Chitosan nanoparticles generation using CO2 assisted processes

(1)

HAL Id: hal-01611626

https://hal.archives-ouvertes.fr/hal-01611626

Submitted on 27 Oct 2017

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Chitosan nanoparticles generation using CO2 assisted

processes

Nibal Hijazi, Élisabeth Rodier, Jean-jacques Letourneau, Haithem Louati,

Martial Sauceau, Nicolas Le Moigne, Jean-Charles Bénézet, Jacques Fages

To cite this version:

Nibal Hijazi, Élisabeth Rodier, Jean-jacques Letourneau, Haithem Louati, Martial Sauceau, et al..

Chitosan nanoparticles generation using CO2 assisted processes. Journal of Supercritical Fluids,

Elsevier, 2014, 95, pp.118-128. �10.1016/j.supflu.2014.08.003�. �hal-01611626�

(2)

Chitosan

nanoparticles

generation

using

CO

2 assisted

processes

Nibal

Hijazi

a,b

_,

_Elisabeth

_Rodier

a

_,

_Jean-Jacques

_Letourneau

a

_,

_Haithem

_Louati

a

_,

Martial

Sauceau

a

_,

_Nicolas

_Le

_Moigne

b

_,

_Jean-Charles

_Benezet

b

_,

_Jacques

_Fages

a,∗

a_Université_de_Toulouse;_École_des_Mines_d’Albi;_UMR_CNRS_5302;_RAPSODEE_Research_Centre,_F-81013_Albi,_France b_École_des_Mines_d’Alès,_Centre_des_Matériaux_des_Mines_d’Alès_(C2MA),₆_avenue_de_Clavières,_F-30319_Alès,_France

Thecurrentconcernsofsustainabledevelopmentmakethebiobasedpolymerstheobjectofmanystudies. Chitosanisabiobased,biocompatibleandbiodegradablepolysaccharidewithantibacterialand cytocom-patibleproperties.Inthisstudy,weaimedtogeneratechitosanparticleswithtwoprocessesusingCO2

underpressure,inordertodecreasetheuseoforganicsolventandtoobtainnanoparticles.

Thefirstisasupercriticalanti-solventprocess:CO2actsasananti-solventtowardanaceticacid

aque-oussolutionofdissolvedchitosaninwhichethanolwasaddedtoenhancetheanti-solventeffect.The reciprocalmiscibilityofCO2withthesolventsinducesthereductionoftheirsolvatingpower,leadingto

supersaturationatthecapillaryoutletandcausingthecrystallizationoftheparticles.

Thisprocessledtothegenerationofmoreorlessagglomeratedchitosannanoparticleswithan indi-vidualaveragesizeof378nm.

Inthesecondprocess,thepressurizedCO2isdissolvedinwatertolowerthepH.Thisinturnallows

thechitosantobedissolvedandtheresultingsolutionissprayed,thankstothepressurizedCO2,intoa

hotairstream.Thisnewprocessallowedthegenerationofdriedchitosannanoparticleswithamedian sizeof390nm.

1. Introduction

Nowadays,mankindhastocopewithglobalchallengesinits

attempttoprotecttheenvironmentinthelongterm.Forthis

pur-pose,sustainableindustrialdevelopmenthasbeenencouraged.In

thecaseofengineeringofpolymericmaterials,sustainabilityissues

impactthechoiceofrawmaterials,thedesignofmaterialprocess,

theconsiderationoflifecycleassessment.

Among the studies carried out to date, an approach is

to use biobased polymer materials and structure them at

micro/nanometricscalestoenhancesomeoftheirspecific

proper-tiesinordertocreateanoriginalmaterialwithspecificfunctional

properties.

Severalapproacheshavebeenproposed todevelopbiobased

polymernanocomposites,asthedispersionofinorganicparticles

suchasclaysinapolymermatrix[1].Anoriginalapproachisthe

Abbreviations: AcAc,aceticacid;Chit,chitosan;DSC,differentialscanning calorimetry;EtOH,ethanol;SAS,supercriticalantisolvent;SCASA,supercriticalCO2

assistedsolubilizationandatomization;sc-CO2,supercriticalcarbondioxide;TGA,

thermogravimetricanalysis;XRD,X-raydiffraction.

∗ Correspondingauthor.Tel.:+33563493141;fax:+33563493025. E-mailaddress:[email protected](J.Fages).

synthesisofbiobasedpolymernanoparticlesthatcouldbeusedfor

furtherdevelopmentofnanostructuredbiobasedpolymer

assem-blies. Forthispurpose, ourstudyfocused onthegeneration of

polysaccharideparticles.Polysaccharidesrepresentavastcategory

ofbiobasedpolymerswithspecificfunctionalities,presentlyused

inallsectorsofhumanactivitieslikematerialsscience,nutrition,

healthcareandenergyandwith largeapplicationsinindustry.

Withinthisfamily,chitosan,anamino-polysaccharidederivedfrom

chitinbydeacetylation(deacetylationdegreeofatleast50%)which

isthesecondmostbiosynthesizedpolymeraftercelluloseobtained

mainlyfromcrustaceansexoskeleton [2],waschosento bethe

materialfornanoparticleelaboration.

Chitosanisnotonlyreadilyavailablebutalsobiocompatible

andbiodegradableduetoitstwochemicalfunctionalities,

mucoad-hesivewithantibacterialandcytocompatibleactivities[3–5].Itis

principallyusedinagriculture,watertreatment,cosmetics,

phar-maceuticsandbiomedical,foodpackaging[6].

