流式细胞分析Comparative-Flow-Cytometric-Analysis-of

Flow cytometry Comparative Flow Cytometric Analysis of Immunofunctionalized Nanowire and Nanoparticle SignaturesAdriele Prina-Mello,*Aine M.Whelan,*Ann Atzberger,Joseph E.McCarthy, Fiona Byrne,Gemma-Louise Davies,J.M.D.Coey,Yuri Volkov,andYurii K.Gun’ko*F low cytometry is one of the gold-standard techniques used in clinicalmedicine for quantitative immunoassaying.The continuous development ofits probes,commonlyfluorescent nanoparticles,is tely,theintroduction of quantitative multiplexed immunoassay has challenged theuse of nanoparticles as probes.Functionalizedfluorescent silica-basedmagnetic nanowires are investigated underflow cytometry as a novel probecategory.The preparation and full characterization of these multimodalnanowires is reported and compared to those of silica-based magneticnanoparticles byflow cytometry.Full characterization includes trans-mission electron microscopy andfluorescence microscopy imaging,flowcytometric assaying,superconducting quantum interference device(SQUID)magnetization,and Mo¨ssbauer spectroscopy measurements.Thiswork shows that loaded silica nanowires have intrinsic geometricaladvantages when compared to similar spherical particles due to their unique‘‘flow cytometryfingerprint’’when utilized as magnetic carriers forimmunodetection applications.These advantages account for a17%yield indetecting the functional binding between THP-1and ICAM-1,by utilizing amuch lower concentration than that required for the nanoparticles.1.IntroductionIn recent years,there has been an intense effort to develop nanoparticles forfluorescentflow cytometry.[1,2]The ability to perform a specific and quantitative multiplexed immunoassay to discriminate between targeted species has only recently been demonstrated.[3]The ability offlow cytometry to discriminate between individual micro-and nanoparticles based on size,[Ã]Prof.Y.K.Gun’ko,Dr.A.M.Whelan,J.E.McCarthy,G.-L.Davies Centre for Research on Adaptive Nanostructures and Nanodevices andSchool of ChemistryTrinity College Dublin,Dublin2(Ireland)E-mail:igounko@tcd.ie;ainewhelanster@Dr.A.Prina-Mello,F.Byrne,Prof.J.M.D.CoeyCentre for Research on Adaptive Nanostructures and Nanodevices and School of PhysicsTrinity College Dublin,Dublin2(Ireland)E-mail:prinamea@tcd.ieDOI:10.1002/smll.200901014Prof.Y.VolkovCentre for Research on Adaptive Nanostructures and Nanodevices and Department of Clinical MedicineTrinity College Dublin,Dublin8(Ireland)A.AtzbergerFlow Cytometry Laboratory,Institute of Molecular MedicineTrinity College Dublin,Dublin2(Ireland):Supporting Information is available on the WWW under http:// or from the author.Keywords:antibodiesflow cytometryfluorescent probesnanoparticlesnanowiresfluorescence intensity,and/or fluorescence wavelength allows for fast time-resolved detection and low sample and reagent volume,which make this a cost-effective analytical technique.Previous work described how nanomaterials of different sizes for multiplex assays have been used for different biological samples.[4]Recently,the discrimination between multiple micro-and nanoparticles by fluorescent dyes has been proven.[5]However,the technique is prone to the ‘‘encroachment’’problem,or aggre-gation of the mixed microparticle popula-tions.[6]Several solutions have been pro-posed,based on the structural,electronic,magnetic,optical,and catalytic properties of nanomaterials that are not seen in the bulk matter.[4,7,8]This allows for secondary measurements or further assay of the sample,such as high-resolution imaging and detection.[9]To expand the multiplexed capabilities of flow cytometry there is a need to develop novel nanostructures.Nanometer-size wires made of well-characterized composite materials and uniformly coated with biofunctionalities could then offer new opportunities in this area.The aim of our work is to develop the concept of multiplexed flow cytometry by using an improved preparation of super-paramagnetically loaded silica nanoparti-cles and nanowires.The incorporation ofpolydispersed iron oxide nanoparticles into the silica matrix of both structures allows for secondary biomedical assaying,such as cell separation,[10]biosensing,[11]cell ima-ging,[12]and drug delivery.