Hybrid nanosystems based on natural polymers as protein carriers for respiratory delivery: stability and toxicological evaluation

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ContentslistsavailableatScienceDirect

Carbohydrate

Polymers

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

Hybrid

nanosystems

based

on

natural

polymers

as

protein

carriers

for

respiratory

delivery:

Stability

and

toxicological

evaluation

Susana

Rodrigues

a

_,

_Clara

_Cordeiro

b,c,d

_,

_{Bego ˜}

_na

_Seijo

e

_,

_Carmen

_{Remu ˜}

_nán-López

e

_,

Ana

Grenha

a,∗

a_CBME_–_Centre_for_Molecular_and_Structural_{Biomedicine/IBB}_–_Institute_for_{Biotechnology}_and_{Bioengineering,}_University_of_Algarve,

FacultyofSciencesandTechnology,CampusdeGambelas,8005-139Faro,Portugal

b_Faculty_of_Sciences_and_Technology,_University_of_Algarve,_Campus_de_Gambelas,_8005-139_Faro,_Portugal

c_CEAUL_–_Center_of_Statistics_and_{Applications,}_Faculty_of_Sciences,_University_of_Lisbon,_Campo_Grande,_1749-016_Lisboa,_Portugal d_CESUAlg_–_Centre_for_Research_and_Development_in_Health,_University_of_Algarve,_Portugal

e_NanoBioFar_Group,_Department_of_Pharmacy_and_{Pharmaceutical}_Technology,_Faculty_of_Pharmacy,_University_of_Santiago_de_Compostela,

CampusVida,15782SantiagodeCompostela,Spain

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received18August2014 Receivedinrevisedform 27November2014 Accepted21January2015 Availableonline3February2015 Keywords: Biocompatibility Microencapsulation Nanoparticles Nasaldelivery Proteindelivery Pulmonarydelivery

a

b

s

t

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Chitosan/carrageenan/tripolyphosphatenanoparticleswerepreviouslypresentedasholdingpotential foranapplicationintransmucosaldeliveryofmacromolecules,withtripolyphosphatedemonstratingto contributeforbothsizereductionandstabilisationofthenanoparticles.Thisworkwasaimedat evaluat-ingthecapacityofthenanoparticlesasproteincarriersforpulmonaryandnasaltransmucosaldelivery, furtherassessingtheirbiocompatibilitypatternregardingthatapplication.Nanoparticlesdemonstrated stabilityinpresenceoflysozyme,whilefreeze-dryingwasshowntopreservetheircharacteristicswhen glucoseorsucrosewereusedascryoprotectants.Bovineserumalbuminwasassociatedtothe nanoparti-cles,whichweresuccessfullymicroencapsulatedbyspray-dryingtomeettheaerodynamicrequirements inherenttopulmonarydelivery.Finally,asatisfactorybiocompatibilityproﬁlewasdemonstratedupon exposureoftworespiratorycelllines(Calu-3andA549cells)tothecarriers.Anegligibleeffectoncell via-bilityalongwithnoalterationsontransepithelialelectricalresistanceandnoinductionofinﬂammatory responsewereobserved.

1. Introduction

Severalphysicochemical and biopharmaceutical issues make

systemicadministrationofbiomoleculesachallengingtask(Gupta

&Sharma,2009),compellingtheneedtodevelopadequatedrug

deliverysystemsthatovercomeinherentlimitations.Thesecarriers

aremainlydeveloped toavoid theneed ofparenteral

adminis-tration, instead permittingeffective transmucosal delivery. The

pulmonary and nasal routes are increasingly addressed in this

context, because of the large absorptive surface and the easy

access, respectively, among other characteristics. Carriers are

∗ Correspondingauthorat:CentreforMolecularandStructuralBiomedicine (CBME),FacultyofSciencesandTechnology,Building8,Room3.1,Campusde Gam-belas,8005-139Faro,Portugal.Tel.:+351289800100x7441;fax:+351289818419.

E-mailaddresses:susananasus@gmail.com(S.Rodrigues),ccordei@ualg.pt

(C.Cordeiro),mbegona.seijo@usc.es(B.Seijo),mdelcarmen.remunan@usc.es

(C.Remu ˜nán-López),amgrenha@ualg.pt(A.Grenha).

thusdesignedtoexhibitcharacteristicsthatimprovethe

epithe-lialcontact, while protecting sensitivebioactivematerials from

invivodegradation.Nanoparticlesareﬁrst-linecandidatesforthis

end, successfully associating therapeutic macromolecules (Reis,

Neufeld,Ribeiro,&Veiga,2006).Apreviousstudydemonstratedthe

abilityoftwonaturalpolymers,chitosan(CS)and␬-carrageenan

(CRG),toassembleintonanoparticlesof400–600nmbysimple

polyelectrolytecomplexation(Grenhaetal.,2010).WhileCSisa

verywell-knownmaterial,theabilitiesofCRGarenotsufﬁciently

explored.Apart from theadvantage of using natural materials,

whichfacilitatesbiocompatibility(Malafaya,Silva,&Reis,2007),

thepolyelectrolytecomplexationbetweenCSandCRGusesvery

mildconditions,occurringinhydrophilicmedium(Grenha,2012).

Nevertheless, theintimatecontact betweenthecarrier and the

epithelialsurface that highlyfavours mucosaldeliveryofdrugs

is reported to be maximal for particles of 50–500nm (Desai,

Labhasetwar,Amidon, &Levy, 1996; Jani, Halbert,Langridge, & Florence,1990)andstrongpositivesurfacecharge(Bogatajetal., 2003;Carvalho,Bruschi,Evangelista,&Gremião,2010).Therefore,

http://dx.doi.org/10.1016/j.carbpol.2015.01.048

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morerecentlyweassociatedtripolyphosphate(TPP)totheCS/CRG

nanoparticleformulationasacross-linkingagent,providingasize

decreaseandobtainingmorestablenanoparticles(Rodrigues,Rosa

daCosta,&Grenha,2012).

Notwithstandingtherelevancethatnanoparticleshavegained

inthelastdecade,thefactisthatseverallimitationsarestillto

overcomeregardingtheirapplication.Theinherenttendencyfor

particleaggregation,chemical instabilityof polymersand early

releaseofactivesubstancesaremajorproblemstobeaddressed.

In addition,as nanoparticlesare usually formulatedas

suspen-sions,theyarepronetomicrobialgrowth(Wu,Zhang,&Watanabe,

2011).Therefore,underallaspects,thephysicalandchemical

sta-bilityofthesystemsisexpectedtobeimprovedifwaterisremoved

(Abdelwahed,Degobert,Stainmesse,&Fessi,2006).Theshelf-life

of nanoparticlesis a relevant aspect of theirpotential

applica-tionasdrugdeliverycarriers, especiallyconcerningtheneedto

maintainunaltered physicochemical characteristics along time.

Freeze-dryingappearsinthiscontextasaveryadequatechoice,

ensuringasoftdryingprocedure.Themoreaggressiveconditions

associatedtofreeze-dryingcanbesoftenedbytheuseofspeciﬁc

protectants. These will provide the formation of a solid

amor-phousglassﬁlmaroundthenanoparticlesandtheimmobilisation

ofthecarriers,preventingaggregationandpromotingstabilisation

(Beirowski,Inghelbrecht,Arien,&Gieseler,2011,2012).

Safety issues are another permanent subject adjacent to

nanoparticle applications. In fact, regarding the inhalation of

nanoparticles,theirfateafteradministrationremainsuncertainand

thereis thepossibilityofaccumulation inthelung tissueupon

repeatedadministrations(Rogueda&Traini,2007).Withregardto

theevaluationofmaterialssafety,thereisageneral

recommenda-tionconsideringthataninitialinvitroevaluationshouldcomprise

differentcelllinesandtimesofexposure,whilenotonlycell

via-bilitybutalsotheinﬂammatoryresponseshouldbedetermined

(ISO,2009;Rodrigues,Dionísio,Remu ˜nán-López,&Grenha,2012).

TheCalu-3 andA549 respiratoryepithelial celllines havebeen

frequentlyusedinthiscontexttoevaluatethebehaviourof

sys-temsaimedatrespiratorydrugdelivery,eithernasalorpulmonary

(Foster,Avery,Yazdanian,&Audus,2000;Grenhaetal.,2007;Nahar etal.,2013).

Inthiswork,weaimedatexploringthepotentialofCS/CRG/TPP

nanoparticlesforanapplicationinnasalandpulmonary

transmu-cosaldeliveryofproteins,placingaparticularemphasisontheir

biocompatibilitybehaviour.Additionally,thestabilityof

nanopar-ticlesinpresenceoflysozyme(anenzymepresentinthenasaland

lungmucosa),aswellastheabilityoffreeze-dryingtoprovideadry

formofnanoparticlesuspensions,werealsoassessed.Regarding

pulmonaryadministration,asnanoparticlesareknowntolackthe

adequateaerodynamicdiametertopermitdeeplungdeliverywith

success, which should vary between 1 and 5␮m, thestrategy

ofmicroencapsulatingthenanocarrierswasapplied,following a

previouslydescribedapproach(Grenha,Seijo,&Remu ˜nán-López,

2005).

