagedatday10tocheckthe presenceandthelocalizationintherightstriatumoftheU87MG tumor.Anexampleofthelocalizationofthetumorincoronaland longitudinalsectionsisillustratedinFig.2B.Onday10,U87MG volumewas0.880.11

m

^L.

An ideal glioma model should closely resemble human glioblastoma in its morphology, invasiveness and angiogenic behavior, toallowclinically relevant studiesof tumor behavior andtherapeuticdrugefﬁciency.U87MGtumormodelisknownto presentmoderatetohighinvasiveness,coupledtotumor-induced necrosisandvascularalterations,whichmakethemgoodglioma Fig.1.TimescheduleofcombinedtreatmentsandMRI.

Table2

Physico-chemicalcharacterizationofsiRNA/chitosan-LNCscomplexes(n=3).

siRNA/chitosan-LNCscomplexes Size(nm) PDI z^potential^(mV) siRNAnegativecontrol 91.22.7 0.28 20.00.2

Anti-EGFRsiRNA 95.12.45 0.24 15.20.9

Anti-Galectin-1siRNA 92.92.6 0.28 15.90.6 Anti-EGFR+Galectin-1siRNA 94.21.5 0.26 12.22.6

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modelsparticularlyfor therapeuticagent studies(Doblas etal., 2010). The growth proﬁle of U87MG implanted in the right striatumofnudemice(310⁴cells)wasestablishedtoconstitute arationaleinthechoiceofdifferenttimesaftertumorimplanta- tiontotreatU87MG.Theexponentialcurveshouldbedivedinto twolinearcurves.Day6correspondstothemiddleoftheﬁrstline, day10correspondstotheintersectionbetweenthetwolinesand day14approximatelycorrespondstothemiddleofthesecondline.

3.3.Inﬂuenceofthetimescheduleofadministrationonanti-tumor efﬁcacyofthecombinedtherapy

Survival rates of mice receiving thecombined treatment of siRNA/chitosan LNCs and TMZ, at different time after tumor implantation,isillustratedinFig.3.Fig.3Ashowssurvivalratesof micetreated6daysaftertumorimplantationwithsiRNA-chitosan LNCS+40mg/kgofTMZadministered1weeklyfor3weeks.This dosewaschosenaccordinglytotheliterature (LeMercieretal., 2008).AtthisdoseofTMZ,mostofthemicepresentedof100%

long-term survival, leading to an impossible discrimination betweenthedifferentgroups.Hence,wedecided totest longer timesaftertumorimplantationtoadministertreatmentaswellas a decreased dose of TMZ. Firstly, we administered treatments 10daysaftertumorimplantation. Thistime correspondstothe beginningoftheexponentialgrowthoftumors(Fig.2A).Twodoses ofTMZweretested:20and40mg/kg(Fig.4BandC,respectively).

Althoughallmicedied(no100%long-termsurvival),nosigniﬁcant differencebetweentreatedgroups was observed.Anyway, TMZ treatments(20or40mg/kg)allowsfora1.3to1.8-foldenhance- mentofthemeansurvivaltimesoftreatedmice(dependingofthe treatmentgroup)incomparisonwithuntreatedmice.Finally,we administered treatments 14 days after tumor implantation (Fig.4D).Atthisstage,thetumorgrowthisveryfast(exponential partofthecurve).Untreatedmicepresentedameansurvivaltime of32 days.ThemeansurvivaltimesofmicetreatedwithsiRNA negativecontrol+TMZ, anti-EGFR siRNA+TMZ oranti-Galectin- 1siRNA+TMZwere34daysforallthethreegroups(p>0.05).The meansurvivaltimeofmicetreatedwiththecombinationofthe twosiRNA+TMZwassigniﬁcantlyenhancedto39days(p<0.01).

Oneofthelimitingfactorsintheuseoftherapeuticsforbrain tumors is the presence of the blood–brain barrier (BBB) that consistsofalayeroftightconjunctendothelialcellsandhasstrong

efﬂux pumps, whichactively removetoxicsubstances fromthe brainandreduceeffectivedrugpenetration(Abbottetal.,2010).

Mostdrugsdisplayinhibitedtransporttothebrain,and evenif drug transmission through the BBB is achieved, systemic side effectsmayoccurbeforetherequireddrugconcentrationwithin thetargetbraintumorisobtained(Tsujiuchietal.,2014).Forour current study, we chose intratumoral administration of siRNA/

chitosan-LNCsbyCEDwhichleadtoanadministrationofdrugson apressuregradientwhichallowsahomogeneousdistributionin thetumor.CEDoffersadvantagesinthatittargetslocalareasofthe brain, bypasses the BBB, minimizes systemic toxicity of the therapeutics, and allows for delivery of larger molecules that diffusiondrivenmethodscannotachieve(Tsujiuchietal.,2014).

