Layer-by-layer polysaccharide-coated liposomes for sustained delivery of epidermal growth factor

A three-dimensional layer-by-layer (LbL) structure composed by xanthan and galactomannan biopolymers over dioctadecyldimethylammonium bromide (DODAB) liposome template was proposed and characterized for protein drug delivery.

jo u r n al h om ep a g e :w w w e l s e v i e r c o m / l o c a t e / c a r b p o l

a BioPol, Chemistry Department, Universidade Federal do Paraná, R Coronel F H dos Santos, Curitiba 210–81531-980, PR, Brazil

b CEB, Pharmacy Department, Universidade Federal do Paraná, Av Prof Lothário Meissner, Curitiba 3400–80210-170, PR, Brazil

a r t i c l e i n f o

Article history:

Received 14 September 2015

Received in revised form 31 October 2015

Accepted 7 December 2015

Available online 17 December 2015

Keywords:

DODAB

Xanthan

Galactomannan

EGF

Layer-by-layer

Liposomes

a b s t r a c t

Athree-dimensionallayer-by-layer(LbL)structurecomposedbyxanthanandgalactomannan biopoly-mersoverdioctadecyldimethylammoniumbromide(DODAB) liposometemplate wasproposedand characterizedforproteindrugdelivery.Thepolymersandthesurfactantinteractionweresufficiently strongtocreateaLbLstructureupto8layers,evaluatedusingquartzcrystalmicrobalance(QCM)and zetapotentialanalysis.Thepolymer–liposomebindingenthalpywasdeterminedbyisothermaltitration calorimetry(ITC).Thebilayerofbiopolymer-coatedliposomeswithdiametersof165(±15)nm,measured

bydynamiclightscattering(DLS),and␨-potentialof−4(±13)mV.Thesebilayer-coatednanoparticles increasedupto5timesthesustainedreleaseofepidermalgrowthfactor(EGF)atafirstorderrateof 0.005min−1.Thissystemcouldbeusefulforimprovingthereleaseprofileoflow-stabilitydrugslikeEGF

©2015ElsevierLtd.Allrightsreserved

1 Introduction

Therapiesbasedongrowthfactors(GF)havepromising

poten-tialinbiomedicaltechnology.Incasesofchronicwoundsproactive

treatmentisneededforhealingandGFmightprovidethe

neces-sarystimulitoinducewoundclosure(Behm,Babilas,Landthaler,

&Schreml,2011).Ofthevariousgrowthfactors,epidermalgrowth

factor(EGF)hasbeenfirstandmostsuccessfullyappliedfor

treat-ingwounds.Itisapolypeptidecomposedof53aminoacidsthat

enhanceepidermalandmesenchymalregeneration,cellmotility,

andproliferation(Choietal.,2012)

EGFcouldbeemployedtoacceleratere-epithelialization,

reduc-ingriskofinfectionandshorteninghospitalization.However,due

toshorthalf-life,rapiddilutioninthebody,andthefactthatEGF

receptorsareoverexpressedinmostsquamouscarcinomas,brain

gliomasand breast cancers (Gedda, Olsson, Pontén, &Carlsson,

1996;Chen&Mooney,2003),supplyofexogenousEGFmustbe

appliedina sustainedand localizedfashiontobeeffectiveand

safe.TheencapsulationofEGFinliposomesmightbean

alterna-tivetomaximizetheirstabilityandtoavoidenzymaticdegradation

(De˘gimetal.,2011)

∗ Corresponding author Tel.: +55 4133613260; fax: +55 4133613260.

E-mail address: rilton@ufpr.br (R.A.d Freitas).

