Impact of Magnetic Field Parameters and Iron Oxide Nanoparticle

Journal of Magnetism and Magnetic Materials 387 (2015) 96–106

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Journal of Magnetism and Magnetic Materials

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n CorrE-m

journal homepage: www.elsevier.com/locate/jmmm

Impact of magnetic field parameters and iron oxide nanoparticleproperties on heat generation for use in magnetic hyperthermia

Rhythm R. Shah a, Todd P. Davis b, Amanda L. Glover b, David E. Nikles b,Christopher S. Brazel a,n

a Department of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, AL, USAb Department of Chemistry, The University of Alabama, Tuscaloosa, AL, USA

a r t i c l e i n f o

Article history:Received 5 March 2014Received in revised form2 September 2014Accepted 25 March 2015Available online 30 March 2015

Keywords:Magnetic fluid hyperthermiaIron oxide nanoparticlesMagnetic field strengthMagnetic field frequencySpecific absorption rate

x.doi.org/10.1016/j.jmmm.2015.03.08553/& 2015 Elsevier B.V. All rights reserved.

esponding author. Fax: þ1 205 348 7558.ail address: [email protected] (C.S. Brazel).

a b s t r a c t

Heating of nanoparticles (NPs) using an AC magnetic field depends on several factors, and optimizationof these parameters can improve the efficiency of heat generation for effective cancer therapy whileadministering a low NP treatment dose. This study investigated magnetic field strength and frequency,NP size, NP concentration, and solution viscosity as important parameters that impact the heating effi-ciency of iron oxide NPs with magnetite (Fe3O4) and maghemite (γ-Fe2O3) crystal structures. Heatingefficiencies were determined for each experimental setting, with specific absorption rates (SARs) rangingfrom 3.7 to 325.9 W/g Fe. Magnetic heating was conducted on iron oxide NPs synthesized in our la-boratories (with average core sizes of 8, 11, 13, and 18 nm), as well as commercially-available iron oxides(with average core sizes of 8, 9, and 16 nm). The experimental magnetic coil system made it possible toisolate the effect of magnetic field parameters and independently study the effect on heat generation.The highest SAR values were found for the 18 nm synthesized particles and the maghemite nanopowder.Magnetic field strengths were applied in the range of 15.1–47.7 kA/m, with field frequencies ranging from123 to 430 kHz. The best heating was observed for the highest field strengths and frequencies tested,with results following trends predicted by the Rosensweig equation. An increase in solution viscosity ledto lower heating rates in nanoparticle solutions, which can have significant implications for the appli-cation of magnetic fluid hyperthermia in vivo.

& 2015 Elsevier B.V. All rights reserved.

1. Introduction

Magnetic fluid hyperthermia (MFH) using localized iron oxideNPs offers a significant benefit over whole body and regional hy-perthermia, which can lead to several vascular and cardiac dis-orders [1–3]. MFH has also been shown to kill cells faster ascompared to traditional hyperthermia methods, which can play anessential role in reducing the therapy administration time forcancer treatment [4]. MFH can reduce side effects in patients whileamplifying treatment of cancer using superparamagnetic NPs,which can be specifically targeted using antibodies or peptidesequences [5, 6] and directed to cancerous tissue through theenhanced permeation and retention (EPR) effect [7]. Magneticfields in the kHz to MHz range have been investigated for heatgeneration in various MFH systems using superparamagnetic andferromagnetic iron oxide NPs [8, 9]. To be used effectively forcancer treatment, the least possible dose of NPs should be

introduced in the human body to avoid possible side-effects andbioaccumulation. Thus, it is essential to understand the factorsthat affect heat generation in NP dispersions to maximize thetherapeutic effectiveness of MFH.

Some NPs based on iron oxide have been approved for medicaluse by the US Food and Drug Administration (FDA) and the Eur-opean Medicines Agency (EMA) [6]. While magnetic NPs thatcontain cobalt ferrites and nickel ferrites may have better mag-netic properties for heat generation, the medical use of thesematerials is generally infeasible. NPs made of nickel ferrite havebeen shown to have an adverse effect on cell viability and re-plication, while it was demonstrated that cobalt ferrite NPs can betoxic to mammalian cells at concentrations needed for cancerhyperthermia treatment [10–12]. In addition to their acceptabilityfor medical use, iron oxide NPs feature good colloidal stabilitywhen coated with appropriate surfactants or polymers which canalso provide a linkage to cell-targeting moieties [13]. Iron oxideNPs have been widely investigated for magnetic heating, and havealso proven to be useful as MRI contrast agents [6, 14]. Theseproperties make iron oxide NPs attractive for use in cancertheranostics.

R.R. Shah et al. / Journal of Magnetism and Magnetic Materials 387 (2015) 96–106 97

To characterize the heating of magnetic nanoparticles under ACmagnetic field exposure, specific absorption rate (SAR) values aredetermined from temperature–time profiles and computed as heatgeneration per mass of NPs or iron (Fe) content of NPs in W/g [15–18]. NPs with high SAR are largely favored for cancer treatment asadministration of NPs to patients can be kept to a minimum whileusing brief durations of magnetic field exposure that still achievethe temperature rise essential to induce cell death. SAR is calcu-lated as:

⎛⎝⎜

⎞⎠⎟SAR W g

m c

mTt

( / )(1)

s p

np

ΔΔ

=⁎

⁎

Here ms is the mass of solution, mnp is either the mass of NPs orthe mass of Fe in the NPs, cp is the heat capacity of the solution,and (ΔT/Δt) is the initial slope of the temperature rise vs. timecurve for NP heating. The SAR value serves as guidance for com-paring the heating rates of NPs with different compositions andconcentrations, at different magnetic field settings.

