Magnetic microgels with pH- and thermo-responsive properties were developed from the pectin maleate, N-isopropyl acrylamide, and Fe3O4 nanoparticles. The hybrid materials were characterized by infrared spectroscopy, scanning electron microscope coupled with X–ray energy dispersive spectroscopy, wide angle X–ray scattering, Zeta potential, and magnetization hysteresis measurements.
Trang 1jo 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
Elizângela A.M.S Almeidaa, Ismael C Bellettinid, Francielle P Garciae,
Maroanne T Farinácioa, Celso V Nakamurae, Adley F Rubiraa, Alessandro F Martinsb,c,∗,
Edvani C Muniza,b
a Grupo de Materiais Poliméricos e Compósitos (GMPC), Departamento de Química, Universidade Estadual de Maringá-UEM, 87020-900 Maringá-PR, Brasil
b Programa de Pós-graduac¸ ão em Ciência e Engenharia de Materiais (PPGCEM), Universidade Tecnológica Federal do Paraná (UTFPR), 86036-370
Londrina-PR, Brasil
c Programa de Pós-graduac¸ ão em Engenharia Ambiental (PPGEA), Universidade Tecnológica Federal do Paraná (UTFPR), 86812-460 Apucarana-PR, Brasil
d Departamento de Química, Universidade Federal de Santa Catarina–UFSC, 89065-300 Blumenau-SC, Brasil
e Laboratório de Microbiologia Aplicada aos Produtos Naturais e Sintéticos, Departamento de Ciências Básicas da Saúde, Universidade Estadual de
Maringá–UEM, 87020-900 Maringá-PR, Brasil
a r t i c l e i n f o
Article history:
Received 12 February 2017
Received in revised form 10 April 2017
Accepted 9 May 2017
Available online 11 May 2017
Keywords:
Pectin
Magnetite
Poly(N-isopropyl acrylamide)
Curcumin
Release
a b s t r a c t
MagneticmicrogelswithpH-andthermo-responsivepropertiesweredevelopedfromthepectinmaleate, N-isopropylacrylamide,andFe3O4nanoparticles.Thehybridmaterialswerecharacterizedbyinfrared spectroscopy,scanningelectronmicroscopecoupledwithX–rayenergydispersivespectroscopy,wide angleX–rayscattering,Zetapotential,andmagnetizationhysteresismeasurements.Curcumin(CUR) wasloadedintothemicrogels,andreleaseassayswerecarriedoutinsimulatedenvironments(SGF andSIF)atdifferentconditionsoftemperature(25or37◦C).AslowandsustainabilityCURreleasewas achievedunderexternalmagneticfieldinfluence.LoadedCURdisplayedstability,bioavailabilityand greatersolubilityregardingfreeCUR.Besides,thecytotoxicityassaysshowedthatmagneticmicrogels withoutCURcouldsuppresstheCaco-2cellsgrowth.So,thepectinmaleate,N-isopropylacrylamide, andFe3O4couldbetailoredtoelicithybrid-basedmaterialswithsatisfactoryapplicationinthemedical arena
©2017ElsevierLtd.Allrightsreserved
1 Introduction
Curcumin(CUR)iswidelyappliedinthebiomedicaland
phar-maceutical fields owing to its anti-inflammatory capacity, and
renownedpotentialtotreatcysticfibrosis,Alzheimer’sdiseaseand
manycancertypes(Maheshwari,Singh,Gaddipati,&Srimal,2006)
However,theclinicalapplicationofCURislimitedbecauseofits
lowwatersolubilityandweakoralbioavailability,aswellaslack
stabilityunderneutralandmildalkalineconditions(Tangetal.,
2010).Theseshortcomingsshouldbeovercometoachievean
effi-cientCURapproach.Microencapsulationcouldbeusedtoconceive
CURapplicability,andmanystudieshavebeendepictedtheuseof
liposomesandpolymericcomposites(beads,particles,andfilms)
∗ Corresponding author at: Programa de Pós-graduac¸ ão em Ciência e
Engen-haria de Materiais (PPGCEM), Universidade Tecnológica Federal do Paraná (UTFPR),
86036-370 Londrina-PR, Brasil.
