iron oxide nanoparticle-based radio-frequency thermotherapy for human breast adenocarcinoma cancer...

BiomaterialsScience

PAPER

Cite this: DOI: 10.1039/c3bm60015g

Received 15th January 2013,Accepted 27th March 2013

DOI: 10.1039/c3bm60015g

www.rsc.org/biomaterialsscience

Iron oxide nanoparticle-based radio-frequencythermotherapy for human breast adenocarcinomacancer cells

Thikra Mustafa,a Yongbin Zhang,b Fumiya Watanabe,a Alokita Karmakar,a

Madhu P. Asar,a Reginald Little,c M. Keith Hudson,d Yang Xu*a andAlexandru S. Biris*a

Iron oxide nanoparticles (IONPs) with diameters of 15, 25, and 41 nm were evaluated as mediators of

thermal cytotoxicity under radio-frequency (RF) exposure. The 25 nm IONPs were found to be the most

efficient of the three in killing cancer cells at 350 kHz low-frequency RF irradiation. However, at a higher

frequency of 13.56 MHz, 15 nm IONPs produced the highest percentage of cell death. Moreover, the

killing effect was concentration-dependent in that a higher concentration of IONPs resulted in increased

cellular death. Size-dependent internalization of IONPs in MCF-7 cells was quantified by using inductively

coupled-plasma mass spectrometry (ICP-MS). Dark-field microscopy and transmission electron microscopy

(TEM) revealed that MCF-7 cells internalize IONPs through endocytosis after 24 hours of incubation. In

addition, after RF treatment, the cancer cells underwent the apoptosis process, and the level of reactive

oxygen species (ROS) increased significantly after hyperthermia. Scanning electron microscopy (SEM) and

TEM further established that the ultrastructure morphological changes in the cancer cells originated

from the apoptosis process.

1. Introduction

Hyperthermia is a promising technique for cancer treatmentbecause of tumor cells’ sensitivity to heat1 owing to the narrowtolerance ranges of cancer cells to micro-environmental para-meters, including low pH range, low oxygen levels, and nutri-ent deprivation.2 Basically, in the treatment of cancer byhyperthermia, the tissue’s temperature is increased to approxi-mately 40–43 °C, which is considered to be detrimental fortumor cells3 compared to normal tissue.4 The primary mech-anism of cell death is the irreversible denaturation of proteinswhen the temperature increases above 40 °C.5 Since proteinsare composed of globular or fibrous-form compounds andmany are involved in the fundamental structures of cells, thedenaturation of proteins leads to alterations in vital cell

structures, such as the cytoskeleton, plasma membrane, orenzyme activity.4 Magnetic nanoparticles—with their ability toefficiently convert electromagnetic energy into heat at RF fre-quencies and their small sizes (which enable them to be takenup by the cell)—are attractive for a variety of nanomedicine-related applications,6 such as drug delivery,7 cancer cell detec-tion,8 MRI contrast agents,9 and hyperthermia.10

In 1957, Gilchrist et al. were the first to report the efficiencyof IONPs in elevating the temperature of human canceroustissues in vitro under RF exposure.11 Subsequent in vitro workhas shown that IONPs greatly enhance thermal cytotoxicity incancer cells.12 Nonetheless, the optimal conditions for ther-malization of RF energy by IONPs are still under investigation.Among the variables, the size and concentration of magneticnanoparticles are among the most important parameters thathave been studied.13–16 However, these critical parametersshould be optimized for medical applications in order toachieve the most efficient thermal transfer and, in turn, thehighest efficiency for cancer cell death. Thus far, there havebeen few reports concerning the relationship between radiofrequency and the size of the IONPs for RF-heating efficiency.

In this study, we used human breast adenocarcinomacancer cells (MCF-7) as a model to study the nanoparticle size-dependence of hyperthermia using two different frequenciesof RF. Different sizes of IONPs showed varying abilities to

aCenter for Integrative Nanotechnology Sciences, University of Arkansas at Little

Rock, 2801 S. University Ave, AR 72204, USA. E-mail: [email protected],

[email protected]; Fax: +1-501-683-7601; Tel: +1-501-682-5166, +1-501-683-7458bNanotechnology Core Facility, Office of Scientific Coordination, National Center for

Toxicological Research, US Food and Drug Administration, 3900 NCTR Road,

Jefferson, AR 72079, USAcDepartment of Biological and Physical Science, South Carolina State University,

Orangeburg, SC 29117, USAdDepartment of Applied Science, University of Arkansas at Little Rock,

2801 S. University Ave, AR 72204, USA

This journal is © The Royal Society of Chemistry 2013 Biomater. Sci.

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destroy cancer cells under different frequencies of RF thermalactivation. We found that ROS release levels increased after RFtreatment, which finally induced cellular apoptosis. Theresearch described here will provide key insights into how thesize of IONPs influences their RF thermal energy delivery atvarious frequencies, which will lead to the future developmentof magnetic nanoparticle synthesis and functionalization forcancer research using certain controlled methods.

