Magnetic Hyperthermia Properties of Electrosynthesized Cobalt Ferrite

Article pubs.acs.org/JPCC

Magnetic Hyperthermia Properties of Electrosynthesized Cobalt Ferrite Nanoparticles Eva Mazario,† Nieves Menéndez,† Pilar Herrasti,*,† Magdalena Cañete,‡ Vincent Connord,§ and Julian Carrey§ †

Departamento de Química Física Aplicada, Facultad de Ciencias, Universidad Autónoma de Madrid, C/Francisco Tomás y Valiente, 7, Cantoblanco, 28049 Madrid, Spain ‡ Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, C/Darwin 2, Cantoblanco, 28049 Madrid, Spain § Université de Toulouse, INSA, UPS; Laboratoire de Physique et Chimie des Nano-Objects (LPCNO), 135, Avenue de Rangueil, and CNRS, UMR 5215, LPCNO, F-31077 Toulouse, France ABSTRACT: Using the electrochemical route, cobalt ferrite nanoparticles (NPs) with two different sizes were synthesized and stabilized in water by coating with citric acid. The specific absorption rate (SAR) values of aqueous suspensions of magnetic nanoparticles with crystal sizes of 13 and 28 nm were investigated in the frequency range 32−101 kHz and up to 51 mT. SAR values were higher for the larger NPs and reached 133 W/g. Numerical simulations are used for a quantitative analysis of hyperthermia experiments and seem to indicate that the larger NPs are multidomain. Cytotoxicity analysis was also performed in HeLa tumor cells; a null cytotoxicity of these nanoparticles in cell tissues were obtained.

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INTRODUCTION Hyperthermia, a modality of cancer treatment with elevated temperature, between 41 and 45 °C, and a treatment time of at least 30 min, has been paid considerable attention due to its clinical efficacies, such as minimizing clinical side effects and possibility to selectively destroy a localized or a deeply seated malignant cancer tumor. This method involves the introduction of ferromagnetic or superparamagnetic particles into the tumor tissue and then irradiation with an alternating magnetic field (AMF). The application of ferrofluids for hyperthermia treatment was investigated in the work of Chan et al.1 and Jordan et al.2 in 1993. These studies experimentally prove the high efficiency of the superparamagnetic crystal suspension to absorb the energy of an alternating magnetic field and convert it into heat. Up to the past decade, the research in materials for hyperthermia has focused on superparamagnetic iron oxide such Fe3O43,4 and γFe2O35,6 particles, which display a rather small saturation magnetization. However, pure metals (Fe, Co, or CoFe) possess larger saturation magnetization, relatively moderate anisotropy constant Keff, and large losses.7−9 However, they are highly toxic and extremely sensitive to oxidation so they need an appropriate surface treatment to be relevant for biomedical applications. An alternative to improve the magnetic characteristics of iron oxides is the substitution of the Fe2+ ions of the magnetite structure by ions of other metals such (M = Co, Ni, Mn).10−16 Among them, cobalt ferrite is a hard compound and possesses 90% saturation magnetization (80 Am2/kg) of magnetite but a magnetic anisotropy one order magnitude © XXXX American Chemical Society

larger. This large anisotropy is the origin of its large coercivity. Therefore, cobalt ferrite nanoparticles are expected to exhibit a larger hysteresis area as compared to the other spinel nanoparticles of the same size, even if a larger magnetic field should be required to saturate them.17 The synthesis of magnetic fluids consists in two steps: (1) the preparation of the magnetic nanosized particles (NPs) and (2) the stabilization/dispersion of the nanoparticles in a carrier liquid. In the first step, the nanoparticles can be produced by different methodologies such as coprecipitation, microemulsion, ball milling, sonochemical, sol−gel, thermal decomposition, or electrochemically.18−25 The synthesis of monodispersed single domain magnetic nanoparticles with hydrodynamic radius smaller than 200 nm and a narrow size distribution is essential for effective heating in biomedical applications. In the second step, the nanoparticles should be coated with some substance (shell) that can ensure their stability, biodegradability, and nontoxicity in physiological medium. These surfaces can be made with either organic or inorganic chemicals. Different organic materials have been used such as polymer composites like poly ethyleneglycol (PEG) or dextran to coat the surface of magnetic particles. Functional groups such as aldehyde, hydroxyl, aminosilane, and carboxylic acids Received: March 6, 2013 Revised: April 24, 2013

