Biomimetic nanocrystalline apatites: Emerging perspectives in cancer diagnosis and treatment

To link to this article: DOI : 10.1016/j.ijpharm.2011.07.005

URL:

http://dx.doi.org/10.1016/j.ijpharm.2011.07.005

This is an author-deposited version published in:

http://oatao.univ-toulouse.fr/

Eprints ID: 5434

To cite this version:

Al-Kattan, Ahmed and Girod Fullana, Sophie and Charvillat, Cédric and

Ternet -Fontebasso, Hélène and Dufour, Pascal and Dexpert-Ghys,

Jeannette and Santran, Véronique and Bordère, Julie and Pipy, Bernard

and Bernad, José and Drouet, Christophe Biomimetic nanocrystalline

apatites: Emerging perspectives in cancer diagnosis and treatment. (2012)

International Journal of Pharmaceutics, vol. 423 . pp. 26-36. ISSN

0378-5173

O

pen

A

rchive

T

oulouse

A

rchive

O

uverte (

OATAO

)

OATAO is an open access repository that collects the work of Toulouse researchers

and makes it freely available over the web where possible.

Any correspondence concerning this service should be sent to the repository

administrator:

staff-oatao@listes.diff.inp-toulouse.fr

Biomimetic

nanocrystalline

apatites:

Emerging

perspectives

in

cancer

diagnosis

and

treatment

Ahmed

Al-Kattan

_,

_Sophie

_{Girod-Fullana}

_,

_Cédric

_Charvillat

_,

_Hélène

_{Ternet-Fontebasso}

_,

Pascal

Dufour

_,

_Jeannette

_Dexpert-Ghys

_,

_Véronique

_Santran

_,

_Julie

_Bordère

_,

_Bernard

_Pipy

_,

José

Bernad

_,

_Christophe

_Drouet

a_{CIRIMATCarnotInstitute,UMRCNRS/INPT/UPS,UniversityofToulouse,France}

b_{CEMES,UniversityofToulouse,France}

c_{ICELLTIS,Verniolle,France}

d_{UMR-MD3EA2405,UniversitédeToulouse,France}

Keywords: Apatite Nanoparticles Pectin Composite Cancer Medicalimaging Drugdelivery

a

b

s

t

r

a

c

t

Nanocrystallinecalciumphosphateapatitesconstitutethemineralpartofhardtissues,andthesynthesis

ofbiomimeticanalogsisnowwell-masteredatthelab-scale.Recentadvances inthefine

physico-chemicalcharacterizationofthesephasesenableonetoenvisionoriginalapplicationsinthemedical

fieldalongwithabetterunderstandingoftheunderlyingchemistryandrelatedpharmacological

fea-tures.Inthiscontribution,wespecificallyfocusedonapplicationsofbiomimeticapatitesinthefieldof

cancerdiagnosisortreatment.Wefirstreportontheproductionandfirstbiologicalevaluations

(cyto-toxicity,pro-inflammatorypotential,internalizationbyZR-75-1breastcancercells)ofindividualized

luminescentnanoparticlesbasedonEu-dopedapatites,eventuallyassociatedwithfolicacid,formedical

imagingpurposes.Wethendetail,inafirstapproach,thepreparationoftridimensionalconstructs

asso-ciatingnanocrystallineapatiteaqueousgelsanddrug-loadedpectinmicrospheres.Sustainedreleasesof

afluoresceinanalog(erythrosin)usedasmodelmoleculewereobtainedover7days,incomparisonwith

theceramicormicrospherereferencecompounds.Suchsystemscouldconstituteoriginalbone-filling

materialsforinsitudeliveryofanticancerdrugs.

1. Introduction

Thesetupanddevelopmentoffine-tuneddiagnosisdevicesand biomimeticdrugdeliverysystemsappeartodayasmajorchallenges inmodernmedicine,andspecificallyinoncology.

Nanocrystalline calcium phosphate apatites, responding to the general chemical formula Ca10−x(PO4)6−x(HPO4)x(OH)2−x (0≤x≤2), canbe seen as promising candidatesfor the prepa-rationofnano-biotechnologicaldevices(Reyetal.,2007;Roveri etal.,2008).Indeed,theyfullymimicthecompositionand struc-turalfeaturesofbonemineral,whichconfersthemabiocompatible character. Also, they do not originate from biological sources thus eliminating immunological or ethical issues (as well as a poorreproducibilityincompoundcharacteristics).Their physico-chemicalcharacteristicsandsurfacestatehavebeeninvestigated indetails,aswellastheirpotentialitiesinboneregeneration

appli-∗ Correspondingauthorat:CIRIMATCarnotInstitute,ENSIACET,4alleeEmile

Monso,31030Toulousecedex4,France.Tel.:+33534323411;

fax:+33534323499.

E-mailaddress:christophe.drouet@ensiacet.fr(C.Drouet).

cations(see forexample Reyet al.,2007,2009;LeGeros,2008; Autefageetal.,2009).Incontrasttowell-crystallizedmicron-sized hydroxyapatite, nanocrystalline apatitic systems exhibit a high surfacereactivity and theirpreparation is nowmasteredatthe lab-scale(Drouetetal.,2009).Also,recentresultshaveshownthe possibilitytoretaintheadvantageousphysico-chemicalfeatures ofapatitenanocrystalsbyusingsoftprocessingroutes(e.g.Grossin etal.,2010).

Inthisview,twodomainsofinterestforapatite-basedsystems canbemorespecificallyconsidered:thepreparationof individual-izednanoparticulatesystemsandtheelaborationoftridimensional biomimeticconstructs.

Thedevelopmentofnanoparticle-based systemsinmedicine has been widely described. Examples of individualized nano-particulatesystemscurrentlyinvestigatedincludemetalormetal oxide nanoparticles (e.g. gold, magnetite, gadolinium oxide), semiconductors(e.g.quantumdots),polymericnanoparticlesor nanocapsules,liposomes,etc.(Bruchezet al.,1998;Paraketal., 2003; Ow et al., 2005; Fizet et al., 2009).These nano-systems maybeusedascirculatingentitiesinbodyfluids,associatedwith limitedrecognitionbythereticulo-endothelialsystem(Moghimi etal.,2001;MoghimiandBonnemain,1999;Couvreuretal.,2006),

inviewofspecificcelladdressingandinternalization(Schroeder etal.,2007),possiblywiththehelpofacell-targetingagentsuch asfolic acid(FA).Theuseof apatites for cellulardrugdelivery (e.g. for nonviral transfection applications) or medical imaging hasalsolatelyraisedinterestinthescientificcommunitydueto thepossibilitytofunctionalizethesurfaceofbiomimeticapatite nanocrystalswithvaryingmoleculesofinterest(Mondejaretal., 2007;Bouladjineetal.,2009;Sahaet al.,2009;Al-Kattanetal., 2010).Veryrecently,weshowedthepossibilitytopreparecolloidal apatite-basedsuspensionsfromionicsaltsfortheelaborationof luminescentnanoparticles(Al-Kattanet al.,2010), openingnew perspectivesinmedicaldiagnosis.

The association of apatitic tridimensional constructs with biologicallyrelevant ionsor drugs, includinganticancer agents, hasalsobeen pointed out(Lebugle et al.,2002; Barroug et al., 2004;Josseetal.,2005;Gautieretal.,2010;Autefageetal.,2009; Roverietal.,2008;Drouetetal.,2008).Thepossibilitytocontrol thenanocrystallinecharacterofsuchsystemsmakesitpossibleto takeadvantageofthesurfacepropertiesandenhancedbioactivity (Cazalbouetal.,2005;Reyetal.,2007;Tranetal.,2009).Also,new processingroutesavoidinghigh-temperaturesinteringstepsopen new perspectivesin elaborating drug-loaded scaffolds, as low-temperatureprocessesareneededtoretaintheactivestructureof numerousdrugs.Intheabsenceofspecificapatite–drug interac-tion,thesimpleincorporationofthedrugwithintheapatiticmatrix oftenleadstouncontrolledburstrelease.Inthiscontext,including drug-loadedpolymermicroparticlesintoceramicscouldbeaway toimprovedrugreleaseproperties.Polymermicroparticles incor-poratedintocalciumphosphatecements(whosefinalcomposition isapatitic)have alreadybeendescribedandshowedinteresting delayed release properties (Ruhe et al., 2005; Habraken et al., 2008;Girod-Fullanaetal.,2010).However,despiteacontrolled drugrelease,thistypeofformulationsbearspotentialdrawbacks dueinparticulartotheusualexothermiceffectand/orpHchange occurring upon cement hardening (which could prove to be problematicforsomedrugs)andtothemodificationofthecement hardening kinetics as a function of the amount of associated microspheres.