E-mail address: afmartins50@yahoo.com.br (A.F Martins).
toimproveCURefficacy(Chenetal.,2009;Li,Ahmed,Mehta,& Kurzrock,2007;Martinsetal.,2013;Shietal.,2007)
Polymericmaterialsoftenimpartbiocompatibility, biodegrad-ability, and non-toxicity (Abolmaali, Tamaddon, & Dinarvand,
2013) to the composite materials Regarding the drug deliv-eryapproaches,polymericcompositesactasprotectivematrices, repressnon-specificinteractionswithbiologicalmolecules,foster controlledandsustainedrelease,andenhancethebioavailability and stability of the loadeddrug(Brewer, Coleman, &Lowman,
2011) Composite materials may increase the CUR solubility, decreasingitscrystallinitybecauseoftheestablishmentof new interactionsbetweenpolymerchainsandCUR(Facchietal.,2016) Smartpolymericdevicesaredevelopedfocusingresponsiveness
toanexternalmagneticfieldaswellaschangesintemperature andpH(Patraetal.,2015).Thesepropertiesaremainlyrequired fordrugdeliverypurposes.Biocompatibleandsuperparamagnetic iron nanoparticles(SPIONs) have beenhighlighted due totheir renowned magnetization property A magnetic-based material, containingaloadeddrugcanadjustthereleaserateunderexternal
http://dx.doi.org/10.1016/j.carbpol.2017.05.034
0144-8617/© 2017 Elsevier Ltd All rights reserved.
Trang 2260 E.A.M.S Almeida et al / Carbohydrate Polymers 171 (2017) 259–266
Experimental conditions used to create the microparticles, loaded iron atom levels (g g −1 ) and Fe 3 O 4 encapsulation efficiencies (EE%).
magneticfield influence(Patraetal.,2015).Biopolymersshould
improvetheSPIONsbiocompatibility(Patel,Kumar,Jayawardana,
Woodworth,&Yuya,2014)and stability(Kim,Kim,Kim,&Lee,
2009),featuringpotentialfortargetdelivery(Menegucci,Santos,
Dias,Chaker,&Sousa,2015),magneticresonanceimaging(MRI)
(Songetal.,2015)andhyperthermiapurposes(Kimetal.,2009)
Overall,metallicnanoparticles(MNp)arecoveredbybiopolymer
layersorencapsulatedintoapolymericmatrix.Polymernetworks
stabilizetheMNpbysterichindrance,suppressingtheaggregation
duetothechangesofpHandionicstrength(Tiwari,Mishra,Mishra,
Arotiba,&Mamba,2011).TemperatureandpH-responsivenessare
alsoparamountforacquisitionoftargetdeliverydevices
Poly(N-isopropylacrylamide)(PNIPAAm)isathermo-responsivepolymer
widelystudiedbecausecontainsaLowerCriticalSolution
Temper-ature(LCST)around32◦C,justbelowhumanbodytemperature
Therefore,thePNIPAAmmaybeassociatedwithpolymericsystems
toyieldtargetdeliveryapproacheswithtemperaturesensibility
(Jaiswal,Banerjee,Pradhan,&Bahadur,2010)
Pectin (AP) is a polysaccharide formed by galacturonicacid
units, identified as a structural component of plant cell walls
It is found in leaves, fruits, flowers, roots, and seeds
Pectin-based hydrogels have been used in pharmacy because of the
biocompatibility,biodegradability,non-toxicityandpH-responsive
properties (Maxwell,Belshaw, Waldron, & Morris, 2012) Also,
theAPdecreasesthebloodcholesterollevel(Brounsetal.,2012;
Terpstra,Lapre,deVries, &Beynen, 1998), reducestheglucose
uptake (Kim, 2005), and supplies anti-tumor activity (Maxwell
etal.,2012;Zhang,Xu,&Zhang,2015).Inanaturalcondition,
galac-turonicacidunitsoverAP-basedhydrogelsareionized,permitting
repulsionamongthem,andhenceswellingandreleaseofdrugsby
diffusion.So,theseAP-systemsmaypromoteasatisfactorydrug
deliveryinthecolonplace,favoringtheuptake(Liu,Fishman,Kost,
&Hicks,2003;Wong,Colombo,&Sonvico,2011)
ThedevelopmentofmagneticmaterialswithpHandthermal
responseswasproposedinthis study.ThePNIPAAmgraftedon
thepectinmaleate(AP–MA),andFe3O4 wereassociatedtoelicit
microgels usingoil/water emulsion (O/W) and poly(vinyl
alco-hol)(PVA)as a stabilizingagent TheAP-MA synthesis, aswell
asitsphysicochemicalproperties,werepreviouslyreportedina
recentpaperpublishedbyourresearchgroup(Almeidaetal.,2014)
