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RESEARCHOpenAccessInvestigationofmagneticallycontrolledwaterintakebehaviorofIronOxideImpregnatedSuperparamagneticCaseinNanoparticles(IOICNPs)AnamikaSingh,JayaBajpaiandAnilKumarBajpai*AbstractIronoxideimpregnatedcaseinnanoparticles(IOICNPs)werepreparedbyin-situprecipitationofironoxidewithinthecaseinmatrix.
Theresultingironoxideimpregnatedcaseinnanoparticles(IOICNPs)werecharacterizedbyScanningelectronmicroscopy(SEM),Transmissionelectronmicroscopy(TEM),X-rayphotoelectronspectroscopy(XPS),Fouriertransforminfrared(FTIR),Vibratingsamplemagnetometer(VSM)andRamanspectroscopy.
TheFTIRanalysisconfirmedtheimpregnationofironoxideintothecaseinmatrixwhereasXPSanalysisindicatedforcompleteoxidationofiron(II)toiron(III)asevidentfromthepresenceoftheobservedrepresentativepeaksofironoxide.
Thenanoparticleswereallowedtoswellinphosphatebuffersaline(PBS)andtheinfluenceoffactorssuchaschemicalcompositionofnanoparticles,pHandtemperatureoftheswellingbath,andappliedmagneticfieldwasinvestigatedonthewaterintakecapacityofthenanoparticles.
Thepreparednanoparticlesshowedpotentialtofunctionasananocarrierforpossibleapplicationsinmagneticallytargeteddeliveryofanticancerdrugs.
Keywords:Casein,IOICNPs,Swellingbehaviour,pHsensitive,MagneticdrugtargetingIntroductionTheconceptofmagneticdrugtargeting(MDT)simplyentailsretainingspeciallydesignedmagneticdrugnano-carriersataspecificsiteinthebodyusinganexternallyappliedmagneticfield.
Thetwokeypropertiesforaneffectivenanocarriersare(a)efficienttargetingtospecifictissueandcells,and(b)avoidingrapidclearance(i.
e.
re-mainingincirculation)forasignificantamountoftimetoincreaseparticleuptakeintargettissue.
Circulationtime,targeting,andtheabilitytoovercomebiologicalbarriersdependontheshape(e.
g,aspectratio),chemicalcoatingandsizeofthenanoparticles[1].
Thusaprecisecontrolovertheshapeandsizeofnanoparticlesisachallengingtaskandmustbeaddressedinordertoachievehighper-formancedrugdeliverysystems.
InordertoachieveanefficientMDT,variouspoly-meric/inorganichybridmaterialshavebeensuggestedthatofferuniquepropertiesbecauseoftheirsmallsize,limitedtoxicity[2],lowproductioncost,easeofseparationanddetection[3].
Themagneticnanoparticlesoftentendtoformlargeaggregatesowingtothestrongmagneticdipole–dipoleattractionsamongparticles.
Toimprovetheirchemicalstabilityandbiocompatibility,thesurfaceofmagneticnanoparticleshavebeenmodifiedwithvarioussurfactants[4]orbiopolymercompoundshavingmultiplefunctionalgroupscapableofbindingtotheparticlesur-faces(multidentateligands).
Therationalforusingmag-neticnanoparticlestotumortargetingisbasedonthefactthatnanoparticleswillbeabletodeliveraconcentratedoseofdruginthevicinityofthetumortargetsviatheenhancedpermeabilityandretentioneffectoractivetargetingbyligandsonthesurfaceofnanoparticles[5].
Moreover,theextentofdrugloadingontothenanopar-ticlesgreatlydependsonthehydrophilicnatureofthebiopolymeralsoand,therefore,proteinscouldbeanexcel-lentoptiontodesignsuchmagneticnanocarriers.
Useofmilkproteins,likecaseins,indrugdeliveryapplicationsisrelativelyanewtrendandthisismainly*Correspondence:akbmrl@yahoo.
co.
inBMRL,DepartmentofChemistry,GovernmentModelScienceCollege,Jabalpur,India2014Singhetal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Singhetal.
JournalofNanobiotechnology2014,12:38http://www.
jnanobiotechnology.
com/content/12/1/38duetoitsamphiphilicnaturethatallowsthemtointeractwithboththedrugandsolvent.
Infact,caseinsmayberegardedasblockcopolymerswithhighlevelofhydro-philicandhydrophobicaminoacidresiduesandthustheyexhibitastrongtendencytoselfassembleintosphericalmicelles.
Moreover,thierbiodegradability,not-toxicity,metabolizablityandfeasibilitytosurfacemodificationenablethemtointeractwiththetargetingligand.
Inanstudy,thecomplexationofcurcuminwithβ-caseinmi-cellesincreasedthesolubilityofcurcuminatleast2500-foldwithenhancedcurcumincytotoxicitytoahumanleukemiacellline[6].
Shapiraetal.
[7]showedthatβ-CASmicellescouldentrapanddeliverhydrophobicchemotherapeuticssuchasmitoxantroneandpaclitaxel,allowingthemtobethermodynamicallystableinaqueoussolutionsfororaldeliveryapplications.
Thus,thehydro-phobicandhydrophilicdomainsofcaseinareresponsiblenotonlyfortheirwatersorptioncapacity,butalsoforthenatureandtypeofdrugtobeencapsultedinthecaseinnanoparticles.
Thus,motivatedbythepharmaceuticalspecialitiesofthecaseinproteintheauthorswerepusedtoundertakeasystemicinvestigationofsynthesisandcharacteriztionofmagneticcaseinnanoparticlesandtheirwatersorptionbehaviortojudgetheirsuitabilityindesigningswellingcontrolledandmagneticmediateddrugdeliverysystem.
Thepresentstudyaimsatdesigningcontrollablesizeironoxideimpregnatedcaseinnanoparticles(IOICNPs)byco-precipitationofironsaltswithinthecaseinnanoparticlesmatrix.
AstheItisalsoproposedtocharacterizedthesopreparedIOICNPsbyvariousanalyticaltechniquesandinvestigatetheirwatersorptionpotentialinthepresenceofappliedmagneticfield.
ExperimentalMaterialsCaseinwaspurchasedfromMerck,Mumbai,Indiaandusedwithoutanypretreatment.
FeCl2.
H2O,FeCl3.
6H2O,glutaraldehyde(usedasacrosslinker)wereobtainedfromLobaChemie,Mumbai,India.
ToluenewasobtainedfromSigmaAldrichCo.