Liposomeshavealreadybeenstudiedasdeliverysystemsfor

GF(De˘gimetal.,2011).Nevertheless,liposomeshavethe limita-tionsofspillingtheircontentsovertimeandaggregating(Taylor, Davidson,Bruce,&Weiss,2005).Inordertopreventtheseevents, theliposomescanbecoatedwithpolymers,modulatingthedrug deliverytoachievethedesiredreleasekinetics

Averyefficienttechniquetoformpolymericcoatingson two-dimensionalandthree-dimensionalsystems,suchasliposomes,is Layer-by-layer(LbL),introducedbyDecher(1997).Thistechnique consistsin thealternating depositionsof polycationsand poly-anions,generatingamultilayercoatingsupportedmainly,butnot exclusively,bytheelectrostaticinteractionsorofhydrogenbonds (Wangetal.,1997),covalentbonds(Sunetal.,1998),hydrophobic interactions(Lojou&Bianco,2004)andvanderWaalsforces(Sato

&Sano,2005)

Inordertocoatthedioctadecyldimethylammoniumbromide (DODAB)cationicliposomes,usingtheLbLtechnique,two biopoly-merswerechosen.Xanthan(XAN)ananionicbiopolymerproduced

byXanthomonascampestrisandcomposedofa(1→4)-␤-d-glucan cellulosebackbonesubstitutedwithanacidtrisaccharideinthe sidechain(Jansson,Kennark,&Lindberg,1975),and galactoman-nan(GMC),aneutralbiopolymerfromCeratoniasiliquaseedsthat

iscomposedofa(1→4)-␤-d-mannanbackbonewith(1→6)- ␣-d-galactosesubstitutions(Dea&Morrison,1975).Thesepolymers interactpositivelyandsynergistically,aspreviouslydescribedand http://dx.doi.org/10.1016/j.carbpol.2015.12.014

0144-8617/© 2015 Elsevier Ltd All rights reserved.

scattering(Bresolin,Milas,Rinaudo,&Ganter,1998;Khouryieh,

Herald,Aramouni,&Alavi,2007)

Inthismanuscript,weevaluateanewapproachfor

LbL-three-dimensionalsystemsstructuredwithXANandGMC,forsustained

releaseofEGF.WeemployedDODAB,acationiclipidwitha

quater-naryammoniumsaltasitspolarheadandtwo18-carbonsaturated

chains,toformpositivelysuperficialchargedliposomes

potenti-atingthebiopolymerLbLcoating.Theliposomes,includingthose

withcoatings,werecharacterized,andtheEGFreleaserateswere

determinedinvitro

2 Material and methods

2.1 Polymerpreparation

2.1.1 Polymerpurification

XANgum(Sigma-Aldrich)waspurifiedbydialysisthrougha

cel-lulosemembrane(Sigma-Aldrich),firstagainst0.1molL−1 acetic

acidfor3daystoensurethatallthemoleculeswouldbeinthe

molecularconformation,andthenagainstultrapurewater for2

daystoremovetheaceticacid.GMC,fromlocustbeangumfromC

siliquaseeds(Sigma-Aldrich),waspurifiedbydispersionin

ultra-purewaterat40◦Covernightandcentrifugationat10,000×gfor

20minat40◦Cina4K15C(Sigma,OsterodeamHarz,Germany)

centrifugetoprecipitateinsolubleimpurities.Ethylalcohol(99%)

wasaddedtothesupernatanttoachievea70%alcohol

concentra-tion,andthissuspensionwascentrifugedat10,000×gfor20min

at5◦CtoprecipitatethepurifiedXANandGMC.Thepolymerswere

washedwith99%ethylalcohol,centrifugedasdescribedaboveand

driedat40◦C

2.1.2 Polymercharacterization

Thepolymerswerecharacterizedat0.5mgmL−1byhigh

per-formancesizeexclusionchromatography(HPSEC)with0.1molL−1

NaNO3 and200ppmsodiumazideat0.4mLmin−1asthemobile

phase at 40◦C The system was composed of a UV/vis

detec-tor, refractometer, light scattering detector at 7◦ and 90◦ and

a differential viscometer detector (Viscotek, Westborough, MA,

USA)withan OHpakSB-806MHQcolumn (Shodex, NewYork,

NY,USA)

The persistence length (Lp) was determined as previously

described for other galactomannans by Salvalaggio, de Freitas,

Franquetto,Koop,andSilveira(2015)

Thezetapotentialwasdeterminedforpolymersdispersionsat

0.5mgmL−1 inultrapurewaterusingtheelectrophoretic

mobil-itymeasuredonaZetasizerNano-ZS(Malvern,Westborough,MA,

USA)at25◦Cwith120sofstabilization

TheproteinquantificationofpurifiedGMCwasdeterminedby

theHartreemethod(Hartree,1972)