Takingintoaccounttheprecedingstatements,weinvestigated in thepresent work thepotentialities of biomimetic nanocrys-tallineapatite-basedsystemsspecificallyinviewofcancer-related applications:either(i)for intracellularmedicalimaging (cancer diagnosis)or(ii)inviewoflocaldrugdelivery(e.g.anticancerdrug) incancerousbonetissue.

Inthefirstcase,wefocusedontheabove-mentioned lumines-centapatitecolloids.Weprovedthepossibilitytofunctionalizethe surfaceoftheapatitenanoparticleswithfolicacid,or“FA”(a poten-tialcell-targetingagent), andweevaluated(i)theircytotoxicity ondifferentcelltypes:breastcancercells(ZR-75-1)andadipose stemcellsfrombreast(AMSC)whichareinvolvedinprogressionof closedtumorcells,(ii)theirpro-inflammatorypotential(by follow-inginteractionswithHumanmonocytes/macrophages),and (iii) theircapacity tobeinternalizedbyZR-75-1breast cancer cells. Suchbiocompatiblesystemsthenshowpromiseasbiocompatible luminescentnanoprobes.

Inthesecondcase,wepresentanovelapproachforthe prepa-ration ofpolymer/apatite composites, involving theprogressive mild drying of an aqueous biomimetic apatite gel containing drug-loadedmicrospheres. Thisstudyexploresthefeasibility of incorporatinglowmethoxyamidatedpectin(LMAP)microspheres intoananocrystallineapatiteceramic,withtheaimtostudythe influenceofpectinmicrospheresontheceramiccrystalline struc-tureandtoevaluatethecompositedrugreleaseability.Todoso, varyingLMAPincorporationratios,upto18%(w/w),weretested anderythrosin(aredfluorescentdye)waschosenasmodeldrug tobereleased.

2. Materialsandmethods 2.1. Samplespreparation

Theapatitecolloidspreparedin thisworkwereobtainedby applyingtheexperimentalprotocolthatwereportedpreviously (Al-Kattanetal.,2010).Aconstanteuropiumdopingrate corre-spondingtothefinalmolarratioEu/(Ca+Eu)=2%wasusedhere. Briefly,thenanocrystallineapatiteprecipitationwascarriedoutin deionizedwater,atroomtemperatureandpH9.5,fromamixture ofcalciumnitrate, europium nitrateandammonium hydrogen-phosphate, with the starting molar ratio (Ca+Eu)/P=3, and in thepresenceof aphospholipidmoiety:2-aminoethylphosphate (denoted“AEP”)playingtheroleofbiocompatiblestabilizingagent through electrostatic interparticle repulsive effects (Bouladjine et al., 2009).Afterprecipitation, the suspensions were aged at 100◦_{Cfor16h.Thestarting AEP/(Ca+Eu)molarratiowasfixed} to1.Thefluidcolloidsobtainedafteragingwerethenpurifiedby dialysisinwater(cellulosemembrane,cutoff6000–8000Da).The pHofthecolloidscouldthenbeadjustedtophysiologicalvalueby additionofsodiumhexametaphosphate,priortobiological appli-cations.Inthecaseofsynthesesperformedinthepresenceoffolic acid(vitaminB9or“FA”),thelatterwasaddedeitherinthereacting mediumbeforeoraftertheagingstep,typicallyataconcentration of0.45mM.

The preparation of the pectin–apatite composite samples involved two successive steps: the preparation of erythrosin-loadedpectinmicrospheres(ERY-LMAP),and theassociationof LMAPmicrosphereswithnanocrystallineapatitegel.Inthiswork, weusedlow-methoxyamidatedpectins(LMAP)withdegreesof esterificationandamidationof30%and19%,respectively,andan averagemolecularweightof228000Da,assuppliedbyCPKelco (Denmark).LMAPmicrosphereswerepreparedasfollows:3%(w/v) pectinweredispersed intoerythrosinBsolutions in phosphate bufferpH8.ERY-LMAPmicrosphereswereproducedbyionotropic gelationusinganelectrostaticbeadgenerator(Inotech encapsu-latorIE50R,Switzerland)equippedwithasyringepumpanda 300mm nozzle.The pectinsolutionsweredropped intoa solu-tionofcalciumchlorideat500mM(cross-linkingsolution)under continuous agitation. The gelled microspheres, instantaneously formed,wereallowedtocureinthecross-linkingsolutionfor24h. Thentheywereseparated byfiltration, washed withdeionized water,dehydratedinagradedseriesofethanol,anddriedfor48h at37◦_C.

Thepreparationofthepectin–apatitebiocompositeswasthen carriedoutintheshapeofdisks(finaldimensions:18mm diame-ter,5mmheight).Inatypicalprocedure,driedLMAPmicrospheres weredispersed,in varyingamounts (50,100,200and 300mg), into a 100ml solution of calcium nitrate Ca(NO3)2·4H2O at a concentrationof160g/l.A50mlammoniumhydrogenphosphate (NH4)2HPO4 solutionat 70g/l wasthen prepared and adjusted to pH10.5 by addition of 5ml of ammonia, and dropped into thecalcium–pectin suspension under continuous stirring.After 1minofreaction,theobtainedgelwasfilteredandwashedwith deionizedwater.Thispectin-loadedgelwasfinallyintroducedinto cylindricalmouldsandpresseduntiltotalevacuationofairbubbles. Thelidsofthemouldwereremovedandthepastewasallowedto dry,invaryingtemperatures,during4days.

2.2. Physico-chemicalcharacterizations

Calcium and europium contents were assessed by induced coupled plasma atomic emission spectroscopy, ICP-AES (rela-tive uncertainty 3%). The amount of mineral phosphate in the sampleswasdeterminedbycolorimetryat=460nmusingthe yellow phospho-vanado-molybdenum complex (relative

uncer-tainty0.5%).TheAEPcontentinapatitecolloidswasdrawnfrom nitrogenmicroanalysis(relativeuncertainty0.4%).

The apatitic crystallographic structure in the samples was checked,afterfreeze-drying, bypowderX-raydiffraction(XRD) usingaCPS120INELdiffractometerandtheKacobaltradiation (=1.79002 ˚A). Fourier transform infrared(FTIR) analyseswere performedonaNicolet5700spectrometer,in thewavenumber rangeof400–4000cm−1_{witharesolutionof4cm}−1_.

Thesizeandmorphologyofthesampleswerefollowed, depend-ingonthesizerangeofthesamples,eitherbyopticalmicroscopy, byscanningelectronmicroscopy(SEM)usingaJEOLscanning elec-tronmicroscope(JSM-6400F)at15kV,orbytransmissionelectron microscopy(TEM)onaJEOLJEM-1011setat100keV.

Theparticlesizeofapatitecolloids(hydrodynamicdiameter) wasdeterminedbydynamiclightscattering(DLS)usinga Nano-sizer ZSapparatus fromMalvern Instruments(=630nm).The dispersionof the data pointsis estimated to 0.5%.Zeta poten-tialmeasurementswerealsocarriedoutusingthisapparatus.The LMAPmicrospheressizedistributionsweremeasuredusingalaser particlesizer(Mastersizer2000; Malvern,UK)basedona laser light-scatteringtechnique.Eachsamplewasmeasuredintriplicate. Theweightaverageofvolumedistribution(D[4;3])wasusedto describetheparticlesize.

Microsphereswellingratiowasevaluatedbyweighing,and cal-culatedaccordingtoEq.(1):

SR=

h

W0

i

×100 (1)

whereWtisthemicrospheresweightatthegiventimepointand W0theinitialweightofthedrymicrospheres.Eachexperimentwas performedintriplicate.