2. Experimental section2.1 TEM characterization of iron oxide nanoparticles

Water soluble carboxylic iron oxide nanoparticles of threesizes (15, 25, and 40 nm) were used in this study. They wereobtained from Ocean NanoTech (Springdale, AR, USA). Thesize and shape of the particles were confirmed by using atransmission electron microscope (TEM), JEM-2100F (JEOLUSA, Peabody, MA, USA) with an accelerating voltage of 80 kV.TEM grids were prepared by depositing a few drops of an IONPsolution onto holey-carbon coated copper grids, which werethen allowed to dry for 15 minutes on filter paper. In addition,the average particle size and particle size distribution of eachsample were determined by EMAN1 and Image J software. Theequivalent circular diameter of each particle was calculatedusing the following equation:

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffimajor�minor

p. The longest

diameter of an ellipse is considered the “major”, and the“minor” is the shortest.17 The mean and standard deviation ofthe particles’ diameters were obtained after measuring over200 particles in random fields of view for each set of particles.

2.2 Hydrodynamic size and zeta potential measurement

Dynamic light scattering (DLS) was performed for the charac-terization of the hydrodynamic size of the IONPs in aqueoussolution using a Zetasizer Nano-ZS instrument (MalvernInstruments, Malvern, Worcestershire, UK). Samples weremeasured after dilution of the iron-nanoparticles stock solu-tion to 50 μg ml−1 suspensions in DI water. The dilution wasbriefly vortexed and sonicated for 5 min in an ultrasonic waterbath to provide a homogenous dispersion. Then 1 ml of thedilute was transferred to a 1 cm2 cuvette for dynamic sizemeasurement. The concentration of the samples and experi-mental methods were optimized to assure the quality of thedata. Ten, thirty, and sixty nm standard gold nanoparticlesfrom the National Institute of Standards and Technology(NIST) were used in the validation of the instrument. The sizewas measured at least three times for each nanoparticlesample. The data were calculated as the average size of IONPs(mean ± SD, N = 3). The zeta potentials of different sizedIONPs were measured by using a Zeta-Reader MARK 21 (ZETAPOTENTIAL Instrument, Inc., NJ, USA).

2.3 X-Ray diffraction (XRD) analysis

The X-ray diffraction profiles of the different sizes of IONPswere recorded on a Bruker AXS D8 Discover advanced diffracto-meter with a 2D General Area Detector Diffraction System

(GADDS). The monochromatic Cu Kα radiation line (wave-length = 0.154 nm) with a Goebel mirror (parallel beam diver-gence of less than 0.03°) were used as an excitation source.

2.4 MCF-7 cell culture

MCF-7, a breast cancer cell line, was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, VA, USA).Dulbecco’s modified Eagle medium (DMEM) supplementedwith 10% fetal bovine serum (FBS), 1% penicillin (500 unitsml−1), and streptomycin (500 units ml−1) were used to nourishMCF-7 cells. Cells were cultured in 75 cm2 cell culture flasksand maintained under aseptic conditions at 37 °C in a 5% CO2

atmosphere until 80% cell confluence was obtained. The cellswere then harvested and counted using a hemacytometer(Fisher Scientific, Pittsburgh, PA, USA).

2.5 Cytotoxicity assays

2.5.1 Lactate dehydrogenase release. Lactate dehydrogen-ase (LDH) release was measured using an LDH colorimetricassay kit (Cayman Chemicals, Ann Arbor, MI, USA) to evaluatethe cell membrane integrity. MCF-7 cells were seeded on a96-well plate at a density of 25 × 103 cells. A final volume of100 μl of culture medium containing various concentrations ofIONPs (1, 5, 20, 50, and 100 μg ml−1) of each sized sample andthe positive control Triton X-100 (1%) were incubated for24 hours at 37 °C. The following day, 60 μl of culture mediumwere transferred into a 1.5 ml centrifuge tube and centrifugedfor 5 min at 400g. 50 μl of the supernatant were transferred tothe corresponding new 96-well plates, followed by the additionof 50 μl of an LDH reaction solution to each well. The platewas incubated for 30 min at room temperature on an orbitalshaker. Absorbance was recorded at 490 nm using a microplatereader for colorimetric detection (Synergy H1, Biotek,Vermont, USA).

2.5.2 WST-1 assay. A Cayman’s WST-1 cell proliferationassay kit was used to study the induction and inhibition of cellproliferation in vitro. This assay is based on the enzymatic clea-vage of tetrazolium salt WST-1 to formazan by cellular mito-chondrial dehydrogenase. This enzyme is present in viablecells. Briefly, MCF-7 cells were seeded on 96-well plates at adensity of 25 × 103 cells per well and treated with various con-centrations of IONPs (1, 5, 20, 50, and 100 μg ml−1) for eachsize. Wells containing cells not exposed to IONPs wereregarded as negative controls. After 24 hours of incubation at37 °C, 10 μl of a reconstituted WST-1 mixture were added toeach well and mixed gently on an orbital shaker for 1 min. Thereaction was set for 2 hours in the incubator at 37 °C. The reac-tion color was homogenized on an orbital shaker for 1 minbefore reading the absorbance using a microplate reader(Synergy H1, Biotek, Vermont, USA) at 450 nm.

