• Aucun résultat trouvé

Development of multifunctional lipid nanocapsules for the co-delivery of paclitaxel and CpG-ODN in the treatment of glioblastoma

N/A
N/A
Protected

Academic year: 2022

Partager "Development of multifunctional lipid nanocapsules for the co-delivery of paclitaxel and CpG-ODN in the treatment of glioblastoma"

Copied!
9
0
0

Texte intégral

(1)

Development of multifunctional lipid nanocapsules for the co-delivery of paclitaxel and CpG-ODN in the treatment of glioblastoma.

Giovanna Lollo

a,b

, Marie Vincent

c,d

, Gabriela Ullio-Gamboa

a,b

, Laurent Lemaire

a,b

, Florence Franconi

e

, Dominique Couez

c,d

, Jean-Pierre Benoit

a,b,

*

aLUNAMUniversité—MicroetNanomédecinesBiomimétiques,F-49933Angers,France

bINSERMU1066,IBS-CHU,4rueLarrey,F-49933AngersCedex9,France

cINSERM,UMR892,F-49933Angers,France

dCNRS,UMR6299,F-49933Angers,France

ePRIMEX-CIFAB,Universite’d'Angers,LUNAMUniversite’,IRIS-IBS,CHUAngersF-49933Angers,France

ARTICLE INFO

Articlehistory:

Received4August2015

Receivedinrevisedform23September2015 Accepted26September2015

Availableonline30September2015

Keywords:

Nanotechnology Lipidnanocapsules Antitumordrugs Paclitaxel CpG Glioblastoma

ABSTRACT

Inthiswork,multifunctionallipidnanocapsules(M-LNC)weredesignedtocombinetheactivityofthe cytotoxicdrugpaclitaxel(PTX)withtheimmunostimulantCpG.Thisnanosystem,consistingofmodified lipidnanocapsulescoatedwithacationicpolymericshellcomposedofchitosan(CS),wasabletoallocate thehydrophobicdrugPTXintheinneroilycore,andtoassociateontothesurfacethegeneticmaterial CpG.TheCS-coatedLNC(CS-LNC),showedanarrowsizedistributionwithanaveragesizeof70nmanda positivezetapotential(+25mV).TheyencapsulatedPTXinahighamount(98%),and,duetothecationic surfacecharge,wereabletoadsorbCpGwithoutlosingstability.Asapreliminaryinvitrostudy,the apoptoticeffectonGL261gliomacellswasinvestigated.Thedrug-loadedCS-LNCexhibitedtheabilityto interactwithgliomacellsandinduceanimportantapoptoticeffectincomparisonwithblanksystems.

Finally,theM-LNCmadeofCS-LNCloadedwithbothCpGandPTXweretestedinvivo,injectedvia conventionenhanceddelivery(CED)inGL261-glioma-bearingmice.Theresultsshowedthattheoverall survivalofmicetreatedwiththeM-LNCwassignificantlyincreasedincomparisonwiththecontrol, Taxol1,ortheseparatedinjectionofPTX-loadedLNCandCpG.Thiseffectwasalsoconfirmedbymagnetic resonanceimaging(MRI)whichrevealedthereductionoftumorgrowthintheanimalstreatedwithCpG andPTX-loadedM-LNC.AllthesefindingssuggestedthatthedevelopedM-LNCcouldpotentiateboth CpG immunopotency and PTX antitumor activity by enhancing its delivery into the tumor microenvironment.

ã2015ElsevierB.V.Allrightsreserved.

1.Introduction

Overthelastyearstheelucidationoftheroleoftheimmune systemincancerhasopenednewfrontiersforthedesignofnovel treatments. Cancer immunotherapy represents an attractive alternativethatbroadlyaimstoharnessandredirectthepatient’s own immune system to recognize and fight tumors with high specificity (Reardon et al., 2013). This strategy is of particular interest for patients suffering from primary or secondary glioblastoma.Indeed, conventionaltherapy which includes sur- gerywhenpossible,followedbyradiationandchemotherapy,is non-specificandoftenresultsincripplingdamagetohealthybrain

tissue.(Suryadevaraetal.,2015;FinocchiaroandPellegatta,2011, 2014). Glioblastomas exhibit a complex, immunosuppressive environment that suppresses endogenous antitumor immune- reactivity and fosters immune tolerance (Jarry et al., 2014).To reversethiseffect,differentapproacheshavebeenexplored.The useofmicrobialmoietiessuchaspathogen-associatedmolecular patterns or PAMPs, have demonstrated to promote immune- mediated tumorrejection through thestimulation of microglia cells(Andaloussietal.,2006;Graueretal.,2007,2008).Microglia, theinnateimmune cellswhich serveas aresidentmacrophage population in the brain, express toll-like receptors (TLRs) and respond toTLR ligands producing pro-inflammatory molecules (Rivest, 2009). Among different TLR ligands, synthetic non- methylatedoligonucleotidessuchasCpGhaverecentlyemerged as a powerful immunostimulator for both innate and specific immunity.CpG,aTRL9ligandexpressedbymostmurineimmune cells, can trigger immune rejection and induce long-term

*Corresponding author at: LUNAM Université, Micro et Nanomédecines Biomimétiques,INSERMU1066,IBS-CHU,4RueLarreyAngers49933,France.

E-mailaddress:jean-pierre.benoit@univ-angers.fr(J.-P.Benoit).

http://dx.doi.org/10.1016/j.ijpharm.2015.09.062 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

(2)

immunityagainstgliomas(UrsuandCarpentier,2012).However, CpGasasingleintratumoral(i.t.)injectionhasalsobeenstudiedin patientswithrecurrentgliomaswithtolerabletoxicityandpartial tumorresponseinafewpatients(Carpentieretal.,2010).Oneof thereasonsofthesetreatmentfailurescouldberelatedtothelow glioblastoma-cellular internalization of CpG following intratu- moraladministration(BadieandBerlin,2013).Onceinjected,CpG needsto betaken up byimmune cells in order toinitiate the immunestimulationcascade.Moreover,CpGcanbetransported awayfrom thesite of injection and interact withboth desired immune cells associated with healthy tissue, thus limiting its therapeuticefficacy(BadieandBerlin,2013).