Dashetal.[7]haverecentlyreviewedthestudieswhichhave

beenconducted to generatechitosan particles fromaceticacid

aqueoussolutiontobeusedascarriersfordrugdelivery[7]:by

spray-drying(2–10!m[8]),emulsion-crosslinking(350–690!m

[9]),coacervation–precipitation(100–250nm[10])...These

pro-cessesarecommonlyusedbuthaveseveraldrawbacksastheuse

(3)

Nomenclature

C(kgm3₎ _solution_{concentration}

DA(%) degreeofacetylation

K(m3_kg−1_),_a _constants_of_{polymer/solvent}_system

K′ _Huggins_constant

Mv(kDa) viscosityaveragemolecularweight

!(mPas) polymersolutiondynamicviscosity

!0(mPas) puresolventdynamicviscosity

!int(m3kg−1) intrinsicviscosity

!rel relativeviscosity

!sp specificviscosity

PSi(MPa) pressureintheseparatori

PV(MPa) pressureinthevessel

pHi pHofthechitosansolutionbeforetheexperiment

pHf pHofthechitosansolutionaftertheexperiment

Qchit(mgs−1) chitosansolutioninjectionrate

QCO2(mgs−1) CO2injectionrate

TV(K) temperatureinthevessel

wi(%) massfractionofthecomponenti

residualpresenceinthegeneratedmaterial,theuseofhigh

temper-atures,theneedofadditionalstepsaspurificationanddrying...In

thiscontext,particularattentionhasbeenpaidtotheuseof

super-criticalcarbondioxide(sc-CO2).Indeed,itsabilitytodissolvein

largequantitiesinmanypolymers[11],enoughtomodifytheir

properties(viscosity,surfacetension...),mayhelptoimprovethe

compositematerialsproducedwhileminimizingtheuseofharmful

solventsthatareusuallydifficulttorecycle.Inaddition,theuseof

sc-CO2iswellestablishedfortheelaborationoffineparticles[12].

Withthe “Supercritical Assisted Atomization” process (SAA)

[13],1%aceticacidaqueouschitosan solutionallowedthe

gen-erationofparticlesrangingmostlybetween0.1and1.5!m.CO2

wasusedasanexpansionagent.Usingacomparableprocesswith

anenhancedmixingdevicebeforedepressurization,the

“Super-criticalfluidAssistedAtomization”introducedby“Hydrodynamic

CavitationMixer”(SAA-HCM)[14],severalmolecularweightsof

chitosanweretestedinordertoevaluatetheirinfluenceonthe

generatedparticles;particleswithadiameterrangingbetween0.2

and5!mwereobtainedfromaqueousacidicsolutions.Particlesize

distributionwasstronglydependentonthemolecularweight.

Inthisstudy,weinvestigatethegenerationofchitosan

parti-clesbytwoCO2basedprocesses,SupercriticalAnti-Solvent(SAS)

andsc-CO2assistedsolubilizationandatomization(SCASA).CO2

isexpectedtoactasananti-solventinthefirstprocessandasa

solubilizationandexpansionagentinthesecondprocess.The

influ-enceofprocessingconditionsisstudiedforbothtechniquesandthe

sizedistribution,crystallinity,degreeofdeacetylationandthermal

stabilityofthegeneratedparticlesarecharacterized.

2. Materialsandmethods

2.1. Materialsandpreliminarycharacterizations

Commercial chitosanextracted fromshrimp shells was

pur-chasedfromFranceChitine(France).Purityisnotgiven;possible

impuritiesusuallyareresidualchitin,inorganiccompounds,

pro-teins,chloride...[15].Itsviscosity,givenbythesupplier,isabout

50mPasfora1%(w/w)chitosan/aceticacidsolution,measured

byLVTBrookfieldviscometerat298K.TheacetylationdegreeDA,

measuredby thesupplierusingIRspectroscopyisaround 10%.

Whenchitosanisputintodemineralizedwater,thepHofthe

result-ingdispersionisaround13,whichindicatesthepresenceofresidual

NaOHinthecommercialpowder.

Table1

Compositionandviscositiesofchitosansolutionsusedforthedeterminationof chitosanMv.

Masscomposition(%) Viscosity(mPas) Water Aceticacid Chitosan mtotal(g)

90.90 9.10 0 22 1.2

89.89 8.99 1.12 22.25 31.3

88.89 8.89 2.22 22.5 114.3

87.91 8.79 3.30 22.75 258.6

Aceticacid(90%), sodiumhydroxideandpotassium hydrox-ide werepurchased from Prolabo (France); ethanol(96%) was

obtained from VWR (France). CO2 (purity 99.995%) was

sup-plied by Air Liquide (France) and used without any further

purification.

TheviscosityaveragemolecularweightMvofchitosanwas

esti-matedusingMark–HouwinkSakuradalaw:

[!int]=KMaV (1)

Kandawereestimatedtoberespectively4.74·10−6_m3_kg−1_and

0.72[16].

Previously,therelative!rel(Eq.(2))andspecific!sp(Eq.(3))

viscositieswereevaluatedforseveralconcentrationsofchitosan

solutions(Table1)inordertodeducethevalueoftheintrinsic

viscosity[!_int]ofchitosanusingHugginsrelation(Eq.(4)):

Relativeviscosity !rel=_!!

0 (2) Specificviscosity !sp=!−_!!0 0 (3) Huggins !sp C =K′[!int] 2 ×C+[!_int] (4)

Theviscosityofchitosansolutionswasdeterminedusinga

rota-tionalrheometer(RheoStress600,ThermoScientific,USA)usinga

60mmdiameterplane–planegeometry,incontinuousmodefor

shearratesbetween0and500s−1_.

InHuggins equation,[!_int] istheintercept pointofthe line

(!sp/C)=f(C)andits valuewasfound to be about 0.13m3kg−1.

Hugginsequationisspecificallyapplicableforverylow

concentra-tionswith[!_int]·C≪1[17].TheextrapolationofthisEq.(4)above

thislimitinduceamaximumvalueof[!_int]·Cof4.5forthetested

concentrations,whichisconsideredasacceptable.Therefore,the

molecularweightconsideredwascalculatedwithHuggins

equa-tionandfoundtobe62kDa.

2.2. Methods

2.2.1. Preparationofchitosansolutions

2.2.1.1. Chitosan solutions forSAS process. Chitosan solutions of

different concentrations were prepared by dispersing chitosan

powderintowater,followedbyadditionunderstirringofdiluted

aceticacidtodissolvechitosan.A50/50w/wethanol/water(Sol3)

solutionwasalsopreparedtoenhancetheanti-solventeffectof

sc-CO2duetotheverygoodmiscibilityofCO2 andethanol.The

compositionandsomepropertiesofthevarioussolutionsare

pre-sentedinTable2.

