1

Domodelpolymertherapeuticssufficientlydiffusethrougharticularcartilagetobeaviable

therapeuticroute?

AlisonPowell1,BruceCaterson1,ClareHughes1,AlisonPaul2,CraigJames2,StephenHopkins2,

OmarMansour3andPeterGriffiths3

1SchoolofBiosciences,CardiffUniversity,TheSirMartinEvansBuilding,

MuseumAvenue,Cardiff,CF103AX

2SchoolofChemistry,CardiffUniversity,MainBuilding,ParkPlace,CF103TB

3DepartmentofPharmaceutical,ChemicalandEnvironmentalSciences,FacultyofEngineering

andScience,UniversityofGreenwich,MedwayCampus,CentralAvenue,ChathamMaritime,

KentME44TB

2

Abstract

Theabilityofapolymertherapeutictoaccesstheappropriatesubcellularlocationiscrucialto

itsefficacy,andisdefinedtoalargepartbythemanyandcomplexcellularbiologicaland

biochemicalbarrierssuchaconstructmusttraverse.Itisshownherethatmodeldextrin

conjugatesareabletopassthroughacartilaginousextracellularmatrixintochondrocytes,with

littleperturbationofthematrixstructure,indicatingthattargetingofpotentialtherapeutics

throughacartilaginousextracellularmatrixshouldprovepossible.Rapidchondrocytictargeting

ofdrugswhichrequireintracellularisationfortheiractivity,anduniformextracellular

concentrationsofdrugswithanextracellulartarget,isthusenabledthoughpolymer

conjugation.

3

Introduction

Articularcartilagelinestheloadbearingjointsofthebodyactingtoreducefrictionandabsorb

mechanicalloads.Itisananeural,avascular,hypocellulartissue,composedofadense

extracellularmatrixcontrolledandsecretedbythechondrocyteswithinit.Thechondrocytes

compriselessthan2%ofthevolumeofmaturearticularcartilagewiththeremainder

comprisingahighlyorganisednetworkofcollagenfibrils,proteoglycansandwater.Thematrix

macromoleculesofarticularcartilagegivethetissueitsuniquestructureandfunction.These

structuralmacromoleculesincludecollagens(mainlytypeII),proteoglycansandnon-

collagenousproteins.ThetypeIIcollagenfibrilmeshworkgivesthecartilageitsformand

tensilestrength.Theproteoglycansandnon-collagenousproteinsofarticularcartilagebindto

thecollagenmeshworkorbecomemechanicallyentrappedwithinit.Themajorproteoglycanof

articularcartilageisaggrecan,whichhasnumerousglycosaminoglycanchainsattachedtoits

coreproteinandformshugemultimolecularaggregateswithhyaluronanandalinkprotein.

Hydrationoftheglycosaminoglycanchainsofaggrecanprovidescartilagewithits

compressibilityfunctions.

MorethantenmillionpeopleintheUKhavelong-termhealthproblemsduetoarthritisora

relatedcondition1.Themostcommonarthriticdiseasesareosteoarthritisandrheumatoid

arthritis,bothofwhichinvolveerosionofthecartilagecushioningtheendsofboneswithinthe

joint.Symptomsincludejoint-painandstiffness,whichcanleadtodisability.Cartilage

degenerationinarthritisisdueultimatelytotheenzymaticdegradationofthecartilage

extracellularmatrix.Thecomplexityanddensityofthecartilageextracellularmatrixresultsina

barriertothepassageofmoleculesthroughthematrix,howeveritsmaintenanceiscrucialfor

tissuefunction.Inthesynovialjointthearticularsurfaceislubricatedandnourishedbysynovial

fluid,afiltrateofbloodplasmacontaininghighlevelsoftheglycosaminoglycanhyaluronanand

theproteoglycanlubricin(SZP)2.Therelativeviscosityofsynovialfluidprovidesanadditional

barriertodiffusionofmoleculesintochondrocytes.

1. Arthritisandwork(03.2017)http://www.arthritisresearchuk.org/policy-and-public-affairs/reports-and-

resources/reports/work-report.aspx

2. Rhee,D.K.,Marcelino,J.,Baker,M,Gong,Y.,Smits,P.,Lefebvre,V.,Jay,G.D.,Stewart,M.,Wang,H.,Warman,M.L.andCarpten,J.D.(2005)Thesecretedglycoproteinlubricinprotectscartilagesurfacesandinhibitssynovialcellovergrowth.TheJournalofClinicalInvestigation.115:622-631

4

Currenttherapiesforarthritismainlytreattheclinicaldiseasesymptomsratherthantargeting

thedegradativeenzymesthemselves.Treatmentssuchassteroidalandnon-steroidalanti-

inflammatorydrugsarestillthemainstayoftreatment3,4andcanhaveadversesideeffects

includinghighbloodpressure5,6,7,osteoporosis8,cataracts9,10andgastrointestinalbleeding11.