2.6 Hyperthermia treatment conditions

Cells were seeded in 35 mm tissue culture dishes at a densityof 200 × 103 cells per dish. The cells were allowed to attach onthe plate for 24 hours. Two concentrations of 20 and 50 μg ml−1

of IONPs were added, and the nanoparticles were allowed

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to interact with cells for another 24 hours. The treated MCF-7cells, as well as MCF-7 cells that had not been exposed toIONPs, were placed in the center of the horizontal coil andsubjected to a magnetic field using a low-frequency RF genera-tor (350 kHz, Cycle-Dyne MK-20, Pillar) for 20 min, individu-ally. The parallel sets of dishes containing identical amountsof IONPs along with unexposed dishes that had not been sub-jected to RF treatment were regarded as the negative control.The RF exposure experiments were repeated using a modifiedhigh-frequency generator at 13.56 MHz. An Alinco modelDX70TH transmitter (Alinco, Inc., Osaka, Japan) was used asan RF exciter source followed by amplification using a YAESUmodel FL-7000 linear amplifier (YAESU, Cypress, CA, USA).Various power levels were tried; however, all the experimentsreported here were performed at a level of 70 watts. It shouldbe noted that the use of the exciter, alone, above the 10 wattlevel, was rejected since power levels dropped off over time,probably due to heat levels in the final stages of the unit.Results obtained using the 600 watt capacity amplifier weremuch more stable, and therefore the combination of theexciter and the amplifier was used to gather all the reporteddata. Here, only one concentration of IONPs, 50 μg ml−1, wasused at this higher frequency to compare with the results ofthe lower frequency RF treatment.

Calorimetric measurements of the IONPs were performedusing both low-frequency and high-frequency RF generators.2 mg ml−1 each of 15 nm, 25 nm, and 41 nm IONP solutionswere placed in an insulated tube for temperature measure-ment. The temperature was measured by a FLIR SC660 IRcamera (FLIR Systems, Inc., MA, USA). The duration of eachmeasurement was 10 min, and data were collected everyminute. The specific loss of power was calculated from the fol-lowing formula: SLP (W g−1) = c (ms/mFe) (dT/dt)

18 where c isthe heat capacity of water, ms is the mass of the sample, mFe isthe mass of IONPs in the sample, and dT/dt is the slope of theheating curve. We used the initial slope of the T(t)-curve forthe calculation to avoid possible errors caused by heat fromthe ferrofluid being conducted to the sample. The dT/dt wascalculated as the initial one-minute rising rate for the IONPssolution in our experiment.

2.7 Flow cytometry analysis

An annexin V-FITC apoptosis detection kit (BioVision,Milpitas, CA, USA) was used to quantify cell viability. Theanalysis method followed the protocol provided with the kit.48 hours post RF treatment, exposed cells along with negativecontrol cells were harvested, and approximately 1–5 × 105 cellswere collected. The collected cells were re-suspended in 500 μlof binding buffer, and 5 μl each of annexin V-FITC and propi-dium iodide (PI) were added to each sample. The sampleswere incubated in the dark for 5 minutes before analyzingthem using a FACSCalibur instrument.

2.8 Dark-field microscopy examination

25 nm (50 μg ml−1) IONPs were incubated with MCF-7 cells oncollagen-coated coverslips lying in a 6-well plate for 12 hours.

The medium was removed, and the MCF-7 cells were rinsedthree times with sterile phosphate-buffered saline (PBS). Thecoverslips containing cells were sealed on the glass slide usingnail polish. Images were captured using a enhanced dark-fieldillumination microscope (Cytoviva, Auburn, AL, USA).

2.9 Scanning electron microscopy (SEM) analysis for cellmorphology changes

MCF-7 cells were grown on a polycarbonate membrane NuncCC Insert (EMS, Hatfield, PA, USA), treated with 50 μg ml−1 of25 nm IONPs for 24 hours, and exposed to RF for 20 minutes.Two days after RF exposure, the cells were fixed primarily with3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, followedby a secondary fixative of 2% OsO4 in 0.1 M phosphate buffer.All of the samples were washed thoroughly with 0.1 M phos-phate buffer, dehydrated with ascending percentages ofethanol solution, and then dried using a critical point dryer(Samdri®-PVT-3D, Rockville, MD, USA). Each dried sample wascoated with a thin film of gold (∼3 nm) and visualized under aSEM JEOL JSM-7000F (JEOL, Peabody, MA, USA) with an accel-erating voltage of 15 kV and a working distance of ∼10 mm.19