,USA,andusedforpreparingoilphase.
Otherchemicalslikeacetone,NaOHetc.
wereofanalyticalreagent(AR)gradeanddoubledistilledwaterwasusedthroughouttheexperiments.
PreparationofIronoxideimpregnatedcaseinnanoparticles(IOICNPs)Preparationofmagneticcaseinnanoparticlesconsistsofatwostepsprocess.
Inthefirststepthecaseinnanopar-ticlesarepreparedbyemulsioncrosslinkingmethodwhileinthesecondoneironoxidenanoparticlesareimpregnatedwithincaseinnanoparticlesmatrixbyinsituprecipitation.
Preparationofcaseinnanoparticles(CNPs)InordertoprepareCNPsthemicroemulsioncrosslinkingmethodwasadoptedasdescribedinliterature[8].
Inbrief,anaqueousphasewaspreparedbydissolvingknownamountofcaseinin1%NaOHwhereastoluenewasusedtopreparetheoilphase.
Theabovetwosolutionsweremixedwithvigorousshaking(shakingspeed1000RPM,5Lcapacity,Remi,India)for30minandtothisemulsion1mLglutaraldehydewasaddedascrosslinkerwithcon-stantstirring.
Thecrosslinkingreactionwasallowedtotakeplacefor30minatroomtemperature(30°C)andH2SO4wasaddedtothesolutionforthesolidificationofparticles.
Thenanoparticleswerecleanedbywashingthemthricewithacetoneandstoredinair-tightpolyethyl-enebags.
ImpregnationofIronoxideintothecaseinnanoparticlesThedriedCNPswereplacedinanaqueousmixtureofFe2+andFe3+chloridesaltsat1:2molarratioandallowedtoswellfor24hsothatbothFe2+andFe3+ionswereen-trappedintothebiopolymermatrix.
Priortoputtingtheminsaltsolution,adrystreamofN2wasflushedforatleast15mintocontrolthereactionkinetics,whichisstronglyrelatedtotheoxidationspeedofironspecies.
Bubblingnitrogengasthroughthesolutionnotonlyprotectscriticaloxidationofthemagnetitebutalsoreducestheparticlesize[9].
Theferrousandferricionsloadedcaseinnano-particleswereaddedtoNaOHsolutionforadefinitetimeperiodsothatmagnetiteisprecipitatedwithinthebio-polymermatrixaccordingtothefollowingchemicalreac-tions[10].
Fe22OH‐→FeOH2Fe33OH‐→FeOH3FeOH22FeOH3NaOH→Fe3O44H2O1Furthermore,forsynthesisofnanoparticleswitheithertheferrousorferricionalone,thechemicalreactionspassthroughdifferentmechanismsasshowninEquations(2)and(3),respectively.
Fe22OH‐→FeOH23FeOH20:5O2→FeOH22FeOOHH2OFeOH22FeOOH→Fe3O42H2O2Fe33OH‐→FeOH3FeOH3→FeOOHH2O12FeOOH→4Fe3O46H2O3Thesetwomechanismsprovidealargerparticlesizeascomparedtothecaseofthemixtureofferrousandferricionsbecausetheparticlesizeofmagnetitealsodependsonthenatureoftheintermediateform.
SincethemethodsadoptedrequirelongerreactiontimesSinghetal.
JournalofNanobiotechnology2014,12:38Page2of13http://www.
jnanobiotechnology.
com/content/12/1/38forthetransformationsofferricionsandferrousionssothattheintermediatescancontinuouslygrow.
However,caremustbetakenwhileaddingNaOHbecauseaccordingtothethermodynamicsofthisreaction,acompletepre-cipitationofFe3O4occursintherangeof9to14pHandinmolarratioof1:2forFe2+:Fe3+underanonoxidizingoxygenfreeenvironment.
Otherwise,Fe3O4mightgetoxidizedas,Fe3O40:25O24:5H2O→3FeOH34Thechangeincolorofthecaseinnanoparticlesfromorangetodarkbrownalsoconfirmstheformationofoxidesofiron.
Thepreparednanoparticleswerewashed,driedatroomtemperatureandstoredinairtightpoly-ethylenebags.
ThechemicalreactionisshowninFigure1.
Thepercentageimpregnationofironoxidewascalculatedusingfollowingequation.
Impregnation%Wimpregnated‐WdryWdry1005Infact,theprocessofformationofironoxideinvolvesthediffusionofferrous/ferricionsintothepolymermatrixandtheirsubsequentinsituprecipitationinanalkalinemedium.
Itisthereforeexpectedthatthehigheristhewateruptakegreaterwillbe,theironoxideforma-tion[11].
CharacterizationFTIRSpectralanalysisTheFTIRspectraofcaseinandIOICNPswererecordedonaFTIR-8400,ShimadzuSpectrophotometer.
Samplesforthespectralanalysiswerepreparedbymixingnano-particlesandKBrin1:10proportionandthespectrawereobtainedintherangeof4000to400cm1witharesolutionof2cm1.
SEManalysisMorphologicalstudiesofcross-linkedCNPsandIOICNPswereperformedusingSEM,Philips515,finecoater(Philips,Eindhoven,TheNetherlands).
Dropsofthepoly-mericnanoparticlessuspensionwereplacedonagraphitesurfaceandfreeze-dried.
Thesamplewasthencoatedwithgoldbyionsputterat20mAfor4minutes,andobserva-tionsweremadeat10kV.
TEManalysisThesizeandmorphologyofthenanoparticlesweredeter-minedbyconductingTEManalysisofcaseinandIOICNPsonMorgagni268-DTransmissionElectronMicroscopewithanacceleratingvoltageof80.
0kV.
ThesamplesforTEMmeasurementswerepreparedbydispersingadropofthesamplesolutiononFormvar-coatedCgrids.
RamanspectralanalysisInordertoinvestigatetheimpregnationofironoxidenanoparticlesintothematrixofcaseinnanoparticles,Figure1SchematicformationofIOICNPs(Step-I)Formationofcaseinnanoparticlesbycrosslinkingofcaseinmacromoleculesbyreactionwithglutaraldehyde.
(Step-II)Impregnationofironionswithinthecaseinnanoparticlsnetworkbyswellinginironsaltssolution.
(Step-III)InsituprecipitationofironoxidewithinthecaseinnanoparticlesmatrixtoyieldIOICNPs.
Singhetal.
JournalofNanobiotechnology2014,12:38Page3of13http://www.
jnanobiotechnology.
com/content/12/1/38Ramanspectroscopywasusedandthespectrawereob-tainedintherangeof200–1800cm1.