[13]In this workwe use human acute monocytic leukemia cell line (THP-1)to determine their immune expression to CD54antibody.The surface chemistry of the nanomaterials allows antibody incubation and functional coating.Furthermore,the shape difference between the two nanostructured materials is clearly detected by the flow cytometry technique.Silica nanowires and nanotubes are a novel class of inorganic structures for biomedical applications [14,15]and gene delivery.[16]Nanowires were first proposed as an effective platform for immunoassays by Nicewarner-Pena et al.,[17]and the proposal was expanded by Tok and co-workers,who used antibody-coated segmented Au–Ag nanowires for the detec-tion of bacterial spores,bacteriophages,and specific pro-teins.[18]It has been shown that linear magnetic nanostructures have the potential to outperform nanoparticles based on their intrinsic properties.[18,19]Recently,carbon nanotube (CNT)–cell complexes were also measured by side-scattering flow cytometry.[8]The reason for using nanowires is their long aspect ratio.Their surface offers a unique substrate for the selective binding of species such as antibodies with the advantage of maximizing the functional binding of the nanomaterials along their principal axis.This is summarized in Figure 1,in which the nanoparticle forms a single bond to a cell/cell receptor whereas the wire binds at several points.2.Results and Discussion Three aspects of the nanoparticles or nanowires arediscussed here:1)the silica shell which represents the substrate for binding the functional material,2)the magnetic load dispersion of the silica nanoparticle and nanowire in solution,and 3)the uniformity of the loaded fluorescent antibody marker.The coating of magnetic nanocomposites with silica has been the subject of many investigations due to the useful properties of silica.[20]Methods for preparing magnetite-loaded silica include dialysis [21]and sol–gel syntheses.[22]To directly compare the nanoparticles with nanowires,a modified sol–gel synthesis was used,which yielded uniform,mono-disperse nanoparticles with narrow size distribution.[20]Transmission electron microscopy (TEM)images of the magnetically loaded nanoparticles and high-resolutionopticalFigure 1.Schematic comparison of magnetic particle and nanowire binding to a cell/cell receptor.ICAM-1¼intracellular adhesion molecule 1,PE ¼phycoerythrin,FITC ¼fluorescein ß2009Wiley-VCH Verlag GmbH &Co.KGaA,Weinheim small 2009,x,No.x,1–9microscopy images of the nanowires are shown in Figure2a and c,respectively.The magnetite-loaded silica nanoparticles were monodisperse and spherical in shape with an average diameter of144Æ15nm.The magnetically loaded silica nanowires are cylindrical in shape,with lengths of8Æ2m m and diameters of 176Æ50nm.In addition,quantitative measurements of each batch of nanoparticles and nanowires were carried out by confocal microscopy(Figure2b and d)to confirm and extend to a larger sample population the TEM analysis.Figure2e and f showsfluorescent antibody load mapping of the nanomaterials.Laser scanning confocal microscopy provided the dimen-sions of both nanowires and nanoparticles.Hence,the surface area,the surface-to-volume ratio of the functionalized carriers, and the surface loading of the antibody were quantified to be uniform;the results are summarized in Table1.Table1also includes relevant bulk,surface,and magnetic properties and the concentrations used in this study for both nanowires and nanoparticles.The magnetic loading of the nanoparticles and nanowires serves to improve the efficacy of immunoassays by facilitating the separation,preconcentration,and washing steps.