Thenewlyreleasedanti-TNFdrugs(Etanercept,InfliximabandAdalimumab)whichblockthe

pro-inflammatorycytokineTNFinrheumatoidandpsoriaticarthritis12,13areefficaciousforonly

asmallcohortofpatientsandareonlyprescribedforUKpatientswithsevereandcrippling

formsofthediseaseastheycarrytheriskofserioussideeffectsduetoimmuno-suppression14.

Inrecentyears,anumberofcompoundshavebeenidentified,ininvitrostudies,thatareable

toinhibittheinitiallossoftheextracellularmatrixcomponentaggrecan,viaoftenunknown

mechanisticinhibitionoftheproteasesADAMTS-4and/or-5.Thesecompoundshavenotably

3. Marini,S.,Fasciglione,G.F.,Monteleone,G.,Maiotti,M.,Tarantino,U.&Coletta,M.(2003)Acorrelation

betweenkneecartilagedegradationobservedbyarthroscopyandsynovialproteinasesactivities.ClinicalBiochemistry36:295-304;

4. Brandt,K.D.&Slowman-Kovacs,S.(1986)Nonsteroidalanti-inflammatorydrugsintreatmentofosteoarthritis.ClinicalOrthopaedics213:84-91

5. Gabriel,S.E.&Wagner,J.L.(1997)Costsandeffectivenessofnonsteroidalanti-inflammatorydrugs.Theimportanceofreducingsideeffects.ArthritisCareResearch10:56-63

6 Hammer,F.&Stewart,P.M.(2006)Cortisolmetabolisminhypertension.BestPracticeandResearchClinicalEndocrinologyandMetabolism20:337-353

7 Reiche,M.L.(2005)Complicationsofintravitrealsteroidinjections.ClinicalCare76:450-460

8 Ledford,D.,Apter,A.,Brenner,A.M.,Rubin,K.,Frieri,M.&Lukert,B.(1998)Osteoporosisinthecorticosteroidtreatedpatientwithasthma.JournalofAllergyandClinicalImmunology102:353-363

9 Moore,P.(1997)Inhaledcorticosteroidsincreasecataractrisk.TheLancet350:120

10 UrbanR.C.&Cotlier,E.(1986)Corticosteroid-inducedcataracts.SurveyofOphthalmology31:102-110

11 Tringham,V.M.&Cochrane,P.(1979)Aspirin,paracetamol,diflunisalandgastrointestinalbloodloss.TheLancet320:1409

12 Punzi,L.,Podswiadek,M.,Sfriso,P.,Oliviero,F.,Fiocco,U.&Todesco,S.(2007)PathogenicandclinicalrationaleforTNF-blockingtherapyinpsoriaticarthritis.AutoimmunityReviews6(8):524-528

13 Berthelot,J-M.,Varin,S.,Cormier,G.,Tortellier,L.,Guillot,P.,Glemarec,J.&Maugars,Y.(2007)25mgetanerceptonceweeklyinrheumatoidarthritisandspondylarthropathy.JointBoneSpine74(2):144-147

14 Maillard,H.,Ornetti,P.,Grimault,L.,Ramon,J-F.,Ducamp,S.M.,Saidani,T.,Tavernier,C.&Maillefert,J.F.(2005)Severepyogenicinfectionsinpatientstakinginfliximab.Aregionalcohortstudy.JointBoneSpine72:330-334

5

includedtheneutraceuticalglucosamine15,16,17andthesulphatedsugarderivativepentosan

polysulphate18,19.Inaddition,thephysiologicalinhibitorofADAMTS-4and-5,TIMP-3,hasbeen

showntoablatetheinitiallossofaggrecanincytokineinduceddegradativemechanisms20.

Theideathatwater-solublepolymersfunctioningascarriersofdrugsthroughconjugationviaa

biodegradablespacer/linkercouldfacilitatetargeteddrugreleasewasfirstputforwardinthe

mid-1970’s21.Thelasttwodecadeshaveseensuccessfulclinicalapplicationofpolymer

conjugatestotargettherapeuticagentsforthetreatmentofarangeofdiseasesincludingage

relatedmaculardegeneration,cancerandliverdisease22,23,24,25.Thesetherapeuticconjugates

haveutilisedanumberofpolymersasdrugdeliveryvehiclesincludingN-(2-hydroxypropyl)

15 Largo,R.,Alvarez-Soria,M.A.,Diez-Ortego,I.&Calvo,E.(2003)GlucosamineinhibitsIL-1b-inducedNFkB

activationinhumanosteoarthriticchondrocytes.OsteoarthritisandCartilage11:290-298

16 Maillard,H.,Ornetti,P.,Grimault,L.,Ramon,J-F.,Ducamp,S.M.,Saidani,T.,Tavernier,C.&Maillefert,J.F.(2005)Severepyogenicinfectionsinpatientstakinginfliximab.Aregionalcohortstudy.JointBoneSpine72:330-334

17 Ilic,M.Z.,Martinac,B.&Handley,C.J.(2003)Effectsoflong-termexposuretoglucosamineandmannosamineonaggrecandegradationinarticulatcartilage.OsteoarthritisandCartilage11:1-10

18 Smith,J.G.,Hannon,R.L.,Brunnberg,L.,Gebski,V.&Cullis-Hill,D.(2002)Amulticentreclinicalstudyoftheefficacyofsodiumpentosanpolysulphateandcarprofenincanineosteoarthritis(osteoarthrosis).