Theluminescencepropertiesofthecolloidswereinvestigated using a Horiba Jobin Yvon Fluorolog 3-11 spectrofluorometer equipped with a 450W xenon lamp. Excitation and emission were measured at room temperature directly on the dialyzed colloidalsuspensions.Excitationspectrawererecordedbetween 350and 600nm, monitoringthe5_D

0→7F2 emissionof Eu3+ at em=612nm(spectralbandwidth=2nm).Emissionspectrawere recordedinthe500–700nmrange,withaspectralbandwidthof 1nm,underselectedexcitationinthe7_F

0→5L6transitionofEu3+ atex=392.8nmorinthe7F0→5D2transitionatex=464.2nm. Thetransientcharacteristicsoftheemittinglevel5_D

0ofEu3+were investigatedwiththephosphorimeterFL-1040,equippedwitha UVxenonflashtube.Emissiondecayswereanalyzedatchosenex andemonatimeintervalof3.5ms.Thetimeresolutionimposed bytheapparatusintheexperimentalconditionsemployedis30ms. FluorescentconfocalmicroscopywasusedtocharacterizeLMAP microspheresafterrelease. Anargon488nmlaserwasusedfor excitation of erythrosin; the emission light between 505 and 530nmwasdetected.UsingaZeiss510confocalmicroscope, opti-calsectionsof46.8mmthickthroughthemicrospheresweretaken. For theLMAP–apatite composite systems, drug loading and encapsulationefficiencyweredeterminedaftercomplete degra-dationofLMAPmicrospheresinpH8phosphatebuffer.Theywere calculatedaccordingtoEqs.(2)and(3),respectively:

Drugloading =

h

totalweightofmicrospheresbybatch−AQ

i

×100 (2)

Encapsulationefficiency (drugentrapmentabilityin %) =



TQ



×100 (3)

inwhichAQistheactualquantityofdrugpresentinthe matri-ces(drugcontent)andTQthetheoreticalquantityofdrug(initial erythrosinBloadingdoseduringmicrospherespreparation).

Erythrosin B release from microspheres, ceramics alone, and composites underinvitro conditionswas performedin SBFpH 7.25(preparedaccordingtoKokuboandTakadama,2006)at37◦_C. Standardmethodsofreleaseexperimentsinpharmacopoeiasare hardlysuitableformulti-particulatedosageformsduetothelarge volumesofthevessels;amodifiedalternativemethod,proposed byresearchgroupsworkingonmulti-particulatedosageformsfor colondeliverywasused(Atyabietal.,2005).Briefly,composites (containingERY-LMAPmicrospheresataweightratioof6%and 9%,respectively)orceramicspecimens(accurateweightof approx-imately 2.80g) or ERY-LMAP microspheres(accurate weight of approximately100mg)wereplacedintesttubescontaining10ml ofSBFpH7.25,at37◦_{Cunderagitationat100rpm.ErythrosinBwas} assayedbyspectrophotometry(PerkinElmer,USA)at527nm,in triplicate,atvarioustimeintervalsupto7days.Cumulatedreleased amounts(inpercentageoftheinitialamounts)wereplottedversus time.Eachinvitroreleasestudywasperformedthreetimes.Allthe studieswereperformedundersinkconditionsforerythrosinB. 2.3. Biologicalassessments

2.3.1. Cytotoxicityevaluationforapatite-basedcolloidal nanoparticles

The cytotoxicityof thecolloids, relativelytotwo cell types: humanbreastcancercellsZR-75-1andadiposetissue mesenchy-mal stem cells (AMSC), was evaluated by way of MTT tests performedbytheICELLTISCompany(Verniolle,France).Inthese tests,cell viabilityis determined onthebasis of mitochondrial activity which leads to a quantifiable emission of light after exposurewiththeMTTreactant (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazoliumbromide).

Inpreparationofthesetests,thecellswerecultivatedin humidi-fiedatmospherewith5%CO2at37◦CinDulbecco’sModifiedEagle’s Medium(DMEM)supplementedwith10%fetalbovineserumand 1%antibiotics(penicillin/streptomycin).Thecellswerethenplaced in96-wellcultureplates(30000cellsperwell)andeachtestwas runintriplicate.After24hofpreliminaryincubation,theculture mediumwasreplacedbyfreshDMEM(exemptofphenolred)and theMTTtestswereinitiated.

Cellviabilitywasassessedforvaryingcontacttimesbetween thecolloidalnanoparticlesandthecellsrangingbetween1and7 days.Additionally,5differentconcentrationsofnanoparticlesin theculturemediumweretested:0,0.1,1,2,5and10mg/ml.For eachMTTtest,50mlofMTTreactant(5mg/ml)wereaddedtoeach wellandthecellswerefurtherincubatedfor4hat37◦_Cfor com-pletingthereaction.Finally,theculturemediumwasreplacedbya solubilizationsolution(H2O66%,DMF20%,SDS5%,TritonX100,at pH7)overnightandmeasurementofthelightabsorptionat570nm wascarriedout,asameasureofcellviability.

2.3.2. Evaluationofthepro-inflammatorypotentialof apatite-basedcolloids–interactionwithHumanmonocytes

Macrophages and theirprecursors, monocytes, are essential cellsoftheimmunologicalsystem,intendedtomediatethe inflam-matoryresponsetoforeignsubstancessuchasnanoparticles.These cellsareinparticularactivatedbyinflammatorysignals,conducing toanincreasedcapacitytoreleasepro-inflammatoryandcytotoxic mediatorssuchasReactiveOxygenIntermediates,or“ROI”(Laskin andLaskin,2001).ThemeasureoftheamountofproducedROIwas thususedinthisworktoevaluatetheintrinsicpro-inflammatory potentialoftheapatitecolloidalnanoparticlesthatweprepared.

For these measurements Human peripheral blood mononu-clear cells were isolated from the blood of healthy volunteers

by a density gradient centrifugation method on Lymphoprep (Abcys).Monocyteswereisolatedbyadherencetoplasticfor2h inMacrophage-SerumFreeMedium(M-SFM)(GibcoInvitrogen)at 37◦_C,5%CO

2.

The monocytes were plated in 96-well Falcon plates (3×105_{monocytes/well). The oxygen dependent respiratory} burst of monocytes was measured by chemiluminescence as previously described (Lefevre et al., 2010) in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione(luminol)using ther-mostatically(37◦_{C)controlledluminometer(Wallac1420Victor2).} Theluminoldetectsbothreactiveoxygenandnitrogen intermedi-ates(O2• −,ONOO−,OH•).Thegenerationofchemoluminescence wasmonitoredcontinuouslyfor1hafterincubationofthecells with luminol (66mM) and after colloids challenge at various concentrations (1.2, 12, 60 and 120mg/ml). The measurements was realized in triplicate. The negative control was based on experimentscarriedoutintheabsenceofnanoparticles.Statistical analysiswasperformedusingtheareaunderthecurveexpressed incounts×seconds.

2.3.3. Evaluationoftheinternalizationofcolloidalapatite nanoparticlesbyZR-75-1breastcancercells

Thepossibleinternalizationofapatitecolloidalnanoparticles bycancercellswascheckedonZR-75-1breastcancercells,fora concentrationofnanoparticlesintheculturemediumof1.5mg/ml. Anegativecontrolwascarriedoutwithoutnanoparticles.

These tests, realized in triplicate, were carried out by the ICELLTISCompany(Verniolle,France).24hafterseeding,the col-loidwasaddedtothecellsduring24h.Then,afterwashingwith PBSbuffer,thecellswerecountedandcentrifuged.Theresidual pel-letsweredissolvedinHCl37%andincubatedfor30mininviewof quantificationoftheintracellularEuandCacontentsbyICP-AES (analyses preformedby theMARION TECHNOLOGIES Company, Verniolle,France).

3. Resultsanddiscussion

3.1. Biomimeticapatitecolloidsinviewofcancer-relatedmedical imaging

3.1.1. Physico-chemicalaspects

Thecolloidalapatite-basedsuspensionspreparedinthiswork, inthepresenceof2-aminoethylphosphate(AEP),correspondtoan experimentaleuropiumcontentof2at.%relativetocalciumas indi-catedbyICP-AEPevaluations.Also,the(Ca+Eu)/PandAEP/apatite molarratioswerefoundtobecloseto1.60and0.63,respectively. ThepossibilitytosubstitutesomeCa2+_ionsbyEu3+_intheapatite structurehasindeedbeenalreadyshown(e.g.Boyeretal.,2000).