2. Materialsandmethods

2.1. Materials

Chitosan (low molecular weight, deacetylation degree=75–

85%), pentasodium tripolyphosphate, bovine serum albumin

(BSA), phosphate buffer saline (PBS) tablets pH 7.4,

Dul-becco’smodiﬁedEagle’smedium(DMEM),penicillin/streptomycin

(10,000units/mL, 10,000␮g/mL), non-essential amino acids,

l-glutamine 200mM, trypsin–EDTA solution (2.5g/L trypsin,

0.5g/L EDTA), trypan blue solution (0.4%), thiazolyl blue

tetra-zolium bromide (MTT), sodium dodecyl sulphate (SDS), N,

N-dimethylformamyde (DMF), glycerol, lipopolysacharide(LPS),

dimethyl sulfoxide (DMSO), lysozyme and glacial acetic acid

weresuppliedbySigmaChemicals(Germany).k-Carrageenanwas

obtainedfromFMCBiopolymer(Norway)andfoetalbovineserum

(FBS)wasfromGibco(USA).Ultrapurewater(Milli-QPlus,

Milli-poreIberica,Spain)wasusedthroughout.

2.2. Celllines

TheCalu-3andA549celllineswereobtainedfromtheAmerican

TypeCultureCollection(Rockville,USA)andusedbetween

pas-sages35–45and20–30,respectively.Cellculturesweregrownin

75cm2_ﬂasks_in_a_humidiﬁed_5%_CO

2/95%atmosphericairincubator

at37◦C.Forbothcelllines,cellculturemedium(CCM)wasDMEM

supplementedwith10%(v/v)FBS,1%(v/v)non-essential amino

acidssolution,1%(v/v)l-glutaminesolution(200mM)and1%(v/v)

penicillin/streptomycin.Mediumwasexchangedevery2–3days

andcellsweresubculturedweeklyinthecaseofA549cellsand

every10–15daysforCalu-3cellline.

2.3. PreparationofCS/CRG/TPPnanoparticles

CS/CRG/TPP nanoparticles were prepared as described

pre-viously (Rodrigues, Rosa da Costa, et al., 2012) based on the

polyelectrolytecomplexationofCSwithCRGandadditionalionic

gelationofchitosanwithTPPanions.Afteraninitialoptimisation,

aﬁnaltheoreticalCS/CRG/TPPratioof4/1/1(w/w)wasselectedto

performthestudies(4/1/0forcontrolnanoparticles).Brieﬂy,CSwas

dissolvedin1%(w/w)aceticacidtoobtainasolutionof1mg/mL.

CRGandTPPweredissolvedinpuriﬁedwaterandmixedtoobtain

asolutionwheretheconcentrationofeachpolymeris0.63mg/mL.

Thespontaneousformationofnanoparticlesoccursupon

incorpo-rationof0.8mLofasolutionofCRG/TPPorCRGinto2mLoftheCS

solution,undergentlemagneticstirringatroomtemperature.

TheassociationofBSAtothenanoparticleswasperformedby

dissolvingtheproteininwaterandmixingwithapreviously

pre-paredmixture of CRG/TPP solution,prior totheaddition toCS

solution,as described above.The amount of BSA associated to

eachbatchwascalculatedtorepresentatheoreticalcontentof30%

(w/w)oftheamountofCS.

Nanoparticleswereconcentratedbycentrifugationat16,000×g

ona10␮Lglycerollayerfor30minat15◦C(HeraeusFresco17

cen-trifuge,Thermoscientiﬁc,USA).Afterdiscardingthesupernatants,

thenanoparticleswereresuspendedin200_␮lofpuriﬁedwater.

Whennofurtherindicationisprovided,‘CS/CRG/TPP

nanopar-ticles’refertomassratios4/1/1.

2.4. Characterisationofnanoparticles

2.4.1. Determinationofproductionyield

Thenanoparticleproductionyieldwascalculatedby

gravime-try.Fixedvolumesofnanoparticlesuspensionswerecentrifuged

(16,000×g,30min,15◦C),andsedimentswerefreeze-driedover

24hat−34◦_C,_followed_by_a_gradual_increase_in_temperature_until

20◦C,usingaLabconcofreezedryer(Labconco,USA)(n=3).The

yieldofnanoparticleproduction(PY)wascalculatedasfollows:

PY=Nanoparticle_Total_solidssediment_weightweight×100 (1)

wherenanoparticle sedimentweightistheweightafter

freeze-dryingandtotalsolidsweightisthetotalamountofsolidsadded

fornanoparticleformation.

2.4.2. Morphologicalobservation

The morphological examination of CS/CRG/TPP

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(JEM-1011,JEOL,Japan).Thesampleswerestainedwith2%(w/v)

phosphotungsticacid(Carvalho,Grenha,Remunan-Lopez,Alonso,

&Seijo,2009)andplacedoncoppergridswithcarbonﬁlms(Ted

Pella,USA)forTEMobservation.

2.4.3. Physicochemicalproperties

Measurements of nanoparticle size and zeta potential were

performed on freshly prepared samples by photon correlation

spectroscopyandlaserDoppleranemometry,respectively,using

aZetasizerNanoZS(MalvernInstruments,Malvern,UK).Forthe

analysisofparticlesizeanddeterminationoftheelectrophoretic

mobility,eachsamplewasdilutedtotheappropriateconcentration

withultrapurewaterandplacedintheelectrophoreticcell.Each

analysiswasperformedat25◦C.Threebatchesofeachformulation

wereanalysed(n=3).

2.4.4. DeterminationofBSAassociationcapacity

The amount of non-encapsulated BSA was determined in

thesupernatantbyreversephase-highperformanceliquid

chro-matography (RP-HPLC; Agilent 1100 series, Germany). The

chromatographicconditions wereadapted fromUmrethia,Kett,

Andrews,Malcolm,andWoolfson(2010)asfollows:Aeris

wide-pore3.6uXB-C18300Acolumn4.6mmi.d.×250mmlengthwith

securityguardcartridge(Phenomenex,UK);runtemperature25◦C;

gradientﬂow(0.1%TFAinwater(A),0.1%TFAinacetonitrile(B);

A/Bfrom95:5to35:65in 10min)mobilephase,total runtime

15min,UVdetectionat280nm;ﬂow rate1.0mL/min;injection

volume20␮L;BSAretentiontime8.8min.Alinearcalibrationplot

forBSAinthedifferentsolvents(aceticacidsolution,PBSpH7.4and

bufferpH6.8)wasobtainedovertherange10–120␮g/mL(n=3).

BSAassociationefﬁciency(AE)andnanoparticleloadingcapacity

(LC)werecalculatedasfollows:

AE= (TotalamountofBSA−freeamountofBSA)

TotalamountofBSA ×100 (2)

LC=(TotalamountofBSA−freeamountofBSA)

Nanoparticleweight ×100 (3)

2.5. Evaluationofnanoparticlestabilityinthepresenceof

lysozyme

ThestabilityofCS/CRG/TPPnanoparticles(4/1/0and4/1/1)was

analysedinpresenceoflysozyme.Todoso,nanoparticle

suspen-sionsataconcentrationof1mg/mLwereincubatedwithlysozyme

(0.2and0.8mg/mL)(Grenhaetal.,2005)at37◦Cundermild

hor-izontalshaking,for120min.Atappropriatetimeintervals(30,60,

90and120min),thesizeandzetapotentialofnanoparticleswere

measured,asdescribedabove(n=3).

2.6. Nanoparticlestabilityuponfreeze-drying

Withtheaimofdevelopingapharmaceuticallyacceptabledry

formofthenanoparticle suspensions,CS/CRG/TPPnanoparticles

weresubmittedtoafreeze-dryingstudy.Differentconcentrations

ofnanoparticles(1,2and4mg/mL)anddifferentcryoprotectants

(trehalose,lactose,sucroseandglucose)atvariedconcentrations

weretestedasvariablesintheoptimisationofnanoparticle

stabi-lisationbyfreeze-drying.Toperformthestudy,1mLofunloaded

nanoparticleswasfreeze-driedinthepresenceof5or10%(w/v)of

cryoprotectant.Thefreeze-dryingprocessoccurredatapressure

of50–100Torrand consistedof 24hof primarydrying,starting

at−35◦_C _and_gradually_increasing_to₋₁₀◦_C,₂₄_h_at₀◦_C_and _a

ﬁnalstepofsecondarydrying(12–24h)at15◦C(LabconcoCorp.,

USA).Afterfreeze-drying,nanoparticlesuspensionswere

reconsti-tutedbyaddingthevolumeofwatercorrespondingtotheinitial

volume(1mL),andtheirsizeandzetapotentialweredetermined

asindicatedabove(n=3).