19 Rogachefsky,R.A.,Dean,D.D.,Howell,D.S.&Altman,R.D.(1994)Treatmentofcanineosteoarthritiswithsodiumpentosanpolysulphateandinsulin-likegrowthfactor-1.AnnaloftheNewYorkAcademyofSciences732:392-394

20 Gendron,C.,Kashiwagi,M.,Hughes,C.E.,Caterson,B.&Nagase,H.(2003)TIMP-3inhibitsaggrecanases-mediatedglycosaminoglycanreleasefromcartilageexplantsstimulatedbycatabolicfactors.FEBSLetters555:431-436

21 Vasey,P.A.,Kaye,S.B.,Morrison,R.,Twelves,C.,Wilson,P.,Duncan,R.,Thomson,A.H.,Murray,L.S.,Hilditch,T.E.,Murray,T.,Burtles,S.,Fraier,D.,Frigerio,E.&Cassidy,J.(1999)PhaseIClinicalandPharmacokineticstudyofPK1[N-(2-Hydroxypropyl)methacrylamideCopolymerDoxorubicin]:Firstmemberofanewclassofchemotherapeuticagents-drug-polymerconjugates.ClinicalCancerResearch5:83-94

22 Duncan,R.(2005)Targetingandintracellulardeliveryofdrugs.InEncyclopediaofMolecularCellBiologyandMolecularMedicine,EditorMeyers,R.A.,PublishedbyWiley-VCH,Verlag,GmbH&Co.KGaA,Weinheim,Germany,pp163-204

23 Duncan,R.(2006)Polymerconjugatesasanticancernanomedicines.NatureReviewsCancer6:688-701

24 Duncan,R.,Ringsdorf,H.&Satchi-Fainaro,R.(2006)Polymertherapeutics–PolymersasDrugs,ConjugatesandGeneDeliverySystems:Past,presentandfutureopportunities.AdvancedPolymerScience192:1-8

25 Yasukawa,T.,Ogura,Y.,Sakurai,E.,Tabata,Y.&Kimura,H.(2005)Intraocularsustaineddrugdeliveryusingimplantablepolymericdevices.AdvancedDrugDeliveryReviews57:2033-2046

6

methacrylamide(HPMA)26,27,poly(ethyleneglycol)(PEG)28,29,30anddextrin31,32.Conjugationof

drugstopolymershasallowedforspecifictargetingofthetherapeuticactivityofadrugtothe

diseasedtissue.Thisresultsinareductioninthedoserequiredforefficacyoftreatmentanda

reductioninpotentialtoxicityofthedrugs.

Expandingthismodalitytothetreatmentofarthriticjoints,requiresthatsuchpolymersin

ordertodelivertheirdrugpayloadmustbeabletopassthroughacartilaginousextracellular

matrixrichinnegativelychargedproteoglycans,eithertoreachtheirchondrocytictargets,orto

achieveevendistributionthroughoutthecartilageextracellularmatrix.Intheselectionof

suitabletargetingpolymersandpolymerconjugatesforthetreatmentofarthriticdisease,

quantifyingwhetherthesecompoundscanindeedpassthroughthebarrierspresentedbythe

synovialjointwithoutinducingtheirdisruptionwillbeofvitalimportance.Ofthepotential

barrierspresentwebelievethattheinteractionsofpolymersandpolymerconjugateswith

proteoglycans,withinthecartilageextracellularmatrixaswellascoatingitssurface,willbethe

mostinfluentialontheirmovement.Thisstudyfocusedoninvestigatingtheinteractionsof

polymersandpolymerconjugateswiththecartilageproteoglycansaggrecanandlubricinas

wellassynovialfluid.

26 Duncan,R.,Vicent,M.J.,Greco,F.&Nicholson,R.I.(2005)Polymer-drugconjugates:towardsanovel

approachforthetreatmentofendocrine-relatedcancer.Endocrine-RelatedCancer12:189-199

27 Nan,A.,Nanayakkara,N.P.D.,Walker,L.A.,Yardley,V.,Croft,S.L.&Ghandehari,H.(2001)N-(2-hydroxypropyl)methacrylamide(HPMA)copolymersfortargeteddeliveryof8-aminoquinolineantileishmanialdrugs.JournalofControlledRelease77:233-243

28 Ould-Ouali,L.,Noppe,M.,Langlois,X.,Willems,B.,Riele,P.T.,Timmerman,P.,Brewster,M.E.,Arien,A.&Preat,V.(2005)Self-assemblingPEG-r(CL-co-TMC)copolymersfororaldeliveryofpoorlywatersolubledrugs:acasestudywithrisperidone.JournalofControlledRelease102:657-668

