Magnetic Force Microscopy of Iron Oxide Nanoparticles and

Their Cellular Uptake

Yu ZhangDept. of Mechanical Engineering, University of California Riverside, Riverside, CA 92521

Mo YangDept. of Health Technology and Informatics, the Hong Kong Polytechnic University, Hong Kong, China

Mihrimah OzkanDept. of Electrical Engineering, University of California Riverside, Riverside, CA 92521

Cengiz S. OzkanDept. of Mechanical Engineering, University of California Riverside, Riverside, CA 92521

DOI 10.1021/bp.215Published online June 26, 2009 in Wiley InterScience (www.interscience.wiley.com).

Magnetic force microscopy has the capability to detect magnetic domains from a closedistance, which can provide the magnetic force gradient image of the scanned samples andalso simultaneously obtain atomic force microscope (AFM) topography image as well asAFM phase image. In this work, we demonstrate the use of magnetic force microscopy to-gether with AFM topography and phase imaging for the characterization of magnetic ironoxide nanoparticles and their cellular uptake behavior with the MCF7 carcinoma breast epi-thelial cells. This method can provide useful information such as the magnetic responses ofnanoparticles, nanoparticle spatial localization, cell morphology, and cell surface domainsat the same time for better understanding magnetic nanoparticle-cell interaction. It wouldhelp to design magnetic-related new imaging, diagnostic and therapeutic methods. VVC 2009American Institute of Chemical Engineers Biotechnol. Prog., 25: 923–928, 2009Keywords: magnetic force microscopy, cancer cell, iron oxide nanoparticle, cell culture, MCF7

Introduction

Magnetic force microscopy (MFM), which belongs toatomic force microscope (AFM) family, is based on the mag-netic interaction between the sharp magnetic tip and the sam-ple. The operating principle of MFM is similar to AFM. Aflexible cantilever beam with a sharp magnetic tip on its endis used as a force sensor. If the tip is brought close enough tothe sample surface, the magnetic interaction between the tipand sample causes the cantilever to change either in deflectionor in resonant frequency. MFM has been used in studyingmicro/nano scale magnetic systems because it can directlyimage the magnetic field distribution of a magnetic sample ona nanometer scale, and no extensive sample preparation isneeded.1–3 Since the original MFM study by Martin and Wick-ramasinghe in 1987,2 a number of experimental and theoreticalworks have been reported based on MFM, including magneticwriting recording media,4–7 magnetic domain wall struc-tures,1,8–12 MFM image quantification, and simulations andmodeling.13–16 For biological application, MFM has also beenused in studying the magnetic properties of biomolecules,17,18

biogenic nanoparticles,19 and magnetotactic bacterium.18

Recently, the application of magnetic nanoparticles in cel-lular biology and medicine has gained much interest because

of their controllable small size as drug carriers and easymanipulation by external magnetic field. Studies have shownthat iron oxide nanoparticles have low cytotoxicity to thecells under certain concentration and can be biocompatiblewith appropriate surface coating even at very high concentra-tion.20 Monitoring the interaction of magnetic nanoparticleswith cells is important for the current cellular applica-tions.21–25 Fluorescence microscopy is the primary method tostudy the cellular uptake process of magnetic nanoparticles.26

However, the nanoparticles need to be initially fluorescencelabeled, and the maximum resolution is limited by half ofthe light wavelength.27 Two photon microscopy (TPM) hasalso been used to study magnetic nanoparticles and cellinteraction because of its improved resolution and low dam-age outside the focal volume,28 but the nanoparticles stillneed to be labeled with two photon dye. Label-free detectionmethod is of considerable interest because of the possibledifficulties and instabilities associated with fluorescencelabeling. Transmission electron microscope (TEM) is one ofthese methods, which can study the cellular uptake of nano-particles at a nanoscale resolution.29 However, the samplepreparation process to get cell sections for TEM analysis istime consuming, and only a small part of cell section can beanalyzed. Other label-free imaging methods such as mag-neto-motive ultrasound and optical coherence tomography(OCT) are generally used in tissue and animal experimentsbecause of its relatively low resolution.30–32 MFM has been

Correspondence concerning this article should be addressed toC. S. Ozkan at cozkan@engr.ucr.edu.