2.10 Transmission electron microscopy (TEM) analysis ofcells

Samples that had been exposed to 25 nm IONPs at a concen-tration of 50 μg ml−1 and then exposed to RF treatment for20 min were processed for TEM analysis. A control sample wasalso processed. Samples were fixed with 3% glutaraldehydefixative in 0.1 M of phosphate buffer, pH 7.2, overnight at 4 °C.After fixation, all samples were intensively rinsed with 0.1 Mphosphate buffer (pH 7.2), post-fixed with 2% OsO4 in 0.1 Mphosphate buffer for 2 h, rinsed in distilled water, and finallydehydrated in an ethanol solution. The resulting dried speci-mens were embedded in Epoxy Resin which was polymerizedat 70 °C overnight. Semi-thin sections with thicknesses of0.5–1.0 μm, along with thin sections with thicknesses of60–100 nm, were sliced for light microscopy and transmissionelectron microscopy analysis, respectively, using an ultramico-tome, EM UC7 (Leica microsystem GmbH, Wetzlar, Germany).Thin sections were mounted on 150 mesh formvar-carboncoated copper grids and stained with heavy metals, i.e., uranylacetate and lead citrate. Stained samples were coated with athin layer of carbon and visualized under a TEM JEOLJEM-2100F (JEOL, Peabody, MA, USA) with an acceleratingvoltage of 80 kV.

2.11 Quantitation of the uptake of the IONPs using ICP-MS

MCF-7 cells were cultured at the density of 1 × 105 cells ml−1

and incubated with three sizes of nanoparticles (100 μg of Feper well) for 24 hours, respectively. The cells were harvestedand stored at −20 °C after extensively washing with PBS toremove the free nanoparticles in the media and cell mem-brane. The cells were thawed at room temperature and recon-stituted in 1 ml of endotoxin-free 18 MΩ H2O. 200 μl of thesamples were hydrolyzed in 3 ml of optima-grade concentratednitric acid (HNO3) and digested using a CEM MARS-Xpress

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system operating at 100% power and 180 °C for 15 minute. Fol-lowing digestion, samples were diluted to 5 ml with reagentwater. Analysis was performed using an Agilent 7700-x Induc-tively Coupled-Plasma Mass Spectrometer (ICP-MS). Data werecollected for the most abundant isotope 56Fe used in quanti-tation. Germanium (Ge) was used as an internal standard tocorrect for instrument response and sample preparationerrors. The instrument was calibrated for iron (0–1000 ppb)using a NIST traceable standard.

2.12 Reactive oxygen species (ROS) measurement

The intracellular ROS experiment was performed in accord-ance with our previous report.20 Briefly, MCF-7 cells (2 × 105)were cultured on the chamber slides (Lab-Tek, NY, USA) untilsub-confluent. Before the intracellular RF cell exposure,carboxy,-2′,7′-dichlorofluorescein diacetate (H2DCFDA) (CellBiolabs, Inc., San Diego, CA, USA) was added and kept for30 min. Samples were then washed 3 times with PBS, loadedwith 50 μg ml−1 of 25 nm IONPs, and re-incubated for24 hours. Cells incubated with IONP-free medium were usedas a negative control. Loaded and unloaded samples wereexposed to RF for 20 min and kept for 24 hours inside theincubator. Before the intracellular ROS fluorescent measure-ment, the cells were washed with PBS and then loaded with20 μM of H2DCFDA for 30 minutes at 37 °C. The cells werewashed with PBS before being observed under a fluorescentmicroscope, Olympus, BX51 (Tokyo, Japan) with a green filter,and the images were captured with a digital camera, Olympus,DP-71 (Tokyo, Japan). 100 μM H2O2 was used as the positivecontrol. Throughout the experiment, the plate was kept in thedark to prevent the dye from being quenched.

2.13 Temperature measurement of chicken tissue by theinjection of IONPs under RF treatment

5 mg ml−1 of the 25 nm IONPs solution was injected into asample of raw chicken tissue as shown in Fig. 8. Chickensamples were inserted into the center of the RF coil. The temp-erature was measured by a FLIR SC660 IR camera (FLIRSystems, Inc., MA, USA). The images were taken before andafter RF heating. The temperature changes for a control of rawchicken tissue without nanoparticles were also measuredduring the RF treatment. All measurements taken included6 minutes of RF heating followed by 4 minutes of cooling.