29 Cheng,J.,Teply,B.A.,Sherifi,I.,Sung,J.,Luther,G.,Gu,F.X.,Levy-Nissenbaum,E.,Radovic-Moreno,A.F.,Langer,R.&Farokhzad,O.C.(2007)FormulationoffunctionalisedPLGA-PEGnanoparticlesforinvivotargeteddrugdelivery.Biomaterials28:869-876

30 Yu,D.,Peng,P.,Dharap,S.S.,Wang,Y.,Mehlig,M.,Chandna,P.,Zhao,H.,Filpula,D.,Yang,K.,Borowski,V.,Borchard,G.,Zhang,Z.&Minko,T.(2005)Antitumoractivityofpoly(ethyleneglycol)-camptothecinconjugate:Theinhibitionoftumorgrowthinvivo.JournalofControlledRelease110:90-102

31 Hreczuk-Hirst,D.,Chicco,D.,German,L.&Duncan,R.(2001)Dextrinsaspotentialcarriersfordrugtargeting:tailoredratesofdextrindegradationbyintroductionofpendantgroup.InternationalJournalofPharmaceutics230:57-66

32 Hreczuk-Hirst,D.,German,L.&Duncan,R.(2001)Dextrinsascarriersfordrugtargeting:Reproduciblesuccinoylationasameanstointroducependantgroups.JournalofBioactiveandCompatiblePolymers16:353-364

7

Materials&Methods

Preparationofsynovialfluidsamples

Synovialfluidsampleswereharvestedfromthemetacarpo/metatarsophalangealjointsof18

montholdcowslegsusinga15gaugeneedle.

Preparationoflubricin

Followingeffusionofthesynovialfluidfromthejointsof18montholdcowslegsasdescribed

abovethejointswerelavagedusing1.6Msodiumchloridetoisolatethelubricincoatingthe

surfaceofthearticularcartilage33.

Preparationofaggrecanaggregates

Bovinearticularcartilageexplantswereestablishedusingpreviousmethodologies34.Following

a48hourprecultureexplantswerewashedintoserumfreeDMEM.Theexplantswerethen

culturedineither(i)serumfreeDMEMor(ii)serumfreeDMEM+IL-1a(10ng/ml).Cultures

wereincubatedforupto96hours;mediaandexplantswereharvestedandfinelydicedpriorto

additionofguanidineextractionbuffer(4MguanidineHCl,50mMsodiumacetatepH5.8-6.8,

0.1M6-amino-hexanoicacid,5mMbenzamidineHCl,10mMethylenediaminetetraaceticacid

(EDTA)(tetrasodiumsalt),1mMphenylmethylsulphonylfluoride(PMSF-10mlpergram

cartilagewetweight)andincubatedfor48hoursat4oCwithconstantagitation.Theextracted

cartilagedebriswasremovedbycentrifugationat15,000rpmfor10minutes,anddiscarded.

TheliquidsupernatantwasthendialysedexhaustivelyagainstMilliQ™water.Thedensityofthe

extractwasadjustedto1.5g/mlbyadditionofCaCl2andaggrecan-hyaluronanaggregate

purifiedbyultracentrifugationinaBeckmanL-60Ultracentrifugeat37,000rpmfor70hoursat

4oC.Theextractwasfractionatedinto4equalpoolsdesignatedA1-A4,thelowestfractionA1

containingthepurifiedaggrecan-hyaluronancomplexandhavingadensity>1.57g/ml.

33 Jones,A.R.C.,Gleghorn,J.P.,Hughes,C.E.,Fitz,L.J.,Zollner,R.,Wainwright,S.D.,Caterson,B.,Morris,E.A.,

Bonassar,L.J.andFlannery,C.R.(2007)Bindingandlocalizationofrecombinantlubricintoarticularcartilagesurfaces.JournalofOrthopaedicResearch25(3):283-92.

34 Hughes,C.E.,Caterson,B.,Fosang,A.J.,Roughley,P.J.andMort,J.S.(1995)Monoclonalantibodiesthatspecificallyrecogniseneoepitopesequencesgeneratedbyaggrecanasesandmatrixmetalloproteinasecleavageofaggrecan:applicationtocatabolisminsituandinvitro.TheBiochemicalJournal305:799-804

8

AnalysisofcellularuptakeofpolymerconjugatesbyFACSandfluorescencemicroscopy

Explantswereharvestedandpre-culturedusingpreviouslyestablishedmethodsError!Reference

sourcenotfound.priortoincubationinthepresenceorabsenceofIL-1a(10ng/ml)for96hours35.

CultureswerethenincubatedinDMEMwith50µg/mlgentamicinand10%(v/v)heat

inactivatedFBSinthepresenceorabsenceofOregonGreenlabelledpolymersatarangeof

concentrations(1-10µg/ml)foranumberoftimepoints(2-24hours).Followingincubationwith

polymersexplantswereeitherwashedinphosphatebufferedsalineandviewedusinga

confocalmicroscope,ordigestedtofreethecellsusingPronase(1%{w/v}inDMEMcontaining

50µg/mlgentamicinand10%{v/v}heatinactivatedFBS)for30minutesat37oC,followedby

collagenasetypeII(0.4%{w/v}inDMEMcontaining50µg/mlgentamicinand10%{v/v}heat

inactivatedFBS)for45minutesat37oC.Theisolatedcellswillbepelletedbycentrifugation

resuspendedinPBSandrunonaFACsCanto(BDBiosciences).