[23]In our work we used magnetic immobilization during the functiona-lization of the surfaces of both nanostructures with amino groups and amide coupling(see Supporting Information, Figure S1).A uniform coating of the antibodies on the particle and wire surfaces was achieved.The magnetically loaded silica nanoparticles and nanowires were found to behave super-paramagnetically and therefore have little tendency to aggregate,as observed from the TEM images.The magnetiza-tion of the particles and wires is diluted by the SiO2matrix,asFigure2.a)TEMimageofmagneticallyloadedsilicananoparticles(144Æ15nm);b)high-resolutionconfocalmicroscopyimageofR-PE-IgGantibody-functionalized silica nanoparticles(red arrow represents direction for rendered image(e)).c)TEM images of magnetically loaded silica nanowire (diameter176Æ50nm;length8Æ2m m);d)high-resolution confocal microscopy image of R-PE-IgG antibody-functionalized silica nanowires(red arrow represents direction for rendered image(f)).e,f)3Dfluorescent antibody load mapping of R-PE-IgG antibody-functionalized on silica nanoparticles and nanowires,respectively.Intensity measurements expressed in arbitrary units and normalized to background(magnificationÂ63oil immersion lens).parative analysis of nanowires and nanoparticles.Sample TEM:dimensionalmeasurements(avgÆstd.dev.)Confocalmicroscopymeasurements(average of100)VSM[a]measurement:saturation magnetization(M s)[A m2kgÀ1],volume fraction ofmagnetite[%]Density[kg mÀ3],mass[pg],area[m m2],surface/volumeratioInjectedconcentration[NP mLÀ1],mass[m g]Antibodysurface loading(R-PE intensitymeasurement)[a.u.]NotesNanowire With silica shell With shellþfluo-Ab[b]coated M s¼0.43r¼2.34[NW]¼1.00Â10748.0Æ13.0Wires uniformlydispersed withuniform antibodycoating alongfull lengthf¼176Æ50nm f¼200Æ50nm fraction¼0.02%m¼0.59NW mLÀ1m NW¼5.87m g L¼8.0Æ2.0m m L¼11.0Æ2.1m m A¼5.09s/v¼20.25Nanoparticle With shell With shellþcoated M s¼1.2r¼2.35[NP]¼6.50Â108NP mLÀ174.0Æ35.0The antibodycoating onparticles has verysharp decay fromthe particle centerf¼144Æ15nm f¼200Æ75nm fraction¼0.69%m¼0.01m NPffi6.00m gA¼0.13s/v¼31.50[a]VSM¼vibrating sample magnetometer.[b]Fluo-Ab¼fluorescent antibody marker.shown in Figure3a and b in the magnetization curves of the nanocomposites.The saturation magnetization of the nano-particles is measured as1.25A m2kgÀ1and for nanowires is measured as0.04A m2kgÀ1.The low magnetization reflects the low volume fraction of g-Fe2O3in the silica nanoparticles and nanowires.Mo¨ssbauer analysis showed no ferrous or mixed-valence components in the iron hyperfine spectrum(Figure3c). During processing the magnetite is oxidized to g-Fe2O3,which has a magnetization of61A m2kgÀ1.Hence,the loading is2.0% for the particles and0.07%for the wires.The error arising from the uncertainty in the assumed value of magnetization isÆ20%.The superparamagnetism of both silica-coated nanoparti-cles and nanowires facilitated preconcentration during the functionalization of the surfaces with amino groups and amide coupling.These groups allowed for the surface binding of primary immunoglobulin G(IgG)antibody R-phycoerythrin (R-PE)conjugate to secondary human CD54(intracellular adhesion molecule1or ICAM-1)fluorescein isothiocyanate (FITC)conjugate.To guarantee uniform surface loading by the antibody,each surface chemistry step for both nanowires and nanoparticles was benchmarked against thefinal totalfluor-escence intensity.In particular,since aminopropyltriethoxysilane(APTES)is often used to immobilize biomolecules on surfaces,[24]it was of interest to determine how the concentration of APTES affected the covalent binding of thefinal antibody.Hence,increasing volumes of APTES were incubated in the presence of a saturated volume of FITC-conjugate-labeled CD54mouse antibody.Interestingly,for the nanowires but not the nanoparticles,the amount of APTES used in coating the nanowires was related to the signal obtained from the fluorescently labeled antibody inflow cytometry(see Figure4). In addition,to further maximize the functional adhesion between the antibodies and the amino-functionalized nano-wires and nanoparticles,an intermediate preparation step was undertaken following the established1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride(EDC)-facilitated coupling method.A full investigation was then carried out by confocal fluorescence microscopy to confirm that the silica nanoparticles and nanowires were successfully functionalized with both primary and secondary antibodies(see Table1and Figure2). Figure2e and f show three-dimensional(3D)rendered images and intensity profiles of nanoparticles and nanowires functio-nalized with R-PE-labeled IgG mouse antibodies.The images of100particles and wires show that the functional-group coating on both particles and wires is uniform.Figure2b and d are images of representative samples,in