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proven to be a useful imaging technique to study magneticnanoparticles.33–37 Compared with the above imaging meth-ods, MFM can simply localize and characterize the magneticnanoparticles at a nanoscale resolution without labeling. Upto date, there have been only few attempts to use MFM toobserve the interaction of magnetic nanoparticles with cells.Shen et al.38 used MFM combined with AFM topographyimaging to monitor cellular apoptosis of HL-60 cells inducedby anti-cancer drug molecule coupled magnetic iron oxidenanoparticle. However, the capacity of MFM has not beenfully explored. Simultaneous observation of cellular uptakeof magnetic nanoparticles using MFM phase, AFM topogra-phy, and AFM phase imaging can provide more detailed in-formation for better understanding the process. In thisarticle, this three-mode imaging technology shows the capa-bilities not only in characterizing the magnetic responses ofnanoparticles after incubated with cells but also in label-freemapping of the spatial localization of magnetic nanoparticlesinside/outside the cell surface to better understand the cellu-lar uptake process. This information has a great potential toimprove the monitoring capability of nanoparticle-cell inter-action, such as evaluating magnetic response, circulationtime, and targeting efficiency of functional magnetic nano-particle in vitro.

Materials and Methods

Cell culture

Human breast carcinoma epithelial cells (MCF7) werepurchased from American Type Culture Collection (ATCC)and were grown in DMEM supplemented with 10% fetal bo-vine serum, 5% penicillin streptomycin glutamine, and 5%sodium pyruvate. All the cells were cultured at 37�C in ahumidified atmosphere with 5% CO2. For MFM microscopycharacterization purpose, cells were grown on cleaned andsterilized silicon substrates.

Sample preparation

Magnetic iron oxide nanoparticles (c-Fe2O3) were pur-chased from Alfa Aesar, and these uncoated nanoparticles’average diameter is around 30 nm. A total of 50 lg/mLmagnetic iron oxide nanoparticles were incubated withMCF7 cells at 37�C in a humidified atmosphere with 5%CO2 for 24 h. Cells incubated with iron oxide nanoparticleswere then washed three times with 1� phosphate bufferedsaline (PBS). Cells after incubation with nanoparticles wereall fixed by 1% formaldehyde in 1� PBS at room tempera-ture for 10 min, washed with 1� PBS extensively for there

times, followed by washing with an increasing graded etha-nol series to dehydrate, and then left in vacuum desiccator.

Magnetic force microscopy imaging

MFM detects the force or force gradient between theMFM tip and magnetic sample.39 The magnetic force actingon the tip is given by

Ft�s ¼Z

d3r0Z

d3r00Mðr0Þr � Hðr00; r0Þ (1)

where M(r0) is the tip magnetization at position r0 inside thetip, while H(r00,r0) is the sample stray field at position r0 gen-erated by the magnetic moment at position r00 inside of thesample.

The deflection of the cantilever that is directly bended bythe force can be used to form MFM images. However, thismode is not commonly used because of its low sensitivity byvibration or electronic noises. Much higher sensitivity can beachieved in tapping mode by driving the cantilever at orclose to its resonant frequency to reduce environmentalnoise.2 In our study, a tapping/lift mode is used, whichallows imaging long-range magnetic interactions while mini-mizing the influence of topography. Figure 1 shows a sche-matic illustration of operating principles for AFM family,and Figure 2 shows a schematic representation of tapping/liftmode of MFM. As shown in Figure 2, in MFM, a tappingcantilever equipped with a special magnetized tip is firstscanning in the tapping mode over the surface of the sampleto obtain topographic information using the cantilever oscil-lation amplitude as feedback. The tip is then raised to thelift scan height. Magnetic image is then obtained in the liftmode scan by monitoring the cantilever’s frequency or phaseshift on rescanning the previously measured topography witha user controlled height offset, h. The interaction between tipand sample includes magnetic interactions, electrostatic inter-actions, van der Waals interactions, short range, and capil-lary forces.4 During MFM lift mode scanning, within the tip-sample separation distance, which usually varies between 10and 100 nm, the magnetic force, electrostatic force, and vander Waals force are the dominant forces. When the MFM tipis sharp, the magnetic force gradient will be much largerthan the van der Waals force gradient and the electrostaticforce gradient. The magnetic contrast and the topographythen could be nicely separated.

AFM phase imaging used in this study is a powerfulextension of AFM tapping mode that provides nanometer-scale information about surface property.40 During the firsttopographic tapping mode scan, the AFM phase lag of the

Figure 1. Schematic illustration of atomic force microscopeoperating principle.

Figure 2. Schematic representation of tapping/lift mode MFM.

The dotted line indicates a single MFM signal scan line at thelift height above the sample. The solid surface indicates thecorresponding AFM topographic scan line in the experiment.