Thecharacteristicpeakpositionofmagnetite(Fe3O4)anditspossibleoxidationproductmaghemite(У-Fe2O3)andhematite(α-Fe2O3)weredeterminedintheRamanregionof100–1200cm1.
Forcorrectassignmentofthebandposi-tionsandphaseidentificationpresentinthesamples,combinedRamandatawereusedforkeyironoxidesbands.
TheRamanspectraofcaseinandIOICNPswererecordedonaMicroRamanSpectrometer,JobinYvonHoribraLABRAM-HR.
VSManalysisThemagnetizationversusmagneticfieldmeasurements(M–Hfirstmagnetizationcurveandhysteresisloop)at300K,fortheIOICNPs(powdersample)weredoneon14TPPMS-vibratingsamplemagnetometer.
XPSanalysisThesampleswerealsoanalyzedbyX-rayphotoelectronspectroscopy(XPS)onamodifiedlaserablationsystem,RiberLDM-32,usingaCamecaMac3analyzer.
Photo-electronspectrawerecollectedbyacquiringdataforevery1.
0eVwithanenergyresolutionof3eV.
Narrow-scanphotoelectronspectrawererecordedforC1s,N1s,O1s,andFe2pbyacquiringdataforevery0.
2eVandtheenergyresolutionwas0.
8eV.
InvitrocytotoxicitytestInordertodetermineinvitrocytotoxicityofthepreparedmaterialstestonextractmethod(ISO10993-5,2009)wasapplied.
Inbrief,atestsampleofthenanoparticles,nega-tivecontrolandpositivecontrolintriplicatewereplacedwithsubconfluentmonolayerofL-929mousefibroblastcells.
Afterincubationofcellswithtestsamplesat37±1°C,for24h,cellculturewasexaminedmicroscopicallyforcellularresponsearoundandunderthetestsamples.
WatersorptioncapacityTheextentofswellingofCNPsandIOICNPsinbothpresenceandabsenceofmagneticfieldweredeterminedbyaconventionalgravimetricprocedureasreportedintheliterature[12].
Theswellingratiowasdeterminedbythefollowingequation:SwellingRatioWeightofswollennanoparticlesWsWeightofdrynanoparticlesWd6Theamountofwaterimbibedbythesampleprovidesinformationaboutthehydrophilicnatureofthematerial,whichisoneofthecriterionsforbiocompatibility.
EffectofpHTheeffectofpHonswellingofthenanoparticleswasstudiedbypreparingsolutionsoverthepHrange1.
8to9.
0,andthedesiredpHwasadjustedwiththehelpof0.
1MHCland0.
1MNaOHsolutions.
ThepHwasdeterminedonadigitalpHmeter(Systronics,No.
362,Ahmadabad,India).
EffectoftemperatureTheeffectoftemperatureonswellingofthenanoparti-cleswasstudiedbyvaryingtemperatureoftheswellingmediumintherangeof10°to40°C.
SwellingstudiesinphysiologicalfluidInordertostudytheswellingofnanoparticlesinsim-ulatedbiologicalmedia,thefollowingaqueousfluids(100mL)wereprepared:Salinewater(0.
9gNaCI),syn-theticurine(0.
8gNaCl,0.
10gMgSO4,2.
0gurea,0.
6gCaCl2),urea5.
0g,andD-glucose5.
0g.
StatisticalanalysisAllexperimentsweredoneatleastthriceandFiguresanddatahavebeenexpressedalongwiththeirrespectiveerrorbarsandstandarddeviations,respectively.
ResultsanddiscussionEffectofcompositiononimpregnationImpregnationofironoxideintothepolymermatrixisaresultofinclusionofferrous/ferricionsintothepoly-mermatrixandtheirsubsequentinsituprecipitationinalkalinemedium.
Theimpregnationprocessbasicallydependsontheswellingcapacityofthebiopolymericnetworkwhich,inturn,variesasafunctionofchemicalcompositionoftheCNPs.
AmongvariousstructuralfactorsinfluencingwatersorptioncapacityofaCNPs,theratioofhydrophilicitytohydrophobicityplaysakeyroleindeterminingswellingcharacteristicofthematrix.
Inthepresentstudy,thepreparedmatrixiscomposedofcaseinandglutaraldehydewhicharehydrophilicbiopolymerandcrosslinker,respectivelyandtheirrelativeamountsintheCNPSareexpectedtoaffectextentofswellingand,consequently,theimpregnationofironoxidealso.
FTIRspectralanalysisTheFT-IRspectraofnativecasein,CNPsandIOICNPsareshownin(Figure2a,bandc),respectively.
Figure2ashowsabsorptionbandsat3455,3100,1661,1530and1235cm1whichcanbeexplainedasfollows:Inthecaseofnativecasein,theamideAbandat3455cm1andamideBat3100cm1areobserved,whichoriginateasaresultofFermiresonancebetweenthefirstovertoneofamideIIandtheN-Hstretchingvibration.
AmideIandamideIIbandsaretwomajorbandsoftheinfraredSinghetal.
JournalofNanobiotechnology2014,12:38Page4of13http://www.
jnanobiotechnology.
com/content/12/1/38spectrumofcasein.
TheobservedintensebandforamideIappearsat1661cm1andismainlyassociatedwiththeC=Ostretchingvibrationanddependsonthebackboneconformationandhydrogenbonding.
TheamideIIbandsobtainedinthe1510and1580cm1regionresultfromtheN-HbendingandtheC-Nstretchingvi-brations.
Theobtainedbandsat1661cm1and1531cm1fortheamideIandamideII,respectivelyalsoconfirmthealphahelicalstructureofthecaseinprotein.
Caseinalsoexhibitsanothercharacteristicbandat1415cm1whichmaybeattributedtothecarboxylategroup(O-C-O).
Asshownin(Figure2b),abandappearsat1683cm1andmaybeassignedtoC=Nstretchingwhichconfirmsthepresenceofcrosslinkingbetweencaseinandglutaraldehyde.
In(Figure2c)theappearanceofpeaksaround450and480cm1maybeassignedtoFe–Obondsofmagnetite,whicharecharacteristicpeaksofironoxide(e.
g.
,polyhedralFe3+–O2)stretchingvibrationsofironoxide,andthusconfirmtheimpreg-nationofironoxideintothematrixofcaseinnano-particles[13,14].