Hybridmaterialswereloadedwithcurcumin(CUR),and release
testsassessedasafunctionofanexternalmagneticfieldat
differ-entconditionsofpHandtemperature.Thisworkwilldemonstrate
thatas-obtainedmicrogelsbasedonAP–MA/PNIPAAm/Fe3O4can
killCaco-2cellsandtoprovideasuitablematrixforCURdelivery
Inthiscase,AP–MA/PNIPAAm/Fe3O4compositemayimprovethe
solubility,stability,andbioavailabilityoftheCUR
2 Materials and methods
2.1 Materials
Apple pectin (Mw=2.9×105gmol−1), sodium
persul-fate (98%), iron oxide (Fe3O4, 98%) of particle size up to
50nm and curcumin (Turmeric, ≥65%) were purchased
from Sigma-Aldrich (Brazil) Maleic anhydride (99%) and
N,N,N’,N’–tetramethylethylenediamine (TEMED, 99%) were
sup-pliedbyVetec(Brazil).Poly(vinylalcohol)(PVA,88%hydrolyzed with Mw=2.2×104gmol−1) and N–isopropyl acrylamide (99%) wereacquiredfromAcrossOrganics(Brazil).Otherreactantsalso utilizedinthiswork,wereofanalyticalgradeandusedasreceived
2.2 Pectinmaleate/poly[N–isopropylacrylamide]/Fe3O4 microparticlessynthesis
Firstly,theAP-MAwithasubstitutiondegreeof24%was syn-thesizedfromapplepectin(Mw=2.9×105gmol−1)accordingto thepreviouslyprocedurepublishedbyourresearchgroup(Almeida
etal.,2014).Then,formicroparticlessynthesis,theaqueousphase (W)containingAP–MA(2.5wt.%)andPVA (3.0wt.%)wasadded
to the oil phase (benzyl alcohol; BnOH) containing an appro-priateFe3O4 content(Table1 keepingthevolumeratioat1/3 (W/BnOH).TheemulsionwaspreparedintoicebathunderN2(g) fromthesonicationmethod(HielscherUltrasonicProcessor,model UP200S)at 100%amplitude for 5min.Then, suitable quantities
ofNIPAAm(2.0or10wt.%,Table1 sodiumpersulfate(1.5wt.%) and TEMED (1.0wt.%)wereadded totheemulsion, maintained thesonication for more 15min.The suspension containingthe AP–MA/PNIPAAm–Fe3O4wasprecipitatedinacetone,centrifuged, washedfivetimesalsowithacetone,stockedundervacuum(24h) and lyophilized (Mauricio, Guilherme, Kunita,Muniz, & Rubira,
2012).Theexperimentalproceduresaresummarized inTable1 ThemicroparticleswillbecalledasAP–MA/PNIPAAm(x)–Fe3O4(y),
where “x” and “y” correlate the NIPAAmand Fe3O4 levels(%), respectively
2.3 Characterization SpectraoftheAP-AMandAP-AMgraphitizedwithPNIPAAm (SupplementaryMaterial;Fig.S1)wereperformedinaVarian Mer-curyPlus300BBNMRspectrometer,operatingat300.06MHzfor
1Hfrequency.10mgofthesamplesweredissolvedin1.0mLD2O, containingTMSas reference.1HNMR spectrawereacquiredat roomtemperatureandthemainacquisitionparameterswereas follows:pulseof90◦,recycledelayof30sandacquisitionof64 transients Measurements of Infrared Spectroscopy (FTIR) were recordedusinga FourierTransform InfraredSpectrophotometer (ShimadzuScientificInstruments,Cary630Model),operatingfrom
500to2000cm−1,ataresolutionof4cm−1after64scans.Scanning ElectronMicroscope(SEM) coupledwith X–Ray Energy Disper-siveSpectroscopy(EDS)wasusedtoevaluatethemorphologyand
topredict thematerial composition, usinga Shimadzu appara-tus(SS550model).Averagediametersofthemicroparticleswere assessedbySEMimagesusingthesoftwareSizeMeter©,version 1.1.TheLCSTwasevaluatedbyZetapotentialmeasurementsat differenttemperatureconditions.Microgelsuspensionswere car-riedoutinaphosphatebuffersolution(pH7.0)at1.0mgmL−1and placedinaZetasizerNanoZSapparatusatdifferenttemperatures
aswell.The magnetizationwasestimatedusing a magnetome-ter (VSM),at 100Hz frequency under33Oe s−1 field scan rate (25◦C).Theamountofironintothemicroparticleswasassessed
byFlameAtomicAbsorptionSpectroscopy(FAAS),throughaVarian AA–175model(USA).WideangleX–rayscattering(WAXS)profiles
Trang 3Fig 1. SEM images (left panel) and size distribution of the microparticles (right panel): (a, b and c) AP–MA/PNIPAAm(10)–Fe 3 O 4 (1), (d, e and f) AP–MA/PNIPAAm(2)–Fe 3 O 4 (10) and (g, h and i) AP–MA/PNIPAAm(10)–Fe 3 O 4 (10).