ThesurfacetensionanddensityweredeterminedbyanILMS

tensiometer (GBX instruments, France) using Wilhelmy plate

methodandthecalibratedfloatmethod,respectively.

Afterwards,thesolutionisfilteredundervacuumconditions

throughanitrocellulosemembranewithacuttingthresholdvalue

of3!mtoremovethenon-solubilizedimpurities.Theamountof

impuritiesretainedonthemembraneswasabout6mgandwas

(4)

Table2

CompositionsoftestedchitosansolutionsduringSASprocess.

wwater(%) wEtOH(%) wAcAc(%) wChit(%) mtotal(g) Surfacetensiona(mNm−1) Densityb(kgm−3)

Sol1 87.9 0 11.0 1.1 45.5 46.94 1.02

Sol2 87.0 0 10.8 2.2 46 – –

Sol3 49.1 49.1 1.2 0.6 81.5 30.05 0.93

a_Surface_tension_is_given_with_±0.5_mN_m−1.

b_Density_is_given_with_±0.001_kg_m−3. Table3

Compositionsofthealkalinerecoverysolutions.

wbase(%) wwater(%) mtotal(g)

Recov1 NaOH 7.4 92.6 21.6

Recov2KOH 2 98 51.0

Recov3KOH 1 99 50.5

2.2.1.2. AlkalinerecoverysolutionsforSASprocess. Alkaline solu-tionsofsodiumandpotassiumhydroxideatvariousconcentrations wereprepared(Table3)forthesolidgenerationandtherecovery

ofchitosanparticles.

2.2.1.3. ChitosandispersionsforSCASAprocess. Ascommercial

chi-tosan contains residual sodium hydroxide, it was previously

washedwithwatertolowertheinitialpHofaqueousdispersions.

Itwasthenfilteredandoven-dried.ThepHwasthusloweredto8.

Chitosansuspensionswithvaryingconcentrationsandvolumes

weretestedusingthisprocess(Table4).Totalquantityorvolume

treatedinanexperimentwasvariedinordertoobserveitsimpact

ontheprocessyield.

2.2.2. Experimentalset-up

2.2.2.1. SASprocess. Theexperimentswerecarriedoutusinga

ver-satilepilotplant(Separex,France).Aschemeoftheapparatusis

giveninFig.1.

Liquid CO2 is pumped up to a supercritical pressure by a

diaphragm pump P1 (Lewa, Germany). Compressed CO2 goes

Table4

CompositionsofchitosansuspensionsusedfortheSCASAprocess.

wwater(%) wChit(%) mtotal(g)

Susp1 99.50 0.50 502.50

Susp2 99.38 0.62 402.50

Susp3 99.37 0.63 503.14

Susp4 99.38 0.62 603.75

throughaheatexchanger(E2)previouslysetat313Kinorderto reachthecriticalconditionsoftheCO2,beforeenteringintothe

1.2dm3_vessel_V_(Parr_Instrument,_USA).

Thevessel(V)hasmultiplesapphirewindows,amagneticstirrer withamaximumtorqueof1.8N.mandaremovableheatingcollar. Thepressureintheautoclaveiscontrolledbyaback-pressure reg-ulator(D).Aparticulatefilter(F)isplacedattheexitofthevessel topreventtheparticlesfromcontaminatingtherestofthe cir-cuit.Threecyclonicseparators(S1,S2andS3)areusedtoseparate thesolventsfromCO2bygradualdepressurization,thepressure

beingsetbymicrometervalves.Anactivecarbonpackedbed(AC) isplacedaftertheseparatorstopurifytheCO2beforecoolingby

(E1)andreturningtostorage(T).HPLCpump(P2)(Gilson,USA) allowstheintroductionofthechitosansolutioninthevessel(V) throughastainlesssteelcapillarytube(O)whoselengthis10mm andinner-diameteris100!m.

2.2.2.2. SCASAprocess. Fig.2displaystheschematicdiagramofthe

SCASAapparatus.

(5)

Fig.2.SchematicrepresentationoftheSCASAapparatus.

TheintroductionofCO2intothepilotplantandthevessel(V)

aresimilartotheSASprocessapparatus.ThevesselVisusedto

preparethechitosansolution.Asprayingorifice(O),thestainless

steelcapillarytubealreadyusedinSASprocessoranozzle,isplaced

attheexitofthevessel(V)andfixedattheinletofafluidized

bedC(Aeromatic-Fielder,Switzerland),whichactsasanatomizing

device.Thefluidizedbedisflowedbyhotairwithadjustable

veloc-ityandtemperatureinordertodrythegeneratedparticlesduring

spraying.Twofilteringdevices(F1andF2)areplacedatbothendsof

thefluidizedbed.F1,astainlesssteelwirescreenallowsa

homoge-neousdistributionofenteringhotair.F2,equippedwithfourfilter

bagsandstainlesssteelwiregauzeontopofthem,allowsretaining

particles.

2.2.3. Chitosanparticlesgeneration

2.2.3.1. SASprocess. Analkalinesolutionisplacedinsidethevessel

(V)toreceiveandstopfurtherevolutionofthegenerated

parti-clesbyneutralizinganyresidualacidityremainingontheparticles

andinduced by CO2.At first, onlysc-CO2 isintroduced in the

vessel(V)andcirculatedinordertostabilizepressure,

tempera-tureandflowinthesystembeforeinjectingthechitosansolution.

Pressures are adjusted by theback-pressure regulatorand the

micrometricvalves.Onceasteadystateisreached,thechitosan

solutionisinjectedthroughthecapillarytubeintothesc-CO2flow

at1mlmin−1_(or_16.7_mm3_s−1_)._The_reciprocal_miscibility_of_CO₂

withaceticacidandethanolofthechitosansolutioninducesan

anti-solventeffectallowingtheparticlecrystallizationattheexit

ofthecapillary.CO2loadedwithsolvents flowsouttowardthe

separators,fromwhichtheliquidphaseatthebottomarepurged

frequentlytorecoversolvents.Attheendoftheinjection,thevessel

isdepressurizedandtheparticlesinsuspensionarecollected.