Early treatment of U87MG tumors (day 6) by TMZ caused healing(100%long-termsurvival).Itcorrespondstothepartofthe growthproﬁlewherethedoublingtimeoftumorwaslow.Onthe contrary,fromday10(timefromwhichthedoublingtimeoftumor drasticallyincreased),tumors treatedwithTMZdidnotpresent total regression. Interestingly,differences betweenmicegroups wereobservedatadvancedtimeaftertumorimplantation(day14), makingthecombinedtherapyofthetwosiRNAs+TMZrevelant from the clinical situation. Indeed, glioblastoma are diagnosed whenthetumorhasreachedaconsistentsizewhichmakesthem difﬁculttotreat(Ahmedetal.,2014).

Otherstudieshaveshownthatthecombinationofinterfering RNA had a beneﬁcialeffect in reducing tumorgrowth. Indeed, Verreaultetal.haveshownthattargetingofEGFRandm-TORby shRNAresultedineradicationoftumorsimplantedinthebrainsof micewhichconﬁrmstheeffectivenessofthisstrategyoftreating glioblastoma(Verreaultetal.,2013).Differentstudieshaveused CED for siRNA delivery. For example Kato et al. showed that treatmentof braintumors by TMZassociated withanti-MGMT siRNA carried by LipoTrust increased the survival of mice by 5weekscomparedtomicetreatedbyanti-MGMTsiRNAandDMSO (Doblasetal.,2010).However,siRNAtreatmentwascarriedout continuouslyat0.5mL/hforaweekthankstoanosmoticpump and TMZ was injected daily at dose of 50mg/kg for 5 days (Katoet al.,2010).ContinuoustreatmentwithsiRNAcan cause saturationoftheRISCcomplexthatishometotherecognitionof targetmRNAsbysiRNAs (Castanotto etal.,2007).Clinicaltrials usingCEDreportedintheliteraturearetrialsinfusinganti-cancer drugslikepaclitaxelora¹³¹I-labeldchimericmonoclonalantibody,

Fig.2.OrthotopicU87MGtumorgrowth.(A)U87tumorgrowthmonitoredbyMRI.Tumorvolumewasassessedtwiceaweekusinga¹Hcryoprobeandarapidacquisition withrelaxationenhancement(RARE)sequence(n=8).VolumeswerecalculatedfrommanuallydrawnROI.Thesetumorvolumegrowthcurvesarethenﬁttedbythemethod ofleastsquareswithanexponentialfunction.(B)T2-weightedimagesofU87MG10daysafterimplantation.LighterzonespointedbyarrowsareU87tumorlocalizedinthe rightstriatumofmice.Coronal(above)andlongitudinal(underneath)sectionsareillustrated.

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Fig.3.Kaplan-MeiersurvivalratesofU87MGtumor-bearingmicetreatedwithHBSS,siRNAnegativecontrol,anti-EGFRsiRNA,anti-Galectin-1siRNAoranti-EGFR+anti- Galectin-1siRNAadministeredbyCEDfollowed3dayslaterbyaTMZtreatmentadministeredintraperitonealy.siRNAtreatmentswereadministeredatdifferenttimesafter thetumorimplantationandtheTMZdosevaried(A)6daysaftertumorimplantation;TMZdoseof40mg/kg1weeklyfor3consecutiveweeks.(B)10daysaftertumor implantation;TMZdoseof20mg/kg.(C)10 daysaftertumorimplantation;TMZdoseof40mg/kg.(D)14daysaftertumorimplantation;TMZdoseof40mg/kg.

Fig.4.(A)ImmunoﬂuorescencedetectionofEGFRexpressioninU87MGtumorcryosections,8dayspost-transfection,ofmicetreatedwithanti-EGFR/Galectin-1siRNA/

chitosanLNCsandnegativecontrolsiRNA/chitosanLNCs.GreencolorrepresentsforEGFRbindingwithstreptavidin-FITC-labeledantibody(green)andbluecolorrepresents fornucleistainedwithDAPI.(B)QuantitativeFITCimmunoﬂuorescenceintensity.ResultsareexpressedasthemeanSD(n=3).Scalebar=100mm*p<0.05.(For interpretationofthereferencestocolourinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

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Cotara¹.Theconceptofgenenanomedicine-basedtherpydeliv- eredbyCEDhasalsobeenclinicallyinvestigated:theSemlikiforest virusvectorcarryingthehumaninterleukin12gene,encapsulated inliposomes;ora retrovirallymediatedHSV-1-tk genetransfer alsoencapsulatedinliposomes.Thesetrialswereveryencourag- ing.However,themain critical aspectof thesestudies wasthe monitoring of the CED injection and not the eﬁcacy of the liposomes.Itwasconcludedthatthedistributionvolumeisstrictly dependentonthephysico-chemicalpropertiesofthenanocarrier (Allardetal.,2009).

Majors advantages of the combination of siRNA-LNCs com- plexesandTMZconsistin:(i)TMZistheﬁrstlinetreatmentof glioblastoma,(ii)EGFRandGalectin-1aretwomajorsrecepetors involved TMZ resistance and are very well described in the literature,(iii) thepreparation ofLNCs is cheap, greenand the scalingupis feasible(Thomasand Lagarce, 2013), (iv) theCED delivey has been yet tested in clinical trials with encouraging resultsandﬁnally(v)therationaleofthiskindofmoreelaborated andexpensive treatmentsshouldthecase offailureof conven- tionnal therapies, where biopsy revealed resistance of TMZ involving appropriate receptors, as we demonstrated that this combination is more effective on well developed tumors in comparisonwithnewlyformedtumors.