Pulsed-GradientSpin-EchoNuclearMagneticResonance

Measurementsonthepurifiedfreeze-driedaggrecan-hyaluronancomplexredispersedinD2O

wereconductedonaBrukerAMX360NMRspectrometerusingastimulatedecho-sequence36.

Thisconfigurationusedeithera5mm(CryomagnetSystems,Indianapolis)or10mm(Bruker)

diffusionprobeinconjunctionwithBrukerorWoodwardgradientspectroscopyaccessories.

Theself-diffusioncoefficientDswasextractedbyfittingtoequation1either(i)theintegralsfor

agivenpeak,or(ii)theindividualfrequencychannelspresentintheentirebandshape(CORE

analysis);

(1)

35 Arner,E.C.,Hughes,C.E.,Decicco,C.P.,Caterson,B.andTortorella,M.D(1998)Cytokineinducedcartilage

proteoglycandegradationismediatedbyaggrecanases.OsteoarthritisandCartilage.6:214-228

36 Davies,J.A.andGriffiths,P.C.(2003)APhenomenologicalApproachtoSeparatingtheEffectsofObstructionandBindingfortheDiffusionofSmallMoleculesinPolymerSolutions.Macromolecules:36,950

( ) [ ]so kDAGA −=Δ exp,,δ

9

whereAisthesignalamplitudeintheabsence(Ao)andpresenceofthefieldgradientpulses

( )Δ,,GA δ )and( ) ( )

⎟⎟

⎠

⎞

⎜⎜

⎝

⎛ +++−+Δ−=

301435301030 232232

22 σσδσδδσδγ Gk

giventhatgisthemagnetogyricratio,Dthediffusiontime,σthegradientramptime,dthe

gradientpulselengthandGthegradientfieldstrength.

Small-angleneutronscattering

SANSexperimentswereperformedontheLOQdiffractometerbasedatthespallationsourceat

theRutherfordAppletonLaboratory,U.K.,whereaQrangeof0.007Å-1to0.3Å-1isaccessible

using2<l<10Å,whereQ=(4π/λ)sin(θ/2)Q = #$%sin )

*.

Sampleswerecontainedin1mmpathlength,UV-spectrophotometergrade,quartzcuvettes

(Hellma)andmountedinaluminiumholdersontopofanenclosed,computer-controlled,

samplechamber.Temperaturecontrolwasachievedthroughtheuseofathermostatted

circulatingbathpumpingfluidthroughthebaseofthesamplechamber.Undertheseconditions

atemperaturestabilityofbetterthan±0.5°Ccanbeachieved.Experimentalmeasuringtimes

wereapproximately40-60min.

Allscatteringdatawerenormalisedforthesampletransmissionandtheincidentwavelength

distribution,correctedforinstrumentalandsamplebackgroundsusingaquartzcellfilledwith

eitherH2OorD2O(thisalsoremovestheincoherentinstrumentalbackgroundarisingfrom

vacuumwindows,etc.),andcorrectedforthelinearityandefficiencyofthedetectorresponse

usingtheinstrumentspecificsoftwarepackage.Thedatawereputontoanabsolutescaleusing

awell-characterizedpartiallydeuteratedpolystyreneblendstandardsample.

Theintensityofthescatteredradiation,I(Q),asafunctionofthewavevector,isgivenby:

𝐼 𝑄 = 𝑁𝑉* 𝛥𝜌 *𝑃 𝑄 𝑆 𝑄 +𝐵678 (2)

10

whereP(Q)describesthemorphologyofthescatteringspecies,S(Q)describesthespatial

arrangementofthespeciesinsolution,Nisthenumberofspeciesperunitvolume,Visthe

volumeofthespecies,Δρisthedifferenceintheneutronscatteringlengthdensity(SLD)ofthe

scattererandthesolventandBincistheincoherentbackgroundscattering.

11

ResultsandDiscussion

PGSE-NMRprovidesaconvenientandnon-invasivechemicallyselectivetechniquefor

measuringtranslationalmotion,andinparticulartheself-,ratherthanmutual-diffusion

coefficient.Therefore,PGSE-NMRcanbeusedbothtoquantifythemobilityofmoleculesand

toassesstheimpactofthepresenceofthemoleculesontheextracellularmatrixcomponents

themselves.Thediffusionofsmallsolutes(includingwaterandNa+)hasbeenshowntobe

impededbyupto40%bythepresenceofanextracellularmatrix,implyingthatthemovement

oflargermoleculessuchaspolymersmaybeverysignificantlyaffectedbythepresenceofa

matrix37.