Infrared spectroscopy (FTIR), using an attenuated total

reflectance(ATR)modewasdeterminedinaVERTEX70(BRUKER)

with4cm−1 of resolution and 4000–600cm−1 (Supplementary

materialS1)

2.2 Liposomepreparation

Liposomeswerepreparedbyamodifiedmethod(Alvesetal.,

2009) DODAB (Sigma-Aldrich, Switzerland) was dispersed in

chloroformat5mmolL−1,thesolventwasremovedbyrotary

evap-orationat40◦C,andthelipidwasresuspendedina5␮gmL−1EGF

(Caregen)solutionat35◦C,similartoAlvesetal.(2009)andDe˘gim

etal.(2011).Thesuspensionwasthensubmittedtosonicationat

25◦Cinpulsemodefor5min

TheEGF-liposomesolutionwasdiluted1:10inultrapurewater

andcentrifugedat10,000×gfor20minat25◦C.Thesupernatant

wasdiscarded,andtheliposomeswereresuspendedin0.5mgmL−1 XANsolution,followedbycentrifugationandwashingwith ultra-purewater.Thesameprocedurewasperformedwitha0.5mgmL−1 GMCsolution

2.3 Liposomecharacterization Thehydrodynamicdiametersofthecoatedliposomeswere ana-lyzedandcomparedwiththoseoftheplainliposomesbydynamic light scattering (DLS) on a NanoDLS (Brookhaven Instruments, Holtsville,NY,USA).Theuncoatedliposomesandpolymer-coated liposomeswerealsocharacterizedbytheir␨-potentialandAFMas describedelsewhere

The zeta potential of the liposomes was determined for

5mmolL−1dispersionofDODABinultrapurewaterusingthe elec-trophoreticmobilitymeasuredonaZetasizerNano-ZS(Malvern, Westborough,MA,USA)at25◦Cwith120sofstabilization 2.4 PolymerandDODAB/DODABvesicleinteraction

2.4.1 Isothermaltitrationcalorimetry(ITC) ExperimentswereperformedinaVP-ITC(Microcal, Westbor-ough,MA,USA)calorimeterwithanormalcell(1.464mL)at25◦C TheDODABvesicledispersionwasinjectedintoultrapurewateror intoapolymersolutionat0.5mgmL−1.Eachtitrationconsistedofa preliminary2␮Linjectionfollowedby29subsequent10␮L injec-tionswith600sintervalsbetweeneachinjection.Thesyringetip actedasablade-typestirrertoensurepropermixingat300rpm DatawerecollectedandprocessedwithOrigin7.0software (Origin-Lab,Northampton,MA,USA)

2.4.2 Quartzcrystalmicrobalance(QCM) AnalyseswereperformedintriplicateinaSRSQCM200usingthe flowcellmode.TheQCM(Gold/Cr5MHz,SRS,Sunnyvale,CA,USA) crystalswerecleanedbyimmersionin1:3(v/v)H2O2:H2SO4 for

5minandfollowedbyrinsingwithultrapurewater.Tomimicthe liposomecoatingprocess,thegoldsurfacewasmodifiedwith hex-anethiol(Sigma-Aldrich)toformahydrophobicsurfaceandthen coatedwithDODABbyimmersionina5mmolL−1chloroform solu-tionsimilartoMorita,Nukui,andKuboi(2006).Onemilliliterof eachpolymersolution(0.5mgmL−1)wasalternatelyinjectedat 0.1mLmin−1withasyringepump(KD100,KDScientific,Holliston,

MA,USA),and1mLofultrapurewaterwasinjectedbetweeneach solution

2.4.3 Atomicforcemicroscope(AFM) ImagesofeachlayerontheQCMcrystalwereobtainedona PicoPlusMolecularImagingmicroscope(Agilent,SantaClara,CA, USA)intheintermittentcontactmodeinairat25◦Cwithsilicon cantilevers,anoscillatingamplitudeof50to100nmanda reso-nancefrequencycloseto300kHz.Thedynamictappingmodewas usedwithanoxide-sharpenedmicro-fabricatedsilicon␮-Masch cantileverwitha4.7Nm−1nominalspringconstantandtip cur-vatureradiusoflessthan10nm.Theimageprocessingandroot meansquareroughness(rms)determinationwereperformedwith Gwyddionsoftware(CzechMetrologyInstitute,Brno-sever,Czech Republic)