ToeliminatethesaltsformedbythereactionsbetweenCO2,

alkalinesolutionsandaceticacidsuchassodium/potassium

car-bonateandsodium/potassiumacetate,thesuspensionsarewashed

withdistilledwater(6<pH<7)andfilteredundervacuumusing

apolypropylenemembranecuttingat0.45!m.Theoperationis

repeatedseveraltimesbeforefreeze-drying(Freeze-dryerChrist

Alpha1-4LDC-1M,Germany)andperformedat3kPawitha

freez-ingtemperatureof261K.

2.2.3.2. SCASAprocess. Thekey-pointofthisprocessistoreplace

dilutedaceticacidbywateracidifiedbypressurizedCO2:chitosan

powderisfirstdispersedindistilledwaterandplacedinsidethe

vessel(V)understirring.Thevesselisthenfilledandpressurized

withCO2.Underhighpressure(20±0.5MPa)andatmoderate

tem-perature(298±2K),theacidifyingpoweroftheCO2 causesthe

dissolutionofthechitosanpowder.Pressureandtemperatureare

chosensoastofavordissolutionofCO2intowater.Calculationsare

basedontheworkofDiamondandAkinfiev[18]showingthatthe

valueoftheCO2molefractioninwateris2.6%(mol/mol)at20MPa

and308K.ThepHoftheCO2-acidifiedwaterwascalculated

accord-ingtoPengetal.[19]:at308Kand20MPa,pHis2.95andat383K

and20MPa,pHis3.26.Theseauthorsproposedamodelwhose

resultswerecomparedtothemeasurementsperformedbyRead

[20];bothresultsetswerefoundtobeconsistent.

Insuchconditions,48hofstirringallowsthedissolutionofthe

testedamountofchitosan.Theresultisaninstableemulsionof

twophases:aheavyphaseofCO2-saturatedaqueouschitosan

solu-tionandalightphaseofwater-saturatedsc-CO2.Itisthenpushed

towardthecapillary(innerdiameter100!m)orthespraying

noz-zle(outletdiameter340!m)byanupstream-pressurizedCO2flow

rate.ThisflowconsistsofheatedCO2(at333K)introducedinthe

vessel(V)continuouslytomaintainpressureat14±1MPa.Itis

thensprayed atthebottomofthefluidizedbed(C)andcarried

upwardbytheflowingairstream,whichisatatmospheric

pres-sure,previouslyheatedupto368±5Kandinjectedwithaflow

rateof2m3_min−1_._During_the_{depressurization,}_CO₂_turns_back_to

itsgaseousstateleadingtoasupersaturationandanatomizationof

chitosan-loadeddroplets;thehotairstreamsimultaneously

elimi-natesliquidwater.TheflowofpreheatedCO2allowsthepreheating

ofthesprayedsolutionfora betterdrying.Theairflowvelocity

iskeptatitshighestlevelandunchangedduringtheprocessto

avoidthesprayedsolutionfromstickingtothefluidizedbedwalls,

whilethestirringrateinthevessel(V)isadjustedtokeepavisually

homogeneousemulsionandtokeepaconstanttemperatureofthe

outletflowleavingthefluidizedbed(C).Afterspraying,particles

arecollectedinthefilters(F2).Aftertheexperiments,thepHofthe

residualsolutioninsidethevesselwasfoundtobearound5after

depressurization,whichindicatesthatwaterwasindeedacidified

bythepressurizedCO2.

2.2.4. Commercialandgeneratedchitosanparticles

characterization

Particle size distributionand meanparticlediameter inthe

suspensions were measured using a Nano Zetasizer (Malvern

Instruments,France)bytheevaluationoftheBrowniandiffusion

coefficient(for SAS process)oraMastersizer 2000HS(Malvern

Instruments,France)bylightdiffractionpatternusingethanolas

dispersingagent(forSCASAprocess).

Drychitosan particleswerevisualized withan

Environmen-talScanning ElectronMicroscope (ESEM XL30 FEG,FEI Philips,

Netherlands).Foreachsample,over500particlesfromatleastfour

pictureswereanalyzedwithanumericalcaliperintegratedinthe

imageacquisitionsoftwareinordertodeterminemeanparticle

diameterandtheparticlesizedistributionweightedinnumber.

Crystalstructuresofcommercialandgeneratedparticleswere

investigatedbyX-raydiffraction(XRD)usinganAXSD8Advance

Brüker diffractometer (Germany) equipped with a Cu cathode

("=1.54 ˚A).Measurementswereperformedintherange2#=5–60◦

under40kVand40mAwithastepsizeof0.007◦_.

AcetylationdegreeofchitosanwasestimatedbyFTIRusinga

BrükerIFS66spectrophotometer(BrükerOptics,Switzerland).

(6)

Table5

ProcessingparametersofthevariousexperimentsperformedusingtheSASprocess.

Test Chitosansolutionused pH Processingparameters Recoverysolutions

Qchit(mgs−1) QCO2(mgs−1) aPS1(MPa) PS2(MPa) PS3(MPa) pHi pHf

M0 Sol1 14.8 – – – – – 2.4

M1 Sol3 4.9 14.0 1500 7.2 6.9 4.2 – 4.6

M2 Sol2 3.4 14.8 1150 11.3 10.4 5.4 12.3 6.9

M3 Sol3 4.9 14.7 1650 7.1 6.6 4.0 13.5 7.9

M4 Sol3 4.9 14.8 1450 7.1 6.6 4.0 13.3 6.3

a_P_S1_,_P_S2_and_P_S3_are_given_with_an_absolute_accuracy_of_±0.5_MPa.

intherange4000–400cm−1_with_a_resolution_of₄_cm−1_and₃₂

scans,for3–5mgofchitosanpowder.

Severalcorrelationshavebeenproposedintheliteratureto

cal-culatetheacetylationdegreebasedonthechangingofthechemical

structureofthematerialobservedontheFT-IRspectra[21].Several

havebeentested;thechosencorrelationbelowistheonethatgave

themostreproduciblevaluesofDA.

DA=31.92×

!

A_A1320

1420

"

−12.20 (5)

whereA1320andA1420arerespectivelytheabsorbanceofthepeak

at1320cm−1_related_to_the_acetylated_amine_function_and_the_peak

at1420cm−1_related_to_the _C _H_bending._This_last_one_revealed_to

remainalmostunchangedfordifferentknownacetylationdegrees

andwasthenusedasthereferencepeak.