Avitalcomponentintheselectionofpolymer-drugconjugatesforthetreatmentofarthritic

diseaseistheselectionofeitherpolymersabletotargetadrugwithintracellularactivityandits

abilitytodiffuserapidlythroughtheextracellularmatrixintothechondrocyte.Accordingly,the

rateofdiffusionofdextrinpolymers(asmodelconjugates)hasbeenquantifiedbothinfree

solutionandthroughcomponentsofthecartilaginousextracellularmatrix(anaggrecan-

hyaluronanaggregategelpurifiedbydensitygradientcentrifugationfrombovinearticular

cartilage),TableOne.FortheMw=10kg/mol-1dextrinsample,theself-diffusioncoefficientwas

slowedfromDs=2.7x10-10m2s-1infreesolution,correspondingtoahydrodynamicradiusofa

fewnanometres,toDs=2.6x10-11m2s-1intheaggrecan-hyaluronangel,(Table1).Thehigher

molecularweightdextrinshowsalargerreduction,thoughthefreesolutionretardationis

consistentwiththeincreaseinmolecularweightofarandomcoilconfiguration.Bydrawing

analogiesofthediffusionofsuchpolymerinrelated(mucin)gels,thissignificantretardationis

probablynotaconsequenceofdirectassociationofthedextrinwiththeaggrecan-hyaluronan

aggregate,butrathersterichindranceduetothepresenceofthehighlyentangledmatrixofthe

aggrecan-hyaluronanaggregate38.Thenon-linearityoftheattenuationfunctionclearly

demonstratesthepresenceofmorethanasinglediffusionrateandtheCOREanalysisidentifies

37 Burstein,D.,Gray,M.L.,Hartman,A.L.,Gipe,R.andFoy,B.D.(1993)Diffusionofsmallsolutesincartilage

asmeasuredbynuclearmagneticresonance(NMR)spectroscopyandimaging.JournalofOrthopaedicResearch.11:465-478

38 Griffiths,P.C.,Occhipinti,P.,Gumbleton,M.,Morris,C.J.,Heenan,R.K.andKing,S.M.(2010)PGSE-NMRandSANSStudiesoftheInteractionofModelPolymerTherapeuticswithMucinBiomacromolecules.11:120-125

12

thesetobedextrin(theinitialdecay)andtheunderlyingmuchslower(twoordersofmagnitude

slower)diffusionoftheaggrecan-hyaluronanaggregates.

Asanaside,wecanalsocommentontheeffectofsuccinoylation–thefirststepindextrin

functionalisation–ontheconformationofthepolymer,ascharacterisedbytheirself-diffusion

coefficients,figure2.Theself-diffusioncoefficientdropssmoothly(increasinghydrodynamic

size)withincreasingmolepercentofsuccinoylation,uptoavalueofaround~10mol%,above

whichtheself-diffusioncoefficientbecomeslargelyconstantat~1x10-10m2s-1,consistentwith

anhydrodynamicradiusofaround50Å.

The(gel)structureofcartilageextract,andvariousderivativematerialshavebeenexaminedby

small-angleneutronscattering(SANS)intheabsenceandpresenceofaddeddextrin,figures

3a&b(representativedataonlypresented).Inthecaseofthe18mdirectcartilageextract,

wherethescatteringfromthegelitselfissignificant(I(Q)>10cm-1atQ<0.01Å)and

dominatesthemixturescattering(fordextrin,I(Q)<0.5cm-1atQ<0.01Å),additionofthetwo

dextrinsamplesinducednonoticeableperturbationinthescattering-andhence,structureof

thedirectcartilageextractgel.InthecaseoftheIL-1systems,andvariousotherderivative

materials(datanotpresented),figure3b,thesameconclusionmaybedrawn,butnotso

directly.Itisobviousthatthescatteringdoesvaryacrossthedifferentmixedsystems,butitis

shownbelowthatthisisaconsequenceoftheweakerandcomparableintensitiesofthe

variouscomponentsratherthanonedominatingcontribution,asinthecaseofthe18mdirect

cartilageextract.

AnumberofstrategiesexistforinterpretingSANSdata.Thesimplestistoconsidertheslopeof

thedatawhenplottedonadoublelogarithmicrepresentation,extractingtheso-calledQ

dependency,I(Q) µQ-n,illustratingthemostprobableshapeofthescatterer;n=1indicatesa

rod,n=2aflatstructure,increasingallthewayton=4foralarge,solidobject.Forsystems

likegels,onemightanticipaten=5/3tothen=3,reflectingatransitionfromamasstovolume

fractalcharacterassociatedwiththedifferinglength-scalespresentinthegel.Thisisindeedthe

case.

Alternatively,onemaynumericallyanalysethedataintermsofspecificmodels,basedonsome

aprioriknowledgeofthelikelyconformationorarrangementofmoleculesinsolution.For

13

dextrin,asimplepolydisperseGaussiancoilmodeladequatelydescribesthedataforMw=51K

g/mol-1dextrinsample,withRG=80Å(consistentwiththetheoreticalpredictionfortheratioof

theradiusofgyrationandhydrodynamicradius,RG/RH=1.5).