(2=5–70◦)wereobtainedfromaShimadzuXRD–600apparatus
(Japan),equippedwithaNi-filteredCu-K␣radiation
2.4 Curcuminloading
Curcumin(CUR; 1.0mg)wassolubilized in ethanol (100mL)
and, then 1.0g AP–MA/PNIPAAm(10)–Fe3O4(1) was added into
theCUR-solution(Martinsetal.,2013).Thesuspensionwaskept
under stirring for 20h (25◦C), avoiding light exposure After,
the loaded microparticles were centrifuged, dried under
vac-uum (24h) and lyophilized The entrapped CUR was assessed
at=430nm,usinganUV–visspectrophotometer(Femto800Xi
model)fromastandardcurverangingfrom0.080to5.0mgL−1;
y=−9.9044×10−4+0.13384x(R2=0.999)
2.5 Curcuminrelease
DifferentenvironmentswereusedtoevaluatetheCURrelease:
simulatedintestinalfluid(SIF;pH6.8)andsimulatedgastricfluid
(SGF;pH1.2)(Buenoetal.,2015).Thestudieswereperformedinan
USPtypeIdissolutorapparatus(EthikTechnology,299–6TSmodel
−Brazil)undermechanicalstirring(40rpm)at25◦Cor37◦Catthe
presenceorabsenceofthemagneticfield(63.8Oe).So,0.10gof
driedAP–MA/PNIPAAm(10)–Fe3O4(1)/CURwasdepositedin
cellu-losemembranes(12kDasizepore),containingSGForSIF(30mL)
Thesystem wasstored in a sealed flask,containing220 mLof
SGForSIF,avoiding thelight exposure For assessingthe
mag-neticfieldinfluence,anexternalmagneticfieldwascreatedbya
magnetplacedoutsidethesealedflaskcontainingthedialysisbag
ThereleasedCURwasdeterminedbyUV–visabsorbance(=430)
removing3.0mLaliquotsatappropriatetime intervals(Martins
etal.,2013).ThefractionofreleasedCURwasevaluatedfromEq
(1)
Relesead fraction= relesead amount
2.6 Cytotoxicityassay CytotoxicitytestswereassessedagainstVEROcells(kidneycells
ofAfricangreenmonkey)and Caco-2cells(humancolonic ade-nocarcinomacells),fromthesulforhodamineBmethod(Almeida
etal.,2014).ThecellswereculturedandmaintainedinDulbecco’s modifiedEagle’smedium(DMEM;Gibco®,GrandIsland,USA), sup-plementedwith10%heat-inactivatedfoetalbovineserum(FBS; Gibco®)and50gmL−1gentamycin,inanincubatorat37◦C,with 5%CO2and95%relativehumidity.Cellswereobtainedatadensity
of2.5×105 cellsmL−1aftertrypsinizationandaddedto96–well platefor24hatthesameconditionsdescribedpreviously.Afterthe adherence,afixedvolumeofeachmicroparticlesuspensionat dif-ferentconcentrations(10,100,500and1000gmL−1)wasadded intothe96–wellplateandincubatedfor48h.Thecellulartoxicity (CC50)isdefinedwhenthesamplelevelkills50%ofthetreatedcells regardingthecontrol(untreatedcells)
3 Results and discussion
3.1 Characterization Themethodologyusedtosynthesizethemicrogelswasbasedon inversepolymerizationtechnique(Mauricioetal.,2012).1HNMR analysisindicatedthatPNIPAAmwasgraftedintheAP–MAchains Theresonancesignalsrangingfrom6.5to6.2ppmonAP–MA1H NMRspectrum wasattributedtothehydrogenatomsinvinylic
Trang 4262 E.A.M.S Almeida et al / Carbohydrate Polymers 171 (2017) 259–266
Fig 2. (a) FTIR spectra of the AP–MA/PNIPAAm(2), AP-MA and AP–MA/PNIPAAm(2)–Fe 3 O 4 (10) (left panel) (b and c) EDS spectra and WAXS profiles, respectively: (i) AP–MA/PNIPAAm(10)–Fe 3 O 4 (1), (ii) AP–MA/PNIPAAm(2)–Fe 3 O 4 (10), and (iii) AP–MA/PNIPAAm(10)–Fe 3 O 4 (10) (right panel).