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dx.doi.org/10.1021/jp4023025 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

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can be also integrated in to the particle surface,26,27 and finally, magnetic nanoparticles can be coated with inert materials like gold, silver, or SiO2.28 The applicability of the ferrofluids to be used in clinical applications is an important task due to their potential toxicity.29,30 Iron and its oxides as NPs should be considered a part of iron physiology: they can be metabolized, stored, and transported through normal physiology by proteins including ferritin, transferrin, hemosiderin, and others, such that the metabolized iron is incorporated into iron pool. The experiment by Simonsen et al.31 explained the safety of iron nanoparticles. They administered 100 mg/kg of Fe to rats and observed no distinguishable side effects. Moreover, increasing the dosage to 600 mg/kg did not cause death. In an ordinary biomedical application, the injected dosages of NPs are substantially lower than these attempts.32 However, there are certain discrepancies in the bibliography about the use of magnetic ferrofluids containing other kinds of ions in the structure, as cobalt, nickel, or manganese.12,33,34 However, there is a criterion for specifying the upper limit for the magnetic field applied to the human body in order to avoid undesirable effects caused by eddy current loss; this criterion is described by the product of amplitude and frequency (H0 f). Although this limit has not yet been investigated extensively, its acceptable value has been reported to be 4.85 × 108 A/ms;35 a less rigid criterion, 5 × 109 A/ms, has also been proposed.36 Therefore, it is necessary to select an appropriate frequency and amplitude of the magnetic field to obtain an effective heating power that will not damage healthy tissues. Synthesis methods mentioned above usually produce nanoparticles with a big distribution of sizes. The main problem is the polydispersity of the nanoparticles obtained, with the request of subsequent treatments after the synthesis. In our laboratory, a new method has been developed, based on the oxidation of metal foils immersed in a solution of an amine surfactant to inhibit the agglomeration of the nanoparticles formed.24,25 In this study, we focus on the comparison between CoFe2O4 NPs of two crystal sizes, 13 nm labeled as sample 1 and 28 nm labeled as sample 2. AC magnetic-field-induced heating was measured under different applied frequencies and magnetic fields and study the heating dependence on both variables. As results, we have observed promising AC magneticfield-induced heating characteristics and biocompatible cytotoxicity of cobalt ferrite nanoparticles.

simple ultrasonic treatment, the suspension was dialyzed for 48 h, in order to remove the excess of mother surfactant and citric acid not physiadsorbed to the nanoparticle surface. Finally, the solution was filtered through a 0.2 μm syringe filter resulting in a final nanoparticle crystal size of 13 or 28 nm as synthesis temperature function. Colloidal properties of the samples were studied in a Zetasizer NanoTM, from Malvern Instruments. The hydrodynamic size of the particles in suspensions was measured by dynamic light scattering (DLS) and the zeta potential was measured as a function of pH at 25 °C, using 10−2 M KNO3 as background electrolyte and HNO3 and KOH to change the pH of the suspensions. Cell Culture. HeLa (A-549) cells were grown in Dulbecco’s modified Eagles’s medium (DMEM) with 50 units/mL penicillin, 50 μg/mL streptomycin, and supplemented with fetal bovine serum (FBS) at a 10% concentration. All media, serum, and antibiotics were provided by Gibco. Cell cultures were performed in a 5% CO2 atmosphere at 37 °C and maintained in a SteriCult 200 (Hucoa-Erloss) incubator. Cells were seeded on 24 multiwell dishes with or without coverslips. Viability of Hela cells was determined using (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay 24 h after incubation with CoFe2O4. The MTT assay is a colorimetric method for measuring the activity of mitochondrial enzymes. It reduces the yellow tetrazolium dye, MTT, to insoluble formazan, giving a purple color. The amount of formazan formed is proportional to the number of living cells. MTT was added to each well (100 μg/mL in medium) for 3 h at 37 °C. Then, the insoluble formazan formed inside the cells was dissolved adding DMSO to each well, and the optical density was measured at 540 nm in a microplate reader (Tecan Spectra Fluor spectrophotometer). Cell survival was expressed as the absorption percentage of treated cells compared to that of control cells (not incubated with NPs). Both the mean value and the standard deviation were obtained from at least six experiments. In order to analyze the internalization of NPs, HeLa cells grown on coverslips were incubated for 24 h with different CoFe2O4 concentrations (0.5, 0.1, and 0.05 mg NPs/mL). After 24 h incubation, cells were washed three times with phosphatebuffered saline (PBS) fixed in cold methanol (5 min) and stained with toluidine blue (TB, 0.05 mg/mL distilled water, 0.5 min), washed with distilled water, and air-dried. Preparations were mounted on DePeX (Serva) and observed under bright field microscopy. Microscopic observations and photographs were performed in an Olympus BX61 epifluorescence microscope, equipped with an Olympus DP50 digital camera Micromax; Princeton Instruments. Magnetic Hyperthermia Measurements. Magnetic characterization was performed in a Quantum Design PPMS XL-5 SQUID magnetometer, the magnetization curves were measured at room temperature and 5 K after applying a maximum magnetic field of 5 T. The saturation magnetization per unit mass (Ms) was evaluated by extrapolating to infinite field the experimental data obtained in the high field range where the magnetization linearly increases with 1/H. The magnetic hyperthermia (MH) properties have been characterized through the measurement of the temperature rise or by measuring high-frequency hysteresis loops. Temperature measurements were performed on a specially designed frequency-adjustable electromagnet.37 This setup is capable to reach magnetic field amplitudes up to 51 mT and in a range of