XRDanalysiscarriedoutonatypicaldialyzedandfreeze-dried colloid(Fig.1a)confirmedtheapatiticnatureofthecrystallized par-ticlesprepared,withnodetectabletracesofsecondaryphases.FTIR spectralfeatures(Fig.1b)corroboratedthesefindings.Thepresence ofAEPmoleculesassociatedwiththeapatitephasewasindicated bythepresenceofanabsorptionbandat754cm−1_{,linkedtoP–O(C)} librationinsystemssuchasCa(AEP)2involvingacloseinteraction betweenAEPandCa2+_{ions,aswasdiscussedinourpreviousworks} (Bouladjineetal.,2009;Al-Kattanetal.,2010).Moreover,inthese studiestheadsorptionoftheAEPmoleculesonthesurfaceofapatite nanoparticleswasevidenced,explainingthecolloidalstabilization effectbywayofelectrostaticrepulsionsamongadjacentparticles throughtheammonium–NH3+terminalgroupsofthegraftedAEP molecules.Thezetapotentialofthecolloidspreparedinthepresent workwasfoundtobecloseto+12±3mV,andthispositivevalue isindeedingoodagreementwiththis statement.Althoughthis zetapotentialmeasuremayappearratherlowinabsolutevalue,

a 70 60 50 40 30 20 0 1000 2000 3000 4000 5000 (004) (213) (222) (202) (211) (112) (300) (102) (210) (200) (111) (310) Intensity (a.u.) 2 theta (degrees) (002) 500 1000 1500 2000 2500 3000 3500 4000 0.0 0.2 0.4 0.6 0.8 1.0 ν(P-O(C)) ν 3(CO3) ν 2(PO4) ν 1(PO4) ν 4(PO4) HOH Absorbance (a.u.) Wavenumbers (cm-1₎ ν(OH) ν 3(PO4) b

Fig.1.(a)XRDpatternsand(b)FTIRspectraforapatitecolloidalnanoparticles.

thecolloidalstabilitycouldbeassessedmacroscopically(by opti-calobservations)andtheas-synthesizedcolloidsdidnotundergo sedimentation.However,itshouldbenotedherethatacomplete follow-upofthestabilityovertimeofsuchcolloidalsuspensions wasoutofthescopeofthepresentstudy,andwillberegardedina detailedupcomingwork.

TEMobservations of such colloids (Fig. 2a) pointed out the homogeneousellipsoidalmorphologyofthenanoparticles,witha meanlengthoftheorderof25±3nmandameanwidthof7±1nm (asdeterminedfromimageprocessingcarriedoutwiththeImageJ software).DLSmeasurements(Fig.2b)confirmedthe nanometer-scaledimensionsoftheparticles,withaunimodalsizedistribution centeredaround30nm.

Theluminescencepropertiesofthecolloidswerealso investi-gated(Fig.3).Excitationspectrarecordedbymonitoringthered luminescenceintensityor Eu3+ _{at612nm (mainemission line)} showedtwo main emission peaksupon excitation at 392.8nm (major peak) and 464.2nm corresponding, respectively, to the 7_F

0→5L6 and 7F0→5D2 intraconfigurational transitions. These low-energyexcitationdomains(ascomparedforexampleto sys-temsexcitablesolelyunderUVwavelengths)thusenableoneto envisiontheinvestigationofbiologicalmaterialwhilepreventinga prematuredeterioration.Luminescenceemissionspectrarecorded underexcitationat392.8nmindicatedthepresenceofthreemain domainsintheranges575–580nm,583–603nm,and605–627nm, corresponding,respectively,tothetransitionsfromthe5_D

0excited statetothe7_F

0,7F1and7F2statesofEu3+.

Theluminescencedecaytime(definedasthetimeforwhich I()=I0/exp(1)) of the europium 5D0 level was also measured, reaching0.74±0.03ms.Thisvalueoftheorderofa millisecond is remarkably lagerthan the typicaldecay timesfor the auto-fluorescence of biological tissues thus adding to the potential

Fig.2. (a)TEMmicrographand(b)DLSdataforapatitecolloidalnanoparticles.

interestofsuchluminescentnano-systemsasbiologicallyoriented nanoprobes.

TheabovedatastressthatEu-dopedapatitecolloidscanbe pre-pared,inaqueousmedium,andthattheymaybeconsideredfor applicationsinthefieldofmedicalimaging,inparticulardue(i)to thebiocompatiblenatureoftheirconstituents(biomimeticapatite andphospholipidmoiety),(ii)tothenanometer-scaledimensions oftheparticles(typically∼30nm)aimingatfacilitating internal-izationbycells,(iii)tothepossibilitytoexcitethesesystemsunder (low-energy)visibleorclose-to-visiblelightdomains,and(iii)to thelongluminescencelifetimeascompared tothe autofluores-cenceofbiologicaltissues.

Aninterestingaspecttoaddresswhennanoparticles–cells inter-actionsareenvisionedconcernsthepossibilitytotargetaspecific typeofcells(ortissue)soastodevelop“intelligent”diagnosisor therapeutictools.Inthiscontext,severalpotentialtargetingagents canbeenvisaged dependingonseveralparameterssuch asthe typeofpathology,thenatureofthetargetedcells,thedegreeof specificityofthetargetingagent,etc.

Folicacid(vitaminB9or“FA”)isanexampleoftargetingagent thathasledtonumerousstudiesworldwideinthefieldofoncology. Indeed,variousworkshaveshownthatcertaintypesofcancercells (e.g.someovarianorbreastcancercellsforinstance)were over-expressingontheirmembranesomespecificfolatereceptorsFR (e.g.Antony,1992;Parkeretal.,2005)whichexhibitahighaffinity forthefolicacidmolecule(about100000timesgreaterthanthatof thereducedfolatereceptorsthatareusuallypresentonthesurface ofnormalcells,(Spinellaetal.,1995)).Anotherinterestingfeature isthatfolic-acid-mediatedendocytosiswasgenerallyobservedfor

640 620 600 580 560 400 300 200 0 25000 50000 75000 100000 125000 280000 320000 Intensity (a.u.) Wavelength (nm) Excitation

Fig.3. Luminescencepropertiesofapatitecolloidalnanoparticles:excitationand

emission(withorwithoutFAfunctionalization)spectralfeatures.

FA-functionalizednano-systems,rightafterrecognitionofFAby theFRreceptors(Kamenetal.,1988).

Inthepresentwork,wethusfocusedonthephysico-chemical feasibilityofFAassociationonourapatitecolloidalnanoparticles. Takingintoaccountthetwo carboxylategroupsoftheglutamic acid-likeheadoftheFAmolecule,wethussuspectedafavorable adsorptionofFAmoleculesonthesurfaceofapatitenanocrystals bywayofCa2+_{accessiblesurfaceions.}

Our attemptstofunctionalizethesurfaceof apatitecolloidal nanoparticlesbyintroducing FAinthereactingmixture (either before or after the aging step), at a typical concentration of 0.45mmol/l,provedtobesuccessful.Apartfroma clearyellow colorforthecolloids(duetothepresenceofFA)themain physico-chemical characteristics of the nanoparticleswere retained. In particular, no secondary crystallized phase was detected from XRD analyses which were similar to the FA-free samples, and the754cm−1 _{IRabsorption bandassignable tothepresence of} AEPmoleculeswasconserved.TheDLS-andTEM-derivedmean particlesize(43±5nm)remainedalsoessentiallyunchanged.C, H,Nelementalanalysesonfreeze-driednanoparticlesledtoan FAcontentof1.03mg/100mgofdriedsuspension.Thislow con-centrationinFAdidnotallowustodetectitspresence byFTIR spectroscopy;howevertheuseofgreaterFAconcentrations(4–40 timesgreater)led totheappearanceofabsorption bands char-acteristic of calcium folate in the region 940–1800cm−1_{, next} tothebandscharacteristicoftheapatitephase, inparticularat 1590 and 1396cm−1 _{(not shown for the sake of brevity). On} the contrary, thetypical bands of free folic acid (especially at 1671cm−1_{) werenot seen. Thesedata evidenced theexistence} of FA–Ca2+ _{interactions and thus confirm the physical} attach-ment of FAmolecules on thenanoparticles surface,as initially planned.

LuminescencespectraontheFA-functionalizedcolloidswere thenrecordedaspreviously,afterexcitationat392.8nm(seeFig.3). AsfortheFA-freesuspensions,thespectrawerecharacteristicof theEu3+_{emittingcentersdespiteabaselinedriftduetothe} pres-enceofthegraftedFAmolecules.Theluminescencedecaytimewas alsodetermined(FA=0.74ms)andwasfoundtobeunmodifiedby thepresenceofFA.