2.4.4 Contactangle Theanglesof thepolymersubstratesweredetermined with OCA15+(DataPhysics,Filderstadt,Germany)deviceequippedwith SCA20softwarebythesessiledropmethodat25◦Cwiththe deliv-eryof10␮LultrapurewaterdropsontotheQCMcrystal-coated surface

2.5 EGF-liposomereleasekinetics

EGFreleaseprofilesweredeterminedbyHPLCquantification

usingaProminence(Shimadzu,Columbia,MD,USA)systemwitha

SymmetryC18(4.6×250mm,5␮mparticles)column.Apreviously

describedmethod(Yang,Huang,Wu,&Tsai,2005)wasadaptedto

use40%isocraticgradeacetonitrile(Fluka)in0.1%TFAat40◦Cwith

1mLmin−1flowrateand210nmUVdetection.TheresultingEGF

retentiontimewas4.8min

Avolumeof10mLofPlainEGF-liposomes,XAN-coated

EGF-liposomesorGMC+XAN-coatedliposomes,allintriplicates,were

placedinadialysisbag(12,000gmol−1cutoff)andimmersedin

90mL of 0.1molL phosphate buffersaline (PBS)at pH 5.5 and

35◦Ctomimicdermaldeliveryconditions(Wagner,Kostka,Lehr,&

Schaefer,2003)withmagneticstirringforupto48h.Onemilliliter

aliquotswerecollected,ateachtimepoint,fromthemediumand

thatvolumewasreplacedwithfreshPBS

3 Results and discussion

3.1 Polymers

Allthepurifiedpolymersexhibitedaunimodaldistributionas

measuredbyHPSEC(datanotshown).Afterpurification,theXAN

andGMCpresentedthemolarmass(Mw),intrinsicviscosityand

radiusofgyration(Rg)asseeninTable1.Thezetapotentials(

␨-potential)(Table1)showedalargedifferencebetweenDODABand

XAN,suggestingthatthesepolymersmayinteractwitheachother

electrostatically.ThenegativechargeontheneutralGMCpolymer

couldbecausedbyattachedproteins,determinedas4%(m/m),that

remainedevenafterpolymerprecipitationwithethanol

3.2 Liposomes-LbLbiopolymercoating

Thesizeoftheliposomesincreased(Fig.1a)from62(±9)nmto

132(±22)nmwhencoatedwithXANandto165(±15)nmwhen

coatedwiththeXANandtheGMClayers.Thesizeoftheliposomes,

after48hwas79nm,140and195nm,respectivelyforplain,XAN

andXAN-GMClayers,suggestingansmallincreaseinthesize

dur-ingstorageoftheliposomes.Althoughtherewasgreateradsorption

fortheGMCcoating,theXANlayerrepresentedalargerincrease

inthediameteroftheliposomes.Thisincreasewasmostlikely

becauseofitslargerpersistencelength(Lp)of125nm,similarto

thefoundbyRinaudo(2001),thanofGMC(9nm)andtheCoulomb

repulsionbetweennegativelychargedXAN,resultinginporeson

thesurface,whichwereoccupiedbyGMC.Theplainandcoated

liposomescanbeseeninAFM(Fig.2)

ItisclearthatXANleftporesonthesurface,becausethefirst

layeronlydecreasedthe␨-potentialoftheliposomesto

approx-imately+35mV(Fig.1b).However,thefirstGMClayershielded

allthepositivechargefromDODABanddecreasedthe␨-potential

of theliposomes toapproximately −4mV, closetothe own

␨-potentialofthepolymer.BecauseGMCfilledtheporesleftbyXAN,

Table 1

Physicochemical characteristics of the polymers and the surfactant: molar mass

(M w ), radius of gyration (R g ) and zeta potential (␨-potential).