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cantilever oscillation could be simultaneously monitored,which is very sensitive to variations in material properties.By mapping the phase shift of the cantilever oscillation,AFM phase imaging could detect variations in composition,adhesion, friction, viscoelasticity, and perhaps, otherproperties.41

Our MFM experiments were performed with a MultimodeV SPM system (Veeco Instruments) using a dynamic tap-ping/lift mode that is described above. The magnetic forceson the tip will shift their original resonant frequency f0 byan amount Df, which is proportional to vertical magneticforce gradients. In our study, phase detection is used todetect the frequency shifts by magnetic forces between thetip and sample, which tracks the cantilever’s phase of oscil-lation relative to the piezo drive. The MFM tips employed inthis research are commercially available (MESP, Veeco,NY). They are 0.01–0.025 Xcm Antimony (n) doped Si tipswith a cobalt/chrome magnetic coating. The nominal springconstant of the cantilever is 2.8 N/m with coercivity�400 Oe and moment 1e13 EMU. All measurements weretaken under open environment and ambient conditions.

Results and Discussion

Using Multimode V SPM system (Veeco Instruments),magnetic iron oxide nanoparticles (NanoArc, Alfa Aesar)were analyzed first. Figure 3 shows the AFM topography

image (Figure 3A), MFM phase image (Figure 3B), andAFM phase image (Figure 3C) of iron oxide nanoparticles,and the MFM image is obtained at lift scan height of 65 nm.The corresponding cross-sections (Figures 3D–F) along theindicated line are shown on the right. The magnetic attrac-tive forces from the nanoparticles are observed in the blackcolor, and magnetic repulsive forces in the white color. Evenat the MFM lift scan height of 65 nm, the amplitude of mag-netic signals is very large. The amplitude of magnetic signalexpressed in the phase shift (phase angle change) is observedat 320�. We notice that the topography information is alsoshown in lifted scan MFM phase image of Figure 3B, whichindicates the possible van der Waals force gradient and elec-trostatic force gradient between the tip and nanoparticles.However, the phase shift by nonmagnetic forces is muchsmaller than magnetic forces. From MFM phase image ofFigure 3B, it is also observed that the magnetic signals ofthe nanoparticles are roughly toward the same direction. Thereason might be the magnetization by the MFM tip duringthe first AFM tapping scan. Besides, the AFM phase imageof these iron oxide nanoparticles shown in Figure 3C demon-strate these physical vapor synthesized nanoparticles by AlfaAesar are solid, as AFM phase image is very sensitive tovariations in material properties.

Figure 4 shows the results of AFM topography, MFMphase, and AFM phase images of MCF7 carcinoma breastepithelial cells after incubated with 50 lg/mL iron oxidenanoparticles for 24 h. All the MFM images are obtained ata lift scan height of 30 nm. Multiple nucleoli in the nuclearenvelope are clearly observed in the AFM image of Figure4A. From the MFM phase image of Figure 4B, a spatiallocalization of magnetic nanoparticles either inside or out-side MCF7 cell membranes is directly obtained. As the AFMphase imaging is very sensitive to surface variations, Figure4C provides complementary information about the localiza-tion of iron oxide nanoparticles. Figures 4D–F are the corre-sponding zoom-in images of the squared areas in AFMtopography image (Figure 4A), MFM phase image (Figure4B), and AFM phase image (Figure 4C). The line profilesare obtained along the cross sections, which are indicated asa red line and a blue line in the zoom-in images. From theline profiles obtained along the red line, an example for ironoxide nanoparticles outside the cell membrane is shown.Strong magnetic signal at around 340� from MFM phaseprofile (Figure 4H) is observed, and this identifies the exis-tence of magnetic iron oxide nanoparticles. Meanwhile, anobvious bump is observed in topography profile (Figure 4G),and AFM phase profile (Figure 4I) also shows a differentsurface feature at the same position, which indicates theseparticles are on the surface. On the contrary, an example foriron oxide nanoparticles located inside the cell membrane isshown by the line profile measurements obtained along theblue line. Strong magnetic signal by magnetic iron oxidenanoparticles at around 350� is also observed from MFMphase image in Figure 4K. However, no obvious bump isobserved in topography profile in Figure 4J, and AFM phaseprofile (Figure 4L) also does not show a different surfacefeature at the same position. This indicates that these nano-particles are inside the cell.

To have a more detailed exploration of nanoparticleuptake behavior by the MCF7 carcinoma breast epithelialcells in a nanoscale resolution, high resolution images with ascan area less than 1 � 1 lm2 are shown in Figure 5. TheAFM topography image (Figure 5A), MFM phase image

Figure 3. AFM topography image (A), MFM phase image (B),and AFM phase image (C) of iron oxide nanopar-ticles (NanoArc, Alfa Aesar).