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EXPERIMENTAL PART Preparation of Magnetic Fluids. Cobalt ferrite nanoparticles were synthesized and characterized with a protocol published elsewhere.24,25 The synthesis temperatures were 25 and 60 °C, and the surfactant used was tetrabutyl ammonium bromide in order to obtain NPs with crystal sizes of 16 and 32 nm, respectively. Surfactant anions present at the particle surface seem to be responsible for the colloidal stability that provides a biocompatible character to the suspensions. However, the sample required further stabilization in water at pH 7 by adding citric acid. Then, 0.02 g/mL of citric acid solution were added to the nanoparticles, the pH was increased by means of KOH until pH 5.2, and finally this solution were heated and stirred during 1 h at 80 °C. The nanoparticles were separated by means of a magnet of 0.6 T and washed several times with distilled water to remove the excess of acid. After that the pH of the aqueous solution was increased until 7.2 with KOH and the colloidal suspensions were directly obtained by B



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In Figure 1, zeta potential measurements on the two types of NPs are shown. It can be clearly seen that no isoelectric point

frequencies between 32 and 101 kHz. The sample and the water reference were introduced into a calorimeter placed in the electromagnet. The measurement time was adjusted between 0 and 100 s. After the magnetic field stops, the sample and water are shaken during roughly 20 s to ensure the ampule thermalization and the homogeneity of the solutions temperature, which is checked by putting two probes at the top and the bottom of the calorimeter. The temperature rise is measured after this process from the mean slope of the ΔT/Δt function. The power generated by the NPs is evaluated by their specific absorption rate (SAR) that describes the energy amount converted into heat per time and mass. SAR values are calculated using eq 1:

SAR =

∑i Cpimi ΔT · mNPs Δt

Figure 1. Electrophoretic mobility as a function of pH for cobalt ferrite suspension coated with citric acid. Sample 1:13 nm and sample 2: 28 nm.

(1)

appears in the pH range measured, which is due to the high absorption of the citric acid around the nanoparticle surface that protects them from the protonation/deprotonation effect. The values of the superficial charge are negative for all the pH range in both cases, but it is evident that higher values are obtained for sample 2, indicative of a major stability. In addition, for this sample, the pH range where the NPs are above the classical stability limit of ±30 mV is very wide, between pH 4 and pH 12. So this ferrofluid is suitable to work above pH 4. However, the range of stability for sample 1 is reduced, so this sample is stable above pH 7. The mean hydrodynamic radius of the colloidal solution was 62 ± 8 nm for 13 nm nanoparticles and 184 ± 14 nm for 28 nm nanoparticles at physiological pH. Cytotoxicity. To ensure the possible use of CoFe2O4 in hyperthermia, we studied the biological effects of these nanoparticles on a standard human tumor cell line (HeLa) to determine their cytotoxicity and internalization. Figure 2 shows