which it is seen that the nanowires remain discrete and unbundled when in solution, whereas the nanoparticles show some tendency to aggregate into small clusters.Inflow cytometry,this is the cause oftheFigure3.a,b)Magnetization curve measured at room temperature for a)magnetite-loaded silica nanoparticles and b)magnetite-loaded silica nanowires obtained using a superconducting quantum interference device(SQUID)magnetometer.c)Mo¨ssbauer spectra of magnetic-particle-loaded silica particles.B HF¼hyperfine magneticfield.Figure4.APTES optimization for antibody loading(ICAM-1).Overlay of fluorescence intensity response of ICAM-1antibody functionalized on silica nanowires using different concentrations of APTES coupling agent.ß2009Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim small2009,x,No.x,1–9‘‘encroachment’’effect.[6]Since it can seriously compromise the immunoassay[2]by the nonspecific binding,coating variation,decreased surface-to-volume ratio,and lack of stability of the biomarkers,there is benefit in using nanowires. In addition,the nanowires show a good antibody uniformity distribution,and therefore less measurement error inflow cytometry(see Figure5c and c1)in the antibody signal detection along their axis,as shown in Table1.To maximize the ICAM-1secondary antibody functional response,when bound to R-PE primary,a concentration optimization was necessary to prevent any nonspecific binding,bundling,and false-positive results.ICAM-1is a member of the immunoglobulin superfamily of receptors and is the ligand for the leukocyte integrins Mac-1and lymphocyte function-associated antigen1(LFA-1;CD11a/ CD18).[25]This molecule is thought to play a critical role in the attachment and migration of immune and inflammatory cells, such as agranular leukocytes(e.g.,monocytes),into and through tissues.[26]Therefore,the measurement of the expres-sion intensity is a reliable indicator of the ongoing immunor-esponse.Determination of the ICAM-1binding saturation was investigated byflow cytometry after incubation of the nanowires and nanoparticles in the presence of THP-1cells for1h.The binding of secondary antibody reached saturation at 1:10dilution of the secondary when compared to its positive control(Figure6).THP-1surface expression of ICAM-1was subsequently evaluated by comparingfully functionalized nanoparticles and nano-wires to their respective controls.Theresults presented give a quantitative indica-tion of the marker response efficiency.Thedesign of negative(Figure5)and positivecontrols is crucial inflow cytometry,toquantify the THP-1cell immunoresponse.Negative controls were unlabeled silicananoparticles and nanowires,which wereinitially tested against THP-1cells(fluor-escently labeled with DNA nuclear staining(40,6-diamidino-2-phenylindole,DAPI)tohighlight cell viability),as shown inFigure5a1.Figure5a shows where the cellsare positioned in the forward scatter(FS)versus side scatter(SS)log–log plots andFITC versus PE log–log plots(R1gate inboth Figure5a and a1).Almost100%of allcells are detected by their DAPI cell nucleusagainst R-PEfluorescent staining(Figure5a1).Comparative gate analysiswith Figure5a and a1was also used todiscriminate between particles(gates R2and R3against R1in Figure5b,with no cellfluorescence signal in Figure5b1)and wires(gates R2and R3against R1in Figure5c,with no cellfluorescence signal inFigure5c1).Figure5b1and c1also highlightthe lack of autofluorescence of the nano-particle and nanowire when unbound in thenegative control.Results are shown in FSversus SS log–log plots to show the relativedifference in size of the objects underdetection(i.e.,only cells,particles,wires,cell with particles,and cell with wires)versus relative granularity.Log scales areused in order to best visualize the separationin size between the free cells and thosebound to nanowires or particles.The positive controls are given bymeasurements of the expression of thecell-surface binding between THP-1(DAPI labeled)and R-PE and ICAM-1when dispersed in solution only(Figure7aFigure5.Negative controls.a)Side scatter versus forward scatter(SS-FS)scatter plot of THP-1 cell.a1)PE-FITC cytogram of THP-1stained with R-PE to highlight cell counts and gate analysis (gate R1).b)SS-FS plot of uncoated particles