Molecules M w (g mol −1 ) * R g (nm) * ␨-potential (mV) **

XAN 1.03 × 10 6 69 −54.1 (±7.7)

GMC 0.26 × 10 6 52 −5.4 (±6.2)

DODAB 630 – + 66.5 (±9.4)

* 0.1 mol L -1 NaNO 3 and 200 ppm sodium azide as the mobile phase at 0.4 mL min −1

at 40 ◦ C through a Shodex OHpak SB-806 HQ column The zeta potential was

deter-mined using the electrophoretic mobility in ultrapure water at 25◦C.

theeffectofitsadsorptiononthestandarddeviationoftheQCM crystalwaslarger(differentamountsofGMCwererequired) 3.3 Physical–chemicaldescriptionofliposomes-LbLbiopolymer coating

QCMwasusedtoverifythattherewereinteractionsbetweenall layers,enablingtheformationandmaintenanceoftheLbL struc-ture.Becausetheliposomeswerethetemplateforthebiopolymers coating,theQCMcrystalshouldbecoatedwithDODABbefore poly-merdeposition.ToassuretheproperDODABcoating,thecrystal was first coated withhexanethiol Thus, thethiol group inter-acted strongly with gold,and itsalkyl chain pointed upwards, allowinghydrophobicinteractionswiththealkyltailsofDODAB Therefore, the cationic amino head onthesurface was able to interact with anionic XAN molecules, the first polymeric layer injected

ThecrystaltopographywasanalyzedbyAFM(Fig.3 andthe surfacecontactanglewasmeasuredwithultrapurewaterdrops after thefirst layer of XANand the first layerof GMC, ontop

ofXAN.QCMshowedtheadsorptionofbothpolymers (Supple-mentarymaterialS2),withgreateradsorptionforGMC.Thegrater irregularityoftheXANcoating,asevidencedbyAFM,canexplain thisfinding.ItappearsthattheLpofXANmoleculesleavespores thatarefilledbyGMC,amuch moreflexiblemolecule.Because GMC has a slightly negative charge, it can also interact with DODAB

Thecontactangleconfirmedthatallthelayerswereproperly fixed.Hexanethiolformedthemostapolar surface,and DODAB themostpolar,consistentwithitsgreater␨-potential.Thecontact angleoftheXANlayerwasslightlygreaterthanthatofDODAB, indi-catingalesspolarsurface.TheGMClayer,aneutralpolysaccharide, raisedthesurfacecontactangle

AstheLbLprocesscontinued,theadsorbedmassproportional

to−F,wasincreased(Fig.1c).Itisunderstandablethatthe elec-trostaticinteractionis greaterthanotherinteractions.Thus, the DODABchargewasresponsibleforthelargeinitialadsorption.As shieldingdecreasedtheeffectivepositivecharge,fewerpolymers wereadsorbed.Althoughtheadsorbedmassdecreased,itwas suf-ficienttochangethe␨-potentialofthenanoparticlesateachlayer

WeconfirmedthattheLbLprocesswasefficientforupto4tested layersofeachpolymer(Fig.1b)

Isothermaltitrationcalorimetry(ITC)wasusedtodeterminethe bindingenthalpybetweenthecoatingpolymers,XANandGMC, andtheDODABvesicles.ITCanalysisresultsinabindingisotherm (Fig.4)thatprovidespreciseanalyticalinformation, suchasthe numberoffreeandboundvesiclesatdifferentstagesofthebinding process.Itisalsopossibletodeterminetheaveragestoichiometry

ofsupramolecularsurfactant-polymerclusters

Fig.4showstherawsignalcurve(top)andtheintegralofthe areaateachinjection(bottom).Theenthalpymeasuredinthecell

isthesumofseveraldifferentenergies,suchastheenergyofthe dilutioneffectandtheenergyofthepolymersbindingontothe surfactantvesicles.Theenergyofthepolymersbindingtothe vesi-clesismoresignificantthanthesumoftheotherenergies,which representthesmallenergychangesthatoccurafterallpolymer moleculesareconsumed

Theenthalpycurvesofbothpolymersexhibitonlyone coopera-tiveendothermiceventateachinjection,attributedtothebinding

ofthepolymerstotheDODABvesicles.TheHcoat(Hattributed

tothecoatingofDODABvesiclesbypolymermolecules)was calcu-latedasthedifferencebetweentheinitialHandtheaverageH plateaureached