where Cpi and mi are specific heat capacity and mass for each component (Cp = 0.670 J/kg·K for CoFe2O4 NPs, Cp = 4186 J/ kg·K for water, and Cp = 720 J/kg·K for the sample holder (glass)), and mNPs is the mass of the pure CoFe2O4 NPs. Since the losses dissipated during hyperthermia experiments are equal to the area of the hysteresis loop in the magnetization curves,17 an alternative method to access SAR is to measure the highfrequency loop of the colloidal solution. Magnetic hysteresis loops were measured in an alternating magnetic field μ0HAC generated by an air-cooled Litz wire coil. Inside this coil, a system comprising a calorimeter and two contrariwise-wounded compensated pick-up coils connected in series is mounted. The two pick-up coils have the same section surface Scoil and number of turns n. The amplitude of the alternating magnetic field is obtained using μ0HAC = (∫ e1dt)/(nScoil), where e1 is the voltage at the terminals of the empty coil. The magnetization per unit mass Ms of the NPs is obtained using Ms = (∫ e2dt)/ (μ0nρSvesselC) where Svessel is the surface of a the section of the vessel containing the colloidal solution, C is the volume concentration of the sample, ρ is the NPs density, and e2 is the voltage at the terminal of the two coils in series.38 The hysteresis loops were measured at f = 52 kHz for various values of μ0HAC varying between 0 and 85 mT. The SAR of the samples (expressed in W/g) are thus estimated by two methods: (1) from magnetic measurements, by integrating the hysteresis loops, released by the NPs during one cycle of the magnetic field, or (2) from temperature measurements, using eq 1. This kind of comparison is possible only if hysteresis loops are measured at the same frequency and amplitude range as the one used during temperature measurements.

Figure 2. HeLa cell viability, measured by MTT assay 24 h after incubation with different concentrations of NPs during 24 h. Data show the average of at least six experiments ± SD.

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RESULTS AND DISCUSSION Colloidal and Powder Characterization. Although the nanoparticles were synthesized by means of an amine surfactant, the product formed does not have the stability required for biomedical applications. For this reason, a new stabilization agent should be used to improve the colloidal stability. In this article, the citric acid was used to stabilize the NPs in aqueous medium at physiological pH. The colloidal stability is directly related to the zeta potential value, which reflects the surface charge density. It depends on the detailed oxide stoichiometry, degree of order at the particle surface, and adsorbed molecules.

the surviving fraction of HeLa cells incubated during 24 h at different concentrations of NPs and evaluated by the MTT assay 24 h after treatment. Treated cells showed a survival fraction similar to controls cells under all experimental conditions. The use of this dye, allowed clearly to observe the contour of cells and nuclei, easing the observation of the NPs location. When HeLa cells were incubated with CoFe2O4 during 24 h and stained by the toluidine blue method 24 h after (in identical conditions of MTT assay), accumulation of NPs was evident inside cells for all three concentrations used (Figure 3). The intracellular distribution pattern consisted of cytoplasmatic C



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× 105 J/m3 are found for samples 1 and 2, respectively. They are of the same order of magnitude than the expected value for the bulk CoFe2O4 (Keff ≈ 1.8 × 105 J/m3), which confirms the good quality of the NPs. Figure 5 displays SAR measurements performed on the two samples as a function of the amplitude of the magnetic field

Figure 3. HeLa cells incubated with CoFe2O4 24 h and stained with AT 24 h after treatments. (a) Control cells, (b) cells incubated with CoFe2O4 0.5 mg NPs/mL, (c) cells incubated with CoFe2O4 0.1 mg NPs/mL, (d) cells incubated with CoFe2O4 0.05 mg NPs/mL. Scale bar 20 μm.

spots always in extranuclear localization. The distribution pattern of NPs in HeLa cells stained with AT was identical to the one observed in not fixed and stained cells (data not shown). The accumulation of NPs seems to be proportional to concentration (Figure 3b−d). In Figure 3, it can also be observed that part of the nanoparticles accumulate in the intracellular space forming aggregates of different color. This is especially evident for 0.5 mg of NPs/mL concentration. In all cases, cell morphology remained similar to that of control cells (Figure 3a), confirming the absence of toxicity of the treatments. Magnetic Characterization and Hyperthermia Measurements. In Figure 4, the magnetization curves measured at

Figure 5. Dependence of applied magnetic field and frequencies on SAR: (a) sample 1 (13 nm) and (b) sample 2 (28 nm).

applied and for various frequencies. The SAR values are larger for the sample with the large NPs size (Figure 5b). In both cases, SAR follows a monotonic increase without saturation. In sample 2, the curve presents an inflection point around 35 mT. The experimental data (Figure 5) were fitted using an allometric function SAR ∝ (μ0HAC)n. The fit results are shown in Table 1 as a function of the frequency and the Table 1. n Exponent Calculated from the Allometric Adjustment in SAR vs H Curves at Different Frequencies and Sample Sizesa n (SAR vs Hn)

Figure 4. Magnetic hysteresis loop of CoFe2O4 powder at room temperature for samples 1 and 2 with crystal sizes of 13 and 28 nm, respectively. The inset shows the hysteresis loop of CoFe2O4 powder at 5 K.