Inthisstudy,ourworkinghypothesiswasthatbycombining chemotherapy and the immune-stimulant agent in a single multifunctional delivery system may provide a more efficient treatmentstrategyavoidinglongtimerelapses,andenhancingthe targetingofbothactivemolecules.Tothisaim,paclitaxel(PTX)was selectedasthedrugmodel.Paclitaxelprimarilyexertsitseffecton tumorcellsbybindingtothe

b

-tubulinsubunitinmicrotubules, preventing depolymerization and increasing their stability and rigidity.Inaddition,itwasshowntostimulateinnateandacquired immune responses (Buchanan et al., 2010; Javeed et al., 2009;

Zitvogeletal.,2008).OurgrouphasalreadydevelopedPTX-loaded LNCwhichhavebeentestedina9Lorthotopicratglioblastoma model(Garcionetal.,2006).Moreover,thesenanocapsuleswere functionalizedwithapeptideinordertoincreasetheiruptakeby theglioma cells (Balzeau etal., 2013).Theresults showed that encapsulationwithinthenanocapsulesprotectthedrugfromthe surroundingmediumandallowsasustainedconcentrationofthe drugandpeptidesatthetumorsite,evenafterasingleinjection.

Based on theseprevious results, here we have developed a multifunctionalnanosystem(M-LNC)adaptedfortheco-delivery ofPTXandCpG.Tothispurpose,PTX-loadedLNCweremodified witha cationicpolymer,chitosan (CS),inordertofunctionalize themwithCpG.

Thegoalwastoassesstheantitumorefficacyofthenanocarrier obtained,followingstereotacticinjectioninGL261glioma-bearing mice, to show the potential of a synergistic effect between chemotherapyandimmunotherapy.

2.Materialsandmethods

Lipoid1S75-3(soybeanlecithinat69%ofphosphatidylcholine), Captex18000(glyceryltricaprylate)andSolutol1HS15(polyethyl- eneglycolesterof12-hydroxystearicacidandpolyethyleneglycol- PEG-SA) were obtained from Lipoid GmbH (Ludwigshafen, Germany),AbitecCorp(Colombus,OH,USA)andBASF(Ludwig- shafen, Germany), respectively. Ethanol solution 96% was pur- chased from Fisher Scientific (Illkirch, France). Taxol1 6mg/ml solutionandPTXpowderweresuppliedbyBristol-MyersSquibb (Rueil-Malmaison, France) and LC Laboratories1 (Woburn, MA 01801, USA), respectively. Sodium chloride, sodium dodecyl sulphate (SDS), chitosan low molecular weight (average Mn 5000Da,>90%deacetylated),propidiumiodide,bis-chloroethylni- trosourea(BCNU) and gel loading buffer werepurchased from Sigma–Aldrich(Saint-Quentin-Fallavier,France).CpG-ODN:Phos- phorothioated-CpG-oligodeoxynucleotide1826,seq (50-30: tccat- gacgttcctgacgtt)(CpG)waspurchasedfromInvivogen(Toulouse, France).AnnexinV-FITCwaspurchasedfromBDPharmingen(San Diego,CA,USA).

2.1.DevelopmentofblankandPTX-loadedLNC

The formulation of LNC was based on the phase inversion processdescribedbyHeurtaultetal.(2002).LNCwerecomposed ofanoilycoreoflipophilicchains(Captex18000,1.2g),lecithin

(Lipoid1 S75-3, 67.2mg), PEG-SA (Solutol1 HS-15,1g) in NaCl (73.3mg) and1.8ml ofwater. Briefly,all thecomponentswere mixedtogetherundermagneticstirring.Threetemperaturecycles werecarriedouttoreachthephase-inversionfromanoil-in-water toa water-in-oil emulsion.After,themixture underwenta fast cooling-dilutionprocessbyadding11.2mlofMilli-Qwaterwhich ledtotheformationofLNC.ToobtainPTX-loadedLNC,15mgPTX werefirstsolubilizedin1mlofethanol andCaptex18000, and thenalltheothercomponentswereadded(Grooetal.,2013).Once obtained,thenanocapsulesuspensionwasfilteredwith0.22

m

m

Ministar1highflowfilters(Sartorius,Aubagne,France)toremove thenon-encapsulateddrugspriortoHPLCquantification.

2.2.DesignanddevelopmentofPTX-loadedCS-LNC

CS-LNC were obtained following electrostatic layer by layer deposition ofthecationic polymer onthenegativesurface oftheLNC.

The negativecharge of LNC arisesfrom the presence of SDS surfactant whichwasaddedtotheinitialemulsionofLNC.Inordertoavoidthe formationofSDSmicelles,alltheconcentrationstestedwerebelow theCMCofthissurfactant(8mM).Then,onceLNCwithSDSwere obtained,analiquotofthesuspension(1ml)wasincubatedwith 0.5ml of CS solution (concentrations from 0 to 5mg/ml) and maintainedundermagneticstirringatroomtemperaturefor1h.

2.3.Physico-chemicalcharacterizationofLNC

The size distribution and surface charge of the prepared nanocarrierswereanalysedusingaMalvernZetasizerNanoSerie DTS1060(MalvernInstrumentsS.A.,Worcestershire,UK).Sample suspensions were diluted in deionized water to ensure a convenient scatter intensity on the detector. The average hydrodynamic diameter, the polydispersity index and the zeta potentialweredeterminedat25Cintriplicate.Thetotalamount ofPTXwasmeasuredintriplicateexperiments,destroyingtheLNC withacetonitrile(ACN).Thefree drugwas quantified following separationoftheLNCbyfiltrationwith0.22

m

mfilter.Infact,the drug encapsulated, PTX, is a very hydrophobic compound and whenitisnotencapsulated,itprecipitatesintheLNCdispersion.If thereissomeprecipitate,filtrationthrough0.22

m

mfilterwould ensureitselimination.

Thealiquotsofbothno-filtratedand filtrated LNCwerethen treatedwithACNandthefollowingequationwasusedtocalculate theencapsulationefficiency:

E:E:¼ð%Þ¼ðB100Þ A

whereAistheexperimentaltotaldrugconcentrationandBisthe drugconcentrationmeasuredinthefiltratedsolution.

ThedeterminedamountofLNCdispersionswasdissolvedina 96:4 (v/v) methanol/tetrahydrofuran mixture and a Waters Alliance2690HPLCsystemwasused(WatersS.A.,Saint-Quentin enYvelines,France)toquantifythedrug.

A20

m

laliquotofthefiltratewasinjectedintriplicateintoan HPLC XTerra RP18 column (5

m

m, 4.6mm150mm) (Waters, Guyancourt,France).Theanalysiswasperformedwithwaterand acetonitrileinthemobilephase,withagradientelutionprogramof 50–80%acetonitrile,ataflowrateof1ml/min.Elutingfractions wererevealedwithaWaters2487Dual-absorbanceDetector,ata wavelengthof227nm.DatawereanalyzedbytheEmpowerPro1 software,version5.00.PTXgavearetentionpeakat7.2min.