The parameters that govern power loss in magnetic hy-perthermia are defined by the Rosensweig equation [19], wherethe power generation (P) in iron oxide NPs when subjected to anAC magnetic field is defined as:

P H ff

f2

1 (2 ) (2)o o2

2πμ χ π τ

π τ=

+

Here, μo is the permeability constant of free space(4πn10�7 T-m/A), χo is the magnetic susceptibility of the particles,H is the magnetic field strength, f is magnetic field frequency, and τis the relaxation time for reorientation of magnetic moments inNPs, either through whole NP motion (Brownian relaxation) orspin relaxation (Néel relaxation) [19]. The power generatedthrough application of an AC magnetic field results in thermalenergy, and for a given set of superparamagnetic NPs the quantityof heating is a function of the square of magnetic field strengthwhen all other factors are held constant. Frequency can also beused to tune the heat generation, as the power generation reachesan asymptote when frequency is increased. The application of theRosensweig equation, and contribution of different relaxationmechanisms to MFH has been well described [19–24], and furtherrelationships between magnetic heating and NP properties aremanifest in the magnetic susceptibility and relaxation time.

By changing the properties of the applied magnetic field(through field intensity and frequency), heating in super-paramagnetic NPs can be optimized. The power input by themagnetic field can also be tuned by adjusting the time course offield application. The field can be applied for different durations oftime or using variable field intensity, for example through the useof a feedback control loop where the field is adjusted to maintain afixed temperature. One such system has been proposed by Tsenget al. using a thermocouple and a temperature processing unit tomaintain a constant hyperthermia temperature [25]. A number ofstudies have investigated MFH to determine preferred parametersthat lead to high SAR values [26–31]. In most published studies,MFH magnetic field frequencies are applied in the range of 80–700 kHz, while field strength usually lies between 1 and 50 kA/m[15, 26–31]. A wide range of SAR values have been reported for NPsof different compositions, sizes, and size distributions, for manydifferent field strengths and frequencies which are often fixed bythe geometry and electrical configuration of the magnetic coils.Additional complications that make comparison of experimentalresults between groups challenging include the reliability of NPcharacterization and differences in SAR reporting, which is nor-malized by either NP mass or the mass of Fe in the NPs, but is oftennot clearly reported due to difficulties in distinguishing the oxi-dation state of Fe in the NPs. These variables make it difficult to

reach conclusions about optimal NP structures and magnetic fieldparameters to achieve effective heating. SAR values for commercialand custom-synthesized iron oxide NPs have been reported cov-ering a range from lower than 10 W/g Fe to higher than 2000 W/gFe [15, 26–31]. Some of the highest reported SAR values of2452 W/g of Fe for cubic iron oxide NPs and 1650 W/g for sphericaliron oxide NPs were obtained by Guardia et al. and Fortin et al.,respectively [26, 27].

Heat generation in magnetic NPs under application of a highfrequency magnetic field is governed by Néel relaxation, Brownianrelaxation, and a hysteresis loss mechanism [19]. Néel relaxationoccurs due to the flipping of magnetic moments inside each NP,whereas Brownian relaxation occurs due to the rotation of entireparticles along with the magnetic moment. Néel and Brownianrelaxations are theorized to be the dominant heat loss mechan-isms for particles that are superparamagnetic in nature, whilehysteresis losses that occur due to movement of domain wallsunder application of magnetic field are responsible for heating inlarger sized ferromagnetic particles [19]. There is, however, dis-agreement over the maximum size for single domain NPs, andwhere this transition occurs. The critical NP size range separatingsuperparamagnetic and ferromagnetic domain varies based onstructure and composition of NPs. In a study by Bakoglidis et al.where NPs investigated were a mixture of maghemite and mag-netite, it has been suggested that particles beyond 13 nm in size liein the ferromagnetic domain, whereas smaller particles lie in thesuperparamagnetic domain [32]. Krishnan has determined bymathematical modeling that the maximum size for a particle to besingle domain and superparamagnetic is in the range of around35 nm for maghemite and 25 nm for magnetite, while that forbeing single domain and ferromagnetic is approximately 90 nmfor maghemite and slightly larger than 80 nm for magnetite [22].Vergés et al. surveyed the results of other researchers and reportedthat the transition range from single domain to multi domain isaround 50 nm for magnetite NPs [33]. Thus, while the mechanismof heating is expected to depend largely on particle size, there aretwo complicating factors that make attributing heat generation toa particular heating mechanism difficult: (1) the particles mayhave single or multiple crystal domains, and (2) the size dis-tribution of NPs can be widely disperse for a given sample.

The viscosity surrounding NPs can also impact magnetic heat-ing, primarily through increasing the relaxation time for Brownianrelaxation, which reduces the Brownian contribution to heatgeneration. As most experimental investigations of MFH are doneon aqueous dispersions of NPs with no significant additives to alterviscosity, the applicability of data to more complex in vivo en-vironments may not be accurately estimated. For example, manyapplications of magnetic NPs involve the deployment of NPs inblood or tissue, where they will be surrounded by proteins [34], orin the core of a drug delivery device where the free motion of NPsis impeded by a polymer [13]. One recent study has shown thatwhen a mixture of 13.9 nm magnetite and maghemite NPs wassubjected to increasing solution viscosity from 0.9 cP to 43.2 cP ata magnetic frequency of 215 kHz and amplitude of 3.8 kA/m, theSAR value decreased to 70% of the original value [35]. Since theenvironment surrounding NP for medical uses will likely be sig-nificantly more viscous than water, experiments to determine theeffect of viscosity on SAR are needed to predict heating in vivo.Thus, this phenomenon can affect the overall feasibility of MFH,particularly if Brownian relaxation is responsible for much of theheating.

While many research groups have contributed to the under-standing of MFH, it is difficult to compare SAR values from dif-ferent groups, as the magnetic induction coils in each laboratoryoften have fixed or narrow operational frequency ranges. Also inmany research studies multiple factors affecting the NP heating

R.R. Shah et al. / Journal of Magnetism and Magnetic Materials 387 (2015) 96–10698

may vary at the same time, making it difficult to isolate variablesresponsible for magnetic heating. We overcame this drawbackusing a power supply and magnetic coil that allows the frequencyto be varied while the field strength is kept constant, and viceversa. We also studied the effect of particle size by using differentsizes of NPs synthesized using identical experimental methods.Based on the importance of material and magnetic field para-meters on particle heating, we designed a comprehensive study totest the heating efficiency of different types of NPs covering arange of field strengths and frequencies, for different NP compo-sitions, concentrations, and sizes, as well as solution viscosities.