AccordingtoDeaconandPhillips[15],thecarboxylateionmaybecoordinatedtoametalatominoneofthefollowingstructures:structureI:unidendatecomplexwhereonemetalionbindswithonecarboxylicoxygenatomstructureII:bidendatecomplexwhereonemetalionbindswithtwocarboxylateoxygensstructureIII:bridgingcomplexwheretwometalionsbindwithtwocarboxylateoxygen's.
TheFTIRspectraindicatedthepresenceoftwobands,1415cm1(Vs:COO)and1538cm1(Vas:COO),whichmaybeattributedtothecarboxylateionofcaseinimmobilizedonthemagnetitesurface.
SEManalysisSEMimagesofCNPsandIOICNPsareshownin(Figure3aandb),respectivelywhichillustratenon-smoothmorphologyofCNPsandformationofironoxideinthecaseinnetworks.
Thecoatingofironoxidenano-particlesbythecaseinproduceslargersizeparticlesduetotheformationofthecaseinlayersonthesurfacesofironoxide.
Duringin-situprecipitationitmaybeinferredthatironoxidesareassembledorattachedinsidethebiopolymericnetworksandonthecaseinsurfaceaswell.
Loadingofironoxideinsidethenetworkaffectsitsmorphologyandstructuralintegrity.
Itislikelythatthepresenceofintermolecularforcesbetweencaseinmacro-molecularunitsfacilitatesformationofanextensivephys-icalnetworkofhydrogenbondsandothervanderwaalforces,whichprovide'nano'domainsforgrowthoftheironoxidenanoparticlesaswellasensuretheirprotectionwithinthecaseinnetwork.
Thesebiopolymericnetworksmaybeconsideredasnanoreactorstoconstructoras-sembleironoxide.
TheresultsmaybeattributabletocontributionsofaFe-Ocoordinationbondonthesur-face,stericeffectandacompartmenteffectofthenetworkstructuresofcasein,whichlimitthegrowthofironoxide,andthusplayanimportantroleintheprocessofthefor-mationofironoxideaggregates[16].
TEManalysisInordertoascertainmoreprecisemorphologyandsizeoftheironoxideparticlesatthenanoscalelevels,TEMstud-ieswereperformed.
Thesizedistributionofmagneticnanoparticlesisanimportantparameterrelatedtotheirbiologicalapplicationsandperformance.
DifferentshapedIOICNPsmaybepreparedbythefacileco-precipitationmethodbyadjustingtheamountsofpolymer,crosslinkerandFe2+/Fe3+ratio,pHsoastoinvestigatetheinfluenceontheshapeandparticlesizeofIOICNPs.
ItwasobservedthatthesynthesizedIOICNPsdisplayedarelativelyspher-icaldistribution,gooddispersionandauniformmorph-ologywithdistinctcrystallinestructure.
FromtheTEMimageofIOICNPs,itcanbeclearlydemonstratedthatmagneticnanoparticlescompriseofcoreshellstructurewithhomogenousincorporationofmagnetiteasacoreofIOICNPs.
Themagneticnanoparticlesarehomogeneouslycoveredbythecaseinshells[17].
TheparticlesizeofIOICNPsmaybecontrolledbytheamountsofcaseinandglutaraldehyde.
TheTEMimagesofIOICNPswithdiffer-entamountsofcaseinandglutaraldehydeareshowninFigure2FTIRspectraofa)nativecasein,b)CNPs,andc)IOICNPs.
Singhetal.
JournalofNanobiotechnology2014,12:38Page5of13http://www.
jnanobiotechnology.
com/content/12/1/38Figure4AandB,respectively,whichindicatethatanaver-agesizeofIOICNPsfallsintherangeof80–90nm.
Astheamountofcaseinincreasesfrom0.
5to2.
5g,thesizeoftheas-preparedIOICNPsincreasesfrom15nmto50nmasshownin(Figure4A).
Theresultsalsoshowthatincreasingamountofcaseintendstoproduceslargererparticlesizebecauseitcanproduceabiggermicelle[10,17].
Thus,onincreasingtheamountofcasein,thesizeofIOICNPsalsoincreases.
Theaverageparticlesizeofthenanoparticles,asafunctionoftheoil/waterratiointheemulsions,decreasesfrom20to8nmwithincreasingoil/waterratiofrom1.
3to7.
0.
Whentheoil/waterratioishigh,themolarratioofcaseintowaterisalsoincreased,resultinginahighsurfacetensionattheoil/waterinter-face.
Thisproducessmallwaterdropletsanddeterminesthesizeofironnanoparticle[18].
Theeffectofglutaralde-hydeconcentrationonthesizeofIOICNPsisshownin(Figure4B),whichrevealthatastheconcentrationofcrosslinkerincreases,thenumberofmorecrosslinkpointsincreasesthusresultinginanincreasethecrosslinkingdensity.
Asaresult,thenetworkvoidsareminimizedandtheparticlesizedecreases.
TheFiguresalsoshowthatthenanoparticlesformedarepresentasaggregatesduetothereasonthattheyhaveanaturaltendencytoundergoclusteringduetovarietyofchargesandfunctionalgroupspresentontheirsurfaces.
RamanspectralanalysisRamanspectraofIOICNPsareshownin(Figure5a),whichshowcharacteristicRamanbandsforcaseinduetoamideI(CONH)at~1666cm1andamideIIIbandat~1245cm1.
BetweentheseRamanbandsanintensepeakisobservedat1450cm1,whichisattributedtoFigure3SEMimagesofa)CNPsandb)IOICNPs.
Figure4TEMimagesofIOICNPscontainingvaryingamountofcasein0.
5g,1.
0g,1.
5g,2.
0g,andglutaraldehyde5mM,10mM,15mMand20mM.
(A)TEMimagesofIOICNPscontainingvaryingamountsofcaseina)0.
5g,b)1.
0g,c)1.
5g,d)2.
0g.
(B)TEMimagesofIOICNPscontainingvaryingamountsofglutaralehydea)5mM,b)10mM,c)15mM,d)20mM.
Singhetal.
JournalofNanobiotechnology2014,12:38Page6of13http://www.
jnanobiotechnology.
com/content/12/1/38theCH2scissoringmode.
IntheRamanspectraweakerpeaksobservedat193cm1,306cm1and538cm1confirmthepresenceofironoxideintheformofmagnet-ite.
Moreover,anadditionalstrongpeakisalsoobservedat668cm1.
ForcorrectassignmentofsamplecombinedRamandatakeycanbeusedasfollows[19,20].