Fig 3.(a) Paramagnetic response based on the hysteresis curves (b) Zeta potential measurements as a function of the temperature Sample codes: (i) AP–MA/PNIPAAm(10)–Fe 3 O 4 (1), (ii) AP–MA/PNIPAAm(2)–Fe 3 O 4 (10), and (iii) AP–MA/PNIPAAm(10)–Fe 3 O 4 (10).
moieties(SupportingMaterial,Fig.S1).Thesesignalsdisappeared
intheAP–MA/PNIPAAm1HNMRspectrum,indicatingthatNIPAAm
graftingonAP–MAwassuccessful.Suchgraphitizationwasalso
confirmedduetotheNIPAAmsignalsintheAP–MA/PNIPAAm1H
NMRspectrumat3.0and2.7ppmascribedtothehydrogenatoms
on CHand CH2,respectively(SupportingMaterial,Fig.S1)(Leal,
DeBorggraeve,Encinas,Matsuhiro,&Muller,2013)
The ultrasound waves ensured the obtaining of
multi-ple and stable oil emulsion seeking to create microparticles
(Reis et al., 2011) The cavitation energy induced by the
sonication led to the formation of rough, polydisperse and
spherical microparticles with small fractures and deformities,
due to the water randomly movements into the oil phase
drops (Fig 1) (Reis et al., 2011) The average diameters of
AP–MA/PNIPAAm(10)–Fe3O4(1), AP–MA/PNIPAAm(2)–Fe3O4(10)
andAP–MA/PNIPAAm(10)–Fe3O4(10)wereca.10,26,and20m,
respectively(Fig.1).Thesizeandpolydispersityofthemicrogels
wereraisedwhenthemagnetitelevelwasalsoimproved(from1.0
upto10wt.%;Table1 whiletheNIPAAmcontentdidnotchange thesefeaturessignificantly
AP–MA/PNIPAAm(2)–Fe3O4(10) FTIR spectrum was assigned
toFe–OstretchingduetotheFe3O4load(Fig.2a)(Mauricioetal.,
2012).Thebandat1740cm−1onAP–MAFTIRspectrumattributed
toC Ostretchingofcarboxylicacidswasshiftedfor1750cm−1
in theAP–MA/PNIPAAm(2)–Fe3O4(10)spectrum, suggestingthe establishmentofnewinteractions(Mauricioetal.,2012).Overall, alltheFTIRspectrapredictedinFig.2aweresimilarbetweenthem Themagnetitestabilizedtheemulsion(daSilvaetal.,2014;Fang
etal.,2014)becausemicroparticlesbasedonAP–MA/PNIPAAm(2) wasnotobtained(SupportingMaterial, Fig.S2).TheFe3O4 may neutralizetheexcessofnegativechargesovertheAP–MAchain segmentsbyhindranceeffectsowingtotheestablishmentofnew bondsamong COO−andFe(Fangetal.,2014).TheEDSspectra
Trang 5Fig 4. Fraction of released curcumin (%) from the AP–MA/PNIPAAm(10)–Fe 3 O 4 (1) at SIF and SGF under or not magnetic field influence: (a) at 25 and (b) at 37 ◦ C (c) Cytotoxicity effects against Caco-2 and VERO cells: (i) AP–MA/PNIPAAm(10)–Fe 3 O 4 (1), (ii) AP–MA/PNIPAAm(2)–Fe 3 O 4 (10) and (iii) AP–MA/PNIPAAm(10)–Fe 3 O 4 (10) Error bars represent the standard deviation of triplicates (n = 3).