only(gate R2)and b1)gated analysis versus THP-1(gate R1)on PE-FITC scatter plot.c)SS-FS plot of uncoated nanowires only(gate R3) and c1)PE-FITC scatter plot gated analysis versus THP-1(R1gate).NP¼nanoparticle,NW¼nanowire.and b).It is possible from these results to gate and measure the response for both R-PE (Figure 7a 2and a 3)and FITC fluorescence emission (Figure 7b 2and b 3).It can be seen that the vast majority of the cells used in the experiments are live (DAPI negative,gate R1,for Figure 7a 1and b 1;gate R12for Figure 7a 2and b 2).Quantitative flow cytometric analysis was then carried out to measure the THP-1cell anti-ICAM-1binding specificity to the functionally prepared nanoparticles and nanowires.The results are presented in Figure 7c and d.Confirmation of the selective THP-1functional binding is provided by the upwards shift,by almost an order of magnitude,of the cell population count (R1)for both particles (Figure 7c 1)and wires (Figure 7d 1).This is also proven by the increased fluorescence intensity counts (R12),which represent the specific binding between nanomaterials and cells,as shown in Figure 7c 2and d 2,respectively.Finally,in the last two cytograms (Figure 7c 3and d 3)the different scattering patterns of the fluorescent particles and wires functionally bound to THP-1cells (measured by the FITC-conjugated fluorescence)can be seen.These plots show that,of the total viable cells,62%bind to nanoparticles and 17%bind to nanowires.Although the use of particles gives a larger yield,the use of nanowires gives a much better readout of the difference between unbound and functionally bound THP-1,as shown from the scatter diagram in Figure 7d 3.In addition,the nanowires are shown to bind more efficiently than the nanoparticles,since the concentration in mL À1of nanowires injected is 65times smaller than the concentration of nanoparticles (the particle and wire surface areas are similar).Furthermore,the lack of clustering and bundling observed with nanowires can be advantageous in multiplexed flow cytometry.3.ConclusionsMagnetic silica nanoparticles and nanowires were success-fully prepared and functionalized for immunological detection and antibody-specific targeting.The specific binding between THP-1monocyte cells and ICAM-1secondary antibody has been measured by flow cytometry for both particles and wires.Our results demonstrate that the loaded silica nanowires have intrinsic advantages when compared to similar particles due to their unique ‘‘flow cytometry fingerprint’’,which allows them to be clearly distinguished when utilized as carriers for immunodetection applications.Their efficiency at binding to the cells is comparable to that of the particles when the surface areas are compared.Therefore,in this study we introduced a novel method based on magnetic silica nanowires for immuno-and bioassay,with future potential to expand into multiplex flow cytometry nanowire-based assay.Therefore,the nanowire aspect ratio could be of advantage for multiplexed immunoassay detection.Future efforts will be focused on their multimodal use as an analytical tool in flow cytometry for rapid assaying of selected antibodies,cytokines,and nucleic acids.4.Experimental SectionNanoparticle and nanowire synthesis:Magnetically loaded silica nanoparticles were prepared by modification of published procedures.[20,27,28]Firstly,magnetite nanoparticles were pre-pared by co-precipitation of FeCl 2Á4H 2O and FeCl 3Á6H 2O in a molar ratio of 1:2with 0.5M NaOH.This alkaline solution was stirred at 808C for 1h yielding a black colloidal suspension of magnetite nanoparticles,which were washed several times with water.The magnetite nanoparticles (9Æ1nm)were then stabi-lized using a 0.01M solution of citric acid and pH-neutralized using tetramethylammonium hydroxide.To coat the magnetite nanoparticles with silica,the citric acid-stabilized magnetite nanoparticles were agitated by using ultrasound for 1h in an ethanol/H 2O solution containing ammonium hydroxide and tetraethylorthosilane (TEOS)and then stirred overnight.The resulting light brown solution containing silica-coated magnetite was washed with water until the pH was neutral.To prepare the magnetite-loaded silica nanowires it was necessary to initially