Theconditionsfoundtobettermaintaintheoriginalproperties

ofunloadednanoparticleswereappliedonBSA-loaded

nanopar-ticles.Inthiscase,nanoparticleconcentrationsof1and2mg/mL

werefreeze-driedwithbothglucoseandsucroseataconcentration

of10%(w/w).

2.7. Preparationofmicroencapsulatednanoparticles

Mannitolwasselectedasmatrixmaterialfortheproductionof

microparticles,aswaspreviouslydescribed(Grenhaetal.,2005).

Anaqueoussolutionoftheexcipientwaspreparedata

concentra-tionof1.6%(w/v).Uponcentrifugation,CS/CRG/TPPnanoparticles

wereresuspendedwiththemannitolsolutioninsteadofwater,in

ordertoobtainamannitol/nanoparticleratioof80/20(w/w).The

ﬁnalsolidconcentrationofthesuspensionswas2%(w/v).

Dry powders were obtainedby spray-dryingthe previously

preparedsuspensionusingalaboratory-scalespray-dryer(Buchi®

MiniSprayDryer,B-290,Buchi,Switzerland).

Thespray-dryingconditionswere:feedrateof2.5mL/min,two

ﬂuidsexternalnozzle0.7mm,inlettemperatureof160±2◦C,

out-lettemperatureof95±3◦C.Theairﬂowrateandtheaspiratorwere

keptconstantat400Nl/hand80%,respectively.Thespray-dried

powderswerecollectedandstoredinadesiccatoratroom

temper-atureuntiluse.

2.8. Characterisationofmicroencapsulatednanoparticles

2.8.1. Determinationofspray-dryingyield

Thespray-dryingyieldwascalculatedbygravimetry,

compar-ingthetotalamountofsolidsinitiallyaddedforthepreparationof

microencapsulatednanoparticles(CS+CRG+TPP+BSA+mannitol)

withtheamountofresultingmicrospheres(n=3).Thefollowing

equationwasusedforthecalculation:

Yield(%)=Microspheresweight

Totalsolidsweight ×100 (4)

2.8.2. Morphologicalandaerodynamiccharacterisation

Themorphologicalcharacterisationofmicrosphereswas

per-formed using a ﬁeld emission scanning electron microscope

(FESEM;FESEMUltraPlus,Zeiss,Germany)at3kVofvoltage,which

isequippedwithasecondaryelectron(SE)detector.Thedry

pow-derswereplacedontometalplatesanda5nmthickgoldpalladium

ﬁlm wassputter-coated onthe samples(Q150T S/E/ES Sputter

Coater,QuorumTechnologies,UK)beforeviewing.

TheparticlesizewasestimatedastheFeret’sdiameter(distance

betweentwotangentsonoppositesidesoftheparticles)andwas

directlydeterminedwithanopticalmicroscope(OlympusIX51,

Japan).Theestimationwasbasedonthemeandiameterresulting

fromtheevaluationof300particles(n=300).

Real density was determined using a Helium Pycnometer

(Micropycnometer, Quanta Chrome, model MPY-2,USA) (n=3).

Apparenttapdensitywasobtainedbymeasuringthevolumeof

aknownweightofpowderina10mLtest-tubeaftermechanical

tapping(30tap/min,Tecnociencia,Spain).Afterregistrationofthe

initialvolume,thetest-tubewassubmittedtotappinguntil

con-stantvolumewasachieved,accordingtoapreviouslydescribed

method(El-Gibaly,2002)(n=3).

TheaerodynamicdiameterwasobtainedusingaTSIAerosizer®

LDequippedwithanAerodisperser®_(Amherst_Process_Instrument

Inc.,Amherst,MA,USA)andassumingrealdensityasthedensity

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2.9. Invitrobiocompatibilitystudy

2.9.1. Evaluationofmetabolicactivity

TheinvitrocytotoxicityofCS/CRG/TPPnanoparticles,

microen-capsulatednanoparticles(mannitol/nanoparticles=80/20,w/w),as

wellasthatoftherawmaterialsinvolvedinnanoparticle

produc-tion,wasassessedbythemetabolicassaythiazolylbluetetrazolium

bromide(MTT)test.Twocelllinesrepresentative ofpulmonary

andnasalepithelia(Calu-3andA549cells)wereused.A549cells

wereseededatadensityof1×104_cells/well_and_Calu-3_cells_at

2×104_cells/well_in_96-well_plates,_in₁₀₀_␮l_of_the_same_medium

usedforcultureincellcultureﬂasks.Thecellsweregrownat37◦C

ina5%CO2atmospherefor24hbeforeuse(Grenhaetal.,2007).

Three different concentrations (0.1, 0.5 and 1.0mg/mL) of

nanoparticles were evaluated for cytotoxicity over 3 and 24h.

Suspensions of unloaded nanoparticles and microencapsulated

nanoparticles,BSAloadednanoparticlesandsolutionsofthe

indi-vidualrawmaterials(CS,CRGandTPP)wereevaluatedseparately

forcytotoxicity.Sodiumdodecylsulphate(SDS,2%,w/v)wasused

asapositivecontrolofcelldeath.Optimalcellseedingdensityand

theabsenceofMTT conversionintheassaymediumwere

con-ﬁrmedinpreliminaryexperiments.Allformulationsandcontrols

werepreparedassolution/suspensionsinpre-warmedCCM

with-outFBSimmediatelybeforeapplicationtothecells.

Toinitiatetheassay,culturemediumofcellsat24hinculture

wasreplacedby100␮LoffreshmediumwithoutFBScontaining

thetest samplesor controls.After3 or 24hof cell incubation,

samples/controlswereremoved and 30␮Lof theMTT solution

(0.5mg/mLinPBS,pH7.4)wereaddedtoeachwell.After2h,any

generatedformazancrystalsweresolubilisedwith50␮LofDMSO.

Uponcompletesolubilisationofthecrystals,theabsorbanceofeach

wellwasmeasuredbyspectrophotometry(InﬁniteM200,Tecan,

Austria)at540nmandcorrectedforbackgroundabsorbanceusing

awavelengthof650nm(Carmichael, DeGraff,Gazdar, Minna,&

Mitchell,1987).

Therelativecellviability(%)wascalculatedasfollows:

Viability(%)= _(CM(A−S)

−S)×100 (5)

whereAistheabsorbanceobtainedforeachoftheconcentrations

ofthetestsubstance,Sistheabsorbanceobtainedforthe2%SDS

andCMistheabsorbanceobtainedforuntreatedcells(incubated

withCCM).Thelatterreadingwasassumedtocorrespondto100%

cellviability.Theassaywasperformedonthreeoccasionswithsix

replicatesateachconcentrationoftestsubstanceineachinstance.

2.9.2. Determinationofinﬂammatoryresponse

The inﬂammatory response generated by the exposure to

nanoparticlesandmicroencapsulatednanoparticleswasevaluated

onCalu-3cells.Toperformtheassay,thecellswereseededon

96-wellplates(2×104_cells/well)_and,_after₂₄_h_incubation_at₃₇◦_C

in5%CO2atmosphere,exposedtonanoparticlesand

microencap-sulatednanoparticlesataconcentrationof1mg/mL.Theexposure

tolipopolysaccharide(LPS;10␮g/mL)wasusedaspositivecontrol

(Alfaro-Morenoetal.,2009;Muraetal.,2011),whilecellsincubated

withCCMwereusedasnegativecontrol.Allformulationsand

con-trolswerepreparedassolutions/suspensionsinpre-warmedCCM

withoutFBSimmediatelybeforeapplicationtothecells.After24h

ofexposure,thecellsupernatantswerecollectedandcentrifuged

(10min,16,000×g,15◦C,centrifuge5804R,Eppendorf,Germany).

ThelevelsofIL-6andIL-8existinginCalu-3cellsupernatantswere

determinedbyquantitativeELISAusinghumanIL-6andIL-8

Quan-tikineELISAkits(R&DSystems,Minneapolis,MN),accordingtothe

manufacturer’sinstructions.Thesupernatantswereappropriately

diluted(approximately 10timesforIL-6and 50timesforIL-8).

Theassaywasperformedonthreeoccasionswithtworeplicates

oftestsubstanceineachinstance.Theconcentrationofindividual

cytokinesforeachsampleisexpressedaspercentageofthecontrol

(incubationwithCCM)fromtriplicateassays.

2.9.3. Evaluationofepithelialintegrity

Forthestudiesofepithelialintegrity,Calu-3cellswerecultured

atanair interfaceaccordingtoapreviously described

method-ology (Grainger, Greenwell, Lockley, Martin, & Forbes, 2006).