displayedtheironincidenceinthemicrogelsbecauseofthepeaks
at6.4and7.1keV(Fig.2b).Evenmore,theWAXSprofilesexhibited
characteristicdiffractionpeaksassigned totheFe3O4 crystalline
planesat2=30◦(220),35◦(311),43◦(400),54◦(422),57◦(511)
and 63◦ (440) (Fig.2c)(Purushotham &Ramanujan, 2010).The
AP–MA/PNIPAAm(10)–Fe3O4(1)WAXSpatternencompassed
low-ereddiffractionpeaksregardingtheothersamplesduetotheless
levelofloadediron(0.58×10−2gg−1;Table1)
Fig 3a displayed the paramagneticbehaviors of the
micro-gels, and in Table 1 was depicted the content of iron
(g−1) loaded in the microparticles The magnetization of the
AP–MA/PNIPAAm(10)–Fe3O4(1), AP–MA/PNIPAAm(2)–Fe3O4(10)
and AP–MA/PNIPAAm(10)–Fe3O4(10) reached0.68 ± 0.01, 6.20
± 0.10 and 4.20±0.05emug−1, respectively Polymernetworks
shouldscreentheironatoms,suppressingthemagneticresponse
(Fangetal.,2014).TheAP–MA/PNIPAAm(2)–Fe3O4(10)achieved
thehighest magnetic response (6.20±0.10emug−1)due tothe
largeFe3O4content(4.7×10−2gg−1;Table1).TGAanalysis
rein-forcedthisfactsincetheAP–MA/PNIPAAm(2)–Fe3O4(10)imparted
greaterresidualmassabove450◦C, owingtotheFe3O4 content
(SupportInformation,Fig.S3)
The thermo-sensitive feature was evaluated by Zeta
poten-tial()analysisatdifferenttemperatureconditions,rangingfrom
25 to 50◦C (Fig 3b) For all samples, the achieved −10 to
−25mVat pH 7.0 owing to the COO sites onAP-MA chains
Thevaluesdecreasedathighertemperaturesbecausethe
sus-pension was collapsed and conceived the LCST event (Pietsch
et al., 2012) The LCST for AP–MA/PNIPAAm(2)–Fe3O4(10)was
allowedat37◦C,whereasforAP–MA/PNIPAAm(10)–Fe3O4(10)and
AP–MA/PNIPAAm(10)–Fe3O4(1)wascloseto43◦C.Theless PNI-PAAmcontentgraftedinAP–MA/PNIPAAm(2)–Fe3O4(10)lowered theLCST.AnLCST-typeabovethebodytemperature(≈37◦C)can
allowremarkableefficiencytothecompositematerialstoactas drugdeliveryvehicles(Fangetal.,2014;Jaiswaletal.,2010)
3.2 Curcumin(CUR)release The AP-MA/PNIPAAm(10)-Fe3O4(1) sample was chosen as a drugcarriermatrixduetoitshigherLCST(43◦C)andsmaller aver-agesize(≈10m).TheCURencapsulationefficiency(EE)reached 60%.Releaseassayswereassessedinsimulatedfluids(SIForSGF)
at25or37◦C,under(ornot)magneticfieldinfluence.InSIF(25◦C) andwithoutmagneticfieldinfluence,theequilibriumwasreached after35hwhen50%CURwasreleased.Ontheotherhand,inthe samecondition,butundermagneticfieldinfluence,the equilib-riumwaspermittedat80h,designing aslowed,modulatedand sustainedCURrelease(90%)(Fig.4a).InSGF(25◦C),theamount
ofCURreleasedwasnothigherthan10% forallevaluated con-ditions(Fig.4a).Theexpositiontothemagneticfieldmayinduce heatinsidetheAP–MA/PNIPAAm(10)-Fe3O4(1),decreasingthe sta-bilityandhenceenhancingCURreleaserate(Kumar&Mohammad, 2011;Patraetal.,2015).At37◦C,themagneticfielddidnot signif-icantlychangetheprofileofCURrelease(Fig.4b).InSIF(37◦C),the releasedfractionachieved95and80%inthepresenceorabsence
ofmagneticfield,respectively.InSGF(37◦C)thecontentofCUR releasedwaslower,featuring20%withoutand6%withmagnetic fieldinfluence
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Kinetic parameters (n, k, ␣, and kr) obtained by application of Ritger-Peppas and Reis et al mathematical models on the CUR release curves evaluated in SIF.
a CUR release under magnetic field influence − 1st and 2nd refer to first- and second-order kinetics.