2. Materials and methods

2.1. Materials

All chemicals were purchased at reagent grade or better fromthe Sigma-Aldrich company (St. Louis, MO), unless otherwise no-ted. Iron (III) oxide nanopowder, less than 50 nm in size, was ex-amined for hyperthermia applications, primarily as a low-cost,abundant nanomaterial. fluidMAG D with starch coating, andfluidMAG PAD with polyacrylamide coating were purchased fromchemicell GmbH (Berlin, Germany), with a reported concentrationof 100 mg/mL (which included the weight of the polymer coating).Both fluidMAG particles had a reported hydrodynamic radius of50 nm. Additional iron oxide NPs dispersed in hexane were syn-thesized as described below, using iron(III) acetylacetonate 97%,1,2-hexadecanediol 90%, oleic acid 90%, oleylamine 70% (Sigma-Aldrich, St. Louis, MO), benzyl ether 99% (Acros Organics, FairLawn, NJ), hexane, and ethyl alcohol. Table 1 lists the types, ab-breviations, sizes, and concentrations of particles used for thisstudy. Alginic acid, sodium salt (Acros Organics, rated at 485 mPa-sfor a 1% solution at 20 °C) was used to modify the viscosity ofaqueous NP dispersions. Calcium chloride was purchased fromFisher Scientific (Fair Lawn, NJ) to cross-link alginic acid solutions.

2.2. Magnetic NP synthesis

Iron oxide nanoparticles of various sizes were synthesized fromiron (III) acetylacetonate and 1, 2 – hexadecanediol, in the pre-sence of oleylamine and oleic acid using the thermal

Table 1Properties of NPs investigated for magnetic heating.

Nanoparticles Particle ID Iron oxide concentra-tions(mg/mL) a

Solvent Iron ometer(nm)b

Magnetite coated withstarch

fluidMAG D 4.370.1 Water 8726.470.18.670.1

Magnetite coated withpolyacrylamide

fluidMAG PAD 8.370.2 Water 972

Magnetite coated witholeic acid

TDMNP8 6.270.1 Hexane 872TDMNP11 10.870.2 1172TDMNP13 6.470.0 1374TDMNP18 8.170.0 1874

Maghemite withoutcoating

Maghemitenanopowder

1.670.0 Water 1679

a Concentration and standard deviation determined by AA spectroscopy.b Mean diameter7one standard deviation for analysis of more than 100 particles uc Number average diameter7one standard deviation measured by DLS.d Z-average diameter reported from DLS Zetasizer software.e Determined using vibrating sample magnetometry.f Sample not evaluated.g Measured using non-dispersed dry maghemite nanopowder.

decomposition method described by Sun et al. [36]. Briefly,2 mmol of iron (III) acetylacetonate was added to 10 mmol of 1,2-hexadecanediol in 20 mL of benzyl ether containing 6 mmol ofoleic acid and 6 mmol of oleylamine. To synthesize particles ofdifferent sizes, this solution was heated to a set nucleation tem-perature for 2 h under a nitrogen environment, with stirring. Thiswas followed by increasing the temperature and refluxing for 1 h.The mixture was then cooled to ambient temperature under ni-trogen, and then precipitated with 40 mL of ethanol. The pre-cipitated particles were separated by centrifugation and the su-pernatant was decanted. The product was then dispersed in hex-ane in the presence of �0.05 mL of oleic acid and �0.05 mL ofoleylamine. Any remaining undispersed product was removed bycentrifugation at 6000 rpm for 10 min followed by decantation.The product was precipitated with ethanol a final time, cen-trifuged, and decanted. The product was then redispersed intohexane.

Particle sizes and distributions were controlled by slight var-iations in the reaction conditions. Slowly approaching but notexceeding the 200 °C nucleation period temperature producedparticles with narrow size distributions. Refluxing gently at�300 °C produced larger particle sizes of around 11 nm, whereasrefluxing less carefully produced NPs that had a size of around8 nm. Still larger particles were produced using the seed growthmethod described by Sun et al. [36]. Using 11 nm particles as theseeds and employing the same nucleation and reflux conditions asabove, 13 nm particles were produced. Larger particles of around18 nm were produced by increasing the nucleation temperature to210 °C for 5 min, then immediately cooling the mixture to 200 °Cfor the remainder of the nucleation period. Although the sizedistributions were slightly larger, this method removed the needfor another seed growth reaction to produce larger particles.

2.3. Maghemite nanopowder dispersions

To make aqueous maghemite nanopowder dispersions, Iron(III) oxidenanopowder was mixed vigorously in DI water at25 mg/mL and allowed to settle undisturbed for 2 days. The su-pernatant, which formed a stable dispersion, was collected with aPasteur pipette and used for magnetic heating experiments. Theconcentration of these particles was determined by atomic ab-sorption spectroscopy, as discussed later.

xide dia-TEM

Hydrodynamic dia-meter DLS (nm)

Magnetization at10,000 Oe M (emu/g)e

Magnetic suscept-ibility oχ

e

1977c;60d 8076 0.1270.03

2979c;55d 4671 0.0570.01

f f f

f 50 0.04f f f

f 67 0.1335710c;54d 57 g 0.11 g

sing Image J software on TEM images (Figure 1).


2.4. NP characterization

Images of each of the iron oxide NPs were collected usingtransmission electron microscopy (TEM, FEI Technai F-20, Hills-boro, OR). The mean particle size of NPs was found by performingsize analysis on TEM images using NIH Image J software. The hy-drodynamic diameters of aqueous NP dispersions were measuredusing a Malvern ZEN3600 dynamic light scattering (DLS) device(Malvern, Worcestershire, UK).

