Different starch derivatives were evaluated as supports for attachment and recovery of macrophages (RAW 264.7 line). Gelatinized starch (G-St), acetate starch (Ac-St), carboxymethyl starch and aminoethyl starch were synthesized and characterized by FTIR, 1H NMR, SEM and static water contact angle.
Trang 1jo u r n al h om ep age :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
Khalil Sakeera, Tatiana Scorzab, Hugo Romerob, Pompilia Ispas-Szaboa,
Mircea Alexandru Mateescua,∗
a r t i c l e i n f o
Keywords:
Alpha-amylase
a b s t r a c t
Differentstarchderivativeswereevaluatedassupportsforattachmentandrecoveryofmacrophages (RAW264.7line).Gelatinizedstarch(G-St),acetatestarch(Ac-St),carboxymethylstarchandaminoethyl starchweresynthesizedandcharacterizedbyFTIR,1HNMR,SEMandstaticwatercontactangle.These polymersarefilmogenicandmaycoatwelltheholderdevicesusedformacrophageadhesion.Theyalso presentasusceptibilitytomildhydrolysiswithalpha-amylase,liberatingtheadheredmacrophages Cellcounts,percentageofdeadcellsandleveloftumornecrosisfactor(TNF-␣)wereusedtoevaluatethe possibleinteractionbetweenmacrophagesandstarchfilms.Thehighpercentageofcelladhesion(90–95%
onG-StandonAc-St)associatedwithenzymaticdetachmentofmacrophagesfromfilm-coatedinserts, resultedinhigherviabilitiescomparedwiththoseobtainedwithcellsdetachedbycurrentmethods scrappingorvortex.Thisnovelmethodallowsafastmacrophageseparation,withexcellentyieldsand highviabilityofrecoveredcells
©2017TheAuthor(s).PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Starch is widely used in food, pharmaceutical and
biomed-ical applications due to its biocompatibility, biodegradability,
non-toxicity and abundant sources (Rowe, Sheskey, Cook, &
Fenton,2009).Starchmodificationisgenerallyachievedthrough
derivatizationsuchascross-linking(Lenaertsetal.,1998),
etherifi-cation,esterification(Calinescu,Mulhbacher,Nadeau,Fairbrother,
&Mateescu,2005;Mulhbacher,Ispas-Szabo,Lenaerts,&Mateescu,
2001)andgrafting(Kaur,Singh,&Liu,2007)offunctionalgroups
onto the carbohydrate structure Such modifications can
pro-foundlyalterthephysicochemical andmorphologicalproperties
ofstarch,itsenzymaticdigestibilityandcanconsequently
mod-ulateitscurrent useasexcipientin drugdeliverydosageforms
(Mulhbacher,Ispas-Szabo,&Mateescu,2004;Massicotte,Baille,&
Mateescu,2008).Aninterestingreportedapplicationofstarchwas
itsuseforenrichmentofmacrophagecellpopulationsby
adhe-siononcross-linkedstarchmicrospheresfollowedbyliquefaction
ofmicrobeadswithalpha-amylase(Desmangles,Flipo,Fournier,&
Mateescu,1992).Macrophagesarecurrentlyinvestigatedin
iousbiochemicalandbiomedicalfieldsaswellasfortherapeutic applications (Kwan, Wu, &Chadban, 2014; Ostuni, Kratochvill, Murray,&Natoli,2015;Wooden&Ciborowski,2014;Youetal.,
2013).Macrophageswithapossibleroleininflammatoryprocesses andmalignancywerereportedasanewtherapeutictarget.There
isagrowinginterestfortechniquesofmacrophageseparation, par-ticularlytoinvestigateanti-macrophagesnovelstrategiesagainst cancer.Macrophagescanbeobtainedinarelativelypureformas primaryculturesforanalyticalandbiochemicalmanipulationsbut theydo notgenerallyreplicateinculture,haverelatively short-lives, and maybe difficultto obtainenough amounts for large scale.Theyareverysensitivetosmallchangesintheir environ-ment andmay bedamaged considerably,even when delicately handledaftercellculture(Adams,1979;Féréoletal.,2006) Detach-ingadherentmacrophagesfroma culturedish isdifficult,since thesecellsadhereavidlytoplasticsurfacesofcellculturedevices (i.e.Petri dishes,microplates) Severalprocedures arecurrently appliedtoregainmacrophagesuchasmechanicaldetachmentby gentlescrapingof macrophageswitha rubberpoliceman(Fleit, Fleit,&Zolla-Pazner,1984;Jaguin,Houlbert,Fardel,&Lecureur,