3.1.2. Cytotoxicityassessments

For a concentrationof nanoparticles in theculture medium between0and1mg/ml,thetestscarriedoutonAMSCcells(see

Fig.4forFA-functionalizedcolloids)indicatedacellviabilityclose to100%,whatevertheincubationtimebetween1and7days.These resultswerefoundtobeindependentonthepresenceorabsence ofFAonthenanoparticles.Forgreaterconcentrationsin nanopar-ticles,thecellviabilitystartedtodeclinethusenablingtoconsider

AMSC cells - presence

of

FA

ZR-75-1 cells - presence

of

FA

Cell viability (%)

Fig.4.MTTcytotoxicitydataforapatite-basedcolloidsfunctionalizedbyFA,forAMSCandZR-75-1cells.

the1mg/mlvalueastheinflexionvalueforthesecolloidsonAMSC cells.

SimilartestswerealsocarriedoutonZR-75-1cancercells(that donotoverexpressfolatereceptors),andthedata(Fig.4)indicated thattheinflexionofthecytotoxicitycurveoccurredintherange 1–2mg/ml.

Thesefindingspointoutthelowcytotoxicityofthesecolloidal systems,atleastforsuchcellswhichdonotspecificallyrespondto folicacid.Thesefindingswillbetakenasreferenceforfutureworks onthesecolloids.Additionalexperimentsareinprogressoncells thatoverexpressfolatereceptors.

3.1.3. Investigationofpro-inflammatorypotential

The pro-inflammatory potential of the apatite colloidal nanoparticlespreparedwasinvestigatedherebyfollowingtheir interaction with Human monocytes, via the evaluation of the amountofROIproducedbythecells(luminol-enhanced chemi-luminescence).

Chemiluminescencemeasurements(Fig.5),carriedoutafter1h ofcontactbetweenthecolloidalnanoparticles(purifiedby dialy-sisandstabilizedatphysiologicalpH)andthemonocytes,andfor concentrationsinnanoparticlesbetween0and120mg/mlshowed nosignificanteffect(i.e.activatororinhibitor)ontheproduction

Fig.5.Luminol-enhancedchemiluminescenceintensitymeasurementsfor

Fig.6.SEMmicrographofLMAPmicrospheresloadedwitherythrosin(ERY-LMAP)(initialmagnification200×),andparticlesizedistributionaccordingtolaserlightscattering measurements.

ofROI.Theseresultsindicatethattheintrinsicpro-inflammatory potentialofsuchcolloidsislowandremainsundetectableafter1h ofcontact.

3.1.4. Studyofapatitecolloidalnanoparticlesinternalizationby ZR-75-1breastcancercells

Thisstudywasaimedatexamining,inapreliminaryapproach, thepossibilitiesofinternalizationofourcolloidalnanoparticlesby cells.Forthesetests,ZR-75-1breastcancercellswereused.The concentrationofnanoparticlesintheculturemediumwassetto 1.5mg/ml,withacontacttimeof24hat37◦_C.ICP-AES measure-mentswerethencarriedoutaftercellwashing,counting,andacidic treatment(see Section2):the quantitative dataobtained indi-catedthattheCaandEucontentsintheacidicaliquotsincreased afterbeingcontactedwiththenanoparticles,leadingtotheratio betweentheincreaseinEu(initiallyabsentofthecytoplasm)and theincreaseinCaofEu/Ca=0.088±0.002massratio.Thisvalue correspondstoaEu/(Ca+Eu)molarratioof0.022±0.006,whichis inperfectagreementwiththeEu/(Ca+Eu)molarratio characteris-ticoftheinitialnanoparticles(2%).

Suchresultscanbeexplainedbytheexistenceofaclose interac-tionbetweenthenanoparticlesandthecellsduringthecontacting time, leading to aninternalization of theparticles. Taking into accountthesmallsizeoftheparticles(oftheorderof30–40nm), cellinternalizationthroughtheendocytosisprocessappearsasthe mostprobablescenario,althoughthisquestionwillrequirefurther examination.

These firstresultsconfirm thepossibility tousesuch nano-systems for biomedical applications requiring an intracellular action(eitherintermsofdiagnosisoroftherapeutics).Additional experimentsareinprogresswithcellsoverexpressingfolate recep-tors,soastopotentiallyevidenceanincreasedinternalizationrate inthecaseofnanoparticlesassociatedwithfolicacid.

Preliminary analyses by optical microscopy coupled with a spectrophotometer,carriedoutonair-driedcolloids,havepointed outtheeuropiumsignatureoftheparticlesbyshowing lumines-cencepeaksaround590and615nm(uponexcitationat365nm). Additional experiments are now planned for investigating the possibilitytodetecttheeuropiumluminescencesignaturefor cell-internalizedparticles.

3.2. Biomimeticapatite–pectinmicrospherescomposites

Inthefollowingsections,we addresstheelaborationofnew biomimeticapatite-basedcompositebiomaterialsinvolvingLMAP microspheresasdrugreservoirsdistributedwithinthebiomimetic apatitematrix.

3.2.1. Physico-chemicalaspects

Generallyspeaking,theassociationofadrugwithmineralbone repair scaffolds is not a straightforward task, in particular due to therelative fragility of theorganic molecules(usually heat-sensitive) constituting thedrug.The processing ofscaffolds for boneengineeringoftenrequiresheattreatments/sinteringathigh temperature.Inthesecaseshowever,thedrug/scaffoldassociation canthenonlybeconsideredasasubsequentstep,aftercooling, forexamplebyfilling(generallyimperfectly)theaccessibleopen porosityofthescaffoldbyimmersioninasolutioncontainingthe drug,followedbydryingsteps.Abettersuitedwayfor perform-ing drug/scaffold associations can nonetheless be consideredif thescaffoldcharacteristicsallowitspreparation andprocessing withouttheuseofelevatedtemperaturesand/ortheapplianceof largemechanicalpressures.Inthisview,alongwiththeirintrinsic biomimetismproperties,nanocrystallineapatitesthenrepresent an interestingclass of materialdue to thepossibilityto obtain robustbiomaterialsatloworambienttemperatures(Grossinetal., 2010)evenwithoutpressingprocedures.Asstatedearlier,the pos-sibilitytoassociateacalciumphosphatecement(CPC)withdrugs hasbeenreported(Ruheetal.,2005;Habrakenetal.,2008; Girod-Fullanaetal.,2010).However,thekineticsofcementhardening, inducingahydrolysisreaction,aswellastheapatite characteris-tics(apatitebeingthefinalstateofhardenedCPC)aregenerally modified bytheamountandnatureof additivesin thesystem. Also,pHvariationsand exothermiceffectsoftenaccompanythe cementhardeningprocess,whichmayalsopotentiallyaltersome drugs.

Inthisstudy,wethusinvestigatedthepossibilitytoassociate LMAPmicrosphereswithabiomimeticapatitephaseobtainedby directprecipitationasagel,withoutanyheatingorhighpressure treatmentintheprocessingroute.

Pectin is a naturally occurring heterogeneouswater-soluble polysaccharide which is foundin thecell wall of most plants. It consists mainly of linearly connected a-(1,4)-d-galacturonic acid monosaccharide units that may be methyl-esterified or amidated to varying extents. Low methoxy (with a degree of esterificationDE<50%)pectins(LMPs)andLMamidatedpectins (LMAPs) can gel with an “egg box” configuration in the pres-enceofmanydivalentcations(Grantetal.,1973), allowingthe formation of microspheres by ionotropic gelation, withoutany use of organic solvents and harsh ingredients. Pectin is bio-compatible,biodegradable (Orhan etal.,2006)and hasrecently demonstrateditspotentialinteresttobeusedinthesurface mod-ification of medical devices and materials (Morra et al., 2004; Ichibouji et al., 2008) and particularly bone implant nanocrys-tallinecoatings(Kokkonenetal.,2007,2008,2010;Bussyetal., 2008).

Time (min) 800 600 400 200 0 Swelling (%) 0 1000 2000 3000 4000

Fig.7. SwellingpropertiesofLMA-pectinmicrospheresloadedwitherythrosinein

SBFpH7.25.

ALMAP withaDE of30 and a degreeofamidation (DA) of 19 waschosen, as we foundit suitable for the elaboration of organic–mineralcomposites(Girod-Fullanaetal.,2010).