2.4.CpGassociationtoPTX-loadedCS-LNC

ToobtaintheM-LNC,CpGwasadsorbedonthesurfaceofCS- LNC at 0.8–0.75–0.5 and 0.25% w/w loadings, defined as the

(3)

percentagebetweenthemassof CpGand thetotalmassofthe formulation.Fortheadsorptionprocedure,theCpGsolution(20

m

l,

concentration10

m

g/ml)wasaddedtotheLNCsuspension(80

m

l)

andmaintainedundermagneticstirringforonehourtoachievean optimal interaction between the CpG and the nanocapsules.

Differentweightratioswereobtainedbymodifyingtheconcen- trationofCpG;meanwhiletheconcentrationofnanocapsuleswas maintained constant. CpG-associated LNC were characterized according to size and zeta potential as detailed previously.

Additionally, the association of CpG to LNC was studied by a conventionalagarosegelelectrophoresisassay.Inordertodisplace the CpG adsorbed in the nanocapsules, an excess of heparin solution(15mg/ml–2.8

m

l)wasaddedtothesuspensionandthe

mixturewasincubatedfor2hat37C(Lozanoetal.,2013).The CpGwasstainedwithSYBR1GreenIbyadding2

m

lofthereagent

diluted 1:50,000 in DMSO, to 100

m

l of the CpG-associated nanocapsules and 9

m

l of gel loading, and left to interact for

15min.Then,thesamplesandthecontroloffreeCpGwereloaded in1.4%agarosegelandrunfor60minat50VinaTAEbuffer(Sub- Cell GT 96/192, Bio-Rad Laboratories Ltd., Marnes-la-Coquette, France).

2.5.StabilityofPTX-loadedCS-LNC

ThestabilityofPTX-loadedCS-LNCwasevaluatedunderstorage conditionsforonemonthat4C.Threesetsofparameterswere assessedatdifferenttimepoints:(i)macroscopicaspect(presence ofaggregated,creamformation,changesincolor);(ii)particlesize, polydispersityand zetapotential;(iii) PTXconcentrationinthe preparationandencapsulationefficiency.Allthesecharacteristics weredeterminedasdescribedabove.

2.6.Cellline

The GL-261 murine glioma cell line (C57BL/6 origin, H2b), kindly provided byDr Paul Walker (Geneva, Switzerland), was culturedinDMEMmediumcontaining10%heat-inactivatedFetal Calf Serum (FCS), 2mM glutamine, 10mM HEPES and 1mM sodiumpyruvate(allfromLonza,Basel,Switzerland).Thecellline wasgrownat37Cunderahumidified,5%CO2atmosphere.

2.6.1.Cellapoptosisassay

TheGL261celllinewasincubatedwith500

m

MBCNUfor24h

withdifferentconcentrationsofTaxol1,PTX-loadedCS-LNC,and thecorrespondingblank formulation. TheM-LNC, consisting of CpG-PTX-loadedCS-LNC, werealso tested.Apoptotic cell death wasrevealedbyAnnexinV-FITC/propidiumiodidestaining.Briefly, cells were washed and incubated for 10min in buffer (10mM HEPESpH7.4,140mMNaCl,2.5mMCaCl2)containing0.6

m

g/ml

Annexin-Vfollowedby1.6

m

g/

m

lpropidiumiodide.Thenumberof

deadcellswasdeterminedbyflowcytofluorometryusingaFACS calibercytofluorometer(BDBiosciences,LePontdeClaixCedex, France)and thedatawereanalyzedusing FlowJoSoftware(BD Biosciences,LePontdeClaixCedex,France)(n=5).

2.7.Animals

Invivoefficacyexperimentswerecarriedoutonsixtoseven- week-oldfemaleC57BL/6JmicepurchasedfromJanvier(France, SaintBerthevinCedex,France).Themiceweremaintainedunder pathogen-freeconditions,andtheexperimentswerecarriedoutin accordance with French laws and regulations. The procedure (number CEEA-2012-148) was approved by the French Ethics Committeeforanimalexperimentationnumber6.

2.7.1.Invivoantitumorefficacyandoverallsurvival(OS)study InvivoanticanceractivitywasevaluatedagainstGL261tumor- bearing C57BL/6J mice.The tumorcell linewas detached with trypsinEDTA,washedtwicewithPBS,counted,andre-suspended tothefinalconcentrationdesired.

Braintumors wereinducedbythestereotaxicinoculationof gliomacellsaspreviouslydescribed(Lemaireetal.,2000).Briefly, C57BL/6Jmicewereanesthetizedwithamixtureof1

m

g/gxylazine

(Rompun1,BayerAG,Leverkusen,Germany)and10

m

g/gkétamine

(Clorketam1, Vétoquinol, Lure, France) before being fixed in a stereotactic holder (Stoelting, Dublin, Ireland). Through a small holedrilledintheskull (anterior0.5mm,lateral 2mm,depth 2.5mmaccordingtothebregma),3

m

lofasuspensionof10,000

murineGL261gliomacellswereinjectedovera6-mintimeperiod intothecaudateputamenoftherighthemisphere.Thesyringewas heldinplaceforanadditionalminuteandwasslowlyremovedto avoidback-fillingofthesolution.

Twelvedaysaftercellimplantation,mice(7–10micepergroup) were intratumorallyadministered with 10

m

lof saline, Taxol1,

blankLNC,PTX-loadedLNC,PTX-loadedCS-LNC,PTX-loadedLNC plus a separate injection of CpG and M-LNC by convection enhanceddelivery(CED)(0.5

m

l/min).

Animals were observed daily and reduced mobility and significantweightloss(20%)wereconsideredasthelimitpoint forsurvivalcurves.

2.7.2.Assessmentoftumorevolutionbymagneticresonanceimaging (MRI)

Priortotherapy,i.e.Day10postGL261inoculation,themice were scanned using a Bruker Biospec 70/20 operating at a magnetic field of 7T (Bruker, Wissembourg, France) equipped with a 1H cryoprobe under isoflurane anesthesia (1.5–0.5%, O2

0.5l/min)toassesstumordevelopment.Mousebodytemperature was maintained at 36.5–37.5C by using a feedback-regulated heatingpadduringtheentireimagingprotocol.Anatomicalproton imageswereobtainedusingagradientechosequence(TR=110ms;

echotime(TE)=3.3ms;

a

=60,FOV=22cm;matrix192192;

9–11 contiguous slicesof 0.5mm, Nex=4)afteri.v.injectionof 10

m

lofDOTAREM1(Guerbet,Roissy,France).

On theseimages, contrast-enhanced regions were manually drawnandthemeasuredareaoneachsliceweremultipliedbythe slicethicknessinordertocalculatetheentiretumorvolume(n=5 forthecontrolandn=3forthetreatedanimals).