2013;Porcherayetal.,2005)orpre-treatmentwithscandicainK, proteinase,orpronase(Malorny,Neumann,&Sorg,1981),whichis limitativeasithasmitogeniceffectsonmacrophages.Frequently
bymechanicaldetachment,abouthalfofcellsmayremainviable
http://dx.doi.org/10.1016/j.carbpol.2017.01.053
Trang 2ofviablecellsaremajorlimitationsforexistingprocedures
Based onourpreviousseparation of macrophages by
reten-tiononacross-linkedstarchcolumnandfurtherdetachmentby
enzymatichydrolysisofthechromatographicsupport(Desmangles
etal.,1992),fourstarchmaterialsnamelygelatinizedstarch
(G-St), acetate starch (Ac-St), carboxymethyl starch (CM-St) and
aminoethylstarch (AE-St)were investigatedfor their abilityto
formfilmssusceptibletoamylolysistobeusedassubstrate/support
for macrophage separation by mild enzymatic amylolysis.This
approachisdifferenttothepreviousreportedmethod(Desmangles
etal.,1992)toseparatemacrophagesusingcross-linkedstarchas
achromatographicsupport.Ingeneral,cross-linkedmaterialsare
adequatetoformmicrospheresbutpresentlowerfilmogenicability
thantheuncross-linkedmaterials(Berezkin&Kudryavtsev,2015;
Krumova,López,Benavente,Mijangos,&Pere ˜na,2000).Amajor
objectiveofthisstudywastounderstandthecriticalroleofsurface
propertiesofstarchmaterialsontheattachmentofmacrophages
andconsequentlytheinfluencesontheirviability
2 Materials and methods
2.1 Materials
High amylose starch (Hylon VII) was supplied by National
Starch (Bridgewater, NJ, USA) Sodium monochloroacetic acid,
3,5-Dinitrosalicylicacid, sodiumpotassiumtartratetetrahydrate
(Sigma-Aldrich, Germany), d-(+)-Maltose monohydrate
(Sigma-Aldrich, Japan), amyloglucosidase (EC 3.2.1.3) from Aspergillus
niger ≥300U/mL (Sigma-Aldrich, Denmark), acetic anhydride
(Anachemia,Montreal,Canada),␣-amylase(EC3.2.1.1)from
Bacil-lus subtilis 402U/mg (Fluka, Switzerland), 2-chloroethylamine
hydrochloride (Fluka, Switzerland) were all used as received
without further purification CellTrackerTM Green CMFDA
(5-chloromethylfluoresceindiacetate)andpropidiumiodide
(Invitro-gen,UK),lipopolysaccharide(LPS,L3012,Sigma-Aldrich),TNFELISA
kitsfromBiolegend(San Diego,CA)were usedformacrophage
cellscharacterization.TheRAWmacrophagecells(ATCCTIB-71)
wereculturedinRPMI-1640mediumsupplementedwith10%fetal
bovineserumandantibiotics(PenicillinandStreptomycin)
Sub-cultureswerepreparedbygentlescrappingandaspirationpriorto
testinginstarchcoatedsupports
2.2 Preparationofstarchfilmogenicmaterials
Anamountof12.50gofHylonVIIwassuspendedforhydration
in 50mLof distilled water at60–70◦C under continuous
verti-cal stirring (ServodyneMixer, 50000-40, IL, USA) A volume of
75mLof5MNaOHwasaddedtothestarchsuspension,
contin-uing thestirringfor 60minat60–70◦C Thenthesolutionwas
cooleddown and neutralized withglacial acetic acid(untilpH
6.8)togetgelatinized starch (G-St).Thegelatinized starchwas
furtherderivatized either bydirect addition of 18.75mL acetic
anhydride,or byadditionof 18.75gsodiummonochloroacetate
or2-chloroethylaminehydrochloride(eachsolubilizedina
min-imalwatervolume)understirringandcontinuingthereactionfor
1hat60–70◦Ctoobtainacetate(Ac-St),carboxymethyl(CM-St),
oraminoethyl(AE-St)starchderivatives,respectively.Then,each
solutionwascooleddownandneutralizedwithglacialaceticacid
(toreachpH6.8).Thederivatizedstarchpowderswereobtainedby
precipitationfromthereactionsolutionwithanequivalentvolume
ofmethanol/water(70:30)v/vsolution.Forallstarchmaterials,the
processwasrepeateduntilafinalconductivityoffiltratedecreased
atabout50S/cm.Then,200mLofmethanol100%wereused,
fol-lowedby200mLofacetone100%forfinaldrying.Thecollected
powderswereleftatroomtemperatureforcompleteairdrying overnightandsievedtoobtainparticlesoflessthan300m 2.3 Evaluationofsubstitutiondegreeofderivatives