Asafirststep, LMAPmicrospheresloadedwitherythrosinB (ERY-LMAP)were prepared by ionotropic gelation in the pres-enceofcalciumions.LMAPmicrospheresloadedwitherythrosinB (ERY-LMAP)wereobtainedinstantaneouslywhenLMAPsolutions weredroppedintocalciumbath.Intermolecularcross-linkswere formedbetweenthenegativelychargedcarboxylgroupsofLMAP andthepositivelychargedcounter-ions,aspreviouslydescribedby

Grantetal.(1973)inthe“egg-boxmodel”.Afterdrying,the result-ingmicrosphereswerecharacterizedintermsofsizedistribution andmorphology.LoadedLMAPmicrosphereswithaweight aver-ageofvolumedistributionD[4;3]of195mmwereobtained,with monomodalandnarrowparticlesizedistribution(polydispersity indexof0.64)(Fig.6).Microspheresappearedsphericaltoovoid withasmoothsurfacewhenobservedbySEM.

The swelling property of ERY-LMAP microsphereswas then studied(Fig. 7).Theweightgain raisedsharplywithinthefirst 40minuntilitreachedaplateau.Nodownwardtrendinthecurve, signof microsphereserosionordegradation couldbeobserved duringtheexperiments.Oncedriedmicrospheresplacedin aque-ousmedia,highcalciumchlorideconcentrationentrappedinthe microspheresexerted an osmosis phenomenon and water was absorbed,inducingswellingofthemicrospheres.Microspheres fur-therstabilitycanbeexplainedbythestrengthoftheirnetwork structure,duetotheformationofstableinterchainjunctionzones betweeneachcalciumionandtwocarboxylgroups,reinforcedby thepresenceofcalciumionsinSBF.

Compositeapatite–LMAP disks were obtainedas detailed in Section2.Gel dryingcarried outat 4◦_{C led,inour} experimen-talconditions,toanamorphouscompound asindicatedbyFTIR andXRDanalyses(notshown).Incontrast,anapatitephasewas obtainedupondryingateither20,37or50◦_{CasshownbyXRD} andFTIRanalyseswhichpointedoutapatiteastheonlycrystallized phaseinthesystem(Fig.8).However,thedriedcompositedisks obtainedat37or50◦_{Cexhibitedapparentflawssuchasfracture} lines(cracks)andthesedrying conditionswerethusconsidered asinappropriate.Thesefindingsmayprobablybelinkedinthese casestoahighdryingrate,involvingstrongmechanicalstrainsover shortperiodsoftime.Incontrast,thecompositedisksobtainedat RT(∼20◦_{C)revealedanundamagedaspectandthisdrying} tem-peraturewasthenusedforsubsequentexperiments.Thevolume decreaseaccompanyingthedryingstepatRTwasfoundtobeof theorderof50%.

Fig.8. (a)XRDpatternsand(b)FTIRspectraforapatite–LMAPcomposites(3%and

18%LMAP)andforceramicalone.

The presence of LMAP microspheres (introduced in varying amounts) dispersed within the apatite matrix was evidenced byopticalmicroscopyimaging.Additionally,XRDandFTIRdata showedthat thephysico-chemicalcharacteristics oftheapatite phaseremainedbasicallyunchangedindependentlyoftheamount ofLMAPmicrospheresassociatedtothemineralphase.Also, chem-icalanalysesindicatedthattheCa/Pratiooftheapatitephasewas essentiallyconstantandcloseto1.53±0.04(characteristicof non-stoichiometricbiomimetic-likeapatites)forLMAPw/wcontents between0%and18%(correspondingtoamassofLMAPbetween 0and300mg).Thesefindingsareparticularlyinterestingasthey show,contrarilytothecementprocessingroute,thatasingle pro-tocolcanbesetupforpreparingvariousapatite–LMAPcomposites, wheretheamountofLMAPmicrospherescanbetailoredasa func-tionofthedesireddose, regardlessofthecharacteristicsofthe apatitephasethatwillremainunchanged.Theupperlimitof micro-spheresincorporationratiointoapatiteistheconsequenceoftheir swellingpropertiesandcouldbemodulatedbyanaccuratechoice ofpectinamidationandmethoxylationdegree.Itwasfoundtobe about10%(w/w)inthecaseofapectinwithaDAof19%andDEof 30%.

Itwasthusinterestingatthispointtostudythepossibilityto usethesesystemsforalocaldrugdeliveryafterimplantation.In thisview,usingerythrosineasamodelmolecule,weinvestigated therelease propertiesoftheapatitematrixaloneandoftypical compositediskscontaining6and9%(w/w)LMAP.

3.2.2. Releasepropertiesofapatite–LMAPmicrospheres composites

Compositesinvitrodrugreleasepropertieswereevaluatedfor 1weekinSBFpH7.25at37◦_{C,inordertomimictheionic} condi-tionsencounteredwhenimplantedinvivo(KokuboandTakadama, 2006).Erythrosin Bwaschosen asmodel drugbecauseitis an anionicdye,fluoresceinanalog,presentinghighersolubilityatbasic pH(itisaweakacidwithapKaof5.04),allowingustostudythe releaseofadrugabletodiffusefreelyoutofthecompositeswithout

Table1

Kineticanalysisoferythrosinreleasefromapatite–LMAPmicrospherescomposites;comparisonwithLMAPmicrospheresandapatitereference.

Cumulatederythrosin

releasedwithinthefirst

24h(%)

Cumulatederythrosin

releasedafter7days(%)

Higuchimodelcoefficient

ofdeterminationr2 Higuchidissolution constantkH(%h−1/2) Apatite/ERY-LMAP6% 6.43±0.92 17.23±0.67 0.994 1.358 Apatite/ERY-LMAP9% 7.88±0.74 20.13±0.76 0.997 1.563 ERY-apatite 97.36±2.63 100.06±0.64 0.992 33.153 ERY-LMAPmicrospheres 50.04±2.61 78.1±0.98 0.923 9.711

presentingspecificinteractionswiththeapatitephaseorLMAP.In ouroperatingconditions,anencapsulationefficiencyof68%and a drugloading of 15%of erythrosinBinto LMAP microspheres wereobtained.Reachingbetterentrapmentefficiencywasnotthe purposeofourstudy,butitcouldbeoptimizedbyplayingwith thecounter-iontypeandconcentration,withthepHofthe cross-linkingbathesandwiththecompositionoftherinsingsolutions beforedrying(Chambinetal.,2006).DryERY-LMAPmicrospheres wereincorporatedintotheapatitematrixatweightratiosof6%and 9%,leadingtocompositeswithanincreasingdrugloading.Release patternswerepresentedbyplottingthecumulatedpercentageof erythrosinBreleasedversustime.

Fig.9displaysthereleaseprofilesoferythrosinBaccordingto LMAPmicrospheresratiointothecomposites.Theywerealmost identicalinshape,presentingaverylimitedinitialburstinthe ini-tial24h(seeTable1)followedbyaslowandsustaineddrugrelease. ThereleasedataweresimulatedusingHiguchitheorywhich inves-tigatedwhethertheerythrosinBcumulativereleasepercentages fromcompositeswereproportionaltothesquarerootoftime.They correlatedwellwiththismodel,suggestingthaterythrosinBwas releasedbyFickiandiffusionfromallthecompositesduringthe experiments.

ReleasesofsimilarlevelsoferythrosinBreleasefroma pure apatitecompound (so-called “ceramic”) and fromLMAP micro-sphereswerecomparedunderthesameexperimentalconditions (Fig.9).Whilethepureapatitesamplereleased100%erythrosinB within24h,clearlyshowingthatthereisnointeractionbetween theapatitephase andthedye,thecompositeand microspheres releasepatternsweresustained,demonstratingtheroleofLMAP microspheresincontrollingdrugdiffusion(Table1).Suchsustained releasecanbeexplainedbyLMAPmicrospheressensitivitytoionic conditions. Theybehave as hydrophilic matrices whose release abilityiscurrentlyrelatedtotheirswellingabilityindissolution

Time (hours) 200 150 100 50 0

Cumulated erythrosin released (%)

0 20 40 60 80 100

Fig.9.Erythrosininvitroreleasefromapatite–LMAPmicrospherescompositesin

SBFpH7.25at37◦_{C(×:apatite/ERY-LMAP6%composite;:apatite/ERY-LMAP9%}

composite;d:ERY-LMAPmicrospheres;N:ERY-apatite).

media(Chambinetal.,2006).ThepresenceofcalciumintheLMAP surroundinghasbeenshowntoenhancecross-linkingand aggre-gationofthepectinchains(Sriamornsak,1999).Inourcase,LMAP microspheresareimmersedin SBFmedium,which contain cal-ciumions,andsurroundedbyapatiteinthecaseofcomposites, thusswellingpatternsandsubsequentlydrugreleasewere lim-itedformicrospheresandcomposites,inagreaterextentforthe latter.Stericconstraintsmightalsohavehinderedmicrospheres swellingwithinthecomposites.Additionaldrugdiffusionthrough

Fig.10. FluorescenceconfocalmicroscopyphotographsofcrosssectionsofLMA-pectinmicrospheresloadedwitherythrosine,retrievedfromthesurfaceofthecomposites

theapatiteporosity,someofthembeingpartiallyblockedbyLMAP microspheres,couldalsoexplainthisslowingdown,whereasno differencescouldbeobservedbetweenthetwoLMAPratiostested duringourexperiments.