Brieﬂy,100␮LofCalu-3cellsuspensionwasseededatadensity

of5×105_cells/cm2 _in _Transwell _inserts_(0.33_cm2_), _with_0.5_mL

mediuminthebasolateralchamber.Thecellswereincubatedat

37◦C,5%CO2for2days.Afterthistime,themediumwasaspirated

fromtheapicalandbasolateralchambersandreplacedonlyinthe

basolateralchamber,theapicalchamberbeingexposedtothe

incu-batoratmosphere.Mediumwasreplacedevery2daysthereafter.

InordertoverifytheeffectofnanoparticlesonCalu-3celllayer

integrity,theformulationsweredispersedin100␮LFBS-freeCCM

ata concentrationof 1mg/mL andincubated withthecell

lay-ers.Transepithelialelectricalresistance(TER)wasmeasuredusing

chopstickelectrodesandanEVOM®_volt_metre_(STX-2_and_Evom_G,

WorldPrecisionInstruments,UK).TERmeasurementsweretaken

30minbeforetheexperimentandat0.5,1,2,4,6,24and48hafter

administration.TERvalueswerecalculatedbysubtractingthe

resis-tanceofcell-freecultureinsertsandcorrectingforthesurfacearea

oftheTranswellinsert.CelllayersincubatedwithCCMwereusedas

thereference(control),fromwhichTERchangesduringexposure

tothesampleswerecalculated.Allexperimentswereperformed

intriplicateonthreeoccasions.

2.10. Statisticalanalysis

Forthegeneralityofresults,thet-testandtheone-way

anal-ysisofvariance(ANOVA)withthepairwisemultiplecomparison

procedures(Dunn’smethod)wereperformedtocomparetwoor

multiplegroups,respectively.

Thefreeze-dryingcasestudydeterminestheeffectofdifferent

cryoprotectants andnanoparticle concentrationonnanoparticle

stability.Duetothenumberofobservationsinvolved(threetofour)

ineachcell/treatmentandtotheexistenceofemptycells,amore

complexstatisticalmethodologywasnotapplied,becauseitwould

compromisethemodelassumptionsandresultinmisleading

infer-ences.Therefore,thestatisticalapproachusedforthisanalysiswas

theone-wayANOVAwithGowes-Howellposthoctests,basedon

samplesizesandvariancehomogeneitybetweengroups.

Thefactorialdesign(FD)isoftenusedformanipulative

exper-iments,suchasthatofthemetabolicactivity(MTTtest).Inthat

case, for both cell lines tested (Calu-3 and A549) the effect of

sampletype(X1)andconcentration(X2)oncellviability(Y).Each

ofthefactors(X1 andX2)hasthreelevels.InthecaseofX2the

levelsare0.1, 0.5and 1.0mg/mL. ForX1 there isa splitin two

groups:(1)when rawmaterialsareconsideredthelevelsof X1

areCS,CRGandTPP;(2)whencarrierformulationsarestudied,

thelevelsareunloadednanoparticles,BSA-loadednanoparticles,

microencapsulatednanoparticles.Foreitherrawmaterialsor

car-rierformulations,theobjectiveistoinvestigatetheinﬂuenceof

factorsX1andX2 ontheresponsevariable(Y).FDwasoriginally

plannedwithequal number of observationsper treatment, but

someoccurrences related torandom lossof observations orto

practicalconstraints,causeinequalityofsamplesizes.Therefore,

forthefourmultifactordesignspresentinthisstudy,threewere

unbalanced with observations in every cell/treatment. For this

complexFD,thetypeIIIsumofsquares(SS)wasused,because

itisnotinﬂuencedbythesamplesizeineachtreatment.Thebasic

multivariateassumptionstobecheckedarethattheerrorsare

(5)

analyseswithposthoctestsHochberg’sGT2(unbalancedFD)and

Tukey,wereusedtodeterminewhichgroupsdiffer.

ThestatisticalanalyseswereperformedwiththesoftwareSPSS

(version 22.0 for windows) considering a signiﬁcance level of

˛=0.05.

3. Resultsanddiscussion

CS/CRG/TPP nanoparticles were previously reported by our

group in a work that evidenced the ability of TPPto decrease

nanoparticlesizeandimprovestability(Rodrigues,RosadaCosta,

etal.,2012).Inthatwork,thepotentialofthecarriersfor

trans-mucosal drug delivery was suggested. More recently we have

observedtheability of thesenanocarrierstoassociatedifferent

proteins,demonstratingthatthepHofthenanoparticleassembly

mediumhasastrongeffectonproteinassociationandthepHofthe

releasemediumaffectsthereleaseproﬁle(unpublishedresults).

Thepresentworkisdedicatedtothestudyofthesenanoparticles

regardinganapplicationinnasalandpulmonarydelivery.

3.1. CharacterisationofCS/CRG/TPPnanoparticles

Afteran initialscreening of varied CS/CRG/TPP mass ratios,

seekingfortheformulationthatproducesnanoparticleswiththe

most suitable characteristics for the objective of transmucosal

delivery(Rodrigues,RosadaCosta,etal.,2012), theformulation

CS/CRG/TPP=4/1/1(w/w)wasselectedforsubsequentstudies.The

correspondingformulationwithoutTPP(CS/CRG/TPP=4/1/0,w/w)

wasusedascontrolinsomeoftheperformedstudiestoshowthe

effectoftheinclusionofTPP.

Fig. 1A depicts the result of the morphological evaluation

of BSA-loaded nanoparticles,which evidence a spherical

struc-turesimilartobothunloadednanoparticles(Rodrigues,Rosada

Costa, et al., 2012) and to polysaccharide-based nanoparticles

ingeneral(Goycoolea,Lollo,Remunan-Lopez,Quaglia,&Alonso,

2009;Oyarzun-Ampuero,Brea,Loza,Torres,&Alonso,2009;Zorzi, Párraga,Seijo,&Sánchez,2011).

AsshowninTable1,thedevelopedBSA-loadednanoparticles

evidenceadequatephysicochemical propertiesfor theobjective

oftransmucosaldelivery,witha sizearound300nmanda

pos-itivezetapotential(+40mV).Infact,thesizeissmallenoughto

permitanintimatecontactwithepithelialsurfaces,whichis

max-imalat50–500nm(Desaietal.,1996;Janietal.,1990).In turn,

thehighpositivezetapotentialfurtherpotentiatestheinteraction

withepithelia, asthis is negatively chargedand, thus, an

elec-trostaticinteractionisenabled.Insummary,thesecharacteristics

areexpected toprovide a prolongedretention of nanoparticles

closetoepithelialsurfaces,potentiatingproteinrelease.

Notwith-standingtheimportanceof thesephysicochemical features,the

presenceofmucusandthemechanismofmucociliaryclearance

alsoneedtobeconsideredregardingthesuccessofnasaland

pul-monarytransmucosaldelivery(Dombu&Betbeder,2013;Marttin,

Schipper, Verhoef,&Merkus, 1998).Nanoparticles described in

thisworkareexpectedtoexhibitbioadhesivepropertiestosome

extent,duetothechitosancontent.Besidestheproperchitosan

abilitytoadheretobiologicalsubstrates(Du,Cai,&Zhai,2014;Lehr,

Bouwstra,Schacht,&Junginger,1992),chitosan-based

nanoparti-cleshavealsodemonstratedthesamecapacityinseveraloccasions

(Bernkop-Schnürch,Weithaler,Albrecht,&Greimel,2006;Sandri

et al., 2010). This will possibly improve the carrier residence

timeclosetotheepithelia,potentiatingdrugabsorption(Dombu

&Betbeder, 2013;Marttinetal.,1998).Additionally,

nanoparti-cleshavebeenreportedtoavoidordelaymucociliaryclearance,

althoughthisabilityishighlydependentontheirsizeandismore

relevantforparticlesbelow100nm(Oberdörster,Oberdörster,&

Oberdörster,2005;Sung,Pulliam,&Edwards,2007).

It is alsoobserved in the tablethat the inclusion of TPP in

theformulationincreasesthenanoparticleproductionyield,while

maintainingthe encapsulation efﬁciency,which is around50%.

A great difference is observed in loading capacity, which is of

70%forCS/CRG/TPP=4/1/0andapproximately30%for4/1/1.This

differenceisexplainedbythefactthat,whilebothformulations

encapsulateasimilaramountofprotein,thenanoparticles

contain-ingTPPdisplayaconsiderablyhigherproductionyield.Therefore,

asviewedinEq.(3),thereisthesameamountofproteindistributed

byahigheramountoftotalsolids,therebyresultinginalowervalue

ofloadingcapacity.Unloadednanoparticlesdisplaysomewhat

dif-ferentphysicochemicalcharacteristics,4/1/0nanoparticleshaving

491nmand+78mVand4/1/1nanoparticlesdisplayinga sizeof

208nmand+47mV(datanotshown).