Forthegels,anappropriatetreatmentistheShibayama-Geisslertwo-lengthscalemodel39,40

whichtreatsthescatteringastheadditionaloftwocomponents,withfractionf;

𝐼 𝑄 = 𝑓𝐼(0)==

=> ?@AB CDEAD

?/D + 1 − 𝑓 𝐼 0 *𝑒𝑥𝑝 −𝑄*𝑎** + 𝐵 (3)

whereDisthescalingexponentand𝑎** ≈NOD

P.TheparametersdescribingtheShibayama-

GeisslerfittothesedataarepresentedinTableTwo.Byandlarge,thefittingismostsensitive

totheLorentziancomponentembodiedinthescalingexponentD,withalengthscale(𝑎=*)of

around100Å,butthe18mdirectcartilageextractalsorequiresaGuinierterm,withlength

scaleslightlyshorter.Mostimportantly,asisevidentfromtherawdatawheresubtle

differencesareonlyobservedathigherQvalues,theparametersarelargelyinsensitivetothe

additionofthedextrini.e.itmaybeconsideredtobenon-perturbing.

Havingshownitispossiblefordextrintodiffusethroughthegelwhilsthavinglittleimpacton

itsstructure,theprogressionof(OregonGreenfluorophorelabelled)dextrinintochondrocytes

wasassessed.Eachofthecultureswasincubatedindextrin-OregonGreenat1,5and10µg/ml

for2-24hours.Cellswerereleasedfrommonolayerculturesbygentletrypsinisationandby

rapidpronaseandcollagenasedigestfromexplantandexvivotranswellgraftscultures.FACS

analysisofthesecellpopulationswith10µg/mldextrin-OregonGreenat2,4,8and24hours

areshowninFigure4.

Cellularuptakeofthedextrinconjugateachievedinthepresenceofacartilaginousextracellular

matrixinbothexplantculturesandinexvivotranswellgrafts(Figure4B&A,respectively).In

cartilageexplantscellularuptakewasachievedinover90%ofcellsfollowing8hoursexposure

(Figure4B).Uptakeofdextrin-OregonGreenwasslowerinexvivotranswellgraftsthanin

39 Shibayama,M.,Toyoichi,T.,andHan,C.C.(1992)Smallangleneutronscatteringstudyonpoly(N-

isopropylacrylamide)gelsneartheirvolume-phasetransitiontemperature.J.Chem.Phys.97(9),6829-6841

40 Mallam,S.,Horkay,F.,Hecht,A.M.,Rennie,A.R.andGeissler,E.,(1991)Microscopicandmacroscopicthermodynamicobservationsinswollenpoly(dimethylsiloxane)networks.Macromolecules24,543

14

explantscultureswithonly66%ofcellscontainingthefluorophorefollowing24hoursexposure

(Figure4A).Intheexvivotranswellgraftsthedextrin-OregonGreenwasonlyaddedtothe

insertmediumtomimicthesituationinvivowhereatherapeuticagentinjectedintothejoint

wouldonlybeabletopenetratethecartilageatthearticularsurface(Figure5).Thisrestricted

accesstoonesurfaceexplainstheapparentlyslowerrateofuptakebychondrocyteswithinthe

exvivotranswellgraftscomparedtochondrocyteswithincartilageexplants(Figures4A&B).

Inordertodeterminewhetherdextrin-OregonGreenwasabletopassrightthrougha

cartilaginousextracellularmatrixitwasonlyaddedtotheinsertmediumoftheexvivo

transwellgraftsandboththeinsertandoutermediumwereanalysedforitspresenceusing

fluorescencespectroscopyfollowinganumberofdifferentincubationtimes(Figure6).

Thedextrin-OregonGreenwasabletopassrightthroughtheextracellularmatrixoftheexvivo

transwellgraftsandwasdetectedintheoutermediumoftheculturesfollowingonly2hoursof

incubationwiththeconjugate(Figure6).Following24hoursincubationonthefilterinsert

culturesover13%ofthetotalfluorescencepresentinthecultureswasdetectedintheouter

medium(Figure6).

15

Conclusions

Combined,thedatapresentedheredemonstratethatdextrinisabletosuccessfullydiffuse

throughacartilaginousextracellularmatrixandintothechondrocyteswithin,inducinglittle

perturbationinthatmatrix,thusindicatingthefeasibilityofdrugdeliveryusingpolymer

conjugatesforthetreatmentofarthritis.

Acknowledgements

EPSRCarethankedfortheprovisionofanEPSRCPlatformGrant(EP/C013220/1)

”Bioresponsivepolymertherapeutics;synthesisandcharacterisationofnovelnanomedicines”,

STFCforaccesstoSANSfacilitiesandbothCardiffUniversityandUniversityofGreenwichfor

financialsupport.

16

Tables

Sample Freesolution Aggrecan-hyaluronan Ratio

Mw=10Kg/mol-1 2.7x10-10m2s-1 2.6x10-11m2s-1 10

Mw=51Kg/mol-1 1.0x10-10m2s-1 5.0x10-12m2s-1 20

Table1;Self-diffusioncoefficientsinfreesolution(concentration1wt%)andaggrecan-

hyaluronangelsmeasuredbyPGSE-NMR,andtheretardationratiofromfreesolutiontogel.