X-ray photoelectron spectroscopy (XPS) was used to determinethe oxidation state of the iron oxide NPs. Here, samples of ironoxide NPs were washed with acetone to remove the surfacecoating of oleic acid and oleylamine, followed by placing themunder vacuum in a vacuum oven set to ambient temperature untilthey dried for 24 h. The samples were then deposited onto coppertape for XPS analysis, which was carried out on a Kratos AXIS 165Multitechnique Electron Spectrometer (Manchester, UK) using amonochromatic aluminum source set to 10 mA, 12 kV. High re-solution scans were performed with the analyzer pass energy setto 20 mA and the dwell time set to 2000 ms. Two sweeps wereperformed for each scan. Because it has been shown that the C 1speak is unsuitable for use as a reference for charge correction oniron oxide samples [37], the O 1s peak at 530.0 eV binding energywas used for this purpose.

High resolution scans of the Fe 2p region of iron oxides havebeen previously shown to be useful for qualitative studies of theionic and oxidation states of iron, and the presence or absence of asatellite peak between the Fe 2p1/2 and Fe 2p3/2 has been used todifferentiate between magnetite and maghemite [37], as onlymaghemite samples produce a satellite peak attributed to Fe 2p3/2.The position of the Fe 2p3/2 peak is typically 711 eV, with the sa-tellite peak located approximately 8 eV higher [37].

2.5. Chacterization of magnetic properties of NPs

Magnetization curves were acquired using a Digital Measure-ment Systems vibrating sample magnetometer (VSM). The mag-netometer was calibrated using a high purity nickel standard.Powder samples were carefully weighed on an analytical balance.The samples were placed on Scotchs tape and the tape folded overto make a sandwich with the particles trapped inside the tape. The

Fig. 1. Hyperthermia coil

excess Scotchs tape was trimmed off with scissors. The sampleswere affixed to a quartz sample holder using silicon vacuumgrease. Samples of fluidMAG D NPs were dried from aqueous so-lution to give free-standing films. The films were weighed on ananalytical balance and then affixed to the quartz sample holder.Full hysteresis curves were obtained with a saturating field of10,000 Oe. The step size was 10 Oe in the range from þ1000 to�1000 Oe.

2.6. Magnetic heating of NPs

All magnetic heating experiments were performed using acustom-designed hyperthermia coil (Induction Atmospheres, Ro-chester, NY) (Fig. 1). A 5 kW power supply (Ameritherm, ModelNovastar 5 kW, Scottsville, NY) is connected to a heat station towhich the coils are attached. The hollow coils are designed to al-low circulating chilled water to pass through, in order to minimizetemperature rise in the coils. A circulating chiller bath (KoolantKoolers Model JT1000, Kalamazoo, MI) was set to circulate waterthrough the coils at 18 °C. A 4 cm inner diameter coil with twoturns in a distance of 1.2 cm was separated from two identicalturns by a gap of 2 cm. This 4-turn coil was used for all heatingexperiments, as it had a sufficiently wide coil diameter to allowinsulation to fit between the coil and samples to minimize non-specific heating of samples due to the heating of the coil surfaces.An additional 6-turn coil (4 cm inner diameter) could be placed inseries with the magnetic heating coil, and was used to modulatethe frequency applied to the NP dispersions (frequency modulatorcoil shown on left side in Fig. 1). A copper bar can be placed acrossany number of the coils to allow a total of seven testing fre-quencies at each voltage setting (including one with the frequencymodulator removed).

Each NP dispersion was taken at a measured volume of 1.2 mLand filled in a plastic micro-centrifuge tube, which was positionedin the axial and radial center of the top two turns of the coil andsurrounded by a layer of insulating foam. The magnetic fieldstrength was adjusted by altering the voltage setting in the heatingstation circuitry, which in turn changed the current supplied to thecoil. The frequency was modified by placing the frequency mod-ulating coil in series with the hyperthermia coil and adjusting thecopper bar so that the circuit would bypass from zero to all six

experimental set-up.

Table 2SAR values for magnetic heating of NPs at fixed field strengths and frequencies.

Nanoparticletype

Fieldstrength, H(kA/m)

Frequency, f(kHz)

SAR (W/g Fe) SAR (W/g NP)a

fluidMAG D(8.6 mg/mL)

15.1 194 3.771.2 2.770.922.8 15.372.9 11.172.135.8 26.872.9 19.472.138.2 29.971.0 21.670.747.7 43.374.4 31.373.238.2 123 15.172.1 10.971.5

143 23.172.8 16.772.0194 29.971.0 21.670.7430 105.675.0 76.473.6

fluidMAG PAD 47.7 194 41.772.3 30.271.7TDMNP8 22.8 194 6.571.5 4.771.1

35.8 14.671.5 10.671.147.7 30.172.1 21.871.5

TDMNP11 47.7 194 33.471.2 24.270.9TDMNP13 47.7 194 52.874.3 38.273.1TDMNP18 47.7 194 75.772.3 54.871.7

38.2 430 325.9716.0 235.8711.6Maghemite NP 38.2 430 249.174.7 174.273.3

a Iron oxide crystal structure determined by XPS for normalizing SAR valuesper gram NP.


loops. For experiments to determine the effect of field strength onmagnetic heating, the frequency was maintained at a constantvalue by using a fixed coil setup and adjusting the voltage suppliedto the coil. In experiments to investigate the effect of field fre-quency on heating, the voltage was appropriately changed so thatthe field strength remained constant while the frequency waschanged due to addition of the frequency modulator coil. Fieldstrength settings ranging from 15.1 to 47.7 kA/m were used forexperiments, while the frequency was varied from 123 to 430 kHz(Table 2). To test the effect of magnetic field strength on NPheating, four different field strength settings of 15.1 kA/m, 22.8 kA/m, 35.8 kA/m, and 47.7 kA/m were obtained at a set frequency of194 kHz by changing the voltage applied to produce the magneticfield. Additional field strengths of 32.9 kA/m and 27.2 kA/m wereobtained at maximum applied voltage when the frequency wasreduced to 143 kHz and 123 kHz, respectively, by addition of thefrequency modulator coil. To study the effect of frequency on hy-perthermia, field strengths were set at 38.2 kA/m and tested atfour different frequencies: 123, 143, 194 and 430 kHz. Severalcombinations of these magnetic field parameters were tested oncommercial fluidMAG D particles, whereas selected parameterswere used to test the heating of other magnetic particles listed inTable 1.