Althoughtheseresultshavetobecompletedbylongerrelease studies,theyyetclearlyshowtheinterestofcombining nanocrys-tallineapatitewithLMAPmicrospherestogeneratetailoreddrug deliverysystemswithimproveddrugreleaseproperties.

After1weekofrelease,LMAPmicrosphereswerestillintactand couldberetrievedfromthebioceramicandobservedbyconfocal lasermicroscopy.Confocalfluorescenceobservation(Fig.10) con-firmedincorporatedmicrospheresintegrityandmoderateswelling afteraweekofimmersioninSBF.ErythrosinBdistributionwithin the microspheres and progressive diminution over time were observable,evenmoreclearlyonmicrosphereslocatednearthe peripheryof thecomposites.A homogenous distributionofthe dyewithinthemicrospheresatday0(D0,Fig.10a)anda fluores-cencediminutionfromthecentertotheouteroftheparticlesatD7 confirmedadiffusionreleasemechanism.

Theseresultsarepromisingandillustratethepotentialinterest ofnanocrystallineceramicsinelaboratinginnovativedrugrelease systemsthatcouldfindapplicationinbonecancertherapy. 4. Conclusions

Thedatareportedinthiscontributionenabled ustopropose andtestnovelapproachesforthepreparation,storageand process-ingofbiomimetic-apatite-basednanosystemsproducedinviewof cancer-relatedapplications.

Weshowedfirstthathybridluminescentcolloidalnanoparticles basedontheassociationofEu-dopedbiomimeticapatite nanocrys-tals,a phospho-lipidmoiety(AEP) andalsopossiblyvitaminB9 (folicacid,apotentialcell-targetingagent)exhibitedalow cyto-toxicityandpro-inflammatorypotentialandcouldbeinternalized byZR-75-1breastcancercells.Thesefindingsthenallowoneto envisionpotentialapplicationsinintracellularimaging by lumi-nescence,whichwillbeaddressedinfutureworks.

In a second type of approach, the incorporation of LMA pectinmicrosphereswithina nanocrystallineapatitematrixled toorganic–mineralcompositeswithoriginalproperties. Interest-ingly, thepresenceand amountofpectinmicrospheresdidnot modifythephysico-chemicalcharacteristicsoftheapatitephase. Intermsofdrugdelivery,sustainedreleaseofourmodelmolecule wasobtained,whereas itdevelopednospecificinteractionwith apatitenorpectin.ThepresenceofLMAPappearedasaparameter contributingtoregulatedrugdiffusion.Byadjustingtheirpectin microspherescontent,itshouldbepossibletodesigntailorable compositeswhosereleaseratescouldbecontrolledupondemand. Toourknowledgenopreviousstudiesconcerningnanocrystalline apatite-polymermicrospherescompositematerialshadbeen pub-lishedpreviously.

These results add to the existing literature on synthetic biomimeticnano-systems,byexploringthesenoveloptionsinthe fieldofcancerdiagnosisandtreatment.

Acknowledgements

TheauthorswishtothankY.ThébaultforassistancewithSEM experiments, as well as R. D’Angelo and the IFR-150 Rangueil (Toulouse,France)confocalmicroscopyplatform.

References

Al-Kattan,A.,Dufour,P.,Dexpert-Ghys,J.,Drouet,C.,2010.Preparationand

physico-chemicalcharacteristicsofluminescentapatite-basedcolloids.J.Phys.Chem.C

114,2918–2924.

Antony, A.C., 1992. The biological chemistry of folate receptors. Blood 79,

2807–2820.

Atyabi,F.,Majzoob,S.,Iman,M.,Salehi,M.,Dorkoosh,F.,2005.Invitro

evalua-tionandmodificationofpectinategelbeadscontainingtrimethylchitosan,asa

multi-particulatesystemfordeliveryofwater-solublemacromoleculestocolon.

Carbohydr.Polym.61,39–51.

Autefage,H.,Briand-Mesange,F.,Cazalbou,S.,Drouet,C.,Combes,C.,Swider,P.,Rey,

C.,2009.AdsorptionandreleaseofBMP-2onnanocrystallineapatite-coated

anduncoatedhydroxyapatite/beta-tricalciumphosphateporousceramics.J.

Biomed.Mater.Res.B91,706–715.

Barroug,A.,Kuhn,L.T.,Gerstenfeld,L.C.,Glimcher,M.J.,2004.Interactionsof

cis-platinwithcalciumphosphatenanoparticles:invitrocontrolledadsorptionand

release.J.Orthop.Res.22,703–708.

Bouladjine,A.,Al-Kattan,A.,Dufour,P.,Drouet,C.,2009.Newadvancesin

nanocrys-talline apatite colloids intended for cellular drug delivery. Langmuir 25,

12256–12265.

Boyer,L.,Piriou,B.,Carpena,J.,Lacout,J.L.,2000.Studyofsitesoccupationand

chem-icalenvironmentofEu3+_{inphosphate-silicatesoxyapatitesbyluminescence.J.}

AlloysCompd.311,143–152.

Bruchez, M., Moronne, M., Gin, P., Weiss, S., Alivisatos, A.P., 1998.

Semi-conductor nanocrystals as fluorescent biological labels. Science 281,

2013–2016.

Bussy,C.,Verhoef,R.,Haeger,A.,Morra,M.,Duval,J.L.,Vigneron,P.,

Bensous-san,A., Velzenberger,E., Cascardo, G.,Cassinelli, C.,Schols, H., Knox,J.P.,

Nagel,M.D.,2008.Modulatinginvitrobonecellandmacrophagebehavior

byimmobilizedenzymaticallytailoredpectins.J.Biomed.Mater.Res.A86,

597–606.

Cazalbou,S.,Eichert,D.,Ranz,X.,Drouet,C.,Combes,C.,Harmand,M.F.,Rey,C.,2005.

Ionexchangesinapatitesforbiomedicalapplications.J.Mater.Sci.:Mater.Med.

16,405–409.

Chambin,O.,Dupuis,G.,Champion,D.,Voilley,A.,Pourcelot,Y.,2006.Colon-specific

drugdelivery:influenceofsolutionreticulationpropertiesuponpectinbeads

performance.Int.J.Pharm.321,86–93.

Couvreur,P.,Gref,R.,Andrieux,K.,Malvy,C.,2006.Nanotechnologiesfordrug

deliv-ery:applicationtocancerandautoimmunediseases.Prog.SolidStateChem.34,

231–235.

Drouet,C.,Carayon,M.T.,Combes,C.,Rey,C.,2008.Surfaceenrichmentofbiomimetic

apatiteswithbiologicallyactiveionsMg2+_andSr2+_{:apreambletotheactivation}

ofbonerepairmaterials.Mater.Sci.Eng.C28,1544–1550.

Drouet,C.,Bosc,F.,Banu,M.,Largeot,C.,Combes,C.,Dechambre,G.,Estournes,C.,

Raimbeaux,G.,Rey,C.,2009.Nanocrystallineapatites:frompowdersto

bioma-terials.PowderTechnol.190,118–122.

Fizet,J.,Riviere,C.,Bridot,J.L.,Charvet,N.,Louis,C.,Billotey,C.,Raccurt,M.,Morel,

G.,Roux,S.,Perriat,P.,Tillement,O.,2009.Multi-luminescenthybrid

gadolin-iumoxidenanoparticlesaspotentialcelllabeling.J.Nanosci.Nanotechnol.9,

5717–5725.

Gautier,H.,Chamblain,V.,Weiss,P.,Merle,C.,Bouler,J.M.,2010.Invitro

character-isationofcalciumphosphatebiomaterialsloadedwithlidocainehydrochloride

andmorphinehydrochloride.J.Mater.Sci.:Mater.Med.21,3141–3150.

Girod-Fullana,S.,Ternet,H.,Freche,M.,Lacout,J.L.,Rodriguez,F.,2010.Controlled

releasepropertiesandfinalmacroporosityofapectinmicrospheres–calcium

phosphatecompositebonecement.ActaBiomater.6,2294–2300.