Fig.4ashowsthateachGMCmoleculeinteractedwith approx-imately25DODABmoleculesinthevesicle,themoleculesinside thevesicleswerenotconsidered(neithertheflip-flopeffect),with

Fig 1. (a) Gaussian distribution of nanoparticles showing the increase in hydrodynamic diameter (Dh) caused by the polymer coating, determined by DLS at 90 ◦ (b) Zeta potential determined by electrophoretic mobility at 25◦C for LbL-coated liposomes after each layer Bars denote the standard deviation (c) Frequency shift tendency determination (n = 3) by QCM for LbL with polymers injected at 0.1 mL min−1 The crystal was washed with ultrapure water between polymer injections.

abindingenthalpyof17.6kJmol−1ofDODAB.At∼50␮molL−1of

DODABallGMCmoleculeswereconsumedandthereisasurplus

offreeDODABvesicles

In the ITC plot of the DODAB vesicles titration in the XAN

solution(Fig.4b),aplateauisobservedbetweensurfactant

con-centrations of 600 and 900␮molL−1 At these concentrations,

all XAN molecules were consumed; each XAN molecule coats

∼1500DODABmoleculesinthevesiclewitha coatingenthalpy

of26.4kJmol−1 ofDODAB.Thesmallremainingenthalpieswith furtherliposomesadditiontendtozeroandmightbeattributedto associationsinotherlargercolloidalsystems

TobettercomprehendthepolymercoatingsoftheDODAB lipo-somes,thenumberofsurfactant (Ntot) moleculesthatformedeach liposomewascalculatedbythefollowingequation:

Fig 2.AFM topography 4 × 4 ␮m images of the LbL process on a QCM crystal (a) Gold surface; (b) Hexanethiol; (c) DODAB; (d) XAN; (e) GMC.

Fig 3.AFM topography image of (a) Plain liposomes; (b) XAN-coated liposomes; (c) GMC + XAN-coated liposomes.

Ntot=



4

d/22

+4

d/2

−h2 a



(1)

wheredistheliposomesdiameter,62nmdeterminedbyDLS,h

isthebilayerthickness,4nm(Correia,Petri,&Carmona-Ribeiro,

2004)andaisthesurfactantheadgrouparea.Itwasfoundthat

approximately31,000DODABmolecules associatetoformeach

colloidalstructure.Therefore,approximately1250GMCmolecules

coatedeachliposomewhile,approximately,only20moleculesof

XAN

Possible explanations for this great difference include the

electrostaticrepulsionbetweenadsorbedXANmoleculesonthe

liposomesurface,hinderingtheadsorptionofnewlyarrivedXAN

molecules.TheXANmoleculesusedinthisresearcharelargerthan

theGMCmolecules,theyhave4timesthemolarmass.Also,the

longerLpofXANmoleculesplayaroleinthelackoforganization

oftheadsorbedmolecules,whiletheGMCmoleculescanadjust

betteronthesurfaceandeveninteractwitheachother

The largeenthalpy observed in our study suggeststhat the chargedsitesofXANmoleculesarestronglyattractedtotheDODAB liposomes Thelargerheatexchange ofDODAB-XAN, compared withthatofDODAB-GMC,islikelyduetotheion-exchange inter-actionsbetweenDODA+andthenegativesitesonXAN

3.4 Releasekinetics Afterconfirmingtheformationandstructureoftheliposomes coatedwithbiopolymersandthephysical-chemicalparametersof thecoating,EGFwasencapsulatedinthesystem.Theentrapment efficiency was72±3%.The efficiencywasdetermined byHPLC quantificationthroughthedirectmethodoflysingliposomeswith 3%TritonX-100,describedbyLiu,Yang,Liu,andJiang(2008)and

anindirectmethodthatmeasuredtheEGFinthesupernatantof centrifugedliposomes

ThekineticsdataweretreatedwiththeBerensandHopfenberg (1978)model(Eq.(2)),suitableforspheres,whichshowed confi-denceintervalsabove99%forthe3systems:

Fig 4.Thermogram (top) and binding isotherm (bottom) The latter resulted from the integration of the ITC peaks in the former, which were at 25 ◦ C (a) DODAB liposomes titrated in a solution of GMC at 0.5 mg mL -1 (b) DODAB liposomes titrated in a solution of XAN at 0.5 mg mL -1