3.2. Evaluationofnanoparticlestabilityinpresenceoflysozyme

Lysozyme is an enzyme that is present in mucosal

sur-faces,includingthenasaland pulmonary(Konstan etal.,1981;

Muzzarelli, 1997), which exhibits the ability to cleave _␤-(1–4)

glycosidicbondsofchitosan(Muzzarelli,1997).Therefore,taking

into account the high content of chitosan present in the

pro-posednanoparticles,evaluatingtheirbehaviourinpresenceofthis

enzymeisimportantregardingafutureinterpretationofresults,

suchasthoseobtainedinvivo.Toperformtheevaluation,

nanopar-ticleswereincubatedwithlysozyme(0.2and0.8mg/mL)at37◦C

andtheirsizeandzetapotentialweremonitoredalongtime.The

enzymeconcentrationof0.2mg/mLwasusedtakingintoaccount

indications foundin the literature regarding physiological

con-centrationsoftheenzyme(Konstanetal.,1981).Furthermore,a

concentrationof0.8mg/mLwaschosentoinvestigatethe

nanopar-ticle behaviour in extreme conditions, as previously reported

(Grenhaetal.,2005).

AsobservedinFig.2,theformulationofnanoparticles

with-outTPP(CS/CRG)showedatendencyforsizedecreasealongtime.

When lysozyme was present at a concentration of 0.2mg/mL,

thedecreasewasnotsigniﬁcant,ashighstandarddeviationsare

obtained. However, at 0.8mg/mL a strong and signiﬁcant size

decrease(p<0.05)occursupto60minofincubation,althoughafter

that a suddenincrease in size is observed,inverting thetrend.

Thisisprobablyduetoaggregation,asaconcomitantincreasein

thestandarddeviationofthemeansizeisobserved,indicatinga

greatdispersionofsizes.Zetapotential,inturn,remainedstable

duringtheassay(datanotshown).Interestingly,theformulation

containingTPPwasnotaffectedbythepresenceoftheenzyme,

nosigniﬁcantsizereductionbeingobservedindependentlyofthe

concentrationoflysozyme.Thisbehaviourrevealsasomewhat

pro-tectingeffectattributedbyTPP.

Anabsenceofeffectoftheenzymeonchitosan-based

nanopar-ticles (alsocontainingTPP)incubated insimilarconditions was

previouslyreportedinanotherstudy(Vila,Sánchez,Tobío,Calvo,&

Alonso,2002).ItispossiblethattheinclusionofTPP,whichactsas

cross-linker,preventsoratleastmodiﬁeschitosanexposure,thus

notenablinganeasyandrapidenzymaticdegradationasisseenfor

nanoparticlesdevoidofTPP.

3.3. Evaluationofnanoparticlestabilityuponfreeze-drying

When nanocarriers are formulated as aqueous suspensions,

bothphysical(aggregation/particlefusion)andchemical

(hydroly-sisofpolymerandchemicalreactivity)instabilityphenomenaare

knowntoappearatsomepoint(Abdelwahedetal.,2006;Chacón,

Molpeceres,Berges,Guzmán,&Aberturas,1999;Kumar, Shaﬁq, & Malhotra, 2012), limiting nanoparticle applications (Sameti

(6)

Fig. 1.(A) TEM microphotograph of BSA-loaded CS/CRG/TPP (4/1/1, w/w) nanoparticles; (B) SEM microphotograph of microspheres composed of manni-tol/nanoparticles=80/20(w/w). 0 30 60 90 120 150 0 20 40 60 80 100 120 140 si ze ( % of ini tia l)

Time of incubation (min)

0 30 60 90 120 0 20 40 60 80 100 120 140 Siz e ( % of ini tia l)

Time of incubation (min) A

B

Fig.2.SizeevolutionasafunctionoftimeuponincubationofCS/CRG/TPP nanopar-ticles()massratio4/1/0and()massratio4/1/1,with(A)0.2mg/mLand(B) 0.8mg/mLlysozyme,at37◦C(mean±SD,n=3).

etal.,2003;Wuetal.,2011).AqueoussuspensionsofCS/CRG/TPP

nanoparticleswerereportedinapreviousworktoremainstable

forupto9months,thepresenceofTPPintheformulation

demon-stratingtohaveasigniﬁcantroleinthiseffect(Rodrigues,Rosada

Costa,etal.,2012).However,thepreferenceinstoringand

trans-portingproductsindrystate,hasledtothedecisionofdeveloping

alyophilisedformulation.Regardingthepresentwork,itis

impor-tanttorecallthatanasalformulationofnanoparticleswillinvolvea

suspension,whilemicroencapsulatednanoparticles(drypowders)

areproposedforlungdelivery.Still,inthelattercasetheremightbe

atimeperiodbetweennanoparticleproductionand

microencapsu-lationwherealyophilisedformofnanoparticleswillbebeneﬁcial

forstorage.

Freeze-dryinghasbeenconsideredanadequatetechniqueto

improvethelong-termstabilityofcolloidalnanoparticles.

Never-theless,itisacomplexprocessthatrequiresamajoroptimisation

of the inherent conditions. Many parameters of the

formula-tion/processmaydecidethesuccessoffreeze-drying(Abdelwahed

etal.,2006).Inthisstudy,nanoparticleswerefreeze-driedusing

differentsugarsascryoprotectants,atvariedconcentrations.The

concentration of nanoparticle suspensions was also tested as

variable.Thedirectfreeze-dryingofnanoparticles(without

cry-oprotectants)didnotpermitthepreservationoftheirproperties

(datanotshown),leadingtostrongaggregation.Thiseffectmight

beexplained byboth thehighconcentrationofparticles

under-goingthefreezingstepandthemechanicalstressinducedbyice

crystallisation(Abdelwahedetal.,2006;Vauthier&Bouchemal,

2009). This observation was reported in a previous work of

ourgroupfor nanoparticlescomposedof otherpolysaccharides

(Dionísio et al., 2013), as well as in other works available on

theliterature(Abdelwahedetal.,2006;Fuente,Seijo,&Alonso,

2008).

Thepurposeofthis studywasthus examiningtheeffects of

type ofcryoprotectant(sucrose,trehalose, lactoseand glucose),

cryoprotectantconcentration(5%and10%,w/w)andnanoparticle

concentration (1, 2 and 4mg/mL) on the nanoparticle

charac-teristicsuponreconstitutionwithwaterfromthecorresponding

lyophilisedpowder.

Fig.3displaysthevariationofnanoparticlesizewhen

compar-ingtheobtainedvalueafterreconstitutionoffreeze-driedmaterial

(Df)withthatinitiallydetermined(Di)beforethefreeze-drying

process.Asaﬁrstapproach,unloadednanoparticleswereused.The

ANOVAexperimentrevealedthattheconcentrationofnanoparticle

suspensionstobefreeze-driedisthesoleparameterevidencinga

relevantandstatisticallysigniﬁcanteffectonthestabilityconferred

bythecryoprotectants.Inthis regard,theposthoctest

(Gowes-Howell)showsthatusingnanoparticleconcentrationsof2mg/mL

or4mg/mLleadstoahigherincreaseofnanoparticlesize(p<0.05)

Table1

Physicochemicalproperties,productionyieldandencapsulationdataofBSA-loadedCS/CRG/TPP(4/1/1,w/w)nanoparticles(mean±SD,n=3).

CS/CRG/TPPformulation Size(nm) Potential(mV) Productionyield(%) Encapsulationefﬁciency(%) Loadingcapacity(%)

4/1/0loaded 226±35 +27±5 15±2 51±12 70±18

(7)

Fig.3.SizevariationofCS/CRG/TPPnanoparticlesuponfreeze-dryingatdifferent concentrations,inpresenceof5%or10%sucrose,trehalose,lactoseandglucose,and furtherreconstitutioninwater(hydrodynamicdiameterofnanoparticlesbefore (Di)andafter(Df)freeze-drying).Resultsrepresentmean±SD(n=3);***indicates formationofprecipitate.

ascomparedwith1mg/mL,whichistheconcentrationpermitting

thedesiredmaintenanceofsizeafterfreeze-drying.

Zetapotentialvaluesdidnotevidencealterationsafter

freeze-drying inany of thetested conditions(datanot shown).Other

worksstudyingthefreeze-dryingofcomparableconcentrationsof

polysaccharide-basednanoparticleshavereportedgood

stabilisa-tionprovidedbythesameorsimilarcryoprotectants(delaFuente

etal.,2008;Dionísioetal.,2013;Hirsjärvi,Peltonen,&Hirvonen, 2009).