3. Results and discussion3.1 Characterization of the IONPs’ sizes and shapes

The TEM images of the carboxylic group IONPs show that theparticles were mono-dispersed and that most of them had aspherical shape. As can be seen in Fig. 1, none of the samplesshowed agglomerations. The average diameters and the stan-dard deviations of 15, 25, and 40 nm IONPs were 14.7 ±0.8 nm, 24.8 ± 2 nm, and 41.3 ± 5.5 nm, respectively, as shownin Table 1. As a result, we labeled them as 15 nm, 25 nm and41 nm. The size distribution graphs showed that both the

particles of 15 nm and 25 nm had overall narrow particle sizedistributions with a spherical shape. However, the 41 nmIONPs revealed a wider size distribution, and some of themhad a non-spherical shape. The deviation percentage from theaverage diameter value was 13.3% for the 41 nm sample, whilethe deviation percentage was 5.4% and 8% for the 15 nm and25 nm IONPs, respectively. Furthermore, as reported in theliterature, spherical particles of similar size have been shownto uptake into cells more readily than other shaped particles.21

The hydrodynamic diameters of IONPs in a water-basedsolution were determined by dynamic light scattering (DLS)analysis. Compared to the IONP sizes obtained from TEM, thecorresponding hydrodynamic diameters were much larger.The hydrodynamic sizes of 15 nm, 25 nm, and 41 nm IONPswere measured to be 28.7 ± 0.1 nm, 43.1 ± 0.2 nm, and 72.0 ±0.1 nm, respectively (Table 1). All of the IONPs were water-soluble, iron oxide nanoparticles with an amphiphilic polymercoating, and they were negatively charged—as confirmed bythe zeta potential analysis. The values were as follows:−38.49 mV, −40.29 mV, and −54.78 mV as shown in Table 1.These carboxylic water-based nanoparticles showed excep-tional stability in aqueous solution.

3.2 XRD

X-ray diffraction studies of IONPs with different sizes werecarried out using Cu Kα radiation. Fig. 1d shows the X-raydiffraction patterns of IONPs with different sizes. The threesizes of IONP patterns and peak positions were very close tothose of the standard data of Fe3O4 alone.22 The diffractionpeaks of all the samples at 2θ values of 30.6°, 35.7°, 41.8°,49.0°, 62.1°, and 66.4° corresponded to the (220), (311), (400),(422), (511) and (440) planes of Fe3O4 magnetite crystal,respectively.

3.3 Heating effect of the IONP size

To evaluate the influence of the IONP size on heating effects,we measured the maximum temperature reached after 10 minof RF (of 350 kHz) exposure for each IONP sample only(without culture cells) starting from room temperature. At350 kHz RF exposure, the 25 nm IONPs reached the highesttemperature of 55.1 °C, and the temperature increased byabout 32 °C within 10 min of heating. Increasing or decreasingthe particle size reduced the maximum temperature. For thehigher frequency of 13.56 MHz RF, the highest temperaturereached for 15 nm IONPs was 52.1 °C which was an increase ofabout 25 °C. As a result, the 25 nm IONPs showed the highestSLP data of 140.5 W g−1 under lower frequency heating asshown in Fig. 2b. However, the 15 nm IONPs showed a higherSLP of 97.8 W g−1 under 13.56 MHz irradiation. The tempera-ture increases under RF treatment with the presence of IONPswere the result of the Néel and Brownian relaxation processes.In the Brownian relaxation process, the heat is generated fromthe friction between oscillating IONPs and the surroundingmedium. Néel relaxation is due to the internal magneticmoment of IONPs. Superparamagnetic nanoparticles “relax” inaccordance with the Néel mechanism and are ideal candidates



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for hyperthermia therapy.18 Smaller sized nanoparticlesshould be cooler, whereas larger particles with an optimumsize should relax in accordance with the Brownian mechanismand should be hotter. The smaller particles in the externalmagnetic field of the RF rotate their magnetic moments moresynchronously with the external magnetic field and therebydissipate less of the RF electromagnetic energy to the environ-ment including the cancer cells. It was to be expected thatthe heating effect or the temperature increase has a strong

relationship with the volume (size) of the nanoparticles as thenanoparticle relaxation mechanism demonstrated.23 Mean-while, the power of the RF also influences the heatingefficiency (figure not shown here). With less power, the

Fig. 1 TEM images showing the morphology and size distribution graphs for the 15 nm (a), 25 nm (b), and 41 nm (c) IONPs used in this study, and the XRDpatterns of three different sizes of IONPs (d).

Table 1 Average IONP size determined by TEM, the hydrodynamic diameter(HD) obtained by DLS, and the zeta potential data of different sizes of IONPs

SamplesCore size (nm,from TEM)

HD size (nm,from DLS)

Zeta potential(mV)

15 nm 14.7 ± 0.8 28.7 ± 0.1 −38.49 ± 1.5425 nm 24.8 ± 2 43.1 ± 0.2 −40.29 ± 1.2141 nm 41.3 ± 5.5 72.0 ± 0.1 −54.78 ± 2.10

Fig. 2 The changes of temperature (a) and specific loss of power (SLP) profile(b) of IONPs with different sizes under the RF heating at a low frequency of350 kHz at 1.2 kW and a high frequency of 13.56 MHz at 70 W. In profile (a),the IONPs size of 0 stands for the control DI water only, containing no IONPs.Values given are means ± SD (n = 3).