prepare a mixture of TEOS (8.2mL),ethanol (1.76g),water (0.66g),and concentrated HCl (one drop),which was stirred for 2.5h to hydrolyze the TEOS.Then cetyltrimethyl-ammonium bromide (CTAB)-stabilized magnetite (0.6mL)was added simultaneously with a condensation mixture of water (2g),ethanol (1.76g),and four drops of HCl.This mixture was placed on a shaker for 30min,and then infiltrated through a porous alumina membrane template (pore diameter 200nm)under vacuum assistance.The gel was dried for 24hs in air.The template was placed in 0.1M NaOH with sonication to dissolve the membrane and release the nanowires.The solution was cen-trifuged at 2000rpm for 5min and then washed with Millipore water five times to remove NaOH;the pH of the nanowire dispersion after the final washing wasneutral.Figure 6.ICAM secondary loading of nanowires.Optimal secondary antibody intensity saturation of ICAM-1coated on silica-coatednanowires.Overlay of fluorescence response compared to negative (cells in suspension in RPMI 1640only)and positive (cells in suspension in RPMI1640with1:10dilutionofICAM-1antibody).Datanormalizedto105cells per sample.ß2009Wiley-VCH Verlag GmbH &Co.KGaA,Weinheim small 2009,x,No.x,1–9Figure7.Gated analysis of interaction between THP-1cells and primary and secondary antibody functionalized onto nanoparticles and nanowires.a)PositivecontrolofviableTHP-1(DAPIstained)toIgGprimaryantibody(R-PEconjugate)inRPMI1640solution;b)positivecontrolofviableTHP-1(DAPI stained)to ICAM-1secondary antibody(FITC conjugate)in RPMI1640solution;c)measurement of62%specific binding between ICAM-1-biofunctionalized nanoparticle to THP-1cell surface;and d)17%detection efficiency on ICAM-1-biofunctionalized nanowire specific binding to THP-1 cellsurface.Pleasenote,plots(d2)and(d3)alsoquantitativelyhighlightthegeometricaldifferencebetweenunboundcellsandcellsboundtonanowire (right-hand side of gated area R12in(d2)and quadrant R5in(d3)).Antibody functionalization of nanoparticles:To further con-jugate antibodies to the nanoparticles,it was necessary to functionalize the particles with amino groups.Therefore,the silica-coated magnetite nanoparticles(0.1g)were dispersed in Millipore water by sonication.Isopropanol(75mL)was added, followed by ammonia(2.4mL)and TEOS(1mL).After sonication for2h,APTES(1mL)was added and the mixture was sonicated for another hour,then the solvent was removed by evaporation.The presence of amino groups was confirmed by the Kaiser test.[29] Ninhydrin was used to detect ammonia and primary or secondary amines.The solid(50mg)was dispersed in phosphate buffer(10mL) by sonication.This solution(500m L)was mixed with a freshly prepared solution of EDC(0.5M)in phosphate buffer.IgG primary antibody(75m L)was added and the mixture was vortexed at room temperature for48h.The particles were then washed three times with an equal volume of phosphate buffer solution(PBS)to remove the unbound antibodies.Functionalization of nanowires and secondary antibodies:To functionalize nanowires with primary antibodies,dispersed nanowire solution(500m L)was added to each of six eppendorf 1.5mL tubes.The samples were centrifuged on an Eppendorf centrifuge for30min to concentrate the wires.The pellet was redispersed in a mixture of isopropanol(500m L)and Millipore water(100m L).A solution of APTES in isopropanol(0.42M)was systematically added to each 1.5mL tube(20,40,60,80, 120m L,respectively),followed by ammonia(50m L).The samples were mixed on a vortex apparatus overnight.They were then centrifuged for20min at10000rpm and the pellet was redispersed in water(500m L).Then,a solution of EDC(0.5M)in phosphate buffered saline(500m L)as added to each eppendorf tube.Antimouse IgG R-PE conjugate primary antibody(Sigma, USA)was added to each sample at a dilution of1:20,as in the supplier specification.Each sample was vortexed and then incubated at48C for2days.Three different concentrations of mouse monoclonal anti-human CD54(ICAM-1)FITC conjugate(Invitrogen,USA)secondary antibody were prepared at1:10,1:50,and1:100dilution as in the supplier specification for flow cytometric analysis.Solutions were systematically added to six1.5mL eppendorf tubes mixed for1h at48C,and then rinsed three times with Millipore water to remove the