Althoughnottoastatisticallysigniﬁcantmanner,other

inter-pretationsmightbesuggestedbyFig.3.Regardingtheperformance

ofthedistinct cryoprotectants thatweretested, itis important

toobserve that lactose (atypical excipient used in lung

deliv-ery)revealed absolutelyinefﬁcientas cryoprotectantagent,not

preventingparticleaggregationafterfreeze-drying.Thispoor

pro-tectiveeffectwasalsoobservedinapreviousworkofthegroupwith

otherpolysaccharide-basednanoparticles (Dionísio etal., 2013)

andwasfurtherreportedintheliterature,beingattributedtoan

ineffective protection during the freezingstep (Hirsjärvi et al.,

2009).Sucrose,glucoseandtrehalose(inthisorder)aresuggested

tohavebetterabilityas cryoprotectants,astheirpresence

pro-videdamoreadequatemaintenanceoftheinitialcharacteristics

ofnanoparticles,atleastundercertainconditions.Sucrose

main-tainedtheexactcharacteristicsofnanoparticleswhenusedat10%

(withthealreadymentionedexceptionofthe4mg/mL

concentra-tionofnanoparticles)orat5%whennanoparticlesarefreeze-dried

atthelowerconcentrationtested.Averysatisfactory

cryoprotec-tiveeffectprovidedbybothglucoseandsucrosewaspreviously

reportedbyourgroupregardingthestabilisationoftwo

formula-tionsofpullulan-basednanoparticles(sulfatedpullulan/chitosan

and carrageenan/aminatedpullulan) (Dionísio et al.,2013).The

concentrationofthecryoprotectantisalsoanimportant

param-eter tooptimise. In this regard, a content of 5% wasgenerally

foundtobe enoughto stabilise nanoparticlesfreeze-dried at a

concentrationof1mg/mL,while anincreaseto10%of

cryopro-tectantisneededwhenthenanoparticleconcentrationincreases

to2mg/mL.

Consideringtheresultsobtainedwithunloadednanoparticles,

BSA-loadednanoparticlesattheconcentrationof1and2mg/mL

were freeze-dried with 10% of both glucose and sucrose. The

physicochemical characteristics of the nanoparticles were still

maintained(datanotshown).However,itwaspreviouslyreferred

thattheuseofglucoseascryoprotectantofproteinformulations

should becautious becauseof its reducing character(Li et al.,

1996; Wang, 2000). Nevertheless, the freeze-drying of protein

nanocarriersinpresenceofglucosehasbeenreportedasefﬁcient,

consideringboththephysicochemicalstabilityofthecarrierand

themaintenanceofproteinproperties(Freixeiroetal.,2013).In

anycase,acomprehensivereviewonthesubjectsuggeststhatany

evaluationshouldbeperformedona case-by-casebasis(Wang,

2000).

Thisstudyevidencesthesuitabilityofusingfreeze-drying to

obtainnanoparticlesina dryform,but itreinforces the

impor-tanceofcarefullyselectingandoptimisingtheconditionsofthe

freeze-dryingprocessinordertoachieveanoptimisedprotective

effect.

3.4. Microencapsulationofnanoparticles

Thesuccessofinhalableformulationsisprimarilydependenton

theiraerodynamicproperties,astheseinﬂuenceboththe

disper-sionandsedimentationpatternofthecarriers(Sungetal.,2007).

Whenasystemicactionisintended,thedrugcarriersshouldbe

smallenoughtopassthroughthemouth,throatandconducting

airwaysandreachthedeeplung,butnotsosmallthattheyfail

todepositandarebreathedoutagain.Inthiscontext,ithasbeen

consideredthatanaerodynamicdiameterbetween1and5␮mis

necessary(Bosquillon,Lombry,Préat,&Vanbever,2001),whichin

somecasesisevennarrowedto2–3␮m(Oberdörsteretal.,2005).

Aspeciﬁcconjugationbetweensizeanddensityofthecarriersis,

thus,necessary.Thepulmonaryadministrationofnanoparticlesis

severelyhinderedbytheirlowinertia,whichmakesalveolar

depo-sition practicallyimpossible, mainlyresulting in theexhalation

ofthecarriers(Sungetal.,2007).Thespray-dryingofCS-based

nanoparticleswasﬁrstreportedbyGrenhaetal.(2005)asa

strat-egytoendowthenanoparticleswiththeabilitytoreachthedeep

lung.Thisstrategywaslateronfollowedbyothergroups(Lietal.,

2010;Pourshahabetal.,2011).Inthepresentwork,the

microen-capsulationofCS/CRG/TPPnanoparticleswasapproachedtoobtain

adrugdeliverysystemthatpermittedefﬁcientadministrationof

thedevelopednanocarriersaimedatthesystemicpulmonary

deliv-eryofproteins.Mannitolwasselectedtocomposethematrixof

microparticles,asithasdemonstratedtosimplyactascarrierof

nanoparticles,providing theirrelease uponreachingthealveoli

(Grenhaetal.,2005).

Microencapsulated nanoparticles were produced by

spray-drying a suspension of mannitol/nanoparticles (80/20, w/w),

evidencing a sphericalshape and a slightly corrugated surface,

as can be observed in Fig. 1B. The nanoparticle carriers were

determinedtohaveaFeretdiameterof2.3±0.6␮m,areal

den-sityof 1.44±0.01g/cm3 _and _a _tap_density _of_0.42_±_0.04_g/cm3_.

Theaerodynamicdiameterwasdeterminedtobe1.80±0.11␮m.

ComparableresultswerereportedformicroencapsulatedCS/TPP

nanoparticles(Grenhaetal.,2005),asystemthatalready

demon-strateditsefﬁcacyinvivofortheadministrationofinsulin(Al-Qadi,

Grenha,Carrión-Recio,Seijo,&Remu ˜nán-López,2012).Good

indi-cationsarethusprovidedonthecapacityofthedevelopedsystem

toactas carrierof nanoparticlestothelungwiththeaimof a

systemicdeliveryofproteins.Nevertheless,usingthisapproachof

microencapsulatingthenanoparticlescouldpossiblyposeanextra

limitation,regardingthehighproportionofexcipientsthatneed

tobeusedand,consequently,theinherentlowerloading.Taking

intoaccount thatadministrationsofaround 160–180mg ofdry

powderhavebeenreported(Hoppentocht,Hagedoorn,Frijlink,&

deBoer,2014;Labiris,Holbrook,Chrystyn,Macleod,&Newhouse,

1999)andconsideringaproteinloadingof27%innanoparticles

andthe20%amountofnanoparticlesintheﬁnalmicroparticle

for-mulation,160mgwouldcarryapproximately9mgofprotein.This

amountisconsideredsuitableforthepurposeofobtaininga

ther-apeuticeffectandwillpossiblenotcompromisethesuccessofthe

formulation,whichwillobviouslybedependentonseveralother

(8)

3.5. Invitrobiocompatibilitystudy

Evaluatingthebiocompatibilityofdrugdeliveryformulationsis

nowadaysamandatoryaspecttoaddress(Gaspar&Duncan,2009).

Whileanaccuratedeterminationofthetoxicityofaformulation

canonlybedeterminedinvivo,avarietyofinvitrotoxicological

assays,performedinadequatelyselectedcelllines,mightprovide

veryusefulinformationandarewidelyacceptedasﬁrstindicators

(ISO,2009;Rodrigues,Dionísio,etal.,2012).Currentinternational

guidelinesrequiretheevaluationtobecontextualisedwitha

spe-ciﬁcrouteofadministrationanddoseofthematerial(Gaspar&

Duncan,2009).Itisalsoreferredthatpolymersandpolymer-based

carriersshouldberegardedasdifferententitiesand,thus,evaluated

separately(Aillon,Xie,El-Gendy,Berkland,&Forrest,2009;Gaspar

&Duncan,2009;Williams,2008).Thisisbasedontheassumption

thatthepropercarrierstructure,amongotherparameters,might

affecttheﬁnaltoxicologicalbehaviour(Aillonetal.,2009;Gaspar

&Duncan,2009).

Toaddresstheabovementionedissues,theevaluationofthe

biocompatibilityofCS/CRG/TPPnanoparticlesandcorresponding

rawmaterials,wasperformedinthisstudybymeansofthree

dif-ferentassays,(1)themetabolicactivity(MTT),(2)theinﬂammatory

response(detectionofIL-6andIL-8)and(3)theepithelialintegrity

(measurementofTER).Calu-3andA549weretheselectedcelllines,

astheyarerepresentativeof therespiratory epithelia.Calu-3 is

animmortalisedcelllineobtainedfromlungadenocarcinomaand

hasbeenextensivelyusedinthestudyofformulationsdesigned

foreither nasalorpulmonary drugdelivery,as it isconsidered

amodeloftheepitheliumofbothregions(Casettarietal.,2010;

Graingeretal.,2006;Zhu,Chidekel,&Shaffer,2010).Importantly,

Calu-3cellsformtightjunctionsinvitro(Wintonetal.,1998)and,

undersuitablecultureconditions,exhibitinvivo-likebarrier

prop-erties(Graingeretal.,2006).A549isacelllinerepresentativeof

thealveolarepithelium,obtainedfromhumanalveolar

adenocar-cinoma,showingnoabilitytoformfunctionaltightjunctionsin

culture(Forbes&Ehrhardt,2005;Foster,Yazdanian,&Audus,2001;

Wintonetal.,1998).Consequently,itcannotbeusedinstudiesof

theepithelialbarrierintegrity,althoughthisdoesnotpreventtheir

useincytotoxicitystudies.