3.4.Invivogenesilencing

To furtherevaluate if thetumorgrowth inhibition observed upontreatmentwithanti-EGFR/Galectin-1siRNA/chitosanLNCsis relatedtoEGFRandGalectin-1genesilencingofcancercells,we examinedEGFRandGalectin-1expressionattheproteinlevelin

excised U87MG tumor tissue, 8 days post-transfection, by immunofluorescence. Streptavidin-FITC and Oregon green 488- labeledsecondaryantibodieswereusedfor EGFRandGalectin- 1 detection, respectively. Anti-EGFR/Galectin-1 siRNA/chitosan LNCs showed significant EGFR (Fig. 4) and Galectin-1 (Fig. 5) silencingcomparedtosiRNAnegativecontrol/chitosanLNCs.The mean fluorescence intensity of the EGFR protein level was significantly decreased when mice were treated with EGFR/

Galectin-1 siRNA/chitosan LNCs, when compared to negative controlsiRNA(26023versus34315arbitraryunit)(p<0.05) (Fig.4B).Inthesameway,themeanﬂuorescenceintensityofthe Galectin-1proteinlevel was signiﬁcantlydecreased when mice were treated withEGFR/Galectin-1 siRNA/chitosan LNCs, when compared to negative control siRNA (32318 versus 44423arbitraryunit)(p<0.01)(Fig.5B).

The EGFRand Galectin-1knockdown in tumors after siRNA treatmentshowedthattheeffectofsiRNAinvivoextendstoaweek conﬁrmingthetransientnatureoftheiractivity(Xueetal.,2014).

Therefore, cellurar organization inside the tumor was different dependingonwhethertumorshavebeentreatedornot.Indeed,on cryosectionsof negativecontrols, cellularheterogenitycouldbe observed(seemagniﬁcationonFigs.4and 5),whilefor treated cryosections, cells are organized homogeneously. This is likely due tothedifferenceof tumorgrowth under theaction of the treatmentand consequentlytothetumorsize.Itis wellknown that growing tumors become heterogenous with a drastic modiﬁcation of the extracellular matrix, number of pericytes, smooth-musclecellsandatortuousanddisorganizedvasculature (Heldin et al., 2004). Previously, LNCs have been loaded with rhenium-188andadministratedbyCEDinaratorthotopicglioma

Fig.5. (A)ImmunoﬂuorescencedetectionofGalectin-1expressioninU87MGtumorcryosections,8dayspost-transfection,ofmicetreatedwithanti-EGFR/Galectin-1siRNA/

chitosanLNCsandnegativecontrolsiRNA/chitosanLNCs.GreencolorrepresentsforGalectin-1bindingwithOregongreen488-labeledantibody(green)andbluecolor representsfornucleistainedwithDAPI.(B)QuantitativeFITCimmunoﬂuorescenceintensity.ResultsareexpressedasthemeanSD(n=3).Scalebar=100mm**p<0.01.(For interpretationofthereferencestocolourinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

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model(Vanpouille-Boxetal.,2011).Theresultsobtainedshowed theverypromising potential of LNCsin glioma treatment.This allowsustoconsiderapossiblecombinationoftheeffectofsiRNA attached tothe surface of LNCs and Rhenium encapsulated in particlesforgreaterefﬁciencyonglioblastoma.

4.Conclusion

EGFRand Galectin-1 targetingby specificsiRNAs carriedby chitosan-LNCs represent a promising strategy to induce anti- tumoreffectsinglioblastoma.ThelocaladministrationofsiRNA allowed their direct delivery to the tumor and prevented the interactionofsiRNA/chitosan-LNCswithplasmaproteinsthatcan destabilizethe complexes,thereby reducing theefficacy of the treatment.Given thesimplicityof formationof siRNA/chitosan- LNCs complexes and the homogeneity of siRNAs structure, it shouldpossiblewithoursystemtoreachanytargetinvolvedinthe resistanceofglioblastomatoanti-cancerdrugsincludingTMZand toadapttreatmenttoeachpatientprofile.

Acknowledgments

Theauthorsareverygratefultothe“Inter-RegionalCommittee, League Against Cancer, Pays de la Loire region” for providing ﬁnancialsupportforthiswork.Thisworkissupportedbygrants from the Université catholique de Louvain (F.S.R) and Fonds NationaldelaRechercheScientiﬁque(F.R.S.-FNRS).F.Danhierisa postdoctoralF.R.S.-FNRSResearchFellow.Thefundershadnorole instudydesign,datacollectionandanalysis,decisiontopublish,or preparationof the manuscript.The authorsare alsograteful to Dr.AnneClavreulforanimalmodelmethodology.

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