(i)Fe3O4:193(weak),306(weak),538(weak),668(strong),(ii)gFe2O3:350(strong),500(strong),700(strong);and(iii)aFe2O3:225(strong),247(weak),299(strong),412(strong),497(weak),613(medium).
VSManalysisThemagnetizationversusmagneticfieldplot(M-Hmagnetizationcurveandhysteresisloop)at300K,fortheimpregnatedcaseinnanoparticleswasmeasuredovertherangeofappliedfieldbetween6000to+6000Oewithasensitivityof0.
1emu/g,usingvibratingsamplemagnetometer.
Theresultsareshownin(Figure5b)whichshowthatthesaturationmagnetizationvalueisaround64emug1,andthehysteresisisveryweak.
Thevalueobtainedislowerthanthereportedvalueof92–100emug1formagnetitenanoparticlesandmaybeat-tributedtothefactthatbelowacriticalsize,nanocrystallinemagneticparticlesmaybeofsingledomainandshowuniquephenomenonofsuperparamagnetism[21,22].
ThereductioninsaturationmagnetizationofFe3O4particlesmaybeattributedtothepresenceofnon-magneticlayeronthesurfaceoftheparticles,chargedistribution,super-paramagneticrelaxationandspineffectbecauseofultra-finenatureoftheparticles.
XPSanalysisInthepresentstudyXPSanalysesweredonetomonitortheironoxidedepositioninthecross-linkedIOICNPs.
TheXPSspectraofthecross-likedIOICNPsisshowninthe(Figure6)WhichrevealthepresenceoftheC1s,O1s,andN1score-levelpeak.
However,afterimpreg-nationofironoxide,thespectrumexhibitstwomorepeaksassociatedwithFe2+andFe3+,duetotheironoxidedeposition.
Itisworthtomentionthatthepeakassignmentisbasedoncharacteristicbindingenergiesreportedintheliterature[23,24].
Furthermore,theO1score-levelspectraofthecross-linkedIOICNPswerefittedusingtwopeaksat532.
3eVand534eV.
Thefirstoneisassociatedwiththebindingenergyofthe[C=O]intheimidegroupandcarboxylicacidgroupwhilethesecondoneatthebindingenergyoftheOHinthecar-boxylicacidgroup.
Theabsenceofthepeakat287.
2eV,associatedwiththebindingenergyofcarboxylicacidgroups,isaccompaniedbyanincreaseintheintensityofthepeakat285.
9eV,duetothecontributionofthecarboxylatespeciesinthecrosslinkedbiopolymer[25].
ThesegroupswerealsoobservedintheFTIRanalysis.
TheO1score-levelspectrumfromtheresultingcom-positewasfittedtopeaksat530.
2eV(g-Fe2O3),531.
4eV(a-FeOOH).
Furthermore,thespectrumdisplaystwopeaksassociatedwiththeironoxideinthecompositewhichareingoodagreementwiththemagnetite,at715.
3eVand725.
4eVforFe2+andFe3+ions.
Figure5RamanspectraandVSM(M-H)curveofIOICNPsat300K.
a)Ramanspectraandb)VSM(M-H)curveofIOICNPsat300K.
Figure6XPSofIOICNPs.
Singhetal.
JournalofNanobiotechnology2014,12:38Page7of13http://www.
jnanobiotechnology.
com/content/12/1/38TheXPSwasappliedtoprovideelementalinformationofsurfacecompositionofIOICNPsafterFe3O4loading.
TherewereC,N,OandFeelementsinthemagneticIOICNPs,whichfurtherprovedthatFe3O4nanoparticleswereinsitusynthesizedintheIOICNPs[26].
ThedifferentoxidationstatesoftheironinthesenanoparticlescanalsobedetectedanddistinguishedfromeachotherbyXPS.
InvitrocytotoxicitytestInordertodetermineinvitrocytotoxicityofthepre-paredmaterialsTestonextractmethodwasperformed(ISO10993-5,2009).
Inthismethodpowdered(0.
2g)materialwassoakedinculturemedium(1mL)withserumandthentheextractwaspreparedbyincubatingthepresoakedtestmaterialwithserumfor24h.
Afterincubation,theextractwasfilteredusing0.
22μmmillexgpfilter.
100%extractweredilutedwithculturemediumtoget50%and25%concentrations.
Differentdilutionsoftestsampleextracts,positivecontroland100%ex-tractsofnegativecontrolintriplicatewereplacedonsubconfluentmonolayerofL-929cells.
Afterincubationofcellswithextractsofthetestsampleandcontrolsat37±1°Cfor24to26h,culturewasexaminedmicro-scopicallyforcellularresponse.
Fornegativecontrolthesamplewaspreparedbyincubating1.
25cm2polyethylenediscwith1mLculturemediumwithserumat37±1°Candpositivecontrolwaspreparedbydilutingphenolstocksolution(13mg/mL)withculturemediumwithserum.
Thecytotoxicityreactivityweregradedbasedonzoneoflysis,vacuolization,detachmentandmembranedisinte-grationas0,1,2,3and4representingnone,slightmild,moderateandsevere,respectively.
ThequantitativeevaluationofreactivityfornegativeandpositivecontrolsandtestsamplearesummarizedinTable1,whilemicro-scopicobservationaredepictedin(Figure7a,bandc),respectively.
Itwasfoundthatthetestsampleshowednonereactivitytofibroblastcellsafter24hofcontact.
Theachievementofnumericalgrademorethan2isconsideredascytotoxiceffect.
Sincethepolymernetworkmaterialinthepresentworkachievedareactivitygradelessthan2,thematerialisconsideredasnotcytotoxic.
EffectofMagneticField(MF)ontheswellingInmagneticdrugtargetingswelling-controlledsystemmusthavesatisfactoryswellingpropertiesandhighcapacityofdrugloading.
Thedegreeandtimeofswellingareimportantcharacteristicsandhavesignificanteffectonthereleasekineticofloadeddrugsfromswelling-controlledsystems.
ToevaluatetheeffectofMFtheontheswellingofIOICNPs,theMFwasvariedintherangeof1000to3000G.
Theresultsaredepictedin(Figure8a)whichrevealthattheswellingratioincreaseswithincreas-ingstrengthofmagneticfield.
TheobservedresultsmaybeattributedtothefactthatthemagneticmomentofamaterialMisproportionaltotheappliedfieldH.
MχmH;7whereχmismagneticsusceptibilityofthematerial.