3.5.1. Evaluationofmetabolicactivity(MTTassay)

TheMTT assayprovidesan evaluation of thecell metabolic

activityupon exposuretothesamplesand hasbeenused very

frequentlyintheassessmentofdrugdeliverycarriers(Rodrigues,

Dionísio,etal.,2012).Itevaluatestheabilityofthecellstoreduce

theMTTreagenttotetrazoliumsalts,anactionthatisdependenton

mitochondrialmetabolism.Areductionofcellularmetabolic

activ-ityisgenerallyacceptedasanearlyindicatorofcellulardamage

(Scherließ,2011).

Inthiswork,A549andCalu-3cellswereculturedandexposed

todifferentconcentrationsofnanoparticlesandrawmaterialsfor

bothashort(3h)andamoreprolongedperiod(24h).Theexposure

for3hdidnotleadtosigniﬁcantdecreaseincellviability,with

val-uesremainingcloseto90–100%inallcases.Theonlyexception

occurredforA549cellsexposedtochitosan(datanotshown),an

effectcommentedlater.Incontrast,theprolongationofexposure

upto24hinducessomevariabilityoncellviability.Inthismanner,

forthecontacttimeof24hafactorialdesignwasapproachedto

examinetheeffectofsampletype(rawmaterials/carrier

formula-tions;X1)andconcentration(X2)oncellviability(Y),foreachcell

line.Thetwofactors(X1,X2)oftheexperimentaldesignhavethree

levelseach,resultinginninefactor/levelcombinations.

Cellviabilityresultsobtainedupon24hexposurearedepicted

inFig.4(Calu-3cells)and5(A549cells).ForCalu-3cells,an

unbal-ancedFDwasusedinthegroupofrawmaterialsandformulations.

Fortherawmaterials(Fig.4A),asigniﬁcanteffect(p<0.05)was

Fig.4. Calu-3cells viability measuredby the MTTassay after 24hexposure torawmaterials(A –CS:chitosan, CRG:carrageenan,TPP: tripolyphosphate) and nanoparticle-based formulations (B – NP: nanoparticles, BSA-NP: BSA-loadednanoparticles,microNP:microencapsulatednanoparticles).Datarepresent mean±SEM(n=3,sixreplicatesperexperimentateachconcentration).

detectedforeachofthemainfactors(X1andX2)andtheir

inter-action(X1*X2).ThemostrelevantobservationisthatTPPinduced

a greater decreaseof cellviability ascompared to CSand CRG

(p<0.05).

With respect to the formulations, the factorial experiment

revealsthatonlyX1(typeofmaterials)andthetwo-way

interac-tionX1*X2arestatisticallysigniﬁcant(p<0.05).Althoughthelowest

viabilityinducedbyrawmaterialswasobservedforthehighest

concentrationofTPP(1.0mg/mL,Fig.4A),cellviabilityupon

con-tactwithCS/CRG/TPPnanoparticleswasabove95%inallinstances

(Fig.4B),noconcentration-dependenteffectbeingobserved.Inthis

manner,asacellviabilityof70%isthethresholdbeyondwhicha

cytotoxiceffectisconsideredtooccuraccordingtoISO10993-5

(ISO,2009),theobservedeffectsaredevoidofphysiological

rele-vance.ThisindicatesthatCalu-3cellsdonotevidenceaparticular

sensitivitytoanyofthetestedsamples,eitherrawmaterialsor

nanoparticle-basedformulations,atthetestedconcentrations.

InA549cells,abalancedFDoftherawmaterialsshoweda

signif-icanteffectforbothmainfactors(X1andX2)andtheirinteraction

(X1*X2).AmongrawmaterialsonlyCRGandTPPshowasigniﬁcant

differencebetweentheconcentrationlevels(p<0.05),cellviability

decreasingwithincreasingconcentrations,particularlyatthe

con-centrationof1mg/mL(Fig.5A).Nevertheless,cellviabilitieswere

higherthan70% inallconditionstested forbothpolymers.The

mostdeleteriouseffectwasobservedforchitosan(p<0.05),which

inducedhighcytotoxicity,aswasalsodetectedupon3h,evenat

thelowerconcentrationof0.1mg/mL.

ThedifferentresponseofbothcelllinestoCSexposure(in

(9)

Fig. 5.A549 cells viability measured by the MTT assayafter 24h exposure torawmaterials(A –CS:chitosan, CRG:carrageenan,TPP: tripolyphosphate) and nanoparticle-based formulations (B – NP: nanoparticles, BSA-NP: BSA-loadednanoparticles,microNP:microencapsulatednanoparticles).Datarepresent mean±SEM(n=3,sixreplicatesperexperimentateachconcentration).

withthedifferentstructuresevidencedbyCalu-3andA549cellsin

cultureandwiththeirsensitivitytopHalterations.Infact,while

Calu-3 cells are rounder, A549 cells growaccording to a more

extendedproﬁle,ascanbeeasilyseeninATCCwebpage(Collection,

2012).Chitosanusedinthisworkisabaseand,therefore,is

dis-solvedinaceticacid(pHaround3.2)toassemblethenanoparticles.

Thesamesolutionwasusedintheassay,althoughitwas

previ-ouslydilutedwithCCM. Uponadditiontothecelllayer,itwas

observedonthemicroscopethatA549cells begintodetach,an

effectnot perceivedforCalu-3 cells. Therefore,thecells are no

longerattachedtotheplateand areeasilyremovedduringthe

washingstepsevenbeforetheMTTreagentisadded,not

permit-tinga reliablemeasure ofthemetabolic activity.Thisindicates

thattheseresultsshouldbetakenwithprecaution.Importantly,

althoughtherearenotmanystudiestestingchitosansolutionsin

A549cells,theliteraturereportsanabsenceofoverttoxicityofthe

polymerinthiscelllineatconcentrationssimilartothoseusedin

thisstudy,althoughadifferentchitosanpolymer(regarding

molec-ularweightandsalt)wasused(Huang,Khor,&Lim,2004).

ThebehaviourofA549cellsregardingthetesteddrugdelivery

systemswasgenerallysimilartothatofCalu-3cells,evidencing

averyfavourableproﬁle.ThestatisticaldetailsoftheFDshowed

thatbothmaineffects(X1andX2)andtheirinteraction(X1*X2)are

signiﬁcant(p<0.05),theinteractionbeingappreciatedinFig.5B.

Althoughwithslightvariations,cellviabilitywasabove80%inall

cases.Nevertheless,itisobservedthatmicroencapsulated

nanopar-ticlesarethosecausingthemoreamenableeffect.

Asawhole,theseresultsindicatethatneithertheloadingofBSA

innanoparticlesnorthemicroencapsulationprocessinduced

phys-iologicallyrelevantalterationsontheresponseofthecellstothe

0 50 100 150 200 250 300 350 400 450 CS/CRG CS/CRG/TPP Micro NP LPS IL am ount (% of c ont ro l) IL-6 IL-8

Fig.6.IL-6(greybars)andIL-8(whitebars)secretionbyCalu-3cellsexposedfor 24htoCS/CRGandCS/CRG/TPPnanoparticles,microNPandlipopolysaccharide.Data showedas%ofreleasecomparingtocontrol(mean±SD,n=3).

systems.NostudiesregardingtheeffectofCS/CRG/TPP

nanopar-ticlesoncellviabilitywerereportedbefore,thushamperingthe

comparisonofthepresentresultswithothers.Themostsimilar

studiesreported theabsenceof overt toxicity ofeither CS/CRG

nanoparticlesinL929ﬁbroblastsattheconcentrationof1mg/mL

(Grenhaetal.,2010)orCS/TPPnanoparticlesinCalu-3andA549

cells(Grenhaetal.,2007),whileseveralotherformulationsof

CS-basednanoparticleswerealsoreportedtonotinduceacytotoxic

effectindifferentcelllines(Caban,Capan,Couvreur,&Dalkara,

2012;Dionísioetal.,2013;Oliveira,Silva,Bitoque,Silva,&Rosada Costa,2013).

Theﬁndingsobtainedinthis workforallnanoparticle-based

formulations in both respiratory cell lines are consideredgood

indicatorsofbiocompatibility.However,theevaluationofa

bio-compatibilityproﬁledemandstheuseofcomplementaryassays

that assessother aspectsof cellresponse, which iscertainly of

importancetoestablishaﬁnaltendency.