MRIexaminationswerecarriedouttwiceagain,onDays17and 23 post-inoculation in order to evaluate tumor growth and thereforetherapeuticefficiency.

3.Statisticalanalysis

Inthepresentstudy,amultifactorialexperimentaldesignwas employedforprocessoptimization.Theeffectoftheindependent variablesasSDSandCSconcentrationonthedependentvariables sizeand zetapotential of thenanoparticleswas evaluated.The numberoflevelsusedwasof5asrepresentedintheTable1.25 runswereperformedintriplicate,whichalsoincludedthecentral point.

Cell data (expressed as meanSEM) were analyzed using GraphPadPrism5.0software(GraphPadSoftware,Inc.,SanDiego,

Table1

Independentvariablesandtheirlevelsforthemultifactorialexperimentaldesign.

CS:chitosanandSDS:Sodiumdodecylsulphate.

Symbol Units 1Level 0.5Level 0Level +0.5Level +1Level

CS mg/ml 0 1.25 2.5 3.75 5

SDS mM 0 1 2 3 4

(4)

CA,USA).AnimalsurvivaldatawereanalyzedusingtheKaplanand Meiersurvivalanalysis.Statisticalanalysisusedthelog-ranktest forsurvivalcurvesandpvaluesoflessthan0.05wereconsidered significant.

4.Results

4.1.DevelopmentandcharacterizationofblankandPTX-loadedCS- LNC

BlankandPTX-loadedLNCweresuccessfullyobtainedfollow- ing the phase-inversion technique. SDS was used to obtain a negativesurfacechargeontheLNC,necessaryforthedepositionof CS.SDSwaslocatedattheinterphasebetweentheoilcoreandthe externalaqueousenvironmentandinteractselectrostaticallywith CS,allowingtheformationofCS-coatedLNC.CS-SDSinteraction wasconductedbyacombinationofelectrostatic ion-dipoleand hydrophobicinteractionsthatoccurwhenthesurfactantisbelow itscriticalmicelleconcentration(CMC=8mM)(Chatterjeeetal.

2012; Thongngam and McClements, 2004). Fig. 1 shows a representationofLNCmodifiedwithSDSandCS.

Pilot experiments were performed to define the optimal productionconditionsofCS-coatedLNC.Briefly,usingamultifac- torialexperimentaldesign,westudiedtheinfluenceoftheSDSand CSconcentrations,andtheirinteractions,byvaryingtheamountof bothcomponentsintheformulation.Thestudiedconcentration rangeswerefrom0to4mMandfrom0to5mg/mlofSDSandCS, respectively.

The analysis of the physico-chemical properties of the LNC showedthatalltheformulationsobtainedwereofnanometricsize, between50 and 80nm, withzeta potentialsindicative of their coating(Fig.2AandB).SDS,testedatconcentrationsbetween1 and4mM,didnotsignificantlyaffect(p>0.05)thesizeoftheLNC.

However, once LNC were incubated with CS, there was a logarithmic increasein particlesize. Finally, thesize reacheda plateauwhichsuggeststhattheparticlesurfacewassaturated,and thattheexcesschitosandidnotaffectthestructureofthesystem, remainingdissolvedintotheaqueousmedium.Thesameoccurs withthe surface charge. In fact, when the surface charge was saturated,anyadditionalCSwentintotheaqueousphaseanddid notaffectthesystem.

Usingthemodeltominimizetheparticlesizeintotheevaluated range,thefollowingconditionswereselectedinordertoobtain particlesaround70nm:SDSconcentration4mMandCSconcen- tration2.5mg/ml.

ToachievetheincorporationofPTXintoLNC,SDS-modifiedor CS-LNC,agivenamountof thedrugwasdissolved intotheoily phaseoftheformulation,followedbythephaseinversionandthe coating processes.The percentage of the encapsulateddrug,in referencetothetotalamountofdrugdissolved,wasveryhigh,up

to98%,withoutanyinfluencefromthepresenceofeitherSDSorCS ascomparedtothesimpleLNC(Table2).

4.2.StabilityofPTX-loadedCS-LNC

The stability of coated and uncoated PTX-loaded LNC was assesseduponstorageat4Cforupto4weeks.Theparameters determined at different time points were: physico-chemical properties,PTX releaseand stability.Nochangesintheparticle size and zeta potential were observed which confirm that the surface charge of the nanocarriers did not change over time, anotherindicationofchemicalandphysico-chemicalstabilityof thesystem.Moreover,noleakageofthedrugwasshownduringthe whole experimental period, thus confirming that PTX remains encapsulatedintotheoilycoreofthesystem.

4.3.CpGassociationtoPTX-loadedCS-LNC

OncethebaseofourmultifunctionalLNCwasoptimized,we proceededtoloadtheCpGontheirsurfacebysimpleincubation.

TheformationofcomplexesbetweenCpGandCS-coatedLNCwas confirmedbygel-retardationassay. AsCpGinteracted withCS- coatedLNC,viaelectrostaticinteractions,themigrationofCpGon agarosegelwasretardedduetochargeneutralizationandtothe highmolecularweightoftheformedcomplex(Lozanoetal.,2013).

As shown in Fig. 3, slight migration of CpG molecules was observed, after its association with the CS-LNC, for theoretical loadings of 0.8–0.75–0.5–0.25% w/w of CpG/CS-coated LNC.To verifythereversibilityoftheCpGassociationtonanocapsules,we evaluatedthedisplacementofCpGmoleculesfromLNCsurfaceby challengingthem,usingcompetitiveanionssuchasheparin.For this,anexcessofheparinwasincubatedwithCpG-associatedLNC.

Theappearanceofthebandafterheparintreatmentillustratesthe detachmentofCpGmoleculesfromtheshellofthenanocapsules.

Based ontheseresults, andontheimprovedstability at4C of 0.75% w/w loaded nanocapsules with the respect to the 0.8%

formulation, the firstone was selected for furtherstudies. The physico-chemicalcharacteristicsofthefinalM-LNCsystemwere thesamethattheonesobtainedfortheCScoatedPTX-loadedLNC (75nm,0.2PIand+27mV).

4.4.Inducedapoptoticcelldeathfollowingthetreatmentwithblank andPTX-loadedCS-LNC

GL261 glioblastoma cells were used to study cell apoptosis induced by various PTX formulations. At the earlier events of apoptosis,themembrane proteinphosphatidylserine(PS)trans- locatesfromtheinnersideoftheplasmamembranetothesurface andcanbedetectedbyAnnexinV-FITCstaining.Inlaterstagesof apoptosis,PIcanpermeatethecellmembraneandpassintothe nucleus where it binds to the DNA. Then, a double staining AnnexinV-FITC/PIindicatesthatcellsareinalatestageofapoptotic death.