MFM phase image is obtained at lift scan height of 65 nm.The corresponding cross-sections (D) (E) (F) along the indi-cated lines are shown on the right. The scale bar represents500 nm.

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(Figure 5B), and AFM phase image (Figure 5C) show a fur-ther zoom-in area of part of one MCF7 cell after incubatedwith iron oxide nanoparticles for 24 h. MFM image is alsoobtained at a lift scan height of 30 nm. MFM phase meas-urements displayed in Figures 5B,E show a big phase shiftat around 320� caused by a strong attractive magnetic forcebetween the tip and the sample. The size of magnetic featureis in the range of 100 nm. From the height information dis-played in Figures 5A,D, the topography is pretty flat at thesame position. Furthermore, the AFM phase informationbased on the surface properties shown in Figures 5C,F alsocould not identify this magnetic feature. However, differentcell surface domains are clearly observed in the high resolu-tion AFM phase detection. After combining the informationfrom AFM topography, AFM phase, and MFM phaseimages, the observed magnetic iron oxide nanoparticles byMFM phase image are clearly proven to be under the cellmembrane, which confirms iron oxide nanoparticles areinternalized by the MCF7 cells.

From the above, this three-mode imaging technology dem-onstrates the ability for label-free mapping of the spatiallocalization of iron oxide nanoparticles inside/outside the

cell surface. This high resolution technique is suitable forstudying the interaction of various magnetic nanomaterialswith cells, even as small as single domain superparamagneticnanoparticles with the help of external magnetic field.37

However, there is difficulty to quantify the magnetic nano-particles uptaken in cells using MFM measurements. Thedetection depth of MFM imaging is decided by the accuratemagnetic state of the MFM tip, the tip to sample distanceand the magnetic property of scanned samples. There is evi-dence that aggregation of iron oxide nanoparticles will ex-hibit a greater magnetic dipole than a single iron oxidenanoparticle42 and the aggregation of iron oxide nanopar-ticles after incubated with MCF7 cells was also confirmedby our previous study using TEM characterization.43

Because the aggregation size and depth of iron oxide nano-particles under cell membrane are unknown, they will affectthe magnetic signal recorded even when the accurate mag-netic state of the MFM tip and the accurate tip to cell sur-face distance are known. However, an approximate MFMdetection depth and quantification of magnetic nanoparticlescould be achieved if a model could be established to simu-late the same conditions with further experimental studies on

Figure 4. AFM and MFM imaging of MCF7 cells after incubated with iron oxide nanoparticles for 24 h.

MFM image is obtained at lift scan height of 30 nm. (A) AFM topography image, (B) MFM phase image, and (C) AFM phase image of a zoom-outarea. (D) (E) (F) are the corresponding zoom-in squared area images of (A) (B) (C). (G) (H) (J) are the corresponding cross-sections along the indi-cated red line, and (J) (K) (L) are the corresponding cross-sections along the indicated blue line shown in the above zoom-in images. The scale barrepresents 10 lm.

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the statistical distribution of iron oxide nanoparticles aggre-gation sizes and spatial distributions. As the aggregationsize of iron oxide nanoparticles were shown to be largerthan 1 lm,43 MFM should be able to achieve hundreds ofnanometer detection depth.

Conclusions

In summary, MFM was used to study the cellular uptakeof iron oxide nanoparticles by MCF7 carcinoma breast epi-thelial cells using a three-mode imaging technology includ-ing MFM phase image, AFM topography image, and AFMphase image. The cellular uptake of iron oxide nanoparticleswas observed in nanoscale resolution. The magneticresponses of nanoparticles, nanoparticle spatial localization,cell topography, and cell surface domains could be directlyvisualized at the same time using this technology. Theseobservations offer direct evidence on studying the interactionbetween magnetic nanoparticles and cells, which will pro-vide very useful information for better design of new imag-ing, diagnostic and therapeutic methods especially forapplications using magnetic properties of nanomaterials,such as magnetic resonance imaging, hyperthermia, andmagnetic field directed drug delivery.

Acknowledgments

The authors gratefully acknowledge financial support fromthe Center for Nanotechnology for the Treatment, Understand-

ing and Monitoring of Cancer (NanoTumor) funded by theNational Cancer Institute (NCI).

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Figure 5. AFM topography image (A), MFM phase image (B),and AFM phase image (C) of a further zoom-in areaof one MCF7 cell after incubated with iron oxidenanoparticles for 24 h.

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Manuscript received Aug. 11, 2008, and revision received Jan. 14, 2009.

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