Thepossiblereasonfortheobservedincreasedswellinguponapplicationofexternalmagneticfieldmaybethatundertheappliedfieldthemagneticnanoparticlesgetalignedwiththeappliedfield.
Sincetheparticlesareinconstantmotion,theywillexperiencefluctuatingmagneticfieldwhichmaycauseagitationofthenanoparticles.
Thiswillproduceamotioninthenanoparticlesmatrixandre-sultinrelaxationofpolymerchainsofthenanocomposite,thus,leadingtoagreaterswellingofIOICNPs.
Thismayalsoenlargethenanostructureofthepolymericmatrixtoproduceporouschannelsthatcauseenhanceddiffusionprocessenablingeasyswelling.
Themechanicaldeform-ationgeneratescompressiveandtensilestresses,enhan-cingthepenetrationofwatermolecules.
Similartypeofresulthasalsobeenreportedelsewhere[27].
EffectofchemicalcompositioninCNPsandIOICNPsInthepresentwork,theinfluenceofchemicalcompos-itionofcaseinnanoparticlesontheirswellingratiohasbeeninvestigatedbyvaryingtheamountsofcaseinandcrosslinker(glutaraldehyde),inthefeedmixture,respect-ively.
Theobservedresultsmaybediscussedasbelow:VariationofcaseininCNPsandIOICNPsTheeffectofincreasingbiopolymercontentontheswellingcharacteristicsofCNPsandIOICNPshasbeeninvestigatedbyvaryingtheamountofcaseinintherange0·5–2.
5gwhilekeepingtheconcentrationofglutaralde-hydeasconstant.
Theresultsareshownin(Figure8b),whichclearlyindicatethattheswellingofIOICNPsishigherthanCNPsandinitiallyincreasesupto0.
5gofca-seincontentandthereafterdecreaseswithfurtherincreaseintheamountofcasein.
Theresultsmaybeattributedtothefactthattill1.
5gofcasein,highlycompactnanoparti-clesareformedwhichrestrictstheinwardmovementofwatermoleculesthusresultinginadecreaseinswellingratio.
However,beyond1.
5gofcasein,swellingratioisfoundtoincreasewithincreaseintheamountofcaseinupto2.
5g.
TheobservedresultsareduetothefactthatcaseinisahydrophilicbiopolymeranditsincreasingTable1QuantitativeevaluationofinvitrocytotoxicreactivityofvarioussamplesS.
no.
SampleGradeReactivity1.
Negativecontrol0None2.
Positivecontrol4Severe3.
IOICNPs0NoneSinghetal.
JournalofNanobiotechnology2014,12:38Page8of13http://www.
jnanobiotechnology.
com/content/12/1/38amountintheparticleswillobviouslyenhancethehydro-philicityofthenanoparticlesand,thus,anincreaseintheswellingratioisobtained.
InIOICNPs,thedecreaseinthetotalwatercontent,mightbeattributedtoeffectiveinteractionsbetweentheironoxideandthepolymermatrix.
Theseinteractionsmayarisefromthecarboxylicgroupsofcaseinthatactasiron-bindingsites[28,29].
Takingintoaccountthatthenumberofcarboxylicgroupsincreasesonincreasingtheamountofcasein,andtheinteractionsbetweenironoxideandpolymermatrixadecreaseintheswellingbehaviorofIOICNPsmaybejustified.
Theresultsalsoindicatethatswellingofnanoparticlesinmagneticfieldishigherthanthatintheabsenceofmagneticfield.
Theresultscanbeexplainedbythefactthatduetotheappliedmagneticfield,themagneticmomentsofimpregnatedironoxidenanoparticlestendtogetalignedwiththeexternalmagneticfieldandwhiledoingso,theyproduceamotionofmacromolecularchainsinthecaseinmatrix.
Thusduetothemobilityofironoxidenanoparticles,themacromoleculechainsofcaseingetrelaxedandfacilitategreaterinclusionofwatermoleculesintothebiopolymermatrix[30].
Thisclearlyresultsinanenhancedswellingofnanoparticles.
Figure7MIcroscopicimagesshowingL-929cells.
a)negativecontrol,b)positivecontrol,andc)IOICNPs,respectively.
Figure8Effectofmagneticfield,casein,glutaraldehyde,andH2SO4onswellingofCNPsandIOICNPs.
a)magneticfield,b)casein,c)glutaraldehyde,andd)H2SO4onswellingofCNPsandIOICNPs.
Singhetal.
JournalofNanobiotechnology2014,12:38Page9of13http://www.
jnanobiotechnology.
com/content/12/1/38VariationofglutaraldehydeinCNPsandIOICNPsTheeffectofcrosslinkerontheswellingprofilesofCNPsandIOICNPshavebeeninvestigatedbyvaryingtheconcentrationofglutaraldehydeintherange0.
02to20mM.
Theresultsareshownin(Figure8c),whichclearlyrevealthatthewatersorptionbynanoparticlesconstantlyincreaseswhilebeyond10mMconcentrationadropinswellingratioisobserved.
Theobservedincreasemaybeattributedtothefactthatglutaraldehydeisalowmolecularweightcrosslinkingagent,anditcrosslinksbyreactingwiththeNH2groupsofcaseinatitstwoterminals.
Thus,acrosslinkednetworkisdevelopedasaresultofcrosslinkingreactionbetweencaseinandglutaraldehydethuscreatingawidespaceinitsstructureand,therefore,possessinghighcapacityofac-commodatingwatermoleculesintothenetwork[31].
Inthisway,thecapacityofnanoparticlestoaccommodatelargenumberofwatermoleculesresultsinanincreasedswellingratio.
Thedecreaseobservedbeyond10mMofglutaraldehydemaybeexplainedbythereasonthatmuchhighercrosslinkercontentinthenanoparticlesmatrixreducesthefreespaceinthenanoparticlesnetworkaccessibletothepenetratingwatermoleculesandconse-quentlyresultsinafallintheswellingcapacity.
Someauthorshavealsoreportedthatintroductionofcrosslinkerintothepolymermatrixenhancesitsglasstransition(Tg),whichbecauseofglassybehaviorofpoly-mersrestrainsthemobilityofnetworkchainsand,thusdecreasestheswelling[32].
Theswellingresultsalsoin-dicatethattheappliedmagneticfieldalsoenhancestheswellingofnanoparticleswhichhasalreadybeenex-plainedearlier.
VariationofSulphuricacidIonicallycrosslinkedCNPsweresuccessfullypreparedbyatwo-stepprocess.