FortheCM-StandtheAE-St:thedegreeofsubstitution(DS) wasdeterminedbyback-titrationaspreviouslydescribed(Assaad, Wang,Zhu,&Mateescu,2011; Stojanovi ´c,Jeremi ´c, Jovanovi ´c,& Lechner, 2005) Briefly, 100mg of polymer were solubilized in
10mLof0.05MNaOHandthentheexcessofNaOHwastitrated (n=3)with0.05MHCl usingphenolphthalein asindicator The blank(20mLof0.05MNaOH)wasalsotitratedbythesamemethod ThedegreeofsubstitutionofAc-Stwasdeterminedtitrimetrically, followingthemethodofSodhiandSingh(2005)withminor mod-ifications.Acetylatedstarch(0.1g)wasplacedina25mLflaskand
6mLofDimethylsulfoxide(DMSO)wereadded.Theloosely stop-perflaskwasagitated,warmedto50◦Cfor30min,cooleddownand then4mLof0.05MKOHwereadded.Thealkaliexcesswas back-titratedwith0.05MHClusingphenolphthaleinasanindicator.The amountsof COOH, NH2and COCH3 groupsandtheDSwere calculated(Stojanovi ´cetal.,2005)usingthefollowingequations:
whereVb(mL)isthevolumeofHClusedforthetitrationofthe blank;V(mL)isthevolumeofHClusedforthetitrationofthe sam-ple;CHClistheconcentrationofHCl;162(g/mol)isthemolecular massofglucoseunit;W=(58or44or43)(g/mol)istheincreasein themassofglucoseunitbysubstitutionwithonecarboxymethyl, aminoethylandacetylgrouprespectively,andm(g)isthemassof drysample
2.4 Fouriertransforminfrared(FT-IR)analysis TheFT-IR spectraofsamplesas powderswererecorded(64 scansata4cm−1resolution)usingaThermo-Nicolet6700 (Madi-son, WI, USA) FT-IR spectrometer equipped with a deuterated triglycinesulfate-KBr(DTGS-KBr)detectorandadiamondsmart ATR(attenuatedtotalreflection)platform
2.5 1HNMRmeasurements The1HNMRspectrawerecollectedusingahigh-field600MHz BrukerAvanceIIIHDspectrometerrunningTopSpin3.2software and equipped with a 5mm TCI cryoprobe The temperature of sampleswasmaintainedat27◦C.Thesamplesweredissolvedin deuterateddimethylsulfoxide-d6 (DMSO-d6) withboth methyl groupsdeuterated,thenheatedat65◦Cfor30min,andkeptat4◦C for2h
2.6 Scanningelectronmicroscopy(SEM) Themorphologyoftheparticlesandfilmsurfacewereexamined
byaHitachi(S-4300SE/N)scanningelectronmicroscopewith vari-ablepressure(HitachiHighTechnologiesAmerica,Pleasanton,CA, USA)at5–7kVandmagnificationsof100and1000×forpowders andof500×and1000×forfilmsurface.Samplesweremountedon metalstubsandsputter-coatedwithgold
2.7 Filmcastingandmacrophageculture 2.7.1 Preparationoffilm-formingsolutionsofstarchmaterials Gelatinizedstarch(G-St),acetatestarch(Ac-St),carboxymethyl starch(CM-St)andaminoethylstarch(AE-St)havebeendispersed
Trang 3Scheme 1.Design of device and procedure with adhesion (1) and amylolysis (2) steps for fast recovery of macrophages.
at0.5%(w/v)inpurifiedwaterandheatedto95◦C.Thenthe
solu-tionswerecooleddowntoroomtemperatureandcentrifugedat
5000rpmfor2min.Foreachfilmformingmaterialthesupernatant
wascastonacellcultureinsertdevicewithabasefilterof
polyethy-leneterephthalate(PET)having3.0mporeaperture(BDFalcon
CellCultureInserts,353092,USA).Thesolutionwasevaporatedat
40◦Cfor12htoformthefilmcoatingoftheinsertdevice
2.7.2 Macrophageincubation
Beforeincubation theinsertand plates(Costar® 35166well
plate,USA)weresterilizedbyUV-rayfor15min.Then,macrophage
suspensionsinaRPMI-1640culturemediumcontainingFBS10%
andPenicillin/Streptomycin1x,wereincubatedfor48hina
humid-ifiedatmosphereofairand5%CO2at37◦C.Theculturemediumwas
introducedfromtheoutsideofcellcultureinsert(Scheme1)
2.7.3 Microscopy
Themorphology of macrophage cells was investigated after
incubationfor48hontothecellcultureinsertcoatedwithG-St,
CM-St,Ac-StorAE-St.Macrophageswerelabeledwithfluorescent
stainingCellTrackerTMGreenCMFDAandpropidiumiodide
follow-ingmanufacturerinstructions.CellswerevisualizedusingaNikon
EclipseTimicroscope(NikonCanada,Mississauga,ON)equipped
withphasecontrastandepifluorescenceoptics.Photomicrographs wereacquiredusing aDigital SightDS-Qi1Mccamera and NIS-Elements3.0software(NikonCanada)