Asafirstcontrol,GL261cellsweretreatedwithfreeCpGfor24h tostimulateTLR9mRNA.Asexpected,noTLR9mRNAwasdetected, asTLR9receptorswereessentiallyabsentinthiscellline(Grauer et al., 2008).In contrast, TLR9mRNA was foundin thecentral nervoussystemhomogenate(whichcontainsalltheimmunologi- calcells)usedasapositivecontrol.

GL261 cell death inductionafter 24htreatmentwith BCNU (500

m

M)usedasanotherpositivecontrolwasevaluated.Infact,

BCNUinducedstrongcelldeath:20%of GL261cellsin theearly apoptoticphase (Ann+/P.I)and80%inthelateapoptoticphase (Ann+/P.I+).

Fig.1.SchematicrepresentationofLNCpreparedwithSDSandCS.

(5)

ThenumberofGL261cellsundergoingapoptosiswasevaluated followingdifferenttreatmentswith:PTX ethanolsolution,PTX- loadedCS-LNC,andtherespectiveblanksystems(Fig.4).

At7and10

m

M,PTXethanolsolutioninducesahighproportion

oflateapoptoticcellswith73%and70%,respectively.WhenCS- LNC were used to encapsulate PTX, about 50% of the cells experiencedlate apoptoticcelldeath. Blank LNCand blankCS- coatedLNC,testedatthesameconcentrationofdrug-loadedLNC, inducedalowapoptoticcelldeathwith19%oflateapoptoticcells atanLNCconcentrationof0.621mg/ml.Thiseffectcouldberelated to a different mechanism of LNC uptake. In fact, as already demonstratedforPTX-loadedLNC,theencapsulationofthedrug intotheLNCdidnotleadtoanincreaseofPTXuptakeinGL261 gliomacells.Balzeauetal.(2013)demonstratedthatonlythrough thefunctionalizationoftheLNCcouldthecelluptakebeimproved.

Finally, M-LNC were tested and, due to the absence of TLR9 receptorsonGL261,thesameapoptoticbehaviorofPTX-loadedCS- LNCwasobserved.

4.5.Invivostudy

TheinvivoantitumorefficacyoftheM-LNCwasevaluatedina GL261 glioma mouse model. The OS study was carried out by groupingtheanimalsintosevendifferenttreatment,formulation andscheduletimes(Fig.5).OnDay0,GL261cellswereimplanted intothebrainoftheanimals,andthetreatmentwascarriedout12 dayslater.On Days10,17 and23aftercellimplantation,tumor volumeswereassessedusingMRI.TheOScurveswereanalyzed accordingtoKaplan–Meiermethodandcomparedusingalog-rank test.AsshowninFig.6,whenmiceweretreatedwithTaxol1,PTX- loaded LNC, PTX-loaded LNC plus a separate injection of CpG solution,or PTX-loaded CS-LNCthe medianof survival did not differfromthecontrolanimalsreceivingsalinesolutionorblank LNC (Table 3). However, treatment with M-LNC significantly increasedthemousemediansurvivaltime(34days)comparedto the saline group (28 days). This result shows that the co- administration in thesame system of thecytotoxic agentPTX, andtheimmunoadjuvantCpG,affectedtheOSoftheanimals.

Fig.2.(A)ResponsesurfaceshowingtheeffectofSDSconcentration(mM)andCS(mg/ml)ontheparticlesizeofblankLNC.(B)ResponsesurfaceshowingtheeffectofSDS concentration(mM)andCS(mg/ml)onthesurfacechargeofblankLNC.

Table2

Characteristicsofthreedifferentformulationsfollowingtheoptimizedconditionscalculatedfromthemultifactorialexperimentaldesign.n=3.

Formulation SDS(mM) CS(mg/ml) Size(nm) PI ZetaPotential(mV) %Drugdetected

PTX-loadedLNC 552 <0.1 62 982

PTX-loadedLNC-SDS 4mM 531 <0.1 231 981

PTX-loadedCS-LNC 4mM 2.5 703 <0.2 +316 982

Fig.3.(A)AgarosegelelectrophoresisretardationassayofCpGinteractingwithCS-LNCatdifferentCpG/CSratios.(B)Releaseassay.CpGwasreleasedbyaddinganexcessof heparin(15mg/ml)tothesuspensionpreparedatdifferentCpGratios.

1-nakedCpG,2–0.8%w/wCpGassociatedPTX-loadedCS-LNC,3–0.75%w/wCpGassociatedPTX-loadedCS-LNC,4–0.5%w/wCpGassociatedPTX-loadedCS-LNC,5–0.25%w/

wCpGassociatedPTX-loadedCS-LNC.6–7–8–9–10representsthewells1–2–3–4–5incubatedwiththeexcessheparin.

(6)

4.6.Monitoringoftumorgrowthbymagneticresonanceimaging AlongsidetheOSstudy,MRIinvestigationwascarriedoutto better characterize therapy efficiency, especially in the M-LNC group,whichgavethebestresultsintermsofOSandmedianof survivalascomparedtothecontrolsalinegroup.

Firstofall,duetothelownaturalcontrastbetweenGL261brain tumor and brain parenchyma, both on T1- and T2-weighted images,administrationoftheMRcontrastagentwascompulsory todelineatethebraintumor(Fig.7).Thestereotacticinjectionof salinedidnotrestrain tumordevelopmentwhich was of about 10

m

lonDay10,andreached15

m

lonDay17and83

m

lonDay23

(Fig. 8).However,treatmentwithM-LNC significantly impaired tumorgrowthwithareductioninsizeof50%byDay23.

5.Discussion

Withthepurposeoftakingadvantageofthesynergisticeffect betweenchemoandimmunotherapy,thegoalofthepresentstudy wasthedevelopmentofamultifunctionalnanosystemcapableof delivering the anticancer drug PTX, simultaneously with the immunostimulatingCpGintobraintumors.

Despiteitspromises,theclinicalutilityoffreeCpGstillfaces severalchallengeswhichlimititseffectiveness.Thedevelopment of nanosystems fortheinvivo deliveryof CpGhassuccessfully addressed a number of these shortcomings by providing an efficientmeanstoimprovepK characteristics,toprotectitfrom degradationandprovideadirecttransporttotargetcells.Lipid- basednanoparticleshavebeenshowntoexertimprovedimmu- notherapeutic activityofCpGintoTLR9-rich endosomesinvitro andinvivoindifferenttumormodels(Chikhetal.,2009;Wilson et al., 2009). Also, the ability of nanosystems to enhance the activityof CpGasanimmuneresponsemodifierisofparticular relevanceandhasbeenpreviouslydescribed(UrsuandCarpentier, 2012).