excess of noncovalently bound secondary antibody.Samples were then resuspended in Millipore water to a final volume of 200m L per sample.In total,five batches of silica-coated magnetite nanowires were synthesized,functionalized,character-ized,and tested by the flow cytometry technique against their matched negative and positive controls.Cell culture and antibodies:Human acute monocytic leukemia THP-1is a phagocytic cell line.THP-1cells(ATCC,Rockville,MD) were maintained in RPMI1640cell culture medium(Sigma,St. Louis,MO)containing10%fetal bovine serum,L-glutamine (Sigma,UK),and0.05m M2-mercaptoethanol(Chemicon,USA). To prevent the active uptake of the nanoparticles and nanowires, THP-1cells were used in suspension without any preactivation.Antimouse IgG R-PE conjugate,developed in goat,was used as primary antibody for both confocal and flow cytometry measure-ments.This antibody was conjugated to the secondary CD54at a working dilution of1:20according to the supplier’s protocols.Monoclonal antibody CD54,also known as intracellular adhesion molecule-1(ICAM-1),is a member of the immuno-globulin superfamily of receptors expressed by many cell types. The expression of CD54(ICAM-1)is linked to inflammatory mediators such as interleukin-1(IL-1),IL-6,tumor necrosis factor alpha,and interferon gamma,which are all cytokines linked to chronic disease such as cancer.Confocal microscopy:The surface coating uniformity and IgG primary antibody load were measured by confocal microscopy analysis.High-resolution images were acquired for at least100 particles and100wires by using an Axiovert system(Carl Zeiss, Germany),then surface and geometrical analysis and3D rendering were carried out by using the Axiovert and Zeiss LSM image software(Carl Zeiss,Germany).Confocalfluorescence microscopy was also used to measure the concentration of each nanomaterial in solution.Flow cytometry:THP-1expression of surface ICAM-1was determined byflow cytometry.For each experiment,after THP-1 cells were washed twice with sterile1X PBS,cells were incubated for1h at378C in5%CO2in the presence of an equal mass (approximately6m g)of biofunctionalized nanoparticles or nano-wires.At all times,THP-1cell viability was measured by fluorescently staining the nuclear DNA with DAPI(Sigma,UK). Samples were measured with a Dako CyAn ADP(advanced digital processing)system(Dako Colorado Inc.,USA).Data and gated analysis were performed with Summit software(version4.3;Dako Colorado Inc.,USA).Magnetization measurements:Magnetization measurements on nanoparticle and nanowire samples were carried out at room temperature using a Quantum Design MPMS XL superconducting quantum interference device(SQUID)magnetometer withfields up to5T.The dried nanoparticle samples were weighed and placed in a gel cap holder.The magnetic curves werefitted to Langevin functions and these are reported in Figure3a and b;no hysteresis is shown.Mo¨ssbauer spectroscopy:57Fe Mo¨ssbauer spectra were collected at room temperature(RT)using a constant-acceleration spectrometer equipped with a57Co(Rh)source kept at RT.Velocity calibration was performed using a-Fe and all the isomer shift values are given relative to a-Fe.IS,D E q,e,G/2,B HF,and A denote the isomer shift,quadrupole splitting,quadrupole shift,line width (half width of half maximum),hyperfine magneticfield,and relative absorption area,respectively,of the components used to fit the experimental spectra.The Mo¨ssbauer spectrum of the iron oxide-loaded silica particles is shown in Figure3c.This is a relaxation spectrum typical of superparamagnetic ferric oxide particles,with a relaxation time of approximately10À7s.It suggests that the magnetite has been largely oxidized to maghemite,g-Fe2O3.Transmission electron microscopy:TEM images were obtained on a Jeol JEM-2100,200kV LaB instrument,operated at120kV with a beam current of about65m A.Samples for TEM were prepared by deposition and drying of a drop of the powder dispersed in water,or the appropriate liquid sample,onto a carbon-coated300-mesh copper grid.Diameters were measured using the ImageJ version1.40software program;average values were calculated by counting a minimum of100particles.ß2009Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim small2009,x,No.x,1–9。