An infrared camera (FLIR systems, North Billerica, MA) wasused to record the temperatures of samples contained in the mi-cro-centrifuge tube. The FLIR camera was capable of measuringtemperatures with a resolution of 0.1 °C.

2.7. Measurement of magnetic field strength

The magnetic field strengths generated by the hyperthermiacoil were measured using a magnetic field probe (AMF Life Sys-tems, Auburn Hills, MI). The axial and radial components of the ACmagnetic field were measured as a function of position within thecoil for several input voltages and coil configurations. For magneticheating experiments, samples were placed in the axial and radialcenter of the coils, where the field strength was found to behighest.

2.8. Preparation of viscous alginate NP Solutions

To determine the effect of viscosity on SAR, 2.0 and 4.0 wt%

alginate solutions were prepared by adding measured quantities ofsodium alginate to aqueous NP dispersions. The mixtures werevigorously mixed and formed a viscous solution after leaving itovernight. A cross-linked alginate gel containing dispersed NPswas made by adding 0.5 mL of 2 wt% calcium chloride in water tothe 4% alginate-NP solution. For each of these solutions, care wastaken not to vary the net NP concentration. The viscosities of the2 and 4 wt% alginate solutions were measured by using a Zahncup-style Boekel viscosimeter (Philadelphia, PA). Heating experi-ments were conducted using the same 1.2 mL sample size as fordispersed NPs.

2.9. Experimental data collection

All heating experiments were performed for 10 min, by placingthe micro-centrifuge tube in the center of the coil and applying theselected magnetic field parameters. Experiments were performedin triplicate, starting at room temperature. Non-specific heating ofNP solutions was subtracted from each run by performing iden-tical heating experiments on pure solvents (i.e., water, hexane)using the same magnetic field parameters and subtracting the T(t)profile for the pure solvent from the T(t) profile for the NPsolution.

3. Results and discussion

3.1. Characterization of NPs

TEM and DLS were used to determine mean iron oxide NPdiameters, and the hydrodynamic diameters in aqueous disper-sions, respectively (Table 1). The fluidMAG particles were suppliedas aqueous solutions at 100 mg/mL, with this value including a20 wt% coating of either dextran (fluidMAG D) or polyacrylamide(fluidMAG PAD) according to the manufacturer. Discounting theNP coatings, iron oxide concentrations were determined by AtomicAbsorption Spectroscopy (AA); the AA values are reported for allNP concentrations. The iron oxide concentration determined byAA contrasts with the nominal concentration for the fluidMAGparticles provided by the manufacturer, as the fluidMAG NPscontained a lower percentage of iron oxide than anticipated (Ta-ble 1). The TDMNP particles synthesized in house had an oleic acidcoating, allowing the particles to be dispersed in hexane. Thecommercial maghemite nanopowder was partially dispersible inwater, using the method described above, so particle sizes andmagnetic properties were determined using only the dispersiblefraction.

TEM images of the NPs show a representative sample of eachtype of NP investigated (Fig. 2). The sizes for iron oxide NP dia-meter represent the mean size found for at least 250 NPs using theImage J software area measurement tool; the reported error re-presents one standard deviation for the distribution of particlesizes. The dispersible fraction of maghemite nanopowder andTDMNP18 NPs were the largest of those evaluated with mean sizesof 16 nm and 18 nm, respectively, with the commercial maghemitenanopowder having the widest distribution of particle sizes.Commercially-available fluidMAG D and fluidMAG PAD hadsmaller iron oxide NP sizes of 8 and 9 nm, respectively.

Although fluidMAG D and fluidMAG PAD are sold as havinghydrodynamic diameters of 50 nm each, we found that the num-ber average hydrodynamic size was considerably smaller, at 19 nmand 29 nm, respectively for the fluidMAG D and fluidMAG PADsupplied (Table 1). The increase in diameter compared to the TEMdata is explained by the coating of hydrophilic polymers. The hy-drodynamic diameter of the aqueous maghemite nanopowderdispersion was found to be 35 nm, which is significantly larger

Fig. 2. Representative TEMs of particles investigated: A) FluidMAG D NPs, B) TDMNP11 NPs, C) TDMNP18 NPs, D) maghemite nanopowder.

Fig. 3. XPS spectra used to determine the iron oxide structure of fluidMAG D,maghemite nanopowder, TDMNP 18, TDMNP 13, and TDMNP 11 NPs.


than the 16 nm mean NP diameter determined by TEM (Table 1).Since the maghemite nanopowder particles have no coating, thisresult indicates that there is a small degree of aggregation in thisaqueous dispersion. The Z-average particle size obtained from theDLS Zetasizer software is also reported, with values ranging from54 to 60 nm. From these data, we judge that fluidMAG and ma-ghemite aqueous dispersions did not form substantially large ag-gregates. The custom-synthesized NPs do not have reported hy-drodynamic sizes as hexane dispersions were not measured byDLS.

XPS spectra (Fig. 3) were used to determine that the fluidMAGand TDMNP NPs synthesized in our laboratory were magnetite asthey did not display a satellite peak which would indicate ma-ghemite [37]. However, the satellite peak at 719 eV confirms theγ-Fe2O3 crystalline structure of the maghemite nanopowder.

Saturation magnetization values and the magnetic suscept-ibility of the NPs were determined by VSM (Table 1). All themagnetite particles showed hysteresis curves with similar shapes.The remanent magnetization was zero and at the high appliedfields, up to 10,000 Oe, the curves did not saturate, suggesting theparticles were superparamagnetic. When the data were fit to theLangevin function, which describes the magnetization curve for

Fig. 4. Effect of magnetic field strength on temperature rise for fluidMAG D NPs(f¼194 kHz, [NP]¼8.6 mg/mL).

Table 3Effect on NP concentration on SAR from magnetic heating of fluidMAG D.

