Synthesisofimmunomagneticnanoparticlesandtheirapplicationinthe
separationandpurificationofCD34+hematopoieticstemcells
WeiChena,HebaiShena,*,XingyuLia,NengqinJiaa,JianmingXub
aLifeandEnvironmentScienceCollege,ShanghaiNormalUniversity,Shanghai200234,PRChinabZhongshanHospitalofFuDanUniversity,Shanghai200030,PRChina
Received31October2005;accepted6March2006Availableonline18April2006
Abstract
Thesilica-coatedsuperparamagneticnanoparticleswiththeuniformdiameterofabout60nmweresynthesizedbyreversemicroemulsionsmethod.AndthemagneticnanoparticlesweremodifiedwithN-(2-aminoethyl)-3-aminopropyltrimethoxysilane(AEAPS).Theimmunomagneticnanoparticleswerethensuccessfullypreparedbycovalentlyimmobilizinganti-CD34+monoclonalantibodiestothesurfaceofaminosilanemodifiedmagneticparticles.ThecellseparationresultsshowedthatthesynthesizedimmunomagneticnanoparticlescouldrapidlyandconvenientlyseparatetheCD34+cellswithhighefficiencyandspecificitythannormalones.Thesurfacemorphologyofseparatedtargetcellswasexaminedbyscanningelectronmicroscope(SEM).Atomicforcemicroscope(AFM)alsocharacterizedthemagneticmaterialsonthesurfaceoftheseparatedtargetcellsforthefirsttime,whichfurtherconfirmedthatthetargetcellswereseparatedbytheimmunomagneticnanoparticles.TheviabilityoftheseparatedcellswasstudiedbyculturingmethodandBeckmanVi-cellviabilityanalyst.Therefore,ourexperimentsprovidedanew,direct,rapidmodetoseparatetargetcells.#2006ElsevierB.V.Allrightsreserved.
PACS:75.50.-y;07.79.LH;87.17.E
Keywords:Immunomagneticseparation;CD34+;Monoclonalantibody(MAbs);Atomicforcemicroscopy(AFM);Scanningelectronmicroscopy(SEM)
1.Introduction
Nanoscienceisoneofthemostimportantresearchand
developmentfrontiersinmodernscience[1].Theuseof
nanoparticlematerialsoffersmanyadvantagesduetotheir
uniquesize,physicalproperties,largesurfacearea/volumeand
soon[2,3].Amongallthesematerials,themagneticparticlesare
paidmoreattention.Astheirspecialtraitofmagnetism,they
havebeenwidelyappliedinvariousfieldsofbioseparationand
medicine,suchasproteinandenzymeimmobilization[4,5],
immunoassay,RNAandDNApurification,cellisolationand
recognition[6–8],targetdrug[9–11]andthePCRreaction[12].
Usingmagneticparticlesisadvantageousforfullautomation,
resultinginminimizingmanuallaborandprovidingmoreprecise
results[13].Useofnanomagneticparticlesalsohasthe
advantagesinassaysensitivity,rapidityandprecision[14].
Allthesebioseparationandmedicineapplicationsrequirethat
thesenanoparticleshavehighmagnetizationandsmalldiameterwithnarrowparticlesizedistributionsothattheparticles
haveuniformphysicalandchemicalproperties[15].
Itiswellknownthatthehematopoieticstemcells(HSC)are
thesourceofallbloodcells[16].Theyareveryimportantfor
generatingallkindsofcellsandmaintainingthenumberofcells
inperiphericblood.Sincethefirstsuccessfultreatmentof
Fanconi’sanemiabytheusingofhumanumbilicalcordblood
in1988,moreandmoreresearcheshavebeendoneaboutthe
transplantingofhumanumbilicalcordblood[17].Human
umbilicalcordbloodtransplantinghasbeenappliedincuring
manymalignancyhematonosis,suchasacutelymphocytic
leukaemia,chronicgranulocyteleukaemia,Hodgkin’sdisease
andsoon.Now,ithasbeenrevealedthatthehumanumbilical
cordbloodisrichinHSC.InordertostudytheHSC,thefirst
thingthatshouldbedoneistoseparatetheHSCfrom
hematopoietictissue.Thenormalwayisutilizingthemarker
proteinonthesurfaceofHSC.Nowadays,CD34+isthemost
widelyusedmarkerproteinofHSC.Amongallthosemethods,
theimmunomagneticseparationisaveryattractivemethod
[7].Immunomagneticseparationcanseparatethetargetcells
fromthecrudecellsdirectly,convenientlyandrapidly.Asearly
www.elsevier.com/locate/apsuscAppliedSurfaceScience253(2006)1762–1769
*Correspondingauthor.Tel.:+862164322516;fax:+862164321800.E-mailaddress:shenhb@shnu.edu.cn(H.Shen).
0169-4332/$–seefrontmatter#2006ElsevierB.V.Allrightsreserved.doi:10.1016/j.apsusc.2006.03.012asin1980’s,peoplehadbeguntoresearchthemagneticcell
separation[18–20].Untilnow,themainlyusedmagnetic
particlesaremicroparticlesandtheantibodiesmodifiedonthe
magneticparticlesaremainlysecondantibodies.Thesekinds
ofimmunomagneticparticlesmayhavesomedisadvantages,
suchasfastsedimentationrate,celldamnificationandsoon.
Andalsotheusingofthesecondantibodiesmaybeincreasethe
costoftheexperimentandrequirehigherexperimental
techniquesandconditions.However,thereareverylimited
literaturereportsaboutthedirectapplicationofmonoclonal
antibodymodifiedimmunomagneticnanoparticlesinthe
separationandpurificationofCD34+hematopoieticstem
cells.Byusingthismethod,itcanseparateandpurifythetarget
cellsfromthecomplicatedmixedsystemwithhighefficiency
ratherthanwithcumbersomeexperimentalstepsandhighcost.
Inthisstudy,wehavedevelopedapowerfultechnique,
whichallowsasimpleandfastisolationandenrichmentof
CD34-positivecellsbyusingnano-scalesuperparamagnetic
particleswiththevirtueofcost-effective.Thesuperparamag-
neticnanoparticlesusedtoprepareimmunomagneticnano-
particleshavebeenpreparedandcharacterizedbysome
advancedmeans.Themorphology,structureandcharacterof
thenanoparticleswerestudiedbyTEMandFT-IR.We
modifiedthepreparednanoparticleswiththeactivegroupof–
NH2onthesurfaceofthenanoparticles.Bythisactivegroup,
theCD34+monoclonalantibodiesweredirectlyconnectedto
thesurfaceofthenanoparticles.Themodifiednanoparticles
wereusedtoseparatetheHSCfromthehumanumbilicalcord
blood.Thesurfacemorphology,purityandviabilityofthe
separatedHSCswerecharacterizedbyscanningelectron
microscopy(SEM),atomicforcemicroscope(AFM)and
HematopoieticColonyAssaymethod,respectively.
2.Experiments
2.1.Materials
Theferrouschlorideheptahydrate(FeCl2Á7H2O),ferric
chloridehexahydrate(FeCl3Á6H2O),sodiumhydrate,tetraethyl
orthosilicate(TEOS),ammoniumhydroxide(28%,w/w)were
purchasedfromthesino-pharmchemicalreagentlimited
companyinShanghai.N-(2-aminoethyl)-3-aminopropyltri-
methoxysilane(AEAPS)andglutaraldehydewerepurchased
fromJ&KchemicaCompany.RPMI-1640mediumwere
purchasedfromSino-AmericanBiotechnologyCompany.
Themouseanti-humanmonoclonalantibodyagainst
CD34+cellsurfaceantigenwasobtainedfromBiomeda
Company.Allthechemicals,usedintheexperiment,were
commercialavailableandwereofanalyticalreagentgrade.
Doubledistilledwaterwasusedforalltheexperiments.
2.2.Synthesisofsilica-coatedsuperparamagnetic
nanoparticles
Theg-Fe2O3magneticnanoparticleswerepreparedbyour
reportedmethod[21].Typically,2.5MNaOHsolutionwas
droppedintothesolutionofmixtureofFeCl3(0.2M)andFeSO4(0.12M)withviolentlystirring.Thentheobtained
precipitationwasagedatthetemperatureof608Cfor2hand
washedwithwaterforseveraltimes.Finally,themagnetic
nanoparticlesweredriedat608Cfor24h.Tocoatthemagnetic
nanoparticleswithsilica,weadoptedthewater-in-oilreverse
microemulsionsofwater/TritonX-100/n-hexylalcohol/cyclo-
hexane.0.1gg-Fe2O3wasdispersedinthe60mlabove
microemulsionwith3-minultrasonication,thenthesolution
waspouredintothree-neckedflaskwithvigorousstirringat
228C.Concentratedammoniaandtetraethoxysilane(TEOS)
wereaddeddropwiseintothesysteminturn.Thereaction
endedafter10h.Thenanoparticleswereagedovernightand
thenwashedwithethanolthreetimes.Atlast,thesilica-coated
g-Fe2O3nanoparticlesweregotaftersinteredat6508Cfor2h.
2.3.Modificationofsilica-coatedsuperparamagnetic
nanoparticleswithAEAPS
Twentymilligramsofg-Fe2O3nanoparticlesweredispersed
inthemixtureof25mlmethanoland15mlglycerolwith
30-minultrasonication.Then1.5mlAEAPSwasaddedinto
themixtureanddispersedbyvigorousstirring.Throughthe
wholeexperiment,thetemperaturewasretainedat608C.The
amino-silanemodifiednanoparticleswerewashedwithmeth-
anolanddoubledistilledwaterforthreetimes,respectively.
Thenthenanoparticlesweredriedinthevacuumoven.The
amino-silanemodifiedsilica-coatedmaghemitewasusedto
immobilizethemonoclonalantibodyagainstCD34+cell.
2.4.Characterizationofmagneticnanoparticles
2.4.1.Transmissionelectronmicroscope(TEM)
characterizationofthesilica-coatedmagnetic
nanoparticles
Thesize,distributionandmorphologyofthesilica-coated
magneticnanoparticleswerestudiedbyusingaHitachi-600
TEM.Thesampleofthesilica-coatedmagneticnanoparticles
wasdrop-castontoacarboncoatedcoppergridandairdriedat
theroomtemperature.
2.4.2.Fouriertransformedinfrared(FT-IR)
characterizationofthesilica-coatedmagnetic
nanoparticles
TheFT-IRspectrumwasrecordedinthetransmissionmode
onaNicoletAvatar370spectrometer.Thedriedsampleof
magneticnanoparticleswasgroundedwithKBrandthemixture
wascompressedintoapellet.Thespectrumwasscannedfrom
4000to400cmÀ1.
2.5.Preparationofantibodyimmobilizedamino-silane
modifiedsilica-coatedmagneticnanoparticles
Beforeimmobilizingtheantibody,weadoptedthereported
waytoactivatethenanoparticlesbyglutaraldehydemethod
fortheapplicationinbioseparation[2,3].Theactivated
nanoparticleswerethendispersedinthephosphatebuffered
saline(PBS,0.1M,pH7.4)withidenticalconcentrationsW.Chenetal./AppliedSurfaceScience253(2006)1762–17691763