SampleGuinier

scale

Lorentzian

scale

Radiusof

gyration/

Å

Fractal

dimension

Correlation

length/Å

18m 0.2 18 85(+/-3) 2.2(+/-0.1) 235(+/-30)

18mplusdextrin

10Kgmol-1n/a 18 n/a 2.3 140

18mplusdextrin

51Kgmol-1n/a 14 n/a 2.3 135

IL-1 n/a 0.2 n/a 2.2 105

IL-1plusdextrin

51Kgmol-10.3 1.1 55 2.2 90

Table2:ParametersderivedfromananalysisofthescatteringdataintermsoftheShibayama-

Geisslertwo-lengthscalemodel.

17

18

Figures

Figure1.Experimentaltwo-dimensionalPGSENMRdataset(left)andanalysisofpeakintegrals

usedtoextracttheratesofdiffusion(right)forMw=10kg/mol-1dextrininfreesolutionandin

anaggrecan-hyluronangel,theopensymbolscorrespondtotheattenuationfunctionforthe

peakat~3ppminthespectrumfrom10mg/ml(1wt%)dextrininsolutioni.e.therawdata

presentedabove.Thefilledsymbolscorrespondtotwosuperimposeddatasets,necessaryto

spanthewidedynamicsrangepresentinthesesystems,againforthe3ppmpeak,arisingfrom

thespectrumof10mg/ml(1wt%)dextrininanaggrecan-hyaluronangel(20mg/mlGAG).

k /cm-2s

0.0 5.0e+61.0e+71.5e+72.0e+72.5e+73.0e+73.5e+7

Normalised signal

0.1

1

Ds = 2.7 x 10-10 m2s-1

Ds = 2.6 x 10-11 m2s-1

Ds = 3.0 x 10-12 m2s-1

0 5e+5 1e+6 2e+6 2e+6

0.1

1

19

degree of succinoylation / mol%

0 10 20 30

self-

diffu

sion

coe

ffici

ent /

m2 s-1

1e-10

2e-10

3e-10

4e-10

5e-10

6e-10

7e-10

FigureTwo;Self-diffusioncoefficientsinfreesolutionasafunctionofdegreeofsuccinoylation

measuredbyPGSE-NMRfordextrinMw=51Kg/mol-1at1wt%polymer.

20

Wavevector, Q / ≈ -1

0.01 0.1

Inte

nsity

I(Q

) / c

m-1

0.01

0.1

1

1018m cartilage extract18m cartilage extract and dextrin 10K g mol-1

18m cartilage extract and dextrin 51K g mol-1

dextrin 51K g mol-1

Figure3a.Small-angleneutronscatteringfrom1mmthick(H2O)aqueoussolutionsofdirect

cartilageextractfrom18montholdkneejointsintheabsenceofdextrin,andinthepresenceof

1wt%dextrin,Mw=10kg/mol-1andMw=51kg/mol-1.Thelinesofbestfithavebeenderived

fromtheanalysisusingtheBeaucagemodelasdescribedinthetext.Alsoshownforcomparison

isthescatteringfrom1wt%Mw=51kg/mol-1dextrin(inD2O).Thelinesofbestfitforthis

dextrinsamplehasbeenderivedfromasimpleGaussiancoilmodel,RG=80(±3)Å.

21

Wavevector, Q / ≈ -1

0.01 0.1

Inte

nsity

I(Q

) / c

m-1

0.01

0.1

1IL-1IL-1 and dextrin 51K g mol-1

dextrin 51K g mol-1

Figure3b.Small-angleneutronscatteringfrom2mmthick(D2O)reconstitutedaqueous

solutionsofIL-1intheabsenceofdextrinandinthepresenceof1wt%dextrinMw=51kg/mol-1.

Thelinesofbestfithavebeenderivedfromtheanalysisusingthegelpluscoilmodelas

describedinthetext.Alsoshownforcomparisonisthescatteringfromthedextrinsolution.

22

A.TranswellFilterGraftsB.CartilageExplants

Figure4;FACSanalysisofdextrin-OregonGreenuptakewithtimein:(A)chondrocytesisolated

fromexvivotranswellgraftsfollowingexposuretodextrin-OregonGreenand(B)chondrocytes

isolatedfromarticularcartilageexplantsculturesfollowingexposuretodextrin-OregonGreen.

23

Figure5:Pictureofexvivotranswellgraftsanddiagrammaticrepresentationofthestructureof

thecultures(insert)

TimeZero2Hours4Hours8Hours24Hours

Figure6:Analysisofthedistributionoffluorescenceintranswellfilterculturesbyfluorescence

spectroscopy.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 3 4 5

Pro

pro

tio

n o

f To

tal Flu

orescen

ce (

%)

Cells / matrix / filter

Insert media

Outer media

Insert starting medium

Outer Medium

Cartilage matrix plug

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 3 4 5

Pro

pro

tio

n o

f To

tal Flu

orescen

ce (

%)

24