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heating rate or the maximum temperature reached by IONPsdecreased. Based on our experimental results, iron oxide par-ticles smaller than 15 nm relax in the RF radiation by the Néelrelaxation mechanism and do not heat as well but have stron-ger dynamic magnetic fields around them. On the other hand,as the size of the particles increases beyond 15 nm, the Brow-nian relaxation mechanism begins to dominate. Under Brow-nian relaxation, the particles’ magnetic moments align withthe crystal axis, and the particles rotate as their magneticmoments rotate with the RF field. The rotation of the particlesdissipates more of the RF radiation to heat; as a result, the par-ticles are hotter and their magnetic fields are not as strong.The optimum particle size for rotation in the Brownian relax-ation regime depends on the frequency of the RF: lower fre-quency RF favors larger particle size for optimum rotation andhigher temperature; higher frequency RF favors smaller par-ticle size for optimum rotation and higher temperature. At thefrequency of 350 kHz, it appears that the optimum size for therotation and Brownian heating is about 25 nm for iron oxide.The temperature of the 25 nm iron oxide nanoparticles shouldtherefore be greater and more effective in killing cancer cells.Particles smaller than 15 nm should be cooler and dominatedby the Néel relaxation mechanism; thus, the larger dynamicmagnetic fields they possess as a result of RF excitation shouldmanifest in stronger magnetic fields to kill cancer cells.However, at a frequency of 13.56 MHz, our data show that theoptimum size for Brownian heating is about 15 nm as evi-denced in both the temperature profile and SLP graph inFig. 2b. As the literature reported, if RF heating is performedat frequencies below 1 MHz where the dielectric heating of thewater is negligible,24 only the heating by IONPs should beobserved.

3.4 IONPs cytotoxicity evaluation

WST-1 and LDH assays were used to evaluate the cytotoxicityof IONPs of various sizes. These assays have been used toassess the cytotoxicity of nanomaterials in several studies.25–27

The WST-1 assay is sensitive to cellular mitochondrial activityand thus the proliferation of cells. Five different concen-trations of nanoparticles varying from 1 to 100 μg ml−1 wereused in the WST-1 assay. After 24 hours of incubation withMCF-7 cells, cell viabilities were obtained by comparison withthe control without IONPs. The results showed that there waslittle cytotoxicity (<15%) for the MCF-7 cells using this rangeof concentrations (see Fig. 3a).

The cell death caused by the nanoparticles largely occurredthrough apoptosis or necrosis. One of the most importantenzymes, lactate dehydrogenase (LDH)—which may be used tomonitor cell membrane integrity by evaluating the amount ofthe enzyme released into the culture medium—was measuredin this study. LDH released into the extracellular culturemedium activates coupled enzymatic reactions. The amount offormazan salt developed is directly proportionate to theamount of LDH released from the cell. Treatment of MCF-7with IONPs with different concentrations after 24 hoursrevealed only a 7–15% LDH release when the highest

concentration (100 μg ml−1) was used, suggesting that theIONPs generated very little necrotic or apoptotic damage to theMCF-7 cells.

3.5 Flow-cytometry to determine the cellular death

To examine the thermal ablation capabilities of IONPs oncancer cells in vitro, the flow cytometry cell viability assay wascarried out on MCF-7 cells, and the results are represented inFig. 4. All samples were divided into two groups and exposedto two frequencies of RF for 20 min, individually. The controlcells without any nanoparticles, with and without RF treat-ment, showed a viability of approximately 86–92% for both fre-quencies of RF treatment. As a result, we may conclude thatthe RF treatment had no effect on the control cells—a fact thathas also been demonstrated in our group’s previousworks.28,29 The incubation of MCF-7 with various sizes ofIONPs along with the RF treatments greatly enhanced the per-centage of dead cells, especially at a lower RF frequency. Verysimilar trends in cell damage were seen in the flow cytometry

Fig. 3 Cytotoxicity assays of MCF-7 cells exposed for 24 hours on increasingconcentrations (0–100 μg ml−1) of different sizes of IONPs: (a) WST-1 and(b) LDH assays (Triton X-100 as the positive control). Values given are means ±SD (n = 4).



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data and the SLP temperature profile data, as discussed insection 3.2. The 25 nm IONPs gave us the highest number ofdead cells (∼43.4%) after the low-frequency RF treatment whenthe concentration reached 50 μg ml−1 compared to 33.6% forcells dosed with 20 μg ml−1. The increase in IONP concen-tration was reflected in the greater number of cells killed.12

However, after the high-frequency RF treatment, the 15 nmIONPs produced the highest percentage (∼25.0%) of deadcells.