Magnetite concentration (mg/mL) SAR (W/g Fe) SAR (W/g NP)a

4.3 45.576.4 32.974.66.4 43.973.2 31.872.38.6 43.374.4 31.373.2

a Iron oxide crystal structure determined by XPS for normalizing SAR valuesper gram NP.


superparamagnetic particles, the data deviated from a simpleLangevin function. For nanoscale magnetic particles, particles atthe low end of the distribution will have different magneticproperties from the particles at the high end of the size distribu-tion. The magnetite samples can best be described as a mixture ofsuperparamgnetic particles having a distribution of magneticmoments. Since the magnetization curves did not saturate, thevalues of specific magnetization were reported for an applied fieldof 10,000 Oe. For the maghemite nanopowder, the hysteresis curvewas that of a hard ferromagnet with a small coercivity of 150 Oeand a saturation magnetization of 57 emu/g. This was expected,since maghemite has a modest value of magnetocrystalline ani-sotropy energy density (Ku�104 erg/cc [38 ]).

3.2. Magnetic heating of NPs

Magnetic heating experiments were conducted on each of thenanoparticle types investigated (Table 1) over a range of magneticfield frequencies (123–430 kHz) and intensities (15.1–47.7 kA/m).Here, the temperature rise (T–To), which is the difference betweenthe measured and initial temperatures of each sample, is shown toincrease over a period of 10 min. The samples were well insulatedand the nonspecific heating due to heat transfer from the coilsurface to the solvents used for NP dispersion (which was less than0.6 °C for water and 1.5 °C for hexane over a time period of 10 minfor all coil settings) was subtracted from the data, so that resultsrepresent heat generated by the NPs only (Fig. 4). To test the effectof frequency and field strength independently, other experimentalparameters were held constant. For example, fluidMAG D NP so-lutions were heated using field strengths of 15.1–47.7 kA/m at aconstant 194 kHz frequency, and also tested over a range of fre-quencies from 123 to 430 kHz using the frequency modulator coilwhile holding the field strength constant at 38.2 kA/m. In additionto field intensity and frequency, the effect of NP concentration, NPsize and type, and NP dispersion viscosity on magnetic heatingwere investigated independently.

3.2.1. Effect of magnetic field intensityMagnetic heating was evaluated for 20 mg/mL fluidMAG D NP

dispersions that contained 8.6 mg/mL Fe3O4. These NPs were ex-posed to AC magnetic field strengths ranging from 15.1 to 47.7 kA/m at a fixed frequency of 194 kHz (Fig. 4). Thermal output in-creased with higher magnetic field intensities, with a nearly lineartemperature rise over a period of 10 min. Similar results obtainedby other research groups have shown that heating rates and SARvalues increase with an increase in magnetic field strength [13, 17,23, 24, 27]. Thus, heating can be maximized by using highermagnetic field intensities. However, for medical applications suchas hyperthermia therapy, a patients' tolerance limit of the productof field strength (H) and frequency (f) has been reported by Bre-zovich to be 4.85�108 A/m-s [39].

The temperature–time data were converted to SAR values tonormalize results for the various NP dispersions tested. The initialslopes of the temperature–time graph (in the linear region) wereused to calculate SAR values (W/g of iron content in NPs), withdata normalized by the iron (Fe) mass in each experiment, asdetermined by AA (Table 1). As field strength was increased from15.1 to 47.7 kA/m, the SAR for heating fluidMAG particles at194 kHz increased from 3.7 to 43.3 W/g Fe (Table 2). These datasupport the dependence of SAR on the square of the field intensity,as is discussed in more detail later.

3.2.2. Effect of magnetic field frequencyTo investigate the effect of frequency independent of the field

intensity, the coil was set at field strength of 38.2 kA/m while thefrequency was varied from 123 to 430 kHz for heating experiments

(Fig. 5). Because the frequency depends strongly on coil design(number of turns, diameter, etc.), the frequencies tested were setat values that could be obtained using the frequency modulatorcoil. The same fluidMAG D dispersions used for evaluating theeffect of field intensity (8.6 mg/mL) were used for these experi-ments. The heating rate increased as the frequency increased, witha temperature rise of over 35 °C in five-minutes at 430 kHz, thehighest frequency setting tested. Interestingly, at 430 kHz and38.2 kA/m (Fig. 5), the five-minutes temperature rise is muchhigher than at a lower frequency of 194 kHz but higher fieldstrength of 47.7 kA/m (Fig. 4). This demonstrates the significantrole that AC field frequency has in achieving high heating rates andSAR values. The heating trend is born out in the SAR values forfluidMAG D (Table 2), with a greater than linear relationship be-tween SAR and frequency. This is discussed later, as the data are fitto the Rosensweig equation.

3.2.3. Effect of NP concentrationConcentration is one of the simplest variables to alter when

assessing the therapeutic potential of NP dispersions for MFH. SARvalues for iron oxide concentrations of 4.3 mg/mL, 6.4 mg/mL, and8.6 mg/mL were tested for fluidMAG D NPs. This concentrationrange was selected so that reasonable heating could be achieved ina 10 min time frame, without approaching the boiling point ofwater. Although the more concentrated NP dispersion heatedfaster, when normalized for comparison of SAR values, the valuesobtained were not statistically different (Table 3). This result isconsistent with the concept of SAR, as it is normalized by the massof NPs, and allows comparison between magnetic heating ex-periments of NPs with different concentrations. Similar resultswere obtained by de la Presa et al., who showed that SAR did notvary appreciably with concentration in the range of 6–300 mg/mLof iron, although there was a slight decrease in SAR when theconcentration dropped below 5 mg/mL [18, 33]. Murase et al., haveshown that the temperature rise for Resovists solutions contain-ing 45.5 mM to 115.4 mM Fe (corresponding to approximately 3.5–

Fig. 5. Effect of magnetic field frequency on temperature rise for fluidMAG D NPs(H¼38.2 kA/m, [NP]¼8.6 mg/mL).


9.2 mg/mL iron oxide) follows a linear trend when exposed to2.9 kA/m at 600 kHz [23]; this is also consistent with the SARbeing independent of concentration. Thus, more concentrated NPsolutions (if achievable) can deliver heat effectively, while mini-mizing the need for higher magnetic field strengths and fre-quencies. One study at low NP concentrations (below 1.2 mg/mL)showed a variance in SAR with concentration, but we discountthese results due to lack of analysis for reproducibility [32].