2.7.4 Susceptibilitytoenzymatichydrolysisofstarchfilms Thefilmhydrolysiswasdoneinthreesteps:(a)Hydration step:
Culturemediumwasreplacedby40mMphosphatebufferpH7.4
at37◦Cinsideandoutsideofeachcellcultureinsert;(b) Liquefac-tion step:Asolutionofanalpha-amylase(EC3.2.1.1fromBacillus subtilis)in40mMphosphatebufferpH7.4(1000U/mL)wasused forliquefactionoffilmlayer.(c)Saccharification step:A40mM phosphatebufferpH7.4wasusedtodiluteamyloglucosidasefrom Aspergillusnigerupto(100U/mL)andthenusedfor saccharifica-tionofthestarchfilmspicesresultedfrompartialhydrolysiswith alpha-amylaseundergentleshakingfollowedbyincubationina humidifiedatmosphereofairand5%CO2 at37◦C(Aneja,2009; Lareoetal.,2013)
2.7.5 Determinationofenzymaticactivityonthestarch filmogenicsupports
Enzymaticactivityofalpha-amylasewasmeasuredonthesame filmamylolysisconditionsusingthedinitrosalicylic(DNS)method (Bernfeld,1955)tomeasurethereducingsugargroupsreleasedas
Trang 4Fig 2. 1H NMR spectra of Gelatinized starch (Red), Acetate starch (Green), Carboxymethyl starch (Blue) and Amino-Ethyl starch (Black).
resultofalpha1,4glycosidicgrouphydrolysis.Atdifferenttime
points a hydrolyzedsolution volumeof 0.5mLwaswithdrawn
immediately0.5mLofDNSreagentwasaddedtostopthe
hydrol-ysisreaction.Then,thereactionmediawereboiledfor 5minto
developthecolorofreduced3-amino-5-nitrosalicylicacid
Sub-sequently,after5minpreciselythesolutionswerecooledinan
ice-bathtoroomtemperatureand1mLofeach cooledsolution
wasdilutedwith4mLofdistilled water.Theabsorbanceofthe
finalsolutionafterfiltrationwasmeasuredagainstablank
solu-tionwithoutfilmogenicmaterialat540nm.Maltosesolutionswere
used(asstandardreducingsugar)togenerateastandardcurve.The
requiredtimeforfilmhydrolysiswasobservedvisually
2.7.6 Macrophagecellrecoveryandcounting
Macrophages current recovery approach was the scratching
procedure(usedascontrol)andtherecoverybythenoveldirect
collectionfromstarchcoatedinsertsdevicesafterthemild
enzy-matic filmhydrolysis were compared bycountingdone witha
hemacytometer(NikonTMS-F),andusingTrypanblueasstaining
agent
2.7.7 Macrophageactivation
Follwoing48hincubationanamountof 50ng/50LLPSper
1mLofculturemediumwasaddedandthecellsre-incubatedfor
additional72h
2.7.8 Quantitationoftumornecrosisfactor(TNF-˛)
After 72h incubation, the culture medium over and under
of macrophage layer was gently removed and centrifuged at
12000rpmfor 10min.The amountofTNF-␣wasquantifiedby
theELISAkit (CatalogueNo 430904,Biolegend,Canada) TNF-␣
levelinsamplesweredeterminedaccordingtothemanufacturer’s
instructions Astandardcurvein concentrationsfrom7.8pg/mL
to 125pg/mL was donein duplicate and the level of TNF-␣in thesupernatantswasevaluatedbyuseofthestandardcurveas reference The opticaldensity at 450nm wasmeasured witha microplatereader
2.8 Statisticalanalysis Alltestswereperformedintriplicateanddataarereportedas means±SD.Statisticalanalysisofdatawasperformedusingone wayANOVA,followedbyFisher’sposthoctestswithaminimum confidencelevel(P<0.05)forstatisticalsignificance
0 20 40 60 80 100 120
Trang 53 Results and discussions
3.1 Polymerandfilmcharacterization
Thedegreeofsubstitutionof starchderivativesCM-St, Ac-St
andAE-St,asdeterminedbyback-titrationwereabout0.018,0.022
and0.024,respectively.Thesevaluesrepresenttheaveragenumber
ofcarboxymethyl,acetateoraminoethylgroupsperglucoseunit,
respectively.Thegraftingofeachfunctionalgrouponthestarch
chainswasconfirmedbystructuralanalysis,FT-IRand1HNMR
TheFourier transforminfrared (FT-IR)spectraoftheobtained
starchmaterials(Fig.1)presentabroadbandat3200–3300cm−1
duetothestretchingvibrationsof OH.Smallbandsat2927cm−1
andat2323cm−1attributedtothe−CHstretchingvibrationanda bandat1079cm−1ascribedto CH2 O CH2stretchingvibrations (Ispas-Szabo,Ravenelle,Hassan,Preda,&Mateescu,1999).Incase
ofCM-St,thereareadditionalbandsat1589cm−1andat1323cm−1 ascribedtoCOO−group(Friciu,TienLe,Ispas-Szabo,&Mateescu,