3.6 Examination of cellular uptake of IONPs

In order to confirm that the IONPs were taken up into thecancer cells efficiently, high-resolution, dark-field opticalmicroscope imaging and TEM were performed. Dark-fieldimaging (Fig. 5b) revealed that the IONPs accumulatedefficiently on the surface of the plasma membrane and withinthe cytoplasm of MCF-7 cells after 12 hours of incubation. Weexpected the cells to begin uptaking nanoparticles in less than2 hours, based on previous in vivo experimental work with themurine breast adenocarcinoma cell line.30 TEM visualizationwas also conducted to further confirm the internalization ofparticles within the cell cytoplasm. TEM images of the cellsthat had been exposed to 25 nm IONPs showed that thenanoparticles were sequestered inside the cells after 24 h ofexposure. All TEM images revealed that nanoparticles hadaccumulated within the cell cytoplasm and that most of themhad aggregated inside the late endosome and lysosome after24 hours of incubation. Additionally, more than one vesicleappeared within the cytoplasm of a single cell (Fig. 5c). TheICP-MS data shown in Fig. 5d further confirmed that theIONPs were internalized into the MCF-7 cells after 24 hours ofincubation. The data also indicated that the 41 nm size ofIONPs gave the highest elemental Fe uptake after 24 hours ofincubation. Approximately 17.4 μg per 200 000 cells of Fe wereinternalized, which is more than 2 times higher than the25 nm one and 5 times higher than the 15 nm one. In otherliterature, Chan21 and Cheng et al.31 found that gold nano-particles and silica nanoparticles have size dependent uptakeswhich were consistent with the size-dependent uptake rateof iron oxide nanoparticles in our study. For the uptake

Fig. 4 Flow cytometry measurement of cell viability before and after exposureto a high frequency of 13.56 MHz RF and a low frequency of 350 kHz RF treat-ment using different sizes of IONPs at a concentration of 50 μg ml−1. Valuesgiven are means ± SD (n = 3).

Fig. 5 Dark-field microscope images of (a) control MCF-7 cell; (b) treated cells with 25 nm IONPs for 12 hours. Arrowheads show that IONPs accumulated on thesurface of the cell membrane or within the cytoplasm of MCF-7 cells. Scale bar = 20 μm. (c) TEM images of MCF-7 cell treated with 25 nm IONPs for 24 hours. Redarrows indicate cytoplasmic aggregation of IONPs which indicates that the IONPs were uptaken inside the cells. (d) Total amount of Fe in MCF-7 cells after 24 hoursincubation of different sizes of IONPs quantified by ICP-MS (mean ± SD, N = 3).



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mechanisms, there have been many proposals for the types ofendocytosis induced by nanoparticles: clathrin-mediatedendocytosis, caveolae-mediated endocytosis, and clathrin- andcaveolae-independent endocytosis.32,33 However, the mechan-ism for the carboxylic-functionalized IONPs used in this workis still under investigation. Nevertheless, it can be concludedthat the internalization of IONPs by the MCF-7 cells is size-dependent. In this way, we can conclude that the RF heatingefficiency and its impact on cell viability are not directly depen-dent upon the total Fe concentration taken up by the cells, butthat it is primarily determined by the total concentration ofnanoparticles within the cells and their size, which controlstheir ability to interact with various RF frequencies.

3.7 Cell morphology examination after RF treatment

The morphological alterations of MCF-7 cells after RF treat-ments were observed by SEM. We were able to capture thedynamic plasma membrane blebs as shown in Fig. 6b. Plasmamembrane blebbing is one of the distinct phenomena of apop-tosis. During the final stage of cell death, cells undergo mor-phological changes characterized by the evagination of theplasma membrane from its normal position toward the extra-cellular environment. This change results from the remodelingof actin and myosin which form part of the cytoskeleton struc-ture.34,35 The TEM image (Fig. 6c) of cells after RF exposurerevealed that the cell plasma membrane had become unevenand that the cells showed signs of fragmentation by developingsealed membrane vesicles, termed apoptotic bodies. Mito-chondrial damage was also found after the RF treatments, asshown in Fig. 6d. In addition, the nanoparticles were found tohave been released back into the extracellular environment,

which was another indication of plasma membrane disruptionafter RF treatment.

As seen in the TEM image of Fig. 5, the particles accumu-lated efficiently within the cytoplasm. Moreover, more thanone vesicle filled with IONPs appeared in the cytoplasm of thecells; this also allowed localized heating12,27 corresponding tothe location of the particles when the cells were subjected tothe RF. Heating the cells causes irreversible changes, inacti-vates intracellular metabolic processes, initiates apoptosis andmembrane damage. The subsequent cellular death profiles areshown in Fig. 4 and 6d. It was observed that the aggregatednanoparticles showed much more efficient heat generationwhen compared with the non-aggregated ones, also reportedbefore.36

3.8 ROS (reactive oxygen species) induction

In this experiment, a fluorogenic probe 2′,7′-dichlorodihydro-fluorescein diacetate (H2DCFDA) was used to study the oxi-dative stress potential induced by the RF treatment. H2DCFDAis able to penetrate the cell and convert into a highly fluo-rescent product (DCF) upon oxidation. The generated ROSlevel can be measured from the fluorescence intensity which isproportionally correlated. Generally, the formation of ROS is anatural phenomenon of live cells, and it is a by-product of thecell metabolic activity of oxygen. Under normal conditions, acell can balance a certain amount of increased oxygen-contain-ing molecules through the defense system that most cellspossess, but cells cannot buffer an excessive amount of ROS.Stress or toxic conditions such as heat and pathogenic inflam-mation may lead cultured cells to increase the intra-cellular level of ROS significantly. Moreover, the induction of

Fig. 6 SEM images of MCF-7 (a) and MCF-7 cells exposed to 50 μg ml−1 of 25 nm IONPs that were exposed to RF (350 kHz) for 20 minutes; arrows point to theoutward evagination of cell plasma membrane (b); the inset is an enlarged image showing plasma membrane blebs. (c) TEM image showing cell destruction, frag-mentation, and (d) mitochondrial damage after being exposed to RF.