3.2.4. Effect of NP sizeMagnetite NP dispersions of four different sizes (8, 11, 13 and

18 nm TDMNPs) were heated using a 47.7 kA/m magnetic field at afrequency of 194 kHz (Table 2). Under these conditions, SAR valuesincreased from 30.172.1 to 75.772.3 W/g Fe as the average NPsize increased. When heated using the same 47.7 kA/m, 194 kHzfield, the commercial fluidMAG particles (which have 8–9 nmmagnetite cores coated with polymer) were found to have SARvalues of 41–43 W/g Fe, which lies in between the SAR valuesobtained for TDMNP8 and TDMNP13 NPs.

Larger NPs displayed higher SAR values, regardless of the NPsource or composition. The experimental SAR values for the 16 nmmaghemite nanopowder dispersions were 249.174.7 W/g Fewhile the similarly-sized TDMNP18 dispersions had an SAR of325.9716.0 W/g Fe when both were subjected to magneticheating using a 38.2 kA/m, 430 kHz field. Although 13.5 nm ma-ghemite nanoparticles have been reported to have a somewhathigher SAR than 12.8 nm magnetite NPs [40], the 16 nm maghe-mite nanopowder did not heat as well as the 18 nm TDMNP18magnetite particles. Thus, the size disparity between theTDMNP18 and maghemite nanopowder dispersions is likely moreresponsible for differences in heat generation than the crystallinestructure of the iron oxide NPs.

Nanoparticle size is one of the primary factors investigated foroptimization of SAR. It is commonly observed that for larger sizedparticles (slightly greater than the transition range between par-ticles with single and multiple crystal domains), magnetic heatingis amplified by an increase in the ferromagnetic nature of theparticles as well as contributions of hysteresis losses, althoughthere is yet to be a consensus on particle size optimization. Thelarger particle heating is supported by Hergt et al., who de-termined that the contribution of hysteresis losses in larger par-ticle sizes dominate over heat generation due to Néel relaxation[21]. Ma et al. also tested magnetite NPs covering a wide range ofsizes (from 7.5 nm to 416 nm), but found a mid-range size (46 nm)to yield the highest SAR when heated at 32.5 kA/m and 80 kHz[15]. Li et al. [41] have demonstrated a similar size optimum inmagnetite NPs where they found that 24 nm particles had thehighest SAR values when subjected to magnetic fields of 9.6–23.9 kA/m at 100 kHz (when compared to particles ranging in sizefrom 8 nm to 103 nm) [41]. They attributed the heating of the24 nm particles to a combination of relaxation and hysteresislosses [41].

Nanoparticle size impacts the types of relaxation processes thatcan give rise to magnetic heating. For superparamagnetic particles(typically of smaller sizes), the combination of Néel and Brownianrelaxation cause heating to be optimized for a particular particlecomposition and size, with hysteresis losses dominating relaxationprocesses as particle sizes increase to become ferromagnetic [21,36]. Gonzales-Weimuller et al. used mathematical models to pre-dict SAR for magnetite NPs ranging up to 15 nm, with the optimalNPs size being 12.5 nm at field strength of 24.5 kA/m, and a fre-quency of 400 kHz [24]. According to the authors, reducing thepolydispersity of NPs is essential to obtain higher heating rates,and accurate size-SAR trends [24]. A more recent paper from thesame research group found that 16 nm monodispersed magnetiteNPs give optimal heating for a field of 14 kA/m at 373 kHz [42].

Although the largest particles tested in our experiments(18 nm) displayed the highest SAR (Table 2), there are limitationson the maximum size of NPs that can be administered to humans.For intravenous administration, particles with hydrodynamic dia-meters larger than 100 nm are cleared by phagocytic uptake andthe liver before a significant concentration can accumulate in atarget region for therapy [43]. In addition, larger particles aregenerally infeasible for in vivo applications because they tend tohave lower colloidal stability as surface coatings may not suffi-ciently prevent aggregation [44]. Smaller sized NPs (o10 nm) arealso quickly filtered out of the bloodstream by the kidneys [43],yielding a desirable range of particles sizes for intravenous ad-ministration of approximately 10–100 nm. This size corresponds tothe hydrodynamic diameter, so the particles investigated here arelikely candidates that would avoid first pass clearance and reachtargeted cells and tissues.

3.2.5. Effect of NP typeFor the seven different nanoparticles tested, the highest SARs

were found for the 18 nmmagnetite NPs synthesized in-house andthe maghemite nanopowder, with values from 250–325 W/g Fewhen tested at 430 kHz, the highest frequency achievable in ourexperimental set-up (Table 2). For a set field strength and fre-quency fluidMAG NPs and TDMNP8 NPs (both approximately8 nm) showed a difference in heat generation capabilities. At alltested field strengths and frequencies fluidMAG NPs have higherSAR than TDMNP 8 NPs (Table 2). The slightly larger sized 11 nmTDMNP11 NPs have lower SAR than fluidMAG NPs. fluidMAG Dand fluidMAG PAD NPs that had similar size and composition, butdifferent surface coatings produced similar SAR values. The largermaghemite and TDMNP18 NPs with a similar size range also havea difference in SAR values. This change observed in SAR values canbe a result of difference in composition, polydispersity, and mag-netic properties of these NPs.

3.2.6. Effect of solution viscosity on heatingThe effect of viscosity on SAR values was investigated by pre-

paring NP dispersions in different viscosity alginate solutions, andalso in cross-linked calcium alginate gel in its hydrated state(Fig. 6). For both fluidMAG D particles and maghemite nano-powder, SAR values decreased with higher solution viscositieseven when the NP concentration and magnetic field parameterswere kept constant. The cross-linked alginate solution providedthe most rigid environment surrounding the NPs and had thelowest SAR values. By maintaining the same NP concentration inaqueous and alginate solutions, SAR should only be impacted bychanges in free particle rotation or a change in solution heatcapacity (which was minimal due to the use of dilute alginatesolutions). The trend of decreasing SAR values with higher


viscosities points to a reduction in the Brownian relaxation com-ponent of heating as found by Chen et al. for iron oxide NPs in0.9 cP to 43.2 cP glycerol solutions [35]. Because blood and cellproteins attach rapidly to foreign objects such as magnetic NPthrough opsonization [45], particles that heat primarily throughBrownian relaxation are not likely to be candidates for MFHtherapy.