300 and 5 K in SQUID magnetometer with a maximum applied field of 5 T are shown for the two samples. Both of them showed a ferromagnetic behavior, the main difference being their coercivity μ0HC and the Ms value. Ms = 55 Am2/kg and μ0HC = 27 mT for sample 1, whereas Ms = 63 Am2/kg and μ0HC = 60 mT for the other one at room temperature. In both cases, Ms is lower than the one of the bulk CoFe2O4 (80 Am2/ kg). This reduction is attributed to the existence of a dead surface layer for each particle in which magnetic moment do not contribute to the magnetization in the applied field. The higher coercive field and Ms obtained in sample 2 is coherent with the larger size of the NPs. From the low-temperature data, an estimation of the anisotropy value can be calculated using Keff ≈ ((μ0HCMs)/0.96), where Ms is the saturation magnetization (per unit volume). Keff ≈ 2.7 × 105 J/m3 and Keff ≈ 2.8

a

frequency (kHz)

sample 1

sample 2

32 52 72 88 101

1.61(5) 1.68(3) 1.80(3) 1.94(5) 1.97(3)

2.26(4) 2.32(6) 2.38(5) 2.37(3) 2.40(6)

Standard deviation is included between brackets.

nanoparticle size. The allometric adjustment was made between 0 and 51 mT for sample 1 and between 0 and 35 mT in the case of sample 2. It is clear that sample 2 presents higher exponents than sample 1. The effect of frequency on the exponents cannot be stated unambiguously, even if a slight increase might be observed. These SAR values of cobalt ferrite nanoparticles are similar or slightly higher than others report in the bibliography for similar sizes and hyperthermia experimental conditions.10,14,39−41 Figure 6 shows for the two samples the frequency dependence of the SAR at a fixed magnetic field of 51 mT. The SAR increases mostly linearly with frequency, as generally D



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there is no other simple analytical expression out of this domain of validity; (iii) NPs during hyperthermia form chains and columns of unknown dimensions; this phenomenon is expected to cancel or at least strongly reduce heating due to physical rotation. To illustrate this problem, if we assume that only heating due to physical rotation is present in our samples and use available formula from the linear response theory, we find for μ0HAC = 51 mT that SAR = 337 W/g and SAR = 3769 W/g for samples 1 and 2, respectively, far above the measured values. In the present analysis, we will thus neglect physical rotation and take into account the magnetization reversal only. In many cases, this has been shown to be sufficient to interpret hyperthermia experiments.7,38,45 In this framework, the linear response theory can be used only when ξ < 1, with ξ = (μ0MsHAC)/kBT.17 In our case, calculating ξ leads to 4.03 and 45.9 values for samples 1 and 2, respectively, when μ0HAC = 51 mT. Since this theory is not suitable in the present case, we used numerical simulations of hysteresis loops, which are able to calculate the SAR of magnetically independent randomly oriented single-domain nanoparticles without physical rotation.17 In the following, we always assume that the Néel relaxation time τ0 = 10−10 s. We first ran simulations in which we assume that the nanoparticles display the anisotropy of the bulk CoFe2O4. In this case, the calculated SAR are negligible for both sample (SAR ≪ 1 mW/g). This is easily explained by the fact that the coercive field of such large-anisotropy NPs would be much higher than the applied magnetic field. Then, we studied the reverse problem and looked on the anisotropy value that samples 1 and 2 should have to explain the experimental SAR value. We took as reference point the SAR value obtained for f = 101 kHz and μ0Hmax = 51 mT. We obtain Keff = 3.61 × 104 J/ m3 and Keff = 6.75 × 103 J/m3 for samples 1 and 2, respectively. In Figure 8, the calculated magnetic field dependence of the SAR is plotted along with experimental data. Given the crude

Figure 6. SAR of samples 1 and 2 obtained at the fixed applied field of 51 mT with the frequencies varying from 32 to 101 kHz.

observed experimentally when the applied magnetic field is not in the vicinity of the NP coercive field.9,42 In Figure 7a is the

Figure 7. (a) AC hysteresis loops measured at 52 kHz with a magnetic field varying between 1 and 85 mT of sample 2. (b) SAR calculated as the product of the integrated area of the hysteresis loops and with the slope of the temperature−time curves for sample 2.