Thefirststepinvolvedtheforma-tionofoildroplets(emulsion)byanoil-in-wateremulsionformation.
Thesecondstepwasthesolidificationbyusingsulphuricacidoftheformeddropletsbyioniccrosslinkingofcasein(pH2)envelopingtheoildropletswithglutaral-dehyde.
BydecreasingthepHofcaseinsolutionbelowitsisoelectricpoint(4.
6–4.
8),theaminogroupsofcaseinbe-comepositivelychargedbyprotonationandcanstronglyattractthenegativelychargedaldehydicgroupsofglutaral-dehyde,leadingtotheformationofionicallycrosslinkednanoparticles[33].
Theeffectofsolidifyingagent(sulphuricacid)ontheswellingprofileofCNPsandIOICNPshasbeeninves-tigatedbyvaryingtheconcentrationofH2SO4intherange20–200mM.
Theresultsareshownin(Figure8d),whichclearlyrevealthattheswellingofnanoparticlesconstantlyincreaseswhilebeyond80mMconcentrationadropinswellingbehaviorisobserved.
Theobservedde-creasecouldbeattributedtothefactthatastheamountofH2SO4increasesinthefeedmixture,thenumberofpositivelychargedaminogroupsincreaseswhichenhancestheinteractionbetweenaldehydicgroup(O-+C-H)in-creasesthusresultinghighcrosslinkingdensitybetweencaseinandglutaraldehyde.
VariationofFe2+/Fe3+TheCNPstreatedwithFe2+/Fe3+solutioncontainironionsdispersedthroughoutthecaseinnetwork.
Theelec-trostaticforcesbetweentheironionsandamidegroupsofcaseinareresponsiblefortheincreaseintheswellingofIOICNPs,ascomparedtoCNPswithoutions.
Whentheironions(Fe2+0.
5/Fe3+1M)loadedparticlesaretreatedwithalkali,magnetitenanoparticlesareformedinsidetheCNPs.
Thisresultsinanincreasedswellingcapacityduetoincreasedelectrostaticforcesbetweentheamidegroupsandironoxide.
Theresultsareshownin(Figure9a).
TheswellingcapacityfollowstheorderCNPsIOICNPswhichhassametrendasobservedinPAMhydrogel–silvernanocomposites[34].
Whentheironionsloadednanopar-ticlesaretreatedwithNaOH,magnetitenanoparticlesareproducedinsidethematrix.
Theparticlestreatedwithironsaltshadironionsdispersedthroughoutthepolymericnetwork.
Theelectrostaticforcesbetweentheironionsandamidegroupsofcaseinmatrixareresponsiblefortheincreaseintheswellingofironionloadednanoparticles,ascomparedtonativecaseinnanoparticleswithoutions.
Fe3O4magneticnanoparticleswerepreparedwithdifferentconcentrationsofFe2+,Fe3+inaqueousphase,whileotherpreparationconditionswerekeptsame.
TheaveragesizeofFe3O4magneticnanoparticlesincreasesasconcentrationofironsaltsolutionincreases.
Theparticlesizewasfoundtoincreasewiththeincreaseinconcentra-tionofironsalts[35].
EffectofpHpHsensitivemacromoleculardeviceshavebeenmostfrequentlyusedtodesignswellingcontrolledreleasedrugformulationsfororaladministrationwhichisthemostclinicallyacceptablewayofdrugdelivery.
Inthepresentstudy,theeffectofpHontheswellingratioofCNPsandIOICNPshasbeenstudiedbyadjustingpHofswellingmediumintherangeof1·0to9.
0.
Theresultsaredepictedin(Figure9b),whichclearlyindicatethattheswellingratioofparticlesconstantlyincreaseswithincreasepH.
TheincreaseobservedintheswellingratiooftheparticleswithincreasingpHmaybeexplainedasfollows.
ItisknownthatcaseiniscationicinnatureandthereforeachangeinpHoftheswellingmediumalsoaffectsthechargeprofilesofcaseinaswellasIOICNPs.
TheswellingresultsindicateasignificantdependenceofswellingbehaviorofthenanoparticlesondifferentpHvalues.
InboththecasesofCNPsandIOICNPs,theSinghetal.
JournalofNanobiotechnology2014,12:38Page10of13http://www.
jnanobiotechnology.
com/content/12/1/38resultsrevealthatatpH1.
8,alowerswellingratiowasobserved,becausePKaofcaseinisabout4.
2andmostofthecarboxylgroupsinthecaseinexistintheformofCOOHatlowpHmedium(pH1.
7).
Inthemacromol-ecularnanoparticlenetworkofthehydrogenbondingproducedby–COOHgroupsofcaseinledtothestron-gerinteractionbetweenpolymerchains.
Accordingly,theswellingratioinpH1.
7isrelativelylower.
AthigherpH,thecarboxylicgroupsgetionizedandacquire–COOform.
Thus,weakhydrogenbondingbetweenbiopolymerchainsandelectrostaticrepulsionbetween–COOgroupsresultinthehigherswellingratio[36].
SincepKaofcaseinis4.
2,thespeciesinvolvedintheinteractionsareNH3+andCOOHatpH1–3,NH2andCOOatpH7–13.
Inacidicconditions,theswellingiscontrolledmainlybytheaminogroup(NH2)whichisaweakbasewithanintrinsicpKavalueofabout6.
2.
So,itgetsprotonatedandtheincreasedchargedensityonthebiopolymerenhancestheosmoticpressureinsidethenetworkparticlesbecauseoftheNH3+-NH3+electrostaticrepulsion.
Thisosmoticpressuredifferencebetweentheinternalandexternalsolutionsofthenetworkisbalancedbytheswellingofthenetwork.
However,underveryhighlyacidicconditions(pH8.
5[37].
TheswellingofIOICNPsisgovernedbynegativechargepossessedbyironoxidenanoparticles.
WhenpHoftheswellingmediumis9.
0,thenumberofnegativelychargedgroups(Fe-O)islarge,sotheswellingismax-imumbecauseofthestrongelectrostaticrepulsionbe-tweennegativelychargedgroups.
WhenpHoftheswellingmediumis7.
0,theprotonsfromtheswellingmediumneutralizemostofthenegatively-chargedgroupsand,therefore,theswellingratiodecreasesduetothereducedelectrostaticrepulsion.
At1.
2pH,allnegatively-chargedgroupsareneutralized;instead,therewouldbesomeposi-tivelychargedaminegroupsandironspecies(Fe-OH2+),becausetheaminegroupsintheIOICNPsarefewerthanthecarboxylgroupsandthenetchargeisquitelow,thusthattheswellingisalsoverylow[38].
WhenpHislessthanisoelectricpoint(pI)ofcasein,theextentofincreaseinswellingratioatacidicmediumissmall,becausethereareveryfewaminegroupsexistingalongproteinchainssothatthepositivechargesareveryFigure9Effectofironsalts,pH,temperatureandsimulatedbiofluidsonswellingofCNPsandIOICNPs.
a)ironsalts,b)pH,c)temperature,andd)simulatedbiofluidsonswellingofCNPsandIOICNPs.
Singhetal.
JournalofNanobiotechnology2014,12:38Page11of13http://www.
jnanobiotechnology.
com/content/12/1/38limited.
TheswellingbecomesminimumwhenpHofthemediumisclosetothepIofthecasein(4.
6-4.
8).
ThisisbecausethenetchargeoncaseinmoleculesisclosetozeroatpI,whichmeansalmostnoelectrostaticrepulsionexistbetweenthecaseinchains.
Ontheotherhand,whenpH>pI,therearelotsofnegatively-chargedgroupsonthecaseinmolecule,whichresultsinanincreasedswellingratio.
ThehigherthepH,greateristhesurfacechargeandconsequentlythehigherelectrostaticrepulsiveforcesresultinhigherswellingratio[39].
EffectoftemperatureInthepresentstudy,theeffectoftemperatureonswellingwasstudiedbyvaryingthetemperatureintherange12.
5–37.
5°C.
Theresultsareshownin(Figure9c),whichindicatethatwithincreaseintemperature,theswellingofnanoparticlesincreasesfrom2.
5to4.
5inthewholestudiedrangeoftemperature.
TheobservedincreaseintheswellingofIOICNPsmaybeexplainedbythefactthatariseintemperatureenhancestherateofdiffusionofwatermoleculesandsegmentalmobilityofbiopoly-merchainswhichresultsinagreaterdegreeofswelling[40].
However,beyond30°Ctheswellingratiodecreases,whichmaybeduetothereasonthatathighertempe-raturethehydrogenbondsholdingwatermoleculesandthepolymerchainsgetbrokenand,therefore,theswellingratiodecreases.
EffectofphysiologicalfluidsTheeffectofnatureofphysiologicalfluidsontheswellingofCNPsandIOICNPsinabsenceandpresenceofmag-neticfieldhasbeeninvestigatedbyperformingswellingexperimentsinvarioussimulatedphysiologicalfluidslikeurea,D-glucose,PBS,salinewater,distilledwaterandsyntheticurine.
Theresultsarepresentedin(Figure9d),whichindicatethatswellingratioisquitehighinureaincomparisontootherfluidsandlowerdegreeofwatersorptionisnoticed.
Thepossiblereasonforthehigherswellingratioinureamaybethatthepresenceofureaworksashydrogenbondbreakerincaseinmacromoleculeandthistendstoresultingreaterrelaxationofbiopolymerchainswhicheventuallyleadstohigherswelling.
Inthecaseofotherfluidsthesaltionsinthemediumlowerstheosmoticpressuredifferencebetweenthecaseinmatrixandsolventmediumwhichcausesadecreaseinswellingrationofnanoparticles.
Thechangeinswellingduetothepresenceofelectro-lyteconcentrationhasalsobeenpredictedtheoretically.
Fernández-Nievesetal.
[41]demonstratedthatthead-ditionofsaltsmodifiestheionicdistributioninsidethecaseinmatrix,alteringthenumberofdissociatedgroupsinthenetwork,andchangingthenetcharge.
Thechangeinthenetworkchargestatemakesparticlesswellorshrink,dependingonwhethertheelectricalrepulsionsincreaseordecrease,respectively.
ConclusionsThecontrolledsizemagnetitenanoparticleshavebeensuccessfullypreparedviaaconvenientco-precipitationmethodandcharacterizedbyvariousanalyticaltech-niques,suchasFTIRspectroscopy,TEM,SEM,XPS,VSMandRamananalyseswhichconfirmtheinsituimpregnationofnanosizedironoxidewithinthematrixofcaseinnanoparticles.
ThebiopolymericnanoparticlesclearlyshowthepresenceofcharacteristicfunctionalgroupsofcaseinandironoxideasconfirmedbytheirFTIRspectra.
SEMandTEMofthenanoparticlespro-videinformationabouttheirsizeandmorphology.
TheIOICNPsshowanoptimumswellingwhenthecaseincontentis0.
5gwhileonincreasingcaseincontentfurtherthedegreeofswellingdecreases.
Likewise,whentheconcentrationofcrosslinkerincreasesfrom0.
02-20mM,thequantityofwaterimbibedbythenanoparticlesin-creaseswhilebeyond10mMofcrosslinkerconcentration,theextentofswellingdecreases.
ItisalsofoundthatinalkalinepHthenanoparticlesshowanenhancedswellingwhichthereafterdecreaseswithfurtherincreaseinpH.
Inthecaseofrisingtemperaturetheswellingratioconstantlyincreases.
ThepreparedIOICNPsexhibitpropertyofsuperparamagnetismwhichisasignificantforbiomedicalapplications.
Itisalsoobservedthattheswellingofnanoparticlesisenhancedbytheapplicationofexternalmagneticfield.
Thusthepresentswellingsystemmaybehelpfulindesigningtargeteddrugdeliverycarriersusingexternalmagneticfield.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsAKBsupervisedthestudy,andcontributedtothedraftingofthisarticle,selectionofmethodology,analysis,discussionoftheresultsandfinalizedthemanuscript.
JBconceivedthestudy,participatedinitsdesignandcoordination.
AScarriedoutalltheexperimentalstudies,contributedinthedesignofthestudy,analyzeddata,anddraftedthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
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doi:10.
1186/s12951-014-0038-4Citethisarticleas:Singhetal.
:InvestigationofmagneticallycontrolledwaterintakebehaviorofIronOxideImpregnatedSuperparamagneticCaseinNanoparticles(IOICNPs).
JournalofNanobiotechnology201412:38.
SubmityournextmanuscripttoBioMedCentralandtakefulladvantageof:ConvenientonlinesubmissionThoroughpeerreviewNospaceconstraintsorcolorgurechargesImmediatepublicationonacceptanceInclusioninPubMed,CAS,ScopusandGoogleScholarResearchwhichisfreelyavailableforredistributionSubmityourmanuscriptatwww.
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