A differential scanning calorimeter (TGA-DSC 111 Setaram,

France)wasusedtocharacterizethethermalstabilityofthe

com-mercialandgeneratedchitosanparticles(5–20mg).Thermograms

wereobtained between298and1073Kwith aheating rateof

10Kmin−1_in_a_nitrogen_gas_stream_of₅₀_cm3_min−1_.

3. Resultsanddiscussion

3.1. ParticlesgenerationusingSASprocess

Twomainseriesoftestswereperformed:thefirst(M0andM1)

aimedtotesttheinfluenceofethanolusedinthechitosansolution;

thesecond(M2–M4)wasmeanttostudytheimpactoftherecovery

solutionusedtocollecttheparticlespreviouslyformedandtokeep

theminsuspension.Recoverysolutions1,2and3(seeTable3)were

usedrespectivelyintestsM2,M3andM4.Alltheexperimentswere

madeatTV=308K±2KandPV=17.6±0.3MPa.Theconditionsof

thedifferentexperimentsareshowninTable5.

Intheabsenceofrecoverysolution,theeffectofethanolonthe

particlegenerationwasevaluated.Theexperimentsshowedthat

eitherinthepresenceortheabsenceofethanol(M1andM0tests

respectively),noparticleswereobservedinthevesselattheendof

theexperiment.However,addingethanol,whichishighlymiscible

withCO2athighpressure,lowersthesolutionsurfacetensionand

facilitatedthereciprocaltransferofsolventsintothesc-CO2.This

waseasilyvisualizedthroughthevolumetricexpansionofthe

solu-tiondroplets(notshowninthispaper).Thisusuallypromotesthe

particlegenerationbySASandabetterremovalofaceticacid.

Fur-thermore,thepHofthesolutioncollectedinthevesselafterthese

testswasmeasured.Intheabsenceofethanol,thepHwasfoundto

beabout2.4±0.2whileitwasof4.4±0.2inthepresenceofethanol.

Inbothcases,thepHwasfarlowerthanthepKaofchitosan(≈6.3)

[2].Thisaciditymaycomefrom:(1)thepresenceofresidualacetic

acidinthevesselthatmaynotbedrivenoutbythesc-CO2;(2)

thepresenceofcarbonicacidH2CO3duetothesolubilityofCO2in

waterunderpressure.Aspreviouslymentioned,thevalueofmole

fractionofCO2inwateris2.6%(mol/mol)at20MPaand308K.This

shouldinduceapHof2.95inthesolution.ThepHbeinghigherin

thepresenceofethanolandcloseto4confirmedthebetterremoval

ofaceticacidandtheimprovementoftheanti-solventeffect.

How-everthemediumwasstilltooacidictopreventtheredissolution

ofpreviouslyformedchitosanparticles.Thisshouldexplaintheir

absenceinthesolution.

Itwasalsofoundthatwhentherecoverysolutioncontainedonly

demineralizedwater(notpresentedinthispaper),theresultswere

similartotheexperimentswithoutrecoverysolution.Therefore,a

recoverysolutionwasaddedtoneutralizetheacidexcess(tests

M2–M4).Twokindsofalkalinesolutionswereplacedseparately

inthevesseltobetestedasamediumtocollectparticles:sodium

hydroxidesolutions(M2)andpotassiumhydroxidesolutions(M3

andM4).Forallthetestedalkalinesolutions,suspendedparticles

wereobservedattheendoftheexperimentandthepHwasequalor

higherthan6.3.Nevertheless,afterfreeze-drying,theSEM

obser-vationshowedthepresenceofcrystallayerontheparticlessurface

(Fig.3).

The natureofthesecrystals wasdeterminedby XRD

analy-sis.Itwasfoundthatsodiumacetateandcarbonatecrystalsare

formedbyprecipitationreactionsthatoccurredbycontactofthe

alkalinesolutionswithCO2andaceticacid.Hence,washingthe

sus-pensionsbeforefreeze-dryingwasnecessary.Potassiumhydroxide

basedsolutionswerefoundtobemoresuitableregardingthelower

amountofformedcrystals,whicheasethewashing.

M3andM4testsdifferedintheconcentration ofpotassium

hydroxide.TheresultsofthesetwotestsareshownintheSEM

images(Fig.4) andcomparedto commercialchitosan particles

(Fig.4a).ForM3(Fig.4b),nanoscaledparticles(theaverage

diame-termeasuredbyNanoZetasizeris378±13nm)wereobservedina

porousnetworkofchitosan.M4producedbetterdefined

nanopar-ticles(Fig.4c)withanaveragediameterof820±19nm.Inboth

(7)

Fig.4.SEMphotographsofcommercialchitosan(a)andparticlesgeneratedbytheSASprocess;M3test:chitosannanoparticlesfixedinaporouschitosannetwork(b)and M4test:welldefinedchitosannanoparticles(c).

cases,theaveragediameterofthegeneratedparticleswas

submi-cronic,whichconfirmstheabilityoftheSASprocesstogenerate

nanoparticlesofchitosan.

However, these two different generated morphologies, i.e.

nanoparticlesembeddedinaporouschitosannetworkandwell

definedchitosannanoparticles,couldbeexplainedbythe

coexis-tenceoftwomaingenerationphenomena:(1)particlegeneration

by anti-solventeffect ofthesc-CO2 attheexit ofthecapillary

(Fig.5a)and(2)porousnetworkgeneration(Fig.5b)whichisfirstly

duetothepHtransition(thatinducesaphaseseparation[22]and

particlecoagulationoccurringinbasicmedia)oncetheinjected

chi-tosanisincontactwiththealkalinesolutionandsecondlytothe

CO2desorption.Thesetwocompetingphenomenamayvary

signif-icantlyforevensmallpHvariations;indeed,mainlythefinalpHof

therecoverysolutiondiffersbetweenM3andM4experiments.The

stronginfluenceofthechemistryofthesystemsinvolved,in

par-ticularthepHthatgraduallyevolvesduringthedissolutionand

degassingofCO2 andthewashingphase,appearedtoalter the

organizationofchitosanchannelsduringphaseseparation.