3.3. Rosensweig equation validation with experimental data

The theoretical relationship between SAR and magnetic fieldproperties was used to evaluate magnetic heating data [19]. Ro-sensweig's equation is valid for predicting magnetic heating aslong as the particles remain in the linear portion of hysteresis loopwhen the magnetic field is applied. Here, we investigated fluid-MAG D NPs, and at the high frequencies tested, we assume this tobe true. Since this equation (Eq. 2) shows SAR to be proportional tothe energy generation term, P, and P is proportional to H2, a plot ofSAR against the square of the field strength was fit to a linearequation (Fig. 7A). The correlation coefficient, R2, of the curve fitwas 0.96, confirming that our experimental data follow Ro-sensweig's theory.

As long as the applied frequency is below a threshold fre-quency, the Rosenweig equation predicts that P is proportional tothe square of the applied field frequency, which is confirmed byour experimental results (Fig. 7B). Kallumadil et al., have statedthat when the imaginary component of magnetic susceptibility inthe Rosensweig equation is frequency-independent, SAR is directlyproportional to applied frequency [19, 20]. As a few other researchgroups have shown through simulation and experiments [23, 27],utilizing higher frequencies can be a viable option for improvingthe heating efficiency of NPs for cancer treatment, again with thecaveat that the product magnetic field strength and frequency(Hnf) remains at a tolerable level for patients.

To evaluate the frequency dependence of our experimental SARvalues, the Rosensweig equation was rearranged to show a doubleinverse linear relationship between power, 1/P, and the square offrequency, 1/f2. From Eq. (2), π, μo, χo, H, and τ can be assumed tobe constant and independent of applied frequency. SubstitutingA¼ Ho o

2πμ χ and B¼2πτ , in Eq. (2) we obtain:

P ABf

Bf1 ( ) (3)

2

2=

+

Inverting Eq. (3) we get:

PB f

ABf1 1

(4)

2 2

2= +

which can then be rearranged to relate power and frequency as:

Fig. 6. Effect of viscosity on SAR values for fluidMAG D and maghemite nanopowder dispD]¼8.6 mg/mL, [Maghemite nanopowder]¼1.6 mg/mL). fluidMAG D was not tested at 4

P ABfBA

1 1(5)2

= +

Fitting our data to this equation, we obtain a linear graph witha R2 value of 0.97 (Fig. 7B), confirming that the results are inagreement with Rosensweig's theory.

4. Conclusions

A number of physical and chemical factors impact magneticheating of iron oxide NPs. Magnetic fields with intensities rangingfrom 15 to 48 kA/m and covering a range from 123 to 430 kHzwere investigated, along with seven different nanoparticle for-mulations representing a range of particle sizes (8–18 nm), coat-ings (oleic acid, dextran, polyacrylamide, or none), and composi-tions (Fe2O3 and Fe3O4). The NP concentration was not found toinfluence SAR in the concentration range studied. Thus, thermalpower generation can be maximized by localizing high con-centrations of NPs. For magnetite NPs synthesized in the range of8–18 nm, the largest particles displayed the highest SAR values.Comparing the different types of NPs it was seen that TDMNP18magnetite NPs and maghemite nanopowder had the highest SARvalues. Based on other reports in the literature, the optimal size forradio frequency heating of magnetite NPs is likely in the neigh-borhood of 16 nm [42]. Frequency tuning, whereby differentmagnetic field frequencies could be generated with the same fieldintensity, allowed testing of the same nanoparticle dispersionsover a range of frequencies. Operating at higher frequenciesachieved higher SAR values, as predicted by the Rosensweigequation. Even with the successful heating of magnetite and ma-ghemite at 430 kHz and 38.2 kA/m, patient tolerance of magneticfields will likely require NP optimization to achieve high SAR va-lues while staying below an H–f product of 4.85�108 A/m-s, asdescribed by Brezovich [39], or reaching high local NP con-centrations. Despite limitations due to patient tolerance, it hasbeen suggested that this criterion can be exceeded for treatingmore severe types of cancer when high heating efficiency is nee-ded [29]; there are also reports of commercial hyperthermiatreatment equipment which operate beyond this range [46].

The viscosity of the solution surrounding NPs has a deleteriouseffect on magnetic heating. The SAR values for fluidMAG D NPsand maghemite nanopowder decreased when the NPs were dis-persed in more viscous environments due to a reduction in theBrownian relaxation component of magnetic heating. The reducedheating in viscous solutions (which simulate the extracellularmatrix) has implications for the use of magnetic NPs for hy-perthermia therapy. SAR values were reduced to a fraction of theSARs observed in free solution (which is used by most researchersto evaluate magnetic hyperthermia). Evaluation of NP heating in

ersed in water and various alginate solutions (H¼38.2 kA/m, f¼430 kHz, [fluidMAG81 cP.

Fig. 7. Linear fit of SAR data to fit Rosensweig’s equation. SAR values frommagneticheating data for FluidMAG D experiments conducted at f¼194 kHz were plotted asa function of H2 in Fig. 7A. In Fig. 7B, heating data with variable frequency at a fixedH¼38.2 kA/m were plotted according to Eq. (5).


viscosities matching the desired application is therefore highlyrecommended.

Acknowledgments

The authors gratefully acknowledge the support of the NationalCancer Institute under NIH grant R21CA141388, and The Universityof Alabama through funds supporting graduate student tuition andstipends. They also thank Dr. Yuping Bao for use of dynamic lightscattering equipment in her lab. This work used instruments in theCentral Analytical facility, which is supported by The University ofAlabama.

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