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intracellular ROS levels in live cells is considered one of thecommon mechanisms of nanoparticle toxicity.37 One of themechanisms of IONP-induced cellular death is the generationof reactive oxygen species (ROS) from mitochondria.38 ROSleads to oxidative stress and damage to the mitochondria, aswell as perturbation of mitochondrial activities which inducesapoptosis.39 Accumulation of a great amount of ROS leads tocascades of negative events and finally results in cell death.Cell death results from mitochondrial damage,40 DNAdamage,41 and apoptosis induction.42 In our experiment, theamount of intracellular ROS after 24 hours of incubation with50 μg ml−1 of 25 nm IONPs was negligible compared to thecontrol samples for the concentration used in our study. AfterRF treatments, the ROS induction levels were significantlyincreased. As shown in Fig. 7, the green fluorescence level forthe RF treatment was very close to the one generated by thestandard positive control H2O2 (100 μM). However, only weakfluorescence intensity was observed in the control samplesafter RF treatment. The mechanism for the cell death inducedby RF treatment can largely be attributed to apoptosisin MCF-7 cell lines—primarily through the mitochondrial

pathway by the generation of ROS. This conclusion is alsosupported by the TEM image in Fig. 6d showing that the mito-chondria were damaged by RF treatment.

3.9 Localized heating of chicken tissue by injection of IONPsunder the RF treatment

IONPs were injected into chicken tissue, and the temperaturewas measured before and after exposure to the low frequencyof the RF generator. The heating shown in Fig. 8 only occurredin those portions of the tissue that had been injected withnanoparticles; the part without nanoparticles could not beheated under RF treatment. As reported, low frequency radi-ation, such as approximately 400 kHz, can penetrate into thebiological tissues efficiently at approximately 15 cm depth.43

As a result, we can conduct cancer treatment deep inside thebody. As shown in Fig. 8, the experiment further confirmedthat the tissue can be locally heated efficiently using low-frequency (350 kHz) RF treatment. The results confirmed thepotential for cancer treatment using thermal therapy assistedby nanotechnology in the future. Healthy tissue can be

Fig. 7 ROS-H2DCFDDA assay for intracellular reactive oxygen species with IONPs with (right column) and without (left column) RF treatment. (a) The control and(b) at a concentration of 50 μg ml−1. (c) H2O2 (100 μM) as the positive control.



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preserved, while cancerous tissue is heated and destroyedusing certain sizes of nanoparticles and low-frequency RFheating.

4. Conclusions

Our research demonstrated that IONPs are excellent candi-dates for the hyperthermia treatment of human breast cancercells. Nanoparticle size and RF frequency are critical para-meters that influence the optimum level of the hyperthermiaeffect. For 350 kHz lower frequency RF, the 25 nm IONPsproved to be the most efficient in killing the cancer cells. At ahigher frequency (13.56 MHz) of RF, smaller sized (15 nm)IONPs are the most effective. RF-induced heat treatment wasfound to damage the cells’ plasma membrane and mitochon-dria membrane, thus greatly increasing ROS levels andcausing cellular apoptosis.

Acknowledgements

The support for this research received from the ArkansasScience and Technology Authority (ASTA) through grant no.

08-CAT-03 is acknowledged. The financial support provided bythe US Army TATRC program is acknowledged. The supportsfor this work received from The Office of ExperimentalProgram to Stimulate Competitive Research (EPSCoR) ofNational Science Foundation of USA and South Carolina StateUniversity are acknowledged. The editorial assistance byDr Marinelle Ringer is also acknowledged. Ocean NanoTech isacknowledged for supplying the iron nanoparticle samples forthe XRD analysis.

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Fig. 8 Temperature distribution on chicken tissue by injection of IONPs (5 mg mL−1, 25 nm) under RF (350 kHz) treatment: (a) a photograph of a piece of chickenafter injection of IONPs inside the RF coil; (b) before and (c) after heating under the RF for 10 min which showed that the localized temperature increases to 44.3 °Cfrom room temperature; (d) the temperature changes in chicken tissue with and without IONPs during 6 min of RF heating and 4 min of cooling. Values given aremeans ± SD (n = 3).



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iron oxide nanoparticle-based radio-frequency thermotherapy for human breast adenocarcinoma cancer...

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