LikeSAA[13]andSAA-HCM[14]processes,SASprocessledto

chitosannanoparticlesfromaceticacidsolutionwithalargesize

distribution.However,theprocessusedtoobtaindryparticleswas

different:thefirsttwoprocessesallowedthegenerationofdry

par-ticlesbyheating athightemperatureinthepresenceofCO2 as

expansionagent.TheSASprocessallowedthegenerationof

dis-persedsolidparticlesintherecoverysolutionwhichneedstobe

driedafterwards.Theneedforaposttreatmentandthelackof

controlofthephysicalandchemicalparametersoftheprocessmay

haveinducedlowerreproducibilitythaninSAAprocess.

3.2. ParticlesgenerationusingSCASAprocess

Differentchitosansuspensionsweretestedusingthisprocess

(Table6).Furthermore,theinfluenceofthenatureofthespraying

deviceanditslocationontheparticlegenerationwasevaluated.

TheyieldgiveninTable6istheratiooftheweightofchitosan

particlesandtheweightofchitosanintroducedinthevessel(V).

Notethatsomesolutionremainsinthebottomofthevesselatthe

endoftheexperiment(lessthan20%oftheintroducedsolution).

Notealsothattheparticlecollectingdevice(F2)isnotoptimalyet

andhastobeenhanced;indeedfinestnanometricparticlesmaybe

(8)

Table6

ProcessingparametersofthevariousexperimentsdonebySCASAprocess.

Test Suspension Sprayingdevice Solidgenerationyield(%)

Type Position Diameter

M’1 Susp1 Nozzle 2 Outlet:340!m Notevaluated

M’2 Susp1 Capillary 1 Inner:100!m 21.4

Theuseofanozzle(M’1test)ledtosprayingflowratetoohigh comparedtothedryingairflowrate:awetdepositofthesprayed solutionwasobservedonthedryerwall.Adriedfilmwasthen collectedwithveryfewparticlesonitssurface(Fig.6a).A

capil-laryallowedtoreducesignificantlythesprayingflowrateandtwo

caseswereconsideredregardingthelocationofthecapillary.First,

thecapillarywasplacedinthemiddleoftheaircolumn(position1)

andorientedagainsttheairstream(M’2)inordertohavealonger

trajectoryandabetterdrying:afilmdepositandparticlesof

chi-tosanwerebothobserved.Thefilmdepositwasonthedryerwall

becausethesolutionwassprayedtooclosetothebottompartof

thecolumnbeforetheairstreamcoulddryit.Chitosanparticles

werecollectedonthefiltersF2.AsshowninFig.6b,these

parti-clesarespherical.Someofthemarehollow-shaped,whichcould

beexplainedbythedesorptionoftheCO2duringthesolidification

ofchitosanspheres.

Fig.5.Schematicrepresentationofthemechanismofphaseseparation(a)andparticlegenerationbyanti-solventeffect(b)duringtheSASprocess.

(9)

Fig.7.DifferentscalesofstructuresofchitosangeneratedparticlesbySCASAprocess.

Fig.8.ParticlesizedistributionasdeterminedfromSEMpicturesforcommercial andgeneratedchitosanbySCASAprocess.Dotsrefertothemeasuredvaluesand thelinereferstoalognormallaw.

Then,whenthecapillarywasplacedupwardatthebottomof

thedryingcolumn(position2)(M’3,M’4,M’5),thefilmdepositon

thewallswasnegligiblecomparedtothepreviousconfiguration

andtheparticlesyieldoftheprocesswasenhanced.Thevariation

ofchitosanconcentrationfrom0.5%to0.62%(M’2andM’3tests,

respectively)aswellastheinitialsolutionvolume(M’3toM’5)

didnotseemtosignificantlyaffecttheparticlegenerationnorthe

particlesize(Fig.6b–e).Inallcases,particlesizedistributionislarge

aswillbediscussedbelow.

Generated particles have a bi-modal size distribution:

nanoscaled particles for the most part and microscaled

parti-clesmainlyduetotheagglomerationofnanoparticlesasshownin

Fig.7.Theparticlesizedistributionwasdeterminedbyanalyzing

SEM pictures as follows: a first measurement was done at a

micrometricscalecorrespondingtothelowestmagnificationof

SEMpictures.TheresultsareshownonFig.8.Atthisscale,for

severalexperiments(notshowninthispaper),theSCASAprocess

reducedthe size of the particles compared to the commercial

chitosanpowder.

Asecondmeasurementwasdoneatthehighestmagnification

ofSEMpictures,wherealargenumberofnanometricspherical

par-ticleswasdetectedwithamediansizeof390nmandlessthan15%

ofthemeasuredparticleslargerthan1!m(Fig.9).

Size wasalsoevaluatedby laser diffraction usinga

Master-sizer2000HS:thenumberweightedsizedistributionismonomodal

withameandiameterof721nm.Thisdiameterishigherthanin

Fig.9becauseofsomeremainingaggregatesintheliquiddispersing

mediumbutbothmeasurementmethodsareconsistent.

The particlesize ishardly comparableto thoseobtained by

ReverchonandAntonacci[13]andShenetal.[14]sincetheused

Fig.9.ParticlessizedistributionforgeneratedchitosanbySCASAprocess(M’3test). Dotsrefertothemeasuredvaluesandthelinereferstoalognormallaw.

chitosanmolecularweightsandchitosansolutionconcentrations,

parametersthathighlyimpacttheparticlesizeaccordingtoShen

etal.[14],weredifferent.Theinfluenceofprocessingparameters,

chitosanpropertiesandsolutionconcentrationontheparticlesize

andtheparticlesizedistribution hasnot beenexploreddeeply

yet.Nevertheless,SCASA process generates repeatedlychitosan

nanoparticles.Furthermore,comparedtoprocessesusingorganic

acidsuchasaceticacidandcitricacid,noresidualsolventsare

presentinthe generated chitosan particles. Hence, forspecific

applicationswheretheexcessacidconditionmaynotbe

desir-able,suchascontrolledreleasedrugdelivery[23],SCASAprocess

isadvantageous.