2013).Thehighintensityofthebandat999cm−1forAE-Stcould
beascribedtoC Nstretchingvibrations,whereastheweak shoul-derataround1735cm−1couldbeassignedto NH3 group(Assaad
etal.,2011;Deng,Jia,Zhang,Yan,&Hou,2006).InthecaseofAc-St, theweakshoulderataround1556cm−1correspondsspecificallyto the C Ostretchingofacetylgroups(Bello-Pérez,Agama-Acevedo, Zamudio-Flores,Mendez-Montealvo,&Rodriguez-Ambriz,2010;
Colthup,Daly,&Wiberley,1990)
Trang 6Fig 5.Scanning electron microscopy micrographs of films: Gelatinized starch (G-St), (b) Acetate starch, (Ac-St), (c) Carboxymethyl starch (CM-St) and (d) Amino-Ethyl starch
The 1H NMR spectraof the starch materials(Fig.2) present
protonsignalsat5.3ppmforH1andat3.3–3.9ppmforH2-6on
thestarchbackbone(Yangetal.,2014)whilethepeakat5.6ppm
canbeassignedtoOH3.ThemostsignificantpeaksforAE-Stare
at␦=4.15–4.25,␦=3.16–3.18,whichbelongtothehydrogensof
aminoethylgroup.IncaseofAc-Stthepeaksat␦=1.9–2.1andat
␦=3.5ppmareascribedtomethylprotonsofacetategroups(Xu
andHanna,2005).IncaseofCM-Stsharplesspeaksmaybedueto
thelimitedsolubilityofCM-StinDMSO
Theobtainedzetapotential()chargesvaluesinsolutionwere
−32mVforG-Stand−38mVforCM-St.Thesevaluesareconsistent
withthechemicalmodificationofstarchbycarboxymethylgroups
providingastrongernegativecharge(Wongsagonsup,Shobsngob,
Oonkhanond, & Varavinit, 2005a; Wongsagonsup, Shobsngob,
Oonkhanond, & Varavinit,2005b) Grafting starch with acetate
groups reducedthevalueofzetapotentialforacetatestarch to
−26mV and this can be explained by a decreased polarity in comparisonwithG-St.ThepositivezetapotentialvalueforAE-St +10mVisrelatedtocationicgroupsgraftedonstarchmolecules StaticwatercontactangleFig.3allowedtheevaluationofthe wettability/hydrophilicity of the filmsfor coating of the insert surfaces The CM-St and AE-St films presented a lower angle (67◦ and78◦respectively)incomparisontoG-St(89◦)andAc-St (105◦),meaningthatG-StandAc-Starelesspolarandevenmore hydrophobic
Scanningelectronmicroscopy(SEM)ofstarchmaterialsas pow-dersandfilmsarepresentedinFig.4.Thenativestarch(Hylon VII)hasagranularaspectpredominantlyroundorovalinshape (Fig.4 withsmoothsurfaceanduniformrangeofsizedistribution (5–10m).Thegranularaspectfitswellwiththeknowncrystalline
Trang 7Fig 6. Confocal fluorescence microscopy images showing live cells (green) and dead macrophage cells (red) after incubation 48 h on cell inserts coated with Amino-Ethyl
structureofnativestarch(Friciuetal.,2013)stabilizedby
hydro-genbondsbetweenthehydroxylgroupsofglucopyranoseunits
Theaspectofthefourmaterials:G-St,CM-St,AE-StandofAc-Stis
different,dependingonmodificationoperatedonstarchstructure
TheG-St(Fig.4a)showedaroundandsponge-likeshapewhich
isduetothephysicalmodification(gelatinization)ofnativestarch
Differently,theCM-St(Fig.4b)presentedanirregularshapewith
anunevensurfacelikelyduetotheassociationofnumeroussmall
particlesforminglargergranulessimilarshapeswereobtainedby
Friciuetal.(2013).Thecarboxylicgroupsmayreducethenetwork
self-assemblingbyhydrogenassociationbetweenhydroxylgroups
andpromoterepulsioneffectsloadingtoastructural
reorganiza-tion(Lemieux,Gosselin,&Mateescu,2010).Theacetylation(Fig.4c)
generated a slightly roughsurface of granules which appeared
fusedinakindofaggregate.Theacetylgroupscanalsodecreasethe
starchstabilizationbyhydrogenbondingand,atthesametime,the
glucoseunitswithpolarhydroxylicgroupsandnon-polar(acetate)
functions,mayfavorstarchmacromoleculestocoalescetogether
resultinginakindoffusionofgranules(Bello-Pérezetal.,2010;
Singh,Kaur,&Singh,2004).TheAE-St(Fig.4d)grainsshoweda porousirregularshape,whereaminegroupsmaypromote hydro-genbondingresultingtoareorganizationoftheAE-Stnetwork.As farasfilmsareconcernedtheSEMmicrographsofG-StandCM-St filmsatmagnificationsof500×and1000×(Fig.5aandb)showeda homogeneousandsmoothsurface,whereasAc-StandAE-Stfilms (Fig.5candd)showedcontinuousmatrices,withsmallcracksand lesssmoothsurface