Fig.4.TheapoptosisstudyofGL261cellsafter24hofincubationwith0.6mg/mlofblankLNC(A-1)andblankCS-LNC(A-2),PTX-loadedCS-LNCatPTXconcentrationof7mM (A-3),PTXethanolsolutionatPTXconcentrationof7mM(A-4).B:Cellsincubatedfor24hwithvariousconcentrationsofPTXethanolsolution(*),blankLNC(^),PTX-loaded CS-LNC(~),blankCS-LNC(X)andCpG-PTX-loadedCS-LNC(&)(n=5,meanSEM).

Fig.5.ScheduleofadministrationestablishedtoperformtheOSstudy.OnDay0 GL261cells were implanted.Then,on Day12 thedifferent treatmentswere administeredthroughCED.OnDays10,17and23,MRIimagesofmicetreatedwith salineandM-LNCweretakentofollowtumorevolution.

Fig.6.EvolutionofmousesurvivalfollowingstereotacticinjectionbyCEDof10ml ofblankLNC(^),PTX-loadedLNCs(&),PTX-loadedLNC+CpG(),PTX-loadedCS- LNC(~),andM-LNC(&),Taxol1andsalinesolutionswereusedascontrols.Log- rank(MantelCox)test:**pvalue<0.001n=7–10micepergroup.

(7)

Alltheseeffectivepreclinicalandevenearlyclinicalstrategies havenotbeentranslatedintosuccessfulhumantherapies,which havequestionedthe clinicalutilityof free and/or encapsulated CpG.Fromhere,therationaleofourmultifunctionalnanosystem designedtocombinetheactionofCpGandthecytotoxicdrugPTX wasset.Wedemonstrated thatfunctionalized LNCloadedwith bothPTXandCpGcouldbeusedtosynergizethechemo-immuno responseleading toan improvementof the overalltherapeutic activity.

Withthisaim,suitablePTX-loadedLNCweremodifiedwitha cationicpolymericcoating.Thisstructurewasexpectedtohavea double role: the lipidic core of the LNC would allow an improvement of hydrophobic drug bioavailability and stability, andtheexternalcoatingwouldconferimmunostimulantproper- tiesthrough thepresenceof CpG.CS wasselected asa natural biopolymer to provide a positive charge layer around the oil droplets after immobilization by layer by layer deposition (Démoulins et al., 2014; Vicente et al. 2014). The biophysical properties of chitosan–genetic material complexes and their capacity for transfection have been investigated in detail and beneficialpropertiesincludealowmolecularweight,ahighDD (Degreeof Deacetylation),a small particlesize(100nm),and a moderatepositivesurfacezetapotentialalongwithahigh(+/) chargeratio(Santos-Carballaletal.,2015;Démoulinsetal.,2014).

Inthiswork,lowMw(kDa)CSwithahighDDwaschosentocoat theLNC.Therationale behindthis choicewas that (i)highDD

chitosan results in an increased positive charge and water solubility,thusenablingagreaterCpGbindingcapacity(Démou- linsetal.,2014;Agirreetal.2014),and(ii)lowMWchitosanhas beenassociated withminor cytotoxicitycomparedtohighMW polymers(Alamehetal.,2012;Liuetal.,2007).

The novel multifunctional nanocarrier made of CS-LNC was developed. Theaddition ofthe polycationicCSinduces a slight increase in size (from 55nm to 70nm) but a major changein surfacecharge(from6mVforuncoatedLNCto+30mVin CS- coatedLNC)allowingthelayerdepositionofCpG.Moreover,the incubationofCpGdidnotmodifythephysico-chemicalcharacter- isticsofthesystem,thesizewasinanappropriaterange(70nm), and novariation in zetapotential and no leakageof PTX were observed.This improvedthestability of thedrug-loaded nano- systems,togetherwiththeabilitytoassociateCpGonthesurface, makingthecarriersuitableforfurtherinteractionwiththeTLR9- receptorexpressedinmicrogliacells(Rivest,2009).

In the first set of experiments, we evaluated the relative distribution of early apoptotic and late apoptotic/necrotic cells followingtreatmentwithblankandPTX-loadedLNC(Fig.3).Inour studyweshowedthat blankLNCorCS-LNCdidnotinduceany apoptoticeffect. On thecontrary,due tothehighsensitivityof these cells to the drug, PTX solution induced a considerable apoptoticeffect.ThegreatesteffectwasobtainedwithPTX-loaded CS-LNC,withavalueoflateapoptoticcellsofabout50%.Thiseffect could probably be explained by a different mechanism of internalization of the modified LNC that deserves further investigation.

Then,theinvivoantitumoractivityofthedifferenttreatments wasevaluatedintheGL261tumormicemodel.TheGL261glioma model,whichisthegoldstandardsyngeneicmodelforscreening pre-clinical treatments in glioma, is fullyrelevant since it is a highlyreproducibleandeasy-to-establishmodelsystemanddoes notcompromisetheimmuneresponse(MaesandVanGool,2011).

In ourstudy,theintracranial injectionofM-LNC, enhanced the antitumoreffectofdrug-loadedLNCorCpGalone.

AccordingtoJarryetal.(2014)intratumorinjectionoffreeCpG has pour efficiency in inducing tumor regression, which is in Table3

SummaryofthemediansurvivalofmiceafterGL261implantationandtreatment.p value<0.001.

Treatment Mediansurvival(day)

Control 28

BlankLNC 27

Taxol1 28

PTX-loadedLNC 31

PTX-loadedLNC+CpG 31

PTX-loadedCS-LNC 31

M-LNC 34**

Fig.7.T1-weightedimagesofGL261-bearingmiceafterIVinjectionofDotarem1.FramesAcorrespondtosalinesolution-treatedmiceimagedonDays10, 17and23aftercell inoculation.FramesBcorrespondtoM-LNC-treatedmiceimagedonDays10,17and23aftercellinoculation.

(8)

agreement with clinical trials in recurrent malignant glioma showinglowtherapeuticefficacyofintracerebralCpGadministra- tion.ThesameoccurredwithTaxol1,thecommercialformulation ofpaclitaxel,andwiththeadministrationofPTX-loadedLNCand CpGseparately,evidencingthatonlyfromacombinedapproach couldthetreatmentbeeffective.TheresultsoftheOSshownin Fig. 6 demonstrate the therapeutic effect evoked by the administration of a single system that could allocate PTX and theimmunostimulantagent.Theimmunotherapeuticandsyner- gisticeffectresultingfromtheassociationofPTXandCpGcouldbe ascribed to different factors. Firstly, GL261 tumor cells do not expressTLR9,andhencethetherapeuticeffectobservedintheOS studycouldbeattributedtothedirect stimulationof microglia cells(Graueretal.,2008).Secondly,asreportedintheliterature, PTXantitumoractivitycausesdirectcelldeathwhichleadstothe release of tumor-specific antigens from tumor cells as wellas dangersignals—damage-associatedmolecularpatterns(DAMPs).