Grant,G.T.,Morris,E.R.,Rees,D.A.,Smith,P.J.C.,Thom.,D.,1973.Biological

interac-tionsbetweenpolysaccharidesanddivalentcations:theegg-boxmodel.FEBS

Lett.32,195–198.

Grossin,D.,Rollin-Martinet,S.,Estournes,C.,Rossignol,F.,Champion,E.,Combes,

C.,Rey,C.,Chevallier, G.,Drouet, C.,2010.Biomimeticapatitesinteredat

verylowtemperaturebySparkPlasmaSintering(SPS):physico-chemistryand

microstructureaspects.ActaBiomater.6,577–585.

Habraken,W.J.E.M.,Boerman,O.C.,Wolke,J.G.C.,Mikos,A.G.,Jansen,J.A.,2008.

Invitrogrowthfactorreleasefrominjectablecalciumphosphatecements

con-taininggelatinmicrospheres.J.Biomed.Mater.Res.A91,614–622.

Ichibouji,T.,Miyazaki,T.,Ishida,E.,Ashizuka,M.,Sugino,A.,Ohtsuki,C.,Kuramoto,

K.,2008.Evaluationofapatite-formingabilityandmechanicalpropertyofpectin

hydrogels.J.Ceram.Soc.Jpn.116,74–78.

Josse,S.,Faucheux,C.,Soueldan,A.,Grimandi,G.,Massiot,D.,Alonso,B.,Janvier,

P.,Laib,S.,Pilet,P.,Gauthier,O.,Daculsi,G.,Guicheux,J.,Bujoli,B.,Bouler,

J.M.,2005.Novelbiomaterialsforbisphosphonatedelivery.Biomaterials26,

2073–2080.

Kamen,B.A.,Wang,M.,Streckfuss,A.J.,Peryea,X.,Anderson,R.G.,1988.Deliveryof

folatestothecytoplasmofMA104cellsismediatedbyasurface-membrane

receptorthatrecycles.J.Biol.Chem.263,13602–13609.

Kokkonen,H.E.,Ilvesaro,J.M.,Morra,M.,Schols,H.A.,Tuukkanen,J.,2007.Effectof

modifiedpectinmoleculesonthegrowthofbonecells.Biomacromolecules8,

509–515.

Kokkonen,H.,Cassinelli,C.,Verhoef,R.,Morra,M.,Schols,H.A.,Tuukkanen,J.,2008.

Differentiationofosteoblastsonpectin-coatedtitanium.Biomacromolecules9,

2369–2376.

Kokkonen,H.,Niiranen,H.,Schols,H.A.,Morra,M.,Stenback,F.,Tuukkanen,J.,2010.

Pectin-coatedtitaniumimplantsarewell-toleratedinvivo.J.Biomed.Mater.

Res.A93,1404–1409.

Kokubo,T.,Takadama,H.,2006.HowusefulisSBFinpredictinginvivobone

bioac-tivity?Biomaterials27,2907–2915.

Laskin,D.L.,Laskin,J.D.,2001.Roleofmacrophagesandinflammatorymediatorsin

Lebugle,A.,Rodrigues,A.,Bonnevialle,P.,Voigt,J.J.,Canal,P.,Rodriguez,F.,2002.

Study of implantablecalcium phosphate systemsfor the slowrelease of

methotrexate.Biomaterials23,3517–3522.

Lefevre,L.,Gales,A.,Olagnier,D.,Bernad,J.,Perez,L.,Burcelin,R.,Valentin,A.,

Auwerx,J.,Pipy,B.,Coste,A.,2010.PPARgammaligandsswitchedhighfat

diet-inducedmacrophageM2bpolarizationtowardM2atherebyimproving

intestinalCandidaelimination.PLoSOne5,e12828.

LeGeros,R.Z.,2008.Calciumphosphate-basedosteoinductivematerials.Chem.Rev.

108,4742–4753.

Moghimi,S.M.,Bonnemain,B.,1999.Subcutaneousandintravenousdeliveryof

diag-nosticagentstothelymphaticsystem:applicationsinlymphoscintigraphyand

indirectlymphography.Adv.DrugDeliv.Rev.37,295–312.

Moghimi,S.M.,Hunter,A.C.,Murray,J.C.,2001.Long-circulatingandtarget-specific

nanoparticles:theorytopractice.Pharmacol.Rev.53,283–318.

Mondejar,S.P.,Kovtun,A.,Epple,M.,2007.Lanthanide-dopedcalciumphosphate

nanoparticleswithhighinternalcrystallinityandwithashellofDNAas

fluores-centprobesincellexperiments.J.Mater.Chem.17,4153–4159.

Morra,M.,Cassinelli,C.,Cascardo,G.,Nagel,M.D.,DellaVolpe,C.,Siboni,S.,Maniglio,

D.,Brugnara,M.,Ceccone,G.,Schols,H.A.,Ulvskov,P.,2004.Effectsoninterfacial

propertiesandcelladhesionofsurfacemodificationbypectichairyregions.

Biomacromolecules5,2094–2104.

Orhan,Z.,Cevher,E.,Mulazimoglu,L.,Gurcan,D.,Alper,M.,Araman,A.,Ozsoy,

Y.,2006.Thepreparationofciprofloxacinhydrochloride-loadedchitosanand

pectinmicrospheres–theirevaluationinananimalosteomyelitismodel.J.Bone

JointSurg.:Br.88B,270–275.

Ow,H.,Larson,D.R.,Srivastava,M.,Baird,B.A.,Webb,W.W.,Weisner,U.,2005.Nano

Lett.5,113–117.

Parak,W.J.,Gerion,D.,Pellegrino,T.,Zanchet,D.,Micheel,C.,Williams,S.C.,Boudreau,

R.,LeGros,M.A.,Larabel,C.A.,Alivisatos,A.P.,2003.Biologicalapplicationsof

colloidalnanocrystals.Nanotechnology14,R15–R17.

Parker,N.,Turk,M.,Westrick,E.,Lewis,J.,Low,P.S.,Leamon,C.P.,2005.Folate

recep-torexpressionincarcinomasandnormaltissuesdeterminedbyaquantitative

radioligandbindingassay.Anal.Biochem.338,284–293.

Rey,C.,Combes,C.,Drouet,C.,Sfihi,H.,Barroug,A.,2007.Physico-chemical

proper-tiesofnanocrystallineapatites:implicationsforbiomineralsandbiomaterials.

Mater.Sci.Eng.C27,198–205.

Rey,C.,Combes,C.,Drouet,C.,Glimcher,M.J.,2009.Bonemineral:updateon

chem-icalcompositionandstructure.Osteoporos.Int.20,1013–1021.

Roveri,N.,Palazzo,B.,Iafisco,M.,2008.Theroleofbiomimetismindeveloping

nanostructuredinorganicmatricesfordrugdelivery.ExpertOpin.DrugDeliv.5,

861–877.

Ruhe,P.Q.,Boerman,O.C.,Russel,F.G.M.,Spauwen, P.H.M.,Mikos,A.G.,Jansen,

J.A.,2005. Controlled release of rhBMP-2 loadedpoly(dl-lactic-co-glycolic

acid)/calciumphosphatecementcompositesinvivo.J.Control.Release106,

162–171.

Saha,S.K.,Banerjee,A.,Banerjee,S.,Bose,S.,2009.Synthesisofnanocrystalline

hydroxyapatiteusingsurfactanttemplatesystems:roleoftemplatesin

con-trollingmorphology.Mater.Sci.Eng.C29,2294–2301.

Schroeder,J.E.,Shweky,I.,Shmeeda,H.,Banin,U.,Gabizon,A.,2007.Folate-mediated

tumorcelluptakeofquantumdotsentrappedinlipidnanoparticles.J.Control.

Release124,28–34.

Spinella,M.J.,Brigle,K.E.,Sierra,E.E.,Goldman,I.D.,1995.Distinguishingbetween

folate-receptor-alpha-mediatedtransportandreduced-folate-carrier-mediated

transportinL1210leukemiacells.J.Biol.Chem.270,7842–7849.

Sriamornsak,P.,1999.Effectofcalciumconcentration,hardeningagentanddrying

conditiononreleasecharacteristicsoforalproteinsfromcalciumpectinategel

beads.Eur.J.Pharm.Sci.8,221–227.

Tran,P.A.,Zhang,L.J.,Webster,T.J.,2009.Carbonnanofibersandcarbonnanotubes