3.2 Macrophagecellsattachmentandrecoverybyfilmamylolysis 3.2.1 Morphologyofmacrophagecells
Intact macrophage cultures were treated with two staining agents:CMFDAtoshowlivecells (green)and propidiumiodide
tostaindeadcellswithalteredmembranepermeability(red) Controlculturesonuncoatedinsertdevices appearasplump
orstellate,monolayersroundedandspindle-likewithmajorityof
Trang 80 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Time (min)
0 10 20 30 40 50
0 5 10 15 20 25 30 35 40
5)
0 10 20 30 40 50 60 70 80 90 100
livecells.Macrophagesincubatedoninsertdevicescoatedwith
G-St,Ac-StandAE-Stshowedround,compactandmostlylivecells
Fig.6.Differently,prevalentlydeadcellswereobservedwhen
incu-batedininsertcoatedbyCM-Stfilm,owninground,spindle-like
andtranslucentcytoplasm.Thisbehavioursuggeststhatthe
car-boxymethylfunctionalizedfilmmaycausemembranedisruption
and cell apoptosis.Similar damaged membranes and apoptosis
havebeenobservedwithcertainagentssuchascarboxy-silicalite
(Petushkov,Intra,Graham,Larsen,&Salem,2009)
3.2.2 Determinationofenzymaticactivitywithstarchfilmogenic
supportsassubstrates
Thefilmamylolysisprocesswasinvestigatedbymeasuringthe
enzymaticactivityofalpha-amylasewithvariousfilmsassubstrate
(Fig.7A).ItwasfoundthatG-St,AC-StandAE-Stshowedsimilarfilm
hydrolysisrateoverthefirst40min.Then,theG-sthydrolysiswas
fasterthanthatofAC-StandAE-St.Thisbehaviourwasconsidered
asnormalbecausethereisnochemicalmodificationoftheG-St
ThelowestenzymaticactivitywasobservedwithCM-Stfilm,where
thereleasedamountofmaltoseafter75minwasalmosthalfofthat
liberatedfromG-St.Thefilmhydrolysiswasalsofollowedvisually
Evenwithoutcompleteamylolysis,theCM-Stfilmwasdissolved
inlessthan10min,becauseCM-Stissolubleinalkalinemedium
Differently,G-St filmwaspartially hydrolyzedin 30min,AC-St
andAE-Stin40min.Macrophagesadhereonadequatesurfaces
and floatingcells arecharacteristically dying cells Macrophage countingsuggestedgoodadhesiononG-St,onAc-StandonAE-St materials.Fig.7Bpresentsthenon-adherent(floating)fractionof macrophagesafterincubationofcellcultureoncell-holderdevices (insert)coatedwithCM-St,AE-St,Ac-StorG-St.thehigher per-centagesofdeadmacrophage(floating)wereobservedatinserts coatedwithanionic CM-St(about 32±5%)orwiththecationic AE-St(about32±9%),whereasalowpercentageofdeadcellwas observedwithinsertcoatedwithnon-ionicandneutralpolymers Ac-St (5±2%) and G-St (9±3%)respectively, suggesting higher percentageoflivingcellsfromthisfilms.Theseadhesiondataon non-ionicAc-StandG-Stareinagreementwithourpreviousreport showinggoodadhesionandrecoverybyamylolysisofmacrophage cellsoncross-linkedstarchmicrospheres,notmodifiedwithionic groups(Desmanglesetal.,1992).ThebestretentiononAC-Stfits wellwith a studyof Godek, Michel,Chamberlain, Castner, and Grainger(2009),showingthatmacrophagesadherepreferentially
tohighlyhydrophobicfluorinatedsurfaces(Godeketal.,2009) Similarresults,butnotoncarbohydratematerials,wereobserved
byBrodbecketal.(2002)showingthatthehydrophilicandanionic polyethyleneterephthalatemodifiedsurfacesinhibitadhesionof monocyteandmacrophagecells(Brodbecketal.,2002)
Duetomembranedisruptionandcellinducingapoptosisalong withlowmacrophageviabilityonCM-St,thissupportwasexcluded fromfurtherinvestigationandcellharvestingandcountingwas
Trang 9AE-Stcoated inserts.Cell harvestingwasdoneby scrappingfor
controlcells (culturedon uncoatedinsert devices) or by
enzy-matic hydrolysis for insertscoated withstarch materials.After
incubationfor 48h, cell numbersincreasedabout 3.2times for
controluncoatedinserts,4.2timesforAc-Stand5.3timesfor
G-Stwhereasonly1.5timeswasobservedforAE-Stcoatedinsert