AC hysteresis loops of sample 2 measured at f = 52 kHz for μ0HAC varying between 1 and 85 mT. The magnetization for μ0HAC = 70 mT is Ms = 20 Am2/kg, below the one extracted from quasistatic superconducting quantum interference device (SQUID) measurements (63 Am2/kg). The SAR can be calculated as the product of the integrated area of the hysteresis loop and frequency. These calculated values are represented in Figure 7b and compared with the SAR values deduced from temperature measurements in the same conditions. As expected, both methods are consistent and lead to approximately the same SAR values. Analysis of Hyperthermia Measurements and Numerical Simulations. To analyze quantitatively hyperthermia experiments, one must calculate the area of the MNP hysteresis loop, which results from both the physical rotation of the NPs inside the fluid and to the reversal of their magnetization.17,43 In the present state of the theories, it is very difficult to take into account correctly the physical rotation of the NPs for several different reasons: (i) the linear response theory for the physical rotation is only valid for very small applied magnetic field and its domain of validity is not precisely defined;44 (ii)

Figure 8. Comparison between numerical simulations and experimental SAR values. In numerical simulations, f = 101 kHz, τ0 = 10−10 s, and the density ρ = 5150 kg/m3. For sample 1, the radius r = 6.5 nm, Ms = 55 Am2/kg, and Keff = 36160 J/m3. For sample 2, r = 14 nm, Ms = 63 Am2/kg, and Keff = 6750 J/m3.

approximations of our model (no magnetic interactions, no size distribution, and arbitrary τ0), the data obtained for sample 1 might be acceptable. The obtained anisotropy value is well below the one of bulk CoFe2O4 but is physically reasonable. Its reduction compared to the low-temperature bulk value and to the value deduced from low temperature magnetic measurements could be explained by the strong temperature dependence of anisotropy expected in CoFe204,46 especially in nanoparticles.47 However, in the case of sample 2, the discrepancy between experiments and simulations is very E


The Journal of Physical Chemistry C important: for such a weak anisotropy, the sample should display experimentally a clear Stoner−Wohlfarth behavior,9 which is not observed. Moreover, the anisotropy value found is below the one of bulk magnetite and much below the one estimated for very soft amorphous FeCo nanoparticles.9 This is very unlikely that the NPs under study here could display such a weak anisotropy. Finally, it seems unlikely that the temperature dependence of the anisotropy would be stronger in larger nanoparticles. Another hypothesis to explain the SAR value obtained for sample 2 is the fact that they would be multidomain. To estimate the diameter Dtrans at which the single-domain/ multidomain transition should occur, we use the fact that this transition has been shown to occur around Dtrans ≈ 20 nm in iron nanoparticles7,48 and that the transition is expected to scale approximately with (Tc/Keff)1/2, where Tc is the Curie temperature of the material. It can be deduced from these considerations that Dtrans ≈ 1.35 × 10−7 (Tc/Keff)1/2. Using bulk CoFe204 parameters, we find that Dtrans ≈ 9 nm. Using the reduced anisotropy value found in analyzing data from sample 1 (Keff = 3.61 × 104 J/m3), we find Dtrans ≈ 20 nm. It is thus not unreasonable to interpret the hyperthermia results obtained on sample 2 by the fact that these NPs are multidomain. It is by the way interesting to note that, for this sample, the power exponents in Table 1 are not so far from 2 (i.e., the exponent displayed by superparamagnetic NPs in the linear response regime), whereas we show that the nature of the samples is probably very different. This illustrates that interpretation of hyperthermia experiments cannot be based on these exponents only and that careful quantitative analysis should be performed to understand underlying mechanisms.

ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the Spanish MICINN under projects MAT 2009-14741-C02-02, MAT 2012-37109-C02-02, CAM S2009MAT-176, and the mobility grant (EEBB-I-1203981) of the MINECO Spanish ministry.

(1) Chan, C. F. D.; Kirpotin, D. B.; Bunn, P. A. J. Synthesis and Evaluation of Colloidal Magnetic Iron Oxides for the Site-