Mt

M∞ =1−F

2

n=3

n2



exp

−42n2Dt

d2 −Rexp (−kx)

(2)

whereDisthediffusioncoefficient,kisthefirst-orderrelaxation

constant,FandRarethefractionsofsorptioncontributedby

Fick-iandiffusionandchainrelaxation,respectively,disthediameterof

thenanoparticleandtistime

Eachlayerofcoatingcontributedtothereleasekinetics,asseen

inFig.5.Theplainliposomespresentedaconstantdrugdelivery

rate(k)of0.025min−1,andtheXANlayerreducedthedeliveryrate

to0.016min−1,1.6timeslonger.Fortheliposomescoatedwithone

layerofbothpolymers,thedeliverykdecreasedto0.005min−1,5

timessmallerthantherateoftheplainliposomes

Non-linearleastsquarefittingroutinewasusedtofittherelease

ofEGFfromthenanoparticlesintotheabovemodel.Thismodel

describesthereleasebehaviorintermsofFickianandnon-Fickian

contributions.AsthecoatingtookplacetheRparameterbecame

dominate When the last term of the equation is switched to



−X/

,itprovidesthecharacteristicrelaxationtime()

Thecoatingthicknesses(L)ofthelayersweredeterminedas

thedifference between thenanoparticles hydrodynamic radius

(Fig.1a).The

Mt/M∞

wasplottedagainstthesquarerootoft/L2

incm2toprovidetheEGFdiffusibility(D),theangularcoefficient,in

allnanoparticlesaccordingtoVogt,Soles,Lee,Lin,andWu(2004)

TheandtheDvalueswereplottedtogetheragainstthecoating

layersandcanbeseeninSupplementarymaterial(S3)

0.0 0.2 0.4 0.6 0.8 1.0

Time (min)

Fig 5. The tendency profile in vitro of EGF from the nanoparticles in PBS at pH 5.5 and 35 ◦ C by dialysis • Plain liposomes, 䊏 XAN coated liposomes and  XAN + GMC coated liposomes Bars denote the standard deviation (n = 3).

Theplainliposomesrelaxationtime(t)wasfoundtobe40min, theXANlayerincreasedto60minandtheXANandGMCcoatings increasedto195min.ItcanbenoticedthattheXANlayer con-tributedtoa50%increaseinthenanoparticlessustainedrelease, whiletheGMCovertheXANlayerincreaseditinalmost400%.One additionallayerofeachpolysaccharideXAN-GMCreduced com-pletelythedrugdeliverytozeroduring480minofevaluation

thecoatinglayersbecamemoreimportantforthereleaseofEGF

Thisway,asDdecreased,increasedexponentially

4 Conclusion

Thenatural interactionbetweenXANand GMCwas

demon-stratedtobesufficientlystrongtoprovideLbLstructureforupto8

biopolymerlayers.TheassociationoftheanionicXANandthe

neu-tralGMCforliposomescoatingprovidedasynergisticeffectthat

builtacohesivenanoparticlecapableofincreasing5timesthe

sus-taineddeliveryofEGFwithonlyonelayerofeachpolymer.TheEGF

releasefromthenanoparticleswasattributedtothepolymerchains

relaxationbytheuseofthediffusionandrelaxationmodel.The

useofthesenaturalpolysaccharidesinthree-dimensionaldelivery

systemsprovidesbiocompatibility,easeandabundanceatlowcost

Acknowledgements

We acknowledgetheBrazilian fundingagencies CNPq

(Con-selho Nacional de Pesquisa, process no 477275/2012-5 and

300343/2010-8, Fundac¸ão Araucária, project 23643, convênio

447/2012,andRedeNanobiotec/Capes-Brazil,project34,for

finan-cialsupport.WearegratefultoDra.LeilaBeltramini(Institutode

FísicadeSãoCarlos) forITC analysis,Dr.WatsonLoh(Instituto

deQuímicaUNICAMP)forthermodynamicdiscussionaidandDr

LionelGamarra(InstitutoCérebro,HospitalAlbertEinstein)for

␨-potentialanalysis

Appendix A Supplementary data

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,atdoi:10.1016/j.carbpol.2015.12.014

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