(Fig.7C).Furthermore,129%and164%morecellswererecovered
frominsertsdevicescoatedwithG-StandAc-Stwhencompared
tocontrols(un-coatedinserts),whereasa53%dropoftheyield
wasobtainedforAE-Stcoatedinserts.Thisinhibitoryeffectcould
beexplainedbya toostronginteractionofcationicaminoethyl
groupsofstarchfilmwithmembranephospholipidsofmacrophage
cells (Kurtz-Chalot et al., 2014) Therefore the AE-St was not
retainedforfurtherinvestigation
Macrophage activation by Lipopolysaccharide (LPS) and
quan-titation of induced tumor necrosis factor (TNF-a) allowed the
investigation of the possible effect of starch derivatives with
macrophageactivities.ThecellswerestimulatedwithLPS,a
com-ponentoftheoutermembraneofGramnegativebacteria,which
isapotentactivatorofmonocytesandmacrophages(Mace,Ehrke,
Hori,Maccubbin,&Mihich,1988).LPStriggerstheabundant
secre-tionofcytokinesbymacrophagesincludingtumornecrosisfactor
(TNF-a), interleukin (IL)-1, and IL-6 (Meng & Lowell, 1997) In
our study, the amount of TNF-␣ secreted by macrophages in
responsetoLPSwasinthesamerange asreportedin asimilar
study(Lichtman,Wang,&Lemasters,1998).Moreover,therewere
nodifferences(Fig.7D)inTNF-␣producedbycontrolcells
har-vestedfromuncoatedinserts(91±3.5pg/mL)orbymacrophages
harvestedfromG-St (90±2.3pg/mL)and Ac-St(89±2.9pg/mL)
coatedinserts.Thefunctional groups graftedonpolysaccharide
chainsnotonlyhaveadirecteffectonviabilityofcells,butthey
canimpactmacrophageadhesion.Forinstancethenon-derivatized
starch(G-St)andtheAc-Stwithhydrophobicacetategroups
ori-entedtoward culturemedium,arebettersupportsforadhesion
ofmacrophagecellsthantheanionic(CM-St)andcationic(AE-St)
starchderivativeswhicharelesscompatible.Theminimal
percent-ageofdeadcells(non-adherentfraction)wasobservedwithinserts
coatedwithG-StandAc-St.Therefore,theseGelatinizedstarchand
Acetatestarchmaterialsaffordingabestviability,couldbeagood
choiceassupportmaterialformacrophagecultureduetothehigh
compatibilitywithcells andalsofortheirsusceptibilitytomild
enzymaticamylolysis.ThesefeaturesofG-StandAc-Stallowthe
recoveryofmacrophagecellswithbetterviabilityandhighyields
Furthermore,theactivationbyLPSindicatedthatmacrophagecells
culturedonG-Standonthestarchacetatederivativeare
produc-ingalmostthesamelevelofTNF-␣asthecontrol(uncoatedinsert)
Thisresulttogetherwiththelowpercentageofdeadcellscould
beanevidenceofbiocompatibilityofG-StandAc-Stsupportsas
materialsformacrophagepreparationbythisnovelmildenzymatic
procedure
4 Conclusion
Thepresent studyisproposinganewtypeofapplicationfor
modifiedstarchbasedonitsfilm-formingcapacity.Theproposed
approach,focusedonadhesionofmacrophagecells onAc-St or
G-Stfilms followed bytheirdetachment by enzymatic
amylol-ysis,is faster and the mild condition affords a better viability
ofmacrophagecellsincomparison withtheclassicalprocedure
(mechanicaldetachment).Starchfilmsareeasy toapplyonthe
insertsand theirbiocompatibilityisanimportant characteristic
for cell viability.This study opens new perspectives to obtain
macrophagecellswithahighviability,avoidingsignificantlossof
viablecellswhichstilllimitsthecurrentscratchingprocedures
Fur-therstudieswillbeconductedinordertoevaluatetheimpactof thesubstitutiondegreeofAc-Stontheattachmentandactivityof macrophages
Acknowledgments
ThefinancialsupportfromNSERC(NaturalScienceand Engi-neering Research Council of Canada) Discovery Program is gratefullyacknowledged.ThanksareduetoDr.TienCanhLefor helpfuldiscussions
References
Pvt Ltd Publishers.
of carboxymethyl starch and chitosan as drug carrier for oral administration Carbohydrate Polymers, 84, 1399–1407.
the morphological, physicochemical and structural characteristics of barley starch LWT—Food Science and Technology, 43, 1434–1440.
and growth of polymer films prepared by interfacial polymerization Langmuir,
31, 12279–12290.