TheXRDanalyses(Fig.10)showedthatbothprocessesallowed

thegenerationofrepeatablecrystalstructuresbutdifferentfrom

commercialchitosan:theintensityratiosI/Imaxof2#≈13◦peakand

2#≈20◦_peak_were_{significantly}_altered_during_the_process._In_the

literature,thesetwopeaksareassignedtotwodifferentstructural

formsofchitosan:hydrated(13◦₎_and_anhydrous₍₂₀◦₎ _crystals.

Thepeakaround2#_≈30◦_,_{characteristic}_of_residual_chitin_in

com-mercialchitosan,andthepeakaround2#_≈46◦_disappeared._With

theSASprocess,ahaloat2#≈23◦_that_was_attributed_to_an

allo-morphicformofchitosan[24]andawell-definedpeakat2#≈44◦

peak appeared.Withthesecondgeneration process,two peaks

around27◦_and₂₈◦_,_not_referenced_in_previous_studies,_appeared.

Thus,bothgenerationprocessessignificantlymodifythecrystalline

structureofchitosanandmakeitmoreamorphous.Althoughthe

crystallinestructureofchitinhaswellbeenstudiedinthe

litera-ture,crystallinityofchitosanisstillnotcompletelyexplored,which

(10)

(11)

Shenetal.[14]alsoobservedanamorphizationofchitosane

par-ticleswithSAA-HCMprocess,whichwasattributedtothesudden

supersaturationintheprecipitator,whichledtoarapid

evapora-tionofthesolventfromthedropletsandalowercrystallinityof

thegeneratedparticles.ReverchonandAntonacci[13]attributed

theformationofamorphouschitosanparticlestoa

physicochem-icalmodificationunderthehighoperatingtemperatures,possibly

relatedtodeacetylationand/orcross-linkingofthepolymer.Inour

case,highsupersaturationratesaregeneratedduringthe

depres-surizationthroughthecapillary,whichinducesthegenerationof

unstableamorphoussolidforms.

Forallthechitosanparticles,thethermalstabilityanalyzedby

TGAshowedadegradationaround523K.

Theacetylationdegreewascalculatedaspreviouslypresented.

TheDAwas21_±2%,20_±1%,19_±6%and21_±1%forcommercial

chitosan,M3,M4andM’3,respectively(Fig.11):nosignificant

dif-ferencecouldbeassignedtotheprocess;theslightdifferencesmay

beduetovariouserrorsrelatedtothemeasurementtechnique.A

differencewiththeDAgivenbythesupplierhasbeennoticedbut

norationalexplanationcouldbegiven.

Nosignificantdifference couldbe observedbetweenthe

IR-spectrometerofM’3processedbySCASAandcommercialchitosan.

However,a notable difference could be seen betweensamples

processed by SCASA andby SAS (M3 and M4): several peaks

appearedonlyforSASprocessat825,1245,1506and1556cm−1_.

Thepeaksat1245cm−1_and_1506–1556_cm−1_refer_to_C _N_and_N _H

bondsrespectively,whichmeansthatduringtheSASprocess,a

newchemicalbondappeared:infact,accordingtoNunthanidetal.

[25],dissolvingchitosaninaceticacidinducesthegenerationof

chi-tosanacetatesalts(NH3+_,−_OOC-CH₃₎_that_transform,_after_drying,

toacetylgrouppresentinthe(N-acetyl-d-glucosamine)structure

ofchitin[26].AsimilarresultwasobservedbyShenetal.[14].

4. Conclusion

4.1. Supercriticalanti-solventprocess

In thiswork, a methodfor generatingchitosan particles by

anti-solvent effectusing sc-CO2 hasbeen designed andtested.

Nanometerscaled particleswereproduced with a mean

diam-eterof378±13nm andan amorphizedstructure,whichdiffers

fromthecommercialchitosan.Morphology,aspectandparticle

arrangementstronglydependontherecoverymediumthatseems

tohaveaninfluencethroughthepHonthephaseseparation

mech-anismandtheresultingmorphology,aporouschitosannetworkor

chitosanparticles.Furthermore,particlesobtainedbythese

exper-imentsareinmajorityspherical.Inthiscontext,afurtherstudyon

thestabilityoftheparticlesinsuspensionshouldbeperformed.

4.2. Sc-CO2assistedsolubilizationandatomizationprocess

Anewsc-CO2assistedsolubilizationandatomizationprocess,

SCASA,wassetupandpreliminaryexperimentswereachievedto

checkitsfeasibilityandrepeatability.Threemainconclusionscan

bedrawn:firstly,comparedtotheprocessesalreadyusedto

gen-eratechitosanparticles,thisnewprocessissimpleanddoesnot

requireanyorganicsolvent.Secondly,itisclearthattheacidifying

strengthoftheCO2allowedthedissolutionofchitosanpowder.

Thirdly,theatomizationprocessgeneratedmostlynear-spherical

andsphericalshapedparticlesduringseveralexperiments,which

impliedtherepeatabilityofthisnewprocessanditsrobustness,

asitisnotdestabilizedbysmallparametersvariations.However,

twoparticlesizescaleswerenoticed:nanometricscaledparticles

withamediandiameterof390nmandmicrometricscaledparticles

withameandiameteraround20!mandmadeofagglomerated

nanoparticles.Thepresenceofindividualsphericalparticleslarger

than1!mshouldbementionedbuttheyrepresentedlessthan

10% of the total measurements. Many of the particles seem

hollow-shapedprobablyduetotheCO2desorptionwhileparticles

solidificationtakesplace.Experimentsshowedthatfortheused

chitosanandconfigurations,thebestresultswereobtainedforCO2

pressureinsidethevesselduringsprayingof13–15MPawithanair

flowof140Nm3_h−1_heated_to₃₅₃_K._A_good_compromise_between

thesprayingrateandthedryingairflowrateshouldbefound.A

furtherstepregardingthisprocesswillconsistinoptimizingthe

processparametersasthesolutionflowandtheflowsratio,

dry-ingtemperatureandtheparticle-collectingdevicetofinallyobtain

narrowerparticlesizedistributionwithahighyield.

Infutureworks,thegeneratednanoparticlesandagglomerates

ofnanoparticleswillbedispersedinabiobasedpolymermatrixby

plasticmanufacturingprocessestoproducenanostructured

assem-blies.