TheamountofCURreleasedroseat37◦CduetothePNIPAAm
TheAP–MA/PNIPAAm(10)-Fe3O4(1)LCSTwasroughly43◦C,and
releasetestswerecarriedoutat37◦C(Fig.4b).Thesetemperature
conditionswereclose,andsomePNIPAAmchainscouldbecome
hydrophobic,causingpolymernetwork collapsesandenhancing
thereleasedlevelofCUR,especiallyinSIF(37◦C).Thesefeaturesare
alsoassociatedwithhydrogelpH-responsiveness.AtpH1.2(SGF),
allthecarboxylicgroupsonAP–MAareentirelyprotonated,
hinder-ingtheinteractionswithwatermolecules.Thisfactsuppressedthe
CURrelease.However,atpH6.8(SIF)thecarboxylicsitesarefully
ionized( COO− improvingnegativechargedensity,aspredicted
bymeasurements.Then,thewatermoleculesinteractbetterwith
thehybridmaterial,favoringtheCURdelivery.Fe3O4
nanoparti-clescausetortuosityintoapolymericmatrix,eliminatingtheburst
releaseofCUR(daSilvaetal.,2014)
CURcomprisescrystallinearrangementandhaslackwater
sol-ubility(≈11ngmL−1inabuffersolutionatpH5.0)(Martinsetal.,
2013) In this study, the solubility and stability of the loaded
CURwereimproved AP-MA/PNIPAAm(10)-Fe3O4(1)
microparti-clesprotectedtheCURintegrityallowingsustainedreleaseatpH
closetophysiologicalcondition.Accordingtotheassayperformed
at25◦C(SIFundermagneticfieldinfluence),thesolubilityofloaded
CURreached216gmL−1.Besides,theCURwasnotdegradedatpH
6.8(SIF),anditsbioavailabilitywasalsoenhanced.Thesefindings
opennewstrategiesfordevelopingofCUR-basedmaterialswith
applicationinthemedicalfield
3.3 Transportmechanism
Thereleaseofsolutesfromaflexiblematrixmaybedescribed
bydiffusiontransport(Ritger&Peppas,1987).Ritger-Peppas
pro-posedasemi-empiricalmathematicalmodeltoexplaintherelease
mechanismofsolutes(Eq.(1)(Ritger&Peppas,1987)
Mt
Where Mt/M∞ refers to the solute fraction released in the
desiredtimeinterval (t);k isthekineticconstant,anditsvalue
reliesonthefactorssuchassolvent,typeandshapeofthe
hydro-gel,aswellasdependsontheexperimentalconditions.InEq.(1),n
representsthediffusionexponentbeingassociatedwiththesolute
transport mechanism(Brazel&Peppas, 1999; Guilhermeet al.,
2010)and hydrogelgeometry.For n=1,themechanism is
gov-ernedbyazero–orderkineticandthereleaserateriseswiththe
timelinearlyandreliesonthemacromolecularrelaxation.Whenn
isroughly0.5,thereleasemechanismiscontrolledbyFickian
diffu-sion;fornvaluesrangingfrom0.5to1.0,theanomaloustransport
takesplacebecauseofthesimultaneousdiffusionand
macromolec-ularrelaxation.Regardingthesphericalmatrices,thenparameter
canassumethevalues:0.43(Fickiandiffusion),0.43–0.85
(anoma-lousornon-Fickian),and0.85(zero–orderkinetic)(Ritger&Peppas,
1987)
Thenandkparameterswereassessedfromthereleasecurves
performedinSIF(Table2).Fornvaluescloseto0.5,thereleasewas
explainedthroughnon-Fickiantransport(assayswithfractionsof releasedCURat80,90and95%;Figs.4a–b).However,forthetest with50%ofreleasedCUR,thelowernvalue(0.15)showedthat polymericmatrix/CURsetdidnothaveaffinitybytheSIF(assayat
25◦Cintheabsenceofmagneticfield)
Attheequilibriumcondition,thereleasedfraction(Fr)reachesa maximum(Fmax),andthetransportmechanismcanbetreatedasa diffusion-partitionphenomenon(Reis,Guilherme,Rubira,&Muniz,
2007).Forapplyingthismodel,theFrandFmaxparameterswere alsoassessedfromthecurvescarriedoutinSIF,andthekinetic vari-ables(kr)predictedforfirst–orderkinetic(Eq.(3)andsecond–order kinetic(Eq.(4))(Reisetal.,2007)weremeasuredaswell(Table2) Thepartitionactivity(␣coefficient)isdefinedwhenthesolute con-centrationreachesa constantvalue(Reisetal.,2007).For ␣>0, thesolutediffusiontakesplacebetweenthesolventandhydrogel phases,inferringbetteraffinitybetweensoluteandsolvent.When
␣=0thesolutereleasedoesnotoccur.Thereisnotanysolute parti-tionbetweenthesolventandhydrogelphases(Reisetal.,2007).All studiesshowed␣>0,indicatingthediffusion-partitionoccurrence (Table2)
krt=Fmaxln
max
Fmax−Fr
(3)