3.5.2. Determinationofinﬂammatoryresponse

Inparallelwiththeevaluationofcellviability,theabilityofthe

carrierstoinduceaninﬂammatoryresponseuponcontactwiththe

cellshasalsobeenusedasanindicatorofbiocompatibility(Lim,

Yaacob, Ismail,& Halim,2010; McCarthy, Inkielewicz-St ˛epniak, Corbalan,&Radomski,2012;Muraetal.,2011).Theevaluationof

sucharesponsemight beperformedbyquantifyingtherelease

ofinﬂammatorymarkersbythecellsuponexposuretothedrug

deliverysystems.IL-6andIL-8aretwocytokinessuitableforthis

end,asIL-6isresponsibleforneutrophilactivationandIL-8isa

chemotacticagentforinﬂammatorycells(Lewis&McGee,1992).

In this assay,theeffectof thethree nanoparticle-based

car-riers (CS/CRG/TPP nanoparticles, BSA-loaded nanoparticles and

microencapsulated nanoparticles) was evaluated regarding the

induction ofan inﬂammatory phenotypein theCalu-3 cells by

meansofthedeterminationofreleasedIL-6andIL-8.Theobtained

resultsaredepictedinFig.6.Theassessmentwasconductedupon

24hexposuretothedrugdeliverysystems(1.0mg/mL)usingLPS

asapositivecontrol(10␮g/mL)andCCMasanegativecontrol.The

amountofcytokinesreleasedinresponsetoCCMwasconsidered

100%andalltheotherresultsarepresentedwithrespecttothat

value.Asexpected,abasalconcentrationofcytokinesinthe

super-natantwasdetectedincontrolcells (incubatedonly withCCM)

(Grainger,Greenwell,Martin,&Forbes,2009;Muraetal.,2011)

andIL-8wassecretedatahigherconcentrationthanIL-6(Mura

etal.,2011;Zhuetal.,2010).

Remarkably, no signiﬁcant effect was elicited by either

CS/CRG/TPPnanoparticlesorthemicroencapsulatedcounterparts

(10)

0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 TE R (% of co nt rol) Time (h)

Fig.7. Transepithelialelectricalresistance(TER)ofCalu-3celllayersupon incu-bation with CS/CRG/TPP nanoparticle suspension (1.0mg/mL). Data represent mean±SEMofthreeexperiments(n=3perexperiment).

didnotdifferbetweenthetreatmentgroups,indicatinganabsence

ofeffectofthemicroencapsulationprocess.

NanoparticlesproducedwithoutTPPwerealsotestedas

con-trols,evidencing a response verysimilar tothat ofCS/CRG/TPP

nanoparticles,whichindicatesthattheinclusionofTPPdoesnot

induceadifferenteffect.Finally,asigniﬁcantincreaseintherelease

ofbothcytokineswasonlyfoundupontheexposuretoLPS

(approx-imately220%forIL-8and360%forIL-6;p<0.05).Thiseffectwas

expected,asLPSisknowntoinduceaninﬂammatoryresponse,

cor-respondingtoreportedﬁndings(Alfaro-Morenoetal.,2009;Mura

etal.,2011).

3.5.3. Evaluationofepithelialintegrity

Intercellularstructuresarecrucialforepithelialdevelopment

andfunction.Tightjunctionsarethemostrestrictiveintercellular

junctions,formingacontinuousbandwhichcompletely

circum-ventstheperipheryofepithelialcellsatthemucosalsurface(Matter

&Balda,2003).Asmentionedabove,andcontrarytoA549cells,

Calu-3cellshavetheabilitytoformtightjunctionsinvitro,

display-ingthepropertiescharacteristicofanepithelialbarrier(Grainger

etal.,2006).

Inthisstudy,Calu-3cellswereexposedtoCS/CRG/TPP

nanopar-ticles at 1mg/mL to determinethe effect of the drugdelivery

systemontheepithelialintegrity,asa complementary

biocom-patibilityindicator.TheTERwascontinuouslymonitoredfor48h

andtheresultsareshowninFig.7.ItisobservedthattheTERdid

notsuffersigniﬁcantalterationsaftertreatmentwith

nanoparti-clesoveraperiodof48h.Althoughnotstatisticallysigniﬁcant,a

slightdecreaseofapproximately10%withrespecttocontrolwas

observedatearlytimepoints,butat4htheTERreached100%again

andsurpassedthatvalueatlatertimes.

NosimilarresultswerefoundonCalu-3cells,butanincrease

inTERhasbeenreportedinCaco-2cells (intestinalepithelium)

asaresponseof thecells toanexternalstimulation(Klingberg,

Pedersen,Cencic,&Budde,2005;Sonnier,Bailey,Pritts,&Lentsch,

2011),inonecasebeingassociatedwitharesponsetoan

inﬂam-matorystimulus(Sonnieretal.,2011).Nevertheless,inourstudy

thiscorrelationcannotbeestablishedasnosigniﬁcant

inﬂamma-toryresponsewasobtaineduponcontactwiththenanoparticles,

asevidencedabove.Ontheotherside,althoughadecreaseinTER

wasobservedinmanyoccasionswithchitosansolutionsinseveral

epithelialcelllinesincludingtheCalu-3(Florea,Thanou,Junginger,

&Borchard,2006;Portero,Remu ˜nán-López,&Nielsen,2002;Smith, Wood,&Dornish,2004), anabsenceofeffectofchitosan-based

nanoparticlesis alsodescribed in theliterature(Behrens, Pena,

Alonso,&Kissel,2002;Grenhaetal.,2007).Therefore,this

obser-vationcomprises anextraindicationonthebiocompatibilityof

CS/CRG/TPPnanoparticles.

Althoughtheobtainedresultsaregoodindicatorsthatno

dele-teriouseffects aretobeexpected forshort-termcontact ofthe

pulmonaryornasalmucosawiththedeveloped nanocarriers,it

isimportanttorecallthattheeffectsofaprolonged

administra-tionalsorequireattention.Inthisregard,therapidmucusturnover

inthenasalmucosacertainlypreventsnanoparticleaccumulation,

butthatwillnotbethecaseinpulmonarydelivery,wherethis

con-cerninunavoidable.Theconsequencesoflong-termexpositionto

particulatesarequiteunknown(Dombu&Betbeder,2013),buta

retentionhalf-timeofapproximately700dayswasreportedfor

humans(Oberdörsteretal.,2005).Withtheirabilitytoevade

spe-ciﬁcdefencemechanisms,onceinthelungnanoparticlesmight

eitheraccumulatein thelung tissue,causing inﬂammationthat

cantriggermoresevereeffects,ortheycantranslocatefor

extra-pulmonaryregions,whichisalsoamatterofgreatconcern.This

clearlyevidencestheurgentneedofextrastudiesaddressingthe

raisedconcerns,whicharelackingintheliterature.Itisofutmost

relevancetodeterminethefateofnanoparticleswhenincontact

withrespiratoryepithelia,thusenablinganadequaterisk

assess-mentand,hopefully,theestablishmentofnanoparticlesinclinicto

becomeareality.

4. Conclusion

ThisworkdemonstratesthesuitabilityofCS/CRG/TPP

nanopar-ticlesascarriersforanapplicationinpulmonaryandnasaldrug

delivery, with a particular emphasis on their biocompatibility

potential. Apart from the ability to associate a model

macro-molecule, BSA, the nanoparticlesdemonstrated to be stable in

presence of an amount of lysozyme simulating in vivo

condi-tions.Thenanocarriersalsoevidencedstabilityuponfreeze-drying

in presenceof glucoseor sucrose, usedascryoprotectants. The

protein-loaded nanoparticles were successfully incorporated in

microspheresbymeansofaspray-dryingprocess,resultingina

drypowderwithsuitablepropertiesforlungdelivery.Moreover,

thedevelopedcarriersexhibitedlowornegligibletoxicityin

con-tactwithtwo respiratorycell linesrepresenting boththe nasal

andpulmonaryepithelium,asdemonstratedbytheeffectsoncell

metabolicactivityandtransepithelialelectricalresistance.These

ﬁndingswerereinforcedbytheobservationofnoinﬂammatory

responsegeneratedupon24hofexposureofthecellstothe

car-riers.Altogether,theseresultsareanencouragingindicatorofthe

biocompatibilityandsafetyofCS/CRG/TPPnanoparticlesregarding

nasalandpulmonarydelivery,makingthemsuitablecandidatesfor

theseapplications.

Acknowledgements

This work was supported by National Portuguese funding

throughFCT–Fundac¸ãopara aCiência eaTecnologia,projects

PTDC/SAU-FCF/100291/2008,PTDC/DTP-FTO/0094/2012and

PEst-OE/EQB/LA0023/2013, as well as by the Spanish Government

(ISCIII,AcciónEstratégicadeSalud,PS09/00816).ResearchofClara

CordeiroissupportedbyFCTprojectPEst-OE/MAT/UI0006/2014.

SusanaRodriguesacknowledgesherPhDscholarshipfromFCT

(ref-erenceSFRH/BD/52426/2013),aswellasaShareBiotechmobility

grant.

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