Acknowledgement

Authorsgratefullyacknowledge thetechnicalworkofBruno

Boyer.Thispaperisdedicatedtothe memoryofouresteemed

colleagueElisabethRodierwholeftusmuchtooearly.

References

[1]P.Bordes,E.Pollet,L.Avérous,Nano-biocomposites:biodegradablepolyester/ nanoclaysystems,ProgressinPolymerScience34(2009)125–155.

[2]G. Crini, P.M. Badot, E. Guibal, Chitine et chitosane. Du biopolymère à l’application, Presses Universitaires de Franche-Comté, Besanc¸on, 2009.

[3]M.C.Cruz-Romero,T.Murphy,M.Morris,E.Cummins,J.P.Kerry,Antimicrobial activityofchitosan,organicacidsandnano-sizedsolubilisatesforpotentialuse insmartantimicrobially-activepackagingforpotentialfoodapplications,Food Control34(2013)393–397.

[4]I.Leceta,P.Guerrero,I.Ibarburu,M.T.Due˜nas,K.delaCaba,Characterization andantimicrobialanalysisofchitosan-basedfilms,J.FoodEngineering116 (2013)889–899.

[5]L.Y.Zheng,J.F.Zhu,Studyonantimicrobialactivityofchitosanwithdifferent molecularweights,CarbohydratePolymers54(2003)527–530.

[6]M.N.V.RaviKumar,Areviewofchitinandchitosanapplications,Reactiveand FunctionalPolymers46(2000)1–27.

[7]M.Dash,F.Chiellini,R.M.Ottenbrite,E.Chiellini,Chitosan–aversatile semi-syntheticpolymerinbiomedicalapplications,ProgressinPolymerScience36 (2011)981–1014.

[8]P.He,S.S.Davis,L.Illum,Chitosanmicrospherespreparedbyspraydrying, InternationalJ.Pharmaceutics187(1999)53–65.

[9]J.Akbu˘ga,G.Durmaz,Preparationandevaluationofcross-linkedchitosan microspheres containing furosemide, International J. Pharmaceutics 111 (1994)217–222.

[10]H.Q.Mao,K.Roy,V.L.Troung-Le,K.A.Janes,K.Y.Lin,Y.Wang,J.T.August, K.W.Leong,Chitosan–DNAnanoparticlesasgenecarriers:synthesis, char-acterization and transfection efficiency, J. Controlled Release 70 (2001) 399–421.

[11]G.Weibel,C.K.Ober,ProcessingpolymersinsupercriticalCO2,in:K.H.Buschow,

R.W.Cahn,C.M.Flemings,B.Ilschner,E.J.Kramer,S.Mahajan(Eds.), Ency-clopediaofMaterials:ScienceandTechnology,Elsevier,NewYork,2002,pp. 1–8.

[12]J.Fages,H.Lochard,J.-J.Letourneau,M.Sauceau,E.Rodier,Particlegeneration ofpharmaceuticalapplicationsusingsupercriticalfluidtechnology,Powder Technology141(2004)219–226.

[13]E.Reverchon,A.Antonacci,Chitosanmicroparticlesproductionby supercriti-calfluidprocessing,IndustrialandEngineeringChemistryResearch45(2006) 5722–5728.

[14]Y.B. Shen, Z.Du, Q. Wang, Y.X. Guan, S.J. Yao, Preparation ofchitosan microparticleswithdiversemolecularweightsusingsupercriticalfluidassisted atomizationintroducedbyhydrodynamiccavitationmixer,Powder Technol-ogy254(2014)416–424.

[15]M.X.Weinhold,J.C.M.Sauvageau,N.Keddig,M.Matzke,B.Tartsch,I.Grunwald, C.Kübel,B.Jastorff,J.Thöming,Strategytoimprovethecharacterizationof chi-tosanforsustainablebiomedicalapplications:SARguidedmulti-dimensional analysis,GreenChemistry11(2009)498–509.

[16]D.G.Rao,Studiesonviscosity–molecularweightrelationshipofchitosan solu-tions,J.FoodScienceandTechnology30(1993)66–67.

[17]H.M.Kwaambwa,J.W.Goodwin,R.W.Hughes,P.A.Reynolds,Viscosity, molec-ularweightandconcentrationrelationshipsat298Koflowmolecularweight

(12)

andEngineeringAspects294(2007)14–19.

[18]L.Diamond,N.Akinfiev,SolubilityofCO2inwaterfrom−1.5to100◦_C_and_from

0.1to100MPa:evaluationofliteraturedataandthermodynamicmodelling, FluidPhaseEquilibria208(2003)265–290.

[19]C.Peng,J.P.Crawshawa,G.C.Maitlanda,J.P.MartinTruslera,D.Vega-Mazab, ThepHofCO2-saturatedwaterattemperaturesbetween308Kand423Kat

pressuresupto15MPa,J.SupercriticalFluids82(2013)129–137.

[20]A.J.Read,Thefirstionizationconstantofcarbonicacidfrom25to250◦_C_and

to2000bar,J.SolutionChemistry4(1)(1975)53–70.

[21]M.Kasaai,Areviewofseveralreportedprocedurestodeterminethedegree ofN-acetylationforchitinandchitosanusinginfraredspectroscopy, Carbohy-dratePolymers71(2008)497–508.

demicPublishers,Dordrecht/Boston/London,1997.

[23]S.Puttipipatkhachorn,J.Nunthanid,K.Yamamoto,G.E.Peck,Drugphysicalstate anddrug–polymerinteractionondrugreleasefromchitosanmatrixfilms,J. ControlledRelease75(2001)143–153.

[24]M.Jaworska,K.Sakurai,P.Gaudon,E.Guibal,Influenceofchitosan char-acteristics onpolymer properties.I: crystallographicproperties,Polymer International52(2003)198–205.

[25]J.Nunthanid,A.Laungtana-Anan,P.Sriamornsak,S.Limmatvapirat,S. Puttipi-patkhachorn,L.Y.Lim,E.Khor,Characterizationofchitosanacetateasabinder forsustainedreleasetablets,J.ControlledRelease99(2004)15–26.

[26]M.Rinaudo,Chitinandchitosan:propertiesandapplications,Progressin Poly-merScience31(2006)603–632.