Krt=˛2ln
F
r−2FrFmax+Fmax
Fmax−Fr
(4) TheRitger–Peppasmodelisvalidforatimeintervalinwhich 60%soluteisreleased(Ritger&Peppas,1987),whilethe diffusion-partitionmodelcanpredictthereleasecurveprofileentirely(Reis
etal.,2007).TheRitger–Peppasmodelwasadjustedbettertothe experimentaloutcomesperformedat37◦C(R2≥0.940;Table2and Fig.S4).However,thediffusion-partitionmodelisingood accor-dancewiththeresultsintheintervalof60–160h(Fig.S4(e,f,g, andh))(Reisetal.,2007).Thereleaseassaysat37◦Cwerefollowed forasecond-orderkinetic(R2≥0.900;Table2)andthoseat25◦C werepredictedbyafirst-orderkinetic(R2≥0.964;Table2).All con-ditionscarriedout inSIFpinpointedlowand positivekr values, explainingthesustainedCURrelease(Table2)
3.4 Cytotoxicityassay CellviabilitytestsagainsttheCaco–2coloncancerandhealthy VEROcellswereevaluatedfromthemicroparticleswithoutCUR (Fig.4c).TheresultsindicatedthatAP–MA/PNIPAAm(10)–Fe3O4(1) imparted highest cytotoxic effect on the Caco–2 cells (65gmL−1CC50), while AP–MA/PNIPAAm(2)–Fe3O4(10) and AP–MA/PNIPAAm(10)–Fe3O4(10) CC50 achieved 215 and
435gmL−1, respectively The largest cytotoxicity found for AP–MA/PNIPAAm(10)–Fe3O4(1) towards Caco–2 cells could be attributedtothelowestloadedFe3O4amount(Table2).AP–MA (thepristinepectinmaleate)possessesgreaterinhibitoryactivity (CC50=25gmL−1)forCaco–2cellsaspreviouslyshowninwork publishedbyourresearchgroup(Almeidaetal.,2014).Theraw
APCC50reached140gmL−1.Fe3O4oxideandPNIPAAmcontents playsignificativeroleinthefindingsofcytotoxicity.Higherlevels
Trang 7the AP–MA/PNIPAAm(10)–Fe3O4(10) CC50 was improved for
435g mLư1 Reports suggest theantitumor activityof AP-MA
was related to anhydride hydrolysis, and hence the formation
ofhydrogen–bondedfragments(Gaoetal.,2016;Pascalauetal.,
2016;Zhanget al.,2016).TheFe3O4/PNIPAAmexcessmay
hin-der the establishment of H–bonds among polymer networks,
decreasingtheantitumoractivity(Almeidaetal.,2014;Karakus,
Yenidunya,Zengin,&Polat,2011)
Forhealthy VEROcells,theAP–MA/PNIPAAm(10)–Fe3O4(10),
AP–MA/PNIPAAm(2)–Fe3O4(10)andAP–MA/PNIPAAm(10)–Fe3O4(1)
CC50 were 370, 365and 375g mLư1, respectively So, allthe
microparticleswerenotcytotoxictowardsVEROcells.Itwas
fea-turedthatAP–MA/PNIPAAm/Fe3O4 setcouldbetailoredtoelicit
cytocompatibility for Caco-2 cells, stability, and bioavailability
for CUR, comprising an efficient device for CUR delivery The
cytotoxicityoftheAP–MA/PNIPAAm/Fe3O4/CURcompositeswere
notassessed.Manystudies,includingtwopaperspublishedbyour
group,depictedthat CUR-based composites couldsuppressthe
growthofcancerouscells(Martinsetal.,2013;Pereiraetal.,2013)
4 Conclusions
Anewhybridandmagneticmaterialbasedonmaleatepectin
(AP–MA),PNIPAAm andFe3O4 waspHand thermal-responsive
Thecurcumin(CUR)wasloadedintothemicroparticlesandrelease
findingswerefavoredinSIF.Besides,themagneticfieldinfluenced
thetestscarriedoutat25◦C.Overall,theCURreleasewasslowly
andsustainably.ThestabilityandbioavailabilityoftheCURwere
alsoimproved,andcytotoxicityassaysrevealedthat
microparti-cleswithoutCURwerecytocompatibleforhealthyVEROcells.For
diseasedCaco-2cells,thegrowthwassuppressedmainlyunder
AP–MA/PNIPAAm(10)–Fe3O4(1)presence(CC50=65gmLư1).The
AP–MA/PNIPAAm/Fe3O4featurescanbeusefultoyieldapotential
carrierdeviceforCURdelivery
Acknowledgements
E.A.M.S.A.thankstheCNPqforherMasterfellowship.E.C M
thankstoCNPq(Proc.400702/2012–6and 308337/2013–1)and
Nanobiotec(Proc.851/09)forfinancialsupports.Theauthorsalso
acknowledgeProfessors:L.C.Varanda(IQSCưUSP),D.R.Cornejo
(IFSCưUSP),I.A.dosSantos(DFIưUEM)andE.Radovanovic(DQI
ưUEM)fortheirhelpduringthedevelopingofthiswork
Appendix A Supplementary data
Supplementarydataassociatedwiththisarticlecanbefound,in
theonlineversion,athttp://dx.doi.org/10.1016/j.carbpol.2017.05
034
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