(Vol 1) Academic Press.
monocyte/macrophage cytokine expression in vitro Cytokine, 18, 311–319.
Escherichia coli oral formulations European Journal of Pharmaceutics and Biopharmaceutics, 60, 53–60.
spectroscopy New York: Academic Press.
(II) and lead (II) ions by crosslinked starch graft copolymer with aminoethyl group Chemical Journal on Internet, 8, 68.
separation of macrophages by retention on cross-linked amylose and release
by enzymatic amylolysis of the chromatographic material Journal of Chromatography B: Biomedical Sciences and Applications, 584, 121–127.
Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties Cell Motility and the Cytoskeleton, 63, 321–340.
macrophages and cell lines from tissue culture-treated and -untreated plastic dishes Journal of Immunological Methods, 68, 119–129.
starch and lecithin complex as matrix for targeted drug delivery: I Monolithic mesalamine forms for colon delivery European Journal of Pharmaceutics and Biopharmaceutics, 85, 521–530.
Adsorbed serum albumin is permissive to macrophage attachment to perfluorocarbon polymer surfaces in culture Journal of Biomedical Materials Research Part A, 88A, 503–519.
Structure–properties relationship in cross-linked high-amylose starch for use
in controlled drug release Carbohydrate Research, 323, 163–175.
human M-CSF-generated macrophages and comparison of M1-markers in classically activated macrophages from GM-CSF and M-CSF origin Cellular Immunology, 281, 51–61.
applications In M R Mozafari (Ed.), Nanomaterials and nanosystems for biomedical applications (pp 83–98) Netherlands: Springer.
crosslinking on the mechanical and thermal properties of poly(vinyl alcohol) Polymer, 41, 9265–9272.
with different surface chemical functionalizations: Impact on cytotoxicity Journal of Nanoparticle Research, 16, 1–15.
Roles and therapeutic implications Cellular Immunology, 291, 58–64.
Trang 10Lemieux, M., Gosselin, P., & Mateescu, M (2010) Influence of drying procedure and
of low degree of substitution on the structural and drug release rroperties of
carboxymethyl starch AAPS PharmSciTech, 11, 775–785.
Recent advances Journal of Controlled Release, 53, 225–234.
release of TNF-␣ in RAW 264.7 and peritoneal cells but not in Kupffer cells.
American Journal of Physiology—Gastrointestinal and Liver Physiology, 275,
G39–G46.
tumor necrosis factor in macrophage activation and tumoricidal activity.
Cancer Res, 48, 5427–5432.
procedures on the functional state of cultured murine macrophages.
Immunobiology, 159, 327–336.
starch as pharmaceutical excipients: Structural insights and formulation of
pancreatic enzymes International Journal of Pharmaceutics, 356, 212–223.
activation and signal transduction in the absence of Src-family kinases Hck, Fgr
and Lyn Journal of Experimental Medicine, 185, 1661–1670.
high amylose starch derivatives as matrices for controlled release of high drug
loadings Journal of Controlled Release, 76, 51–58.
amylose starch derivatives for drug release: II Swelling properties and
mechanistic study International Journal of Pharmaceutics, 278, 231–238.
cancer: From mechanisms to therapeutic implications Trends in Immunology,
36, 229–239.
crystal size and surface functionalization on the cytotoxicity of silicalite-1
nanoparticles Chemical Research in Toxicology, 22, 1359–1368.
inflammation Clinical and Experimental Immunology, 142, 481–489.
pharmaceutical excipients (5th ed.) London: Pharmaceutical Press.
corn and potato starches Starch—Stärke, 56, 586–601.
using starches separated from different rice cultivars Journal of Food Engineering, 70, 117–127.
some methods for the determination of the degree of substitution of carboxymethyl starch Starch—Stärke, 57, 79–83.
potential () analysis for the determination of protein content in rice flour Starch—Stärke, 57, 25–31.
potential () and pasting properties of phosphorylated or crosslinked rice starches Starch—Stärke, 57, 32–37.
monocyte derived macrophages Methods, 70, 89–96.
from starch acetate and poly(tetramethylene adipate-co-terephthalate) Carbohydrate Polymers, 59, 521–529.
amphoteric starch-based grafting flocculants for flocculation of both positively and negatively charged colloidal contaminants from water Chemical Engineering Journal, 244, 209–217.
infiltrating macrophages in liver repair after acute injury Biochemical Pharmacology, 86, 836–843.