Alltheseeventscontributetothestimulationandtherecruitment ofimmunocompetentcells(Royetal.,2013;Szajniketal.,2009).

OurfindingshavedirectimplicationsonthedevelopmentofCNS- basedtreatmentformalignantbraintumors.Thestrategybasedon a single in situ administration of both CpG and PTX, can be employedtoovercomealocalimmunosuppressivemicroenviron- mentthroughmodulationsoftumorimmuneresponse.

6.Conclusions

Inthiswork,wereportthedesignofanovelmultifunctional carrierbasedontheincorporationofacytotoxicdrug,PTX,anda TLR9receptoragonist,CpG,onCS-modifiedLNCandtheiruseina chemo-immunotherapy strategy. CS-coated nanocarriers exhib- itedattractivepropertiesfortheencapsulationanddeliveryofboth anticancerdrug and genetic material.The co-administration of CpGandPTXwiththesamenanocarrierintheorthotopicGL261 gliomamousemodelsignificantlyimprovedthesurvivalrateofthe animalsascomparedwithTaxol1andtheseparateadministration ofPXT-loadednanocapsulesandCpG.Consequently,thisstrategy could beof interest for achieving an antitumor effect through stimulationoftheimmunesystem.Combiningchemotherapywith innovative immunotherapy, in the M-LNC, will lead to a novel chemo-immunotherapeutic approach to control the long-term diseaseandhopefullyinducetumorrejection.

Acknowledgments

ThisworkwassupportedbytheRegionPaysdelaLoire(France) Project:CIMATH2.Theauthorswouldliketothankthetechnicians from ‘Service Commun Animalerie Hospitalo-Universitaire

d’Angers’(SCAHU)forthehousingandcareof theanimalsused in thisstudy. WealsothankKarimBeywhocontributed tothe developmentoftheinvitrostudies.

References

Alameh,M.D.D.,Jean,M.,Darras,V.,Thibault,M.,Lavertu,M.,Buschmann,M.D., Merzouki,A.,2012.Lowmolecularweightchitosannanoparticulatesystemat lowN:Pratiofornontoxicpolynucleotidedelivery.Int.J.Nanomed.7,1399–

1414.

Agirre,M.,Zarate,J.,Ojeda,E.,Puras,G.,Desbrieres,J.,Pedraz,J.L.,2014.Low molecularweightchitosan(LMWC)-basedpolyplexesforpDNAdelivery:from Benchtobedside.Polymers6,1727–1755.

Andaloussi,A.E.,Sonabend,A.M.,Han,Y.,Lesniak,M.S.,2006.StimulationofTLR9 withCpGODNenhancesapoptosisofgliomaandprolongsthesurvivalofmice withexperimentalbraintumors.Glia54,526–535.

Badie,B.,Berlin,J.M.,2013.ThefutureofCpGimmunotherapyincancer.

Immunotherapy5,1–3.

Balzeau,J.,Pinier,M.,Berges,R.,Saulnier,P.,Benoit,J.-P.,Eyer,J.,2013.Theeffectof functionalizinglipidnanocapsuleswithNFL-TBS.40–63peptideontheiruptake byglioblastomacells.Biomaterials34,3381–3389.

Buchanan,M.M.,Hutchinson,M.,Watkins,L.R.,Yin,H.,2010.Toll-likereceptor4in CNSpathologies.J.Neurochem.114,13–27.

Carpentier,A.,Metellus,P.,Ursu,R.,Zohar,S.,Lafitte,F.,Barrié,M.,Meng,Y.,Richard, M.,Parizot,C.,Laigle-Donadey,F.,Gorochov,G.,Psimaras,D.,Sanson,M.,Tibi,A., Chinot,O.,Carpentier,A.F.,2010.IntracerebraladministrationofCpG oligonucleotideforpatientswithrecurrentglioblastoma:aphaseIIstudy.

Neuro.-Oncol.12,401–408.

Suryadevara,C.M.,Verla,T.,Sanchez-Perez,L.,Reap,E.A.,Choi,B.D.,Fecci,P.E., Sampson,J.H.,2015.Immunotherapyformalignantglioma.Surg.Neurol.Int.6, S68–S77.

Chatterjee,S.,Salaün,F.,Campagne,C.,Vaupre,S.,Beirão,A.,2012.Preparationof microcapsuleswithmulti-layersstructurestabilizedbychitosanandsodium dodecylsulfate.Carbohydr.Polym.90,967–975.

Chikh,G.,deJong,S.D.,Sekirov,L.,Raney,S.G.,Kazem,M.,Wilson,K.D.,Cullis,P.R., Dutz,J.P.,Tam,Y.K.,2009.SyntheticmethylatedCpGODNsarepotentinvivo adjuvantswhendeliveredinliposomalnanoparticles.Int.Immunol.21,757–

767.

Démoulins,T.,Milona,P.,McCullough,K.C.,2014.Alginate-coatedchitosannanogels differentiallymodulateclass-Aandclass-BCpG-ODNtargetingofdendriticcells andintracellulardelivery.Nanomed.Nanotechnol.Biol.Med.10,1739–1749.

Finocchiaro,G.,Pellegatta,S.,2011.Immunotherapyforglioma:gettingclosertothe clinicalarena?Curr.Opin.Neurol.24,641–647.

Finocchiaro,G.,Pellegatta,S.,2014.Perspectivesforimmunotherapyin glioblastomatreatment.Curr.Opin.Oncol.26,608–614.

Garcion,E.,Lamprecht,A.,Heurtault,B.,Paillard,A.,Aubert-Pouessel,A.,Denizot,B., Menei,P.,Benoît,J.-P.,2006.Anewgenerationofanticancer,drug-loaded, colloidalvectorsreversesmultidrugresistanceingliomaandreducestumor progressioninrats.Mol.CancerTher.5,1710–1722.

Grauer,O.,Pöschl,P.,Lohmeier,A.,Adema,G.,Bogdahn,U.,2007.Toll-likereceptor triggereddendriticcellmaturationandIL-12secretionarenecessaryto overcomeT-cellinhibitionbyglioma-associatedTGF-b2.J.Neurooncol.82, 151–

161.

Grauer,O.M.,Molling,J.W.,Bennink,E.,Toonen,L.W.J.,Sutmuller,R.P.M.,Nierkens, S.,Adema,G.J.,2008.TLRligandsinthelocaltreatmentofestablished intracerebralmurinegliomas.J.Immunol.181,6720–6729.

Groo,A.C.,Saulnier,P.,Gimel,J.C.,Gravier,J.,Ailhas,C.,Benoit,J.P.,Lagarce,F.,2013.

Fateofpaclitaxellipidnanocapsulesinintestinalmucusinviewoftheiroral delivery.Int.J.Nanomed.8,4230–4291.

Heurtault,B.,Saulnier,P.,Pech,B.,Proust,J.E.,Benoit,J.P.,2002.Anovelphase inversion-basedprocessforthepreparationoflipidnanocarriers.Pharm.Res.

19,875–880.

Jarry,U.,Donnou,S.,Vincent,M.,Jeannin,P.,Pineau,L.,Fremaux,I.,Delneste,Y., Couez,D.,2014.TregdepletionfollowedbyintracerebralCpG-ODNinjection inducebraintumorrejection.J.Neuroimmunol.267,35–42.

Javeed,A.,Ashraf,M.,Riaz,A.,Ghafoor,A.,Afzal,S.,Mukhtar,M.M.,2009.Paclitaxel andimmunesystem.Eur.J.Pharm.Sci.38,283–290.

Lemaire,L.,Franconi,F.,Saint-Andre,J.P.,Roullin,V.G.,Jallet,P.,LeJeune,J.J.,2000.

High-fieldquantitativetransverserelaxationtime,magnetizationtransferand apparentwaterdiffusioninexperimentalratbraintumour.NMRBiomed.13, 116–123.

Liu,X.,Howard,K.A.,Dong,M.,Andersen,M.Ø.,Rahbek,U.L.,Johnsen,M.G.,Hansen, O.C.,Besenbacher,F.,Kjems,J.,2007.Theinfluenceofpolymericpropertieson chitosan/siRNAnanoparticleformulationandgenesilencing.Biomaterials28, 1280–1288.

Lozano,M.V.,Lollo,G.,Alonso-Nocelo,M.,Brea,J.,Vidal,A.,Torres,D.,Alonso,M.J., 2013.Polyargininenanocapsules:anewplatformforintracellulardrugdelivery.

J.Nanopart.Res.15,1–14.

Maes,W.,VanGool,S.,2011.Experimentalimmunotherapyformalignantglioma:

lessonsfromtwodecadesofresearchintheGL261model.CancerImmunol.

Immunother.60,153–160.

Reardon,D.A.,Wucherpfennig,K.W.,Freeman,G.,Wu,C.J.,Chiocca,E.A.,Wen,P.Y., Curry,W.T.,Mitchell,D.A.,Fecci,P.E.,Sampson,J.H.,Dranoff,G.,2013.Anupdate Fig.8.Measurementoftumorvolumeevolutionfollowingstereotacticinjectionby

CEDof10ml,saline(&)andM-LNC(&).Datarepresentn=3SME(90%confidence interval).

(9)

onvaccinetherapyandotherimmunotherapeuticapproachesforglioblastoma.

ExpertRev.Vaccines12,597–615.

Rivest,S.,2009.Regulationofinnateimmuneresponsesinthebrain.Nat.Rev.

Immunol.9,429–439.

Roy,A.,Singh,M.S.,Upadhyay,P.,Bhaskar,S.,2013.Nanoparticlemediatedco- deliveryofpaclitaxelandaTLR-4agonistresultsintumorregressionand enhancedimmuneresponseinthetumormicroenvironmentofamousemodel.

Int.J.Pharm.445,171–180.

Santos-Carballal,B.,Aaldering,L.J.,Ritzefeld,M.,Pereira,S.,Sewald,N., Moerschbacher,B.M.,Götte,M.,Goycoolea,F.M.,2015.Physicochemicaland biologicalcharacterizationofchitosan-microRNAnanocomplexesforgene deliverytoMCF-7breastcancercells.Sci.Rep.5,13567.

Szajnik,M.,Szczepanski,M.J.,Czystowska,M.,Elishaev,E.,Mandapathil,M.,Nowak- Markwitz,E.,Spaczynski,M.,Whiteside,T.L.,2009.TLR4signalinginducedby lipopolysaccharideorpaclitaxelregulatestumorsurvivalandchemoresistance inovariancancer.Oncogene28,4353–4363.

Thongngam,M.,McClements,D.J.,2004.Characterizationofinteractionsbetween chitosanandananionicsurfactant.J.Agric.FoodChem.52,987–991.

Ursu,R.,Carpentier,A.,2012.In:Yamanaka,R.(Ed.),ImmunotherapeuticApproach withOligodeoxynucleotidesContainingCpGMotifs(CpG-ODN)inMalignant Glioma.Glioma.Springer,NewYork,pp.95–108.

Vicente,S.,Peleteiro,M.,Gonzalez-Aramundiz,J.V.,Díaz-Freitas,B.,Martínez- Pulgarín,S.,Neissa,J.I.,Escribano,J.M.,Sanchez,Á.,González-Fernández,Á., Alonso,M.J.,2014.Highlyversatileimmunostimulatingnanocapsulesfor specificimmunepotentiation.Nanomedicine1–17.

Wilson,K.D.,deJong,S.D.,Tam,Y.K.,2009.Lipid-baseddeliveryofCpG oligonucleotidesenhancesimmunotherapeuticefficacy.Adv.DrugDeliv.Rev.

61,233–242.

Zitvogel,L.,Apetoh,L.,Ghiringhelli,F.,Kroemer,G.,2008.Immunologicalaspectsof cancerchemotherapy.Nat.Rev.Immunol.8,59–73.

Références

Documents relatifs

In summary, CD4 T ⫹ cell death as a result of HIV-1 infection is mediated by the activation of p53 and the intrinsic mitochondrial apoptotic pathway.. The mech- anism by which

Despite the increase in tumor perfusion and the decreases in tumor vessel area and the number of Ki67 þ cells achieved with SFN-LNCs, these effects were not sufficient to modify

V Anaïs Vaissiière CNRS - Centre de Biologie cellulaire de Montpellier Montpellier, France Lucie Valero Université Paris Descartes - UTCBS Paris, France Mathieu Varache CEA

Most of the studies dealing with glioblastoma cell lines are performed with cells cultured at atmospheric oxygen concentration (i.e. 21% O 2 ), despite the fact that brain

Pharmacokinetic disposition of IVM in different animal species may be affected by formulation design since the vehi- cle in which these compounds are formulated may influence

The GemC 12 -LNC hydrogel reduced signi fi cantly the size of a subcutaneous human GBM tumor model compared to the drug and short-term tolerability studies showed that this system

Experiments were conducted in a parallel study design with the following treatment groups: treatment 1, oral dose of normal saline solution; treatment 2, oral dose of blank

[r]