Oral presentation held at DICNMA 2013. Recently, magnetic nanoparticles (MNP) have attracted much interest for medical applications, like magnetic cell separation, magnetic resonance imaging, drug delivery and hyperthermia [1]. For these purposes, accurate surface coating, including specific targeting ligands, is a necessary task. To achieve specific tagging of biological entities, MNP are frequently attached to antibodies. In this work, 10 nm MNP were functionalized with human α-MICA antibody, via biotin-streptavidin conjugation, due to the importance of MICA receptor with antitumor immunity [2]. MNP have been analyzed by Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and Fourier Transform Infrared spectroscopy (FTIR) before and after functionalization, to check its efficiency. The potentiality of the nanoparticles has been tested in vitro with two different cell lines: HeLa (with MICA ligands on surface) and HL-60 (without MICA ligands). MNP tagged HeLa cells were effectively separated by application of a magnetic field gradient. TEM micrographs were performed after magnetic separation, proving intracellular presence of the functionalized MNP.

1. Goodmorning, I’mDavid Lago-Cachón, fromtheUniversityof Oviedo and todayI’mgoingtotalkaboutourworkinapplyingmagneticnanoparticlestobiologicalapplications.
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3. As thisisa verymultidisciplinarytask, thisworkhas beencarriedout(performed) in thePhysicsDepartmentof theUniversity, theCentral Hospital of Asturias and a companyin thesensoringfield, Healthsens.
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4. In fact, ourobjectiveistodevelopanearlydetectionmethodof illnesesusingmagneticnanoparticlesas tags, and a magneticsensor todetectthem.
Briefly, themethodconsistsof thefollowingsteps:
First, a liquidsamplefromthepatient(i.e. blood) has tobe obtained.
Secondthesampleismixedwithmagneticnanoparticles(MNP). ThoseMNP mustbe functionalizedso thattheyjoinspecificallythatwewanttodetect.
Third, theremanentMNP are thenremoved.
Finally, are detectedbya magneticsensor whichallowsdetectionand quantificationof thebiologicalentity.
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5. So, wehavetwoparalleljobs: Ononehand, thedevelopmentof convenientlyfunctionalizedmagneticnanoparticlesdesignedforthatparticular biologicalissue, whichismainlywhati’mgoingtotalkaboutrightnow.
And ontheother, thedevelopmentof a suitablemagneticsensor todetectthosenanoparticles.
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6. Let’sstartfromthebeginning. Theseare themagneticnanoparticlesthatweworkwith. Theyare magnetitenanoparticlesmadebya co-precipitationmethodand… /next/
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7. … coveredbyPolyAcrilycAcid(PAA).
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8. The mean size of the nanoparticles can be obtained from electronic microscopy like this one, and it is 10 nm. That’s a sufficiently small diameter for the MNP to be superparamagnetic.
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9. The SPM state was confirmed by Field Cooling, Zero Field Cooling experiments, as this one, that shows a Blocking Temperature of about 130 K, far below room temperature.
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Now, as wewantedthenanoparticlestotagspecificallytoa biologicalentity, wehadtofunctionalizethem. Thatmeans, weincorporateanantibodyonthesurfaceof thenanoparticles.

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As youprobablyknow, antibodiesare a veryusefullkindof proteinsthathavetwodetectionregions, at theseends, thatbindotherproteinsormoleculeswitha veryhighspecifity.

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Thestrategywefollowto functionalizetheMNP wasa streptavidin-biotinreaction. Thatproteinand thatmoleculehavea greataffinityforeachotherand are frequentlyusedto conjugatethings. As manyantibodiesare comerciallyavailablewitha biotinaddedto them, weonlyfocusedoncoveringthenanoparticleswithstreptavidin… /next/

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… viaanamida bound.

14. To check if ourfunctionalization has been successful we made several Fourier Transform Infra-Red Spectroscopy of the original nanoparticles, covered with polyacrilicacid, and the final nanoparticles covered with streptavidin and the antibodies. The carbonil(C=O), carboxilategroups and –CH2, disappear in the functionalized nanoparticles, at the same time that nitrogen functional groups appear.
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15. Also, wemeasuredthehidrodinamicsizeof thenanoparticlesbeforeand afterconjugation. As youknow, thehydrodynamicsizehaveintoaccountthesurfactantand theinteractionwiththemoleculesof thesolvent. TheprimaryMNP coveredwithPAA has a mainhydrodynamicsizeof 60 nm, and themagnetitenanoparticlescoveredfirstlywithstreptavidinand secondlywithanantibodyhas a biggersizeof about300 nm.
NowthattheMNP has beencharacterizedI’mgoingtopresentyoutheworkwehavedone withthem.
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16. Weperformed some in vitro experiments with a human tumor cell line, the HeLacells.Why have we chosen that cell line? Two reasons. First one, they are easy growing cells, and the most important is that this kind of cells express in their surface a protein, called MICA, that is mainly related to several illnesses. So, now we do our in vitro experiments, and later it can be done with a patient sample.
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17. I saidthatHeLacells“expressthatprotein”, thatmeanswhichthisproteinislocatedacrossthecellmembrane, witha smallportioninsidethecellbutmostof theproteinisoutsidethecell.
WeusedbiotinilizedantibodyagaintsthatMICA protein, weconjugateditwithourstreptavidincoveredMNP and as a result, theMNP stickonthatprotein, thuslabellingthecells.
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Howdidwelabelthecells?
HeLacellsgrowadheredtosurfaces, and thatsimplifiesthework. Firstweremovethemediuminsidethecellsweregrowingand weaddnew mediumwiththeMNP. Wewaitfora short time, only15 minutes istime enoughtotheantibodiestostickonthecells. Afterthattime weremovethatmedium, wewashusually3 times to ensurethateveryunboundnanoparticlehas beentakenaway. And finnallyweaddednew medium.

19. Afterthelabellingof thecells,wedetacchedthemusingtrypsine-edtaand I’mgoingtoshow youhowthatcellshavea magneticbehaviourthankstothepresenceof MNP. Everyoneof thissmallcirclesare HeLacellsthathavebeentaggedwithMNP. Ifa MFG isapplied, theMNP are goingtobe attracted, and as theyare boundtothecells, we’llseehowthecellsmove.
Thisisimportantbecauseitcouldincreasetheconcentrationand improvethesensibilityof currentdiagnosis methods.
Wecouldseparatethecellsin twofractions, themagneticallytaggedorpossitivefractionand thenegativefraction.
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20. WedoneTEM to thepossitivefractionand wesawhowthenanoparticlesare internalizedinsidethecell. Howcouldthathappen? Thelabellingtime wereshort enoughtoavoidaccidental endocitosisis. In opposite, thatwasa receptor-mediatedendocytosis.
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21. AllthisworkI showedyouhavebeenrepeatedwithnon functionalizedMNP, and no cellmoved. And alsoithas beendone witha differentcellline, thatdo nothaveMICA in theirsurface.
So, weconcludethat…. /next/
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22. … ournanoparticlesspecificallytagMICA expressingcells. As itwasexpected, theantibodyhaveworked, and therewerenon specificinteractionbetweentheMNPsand thecells.
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23. I have to acknowledge all the partners that have made this work possible.
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24. Finally, Iwould like to thank the organization of the congress for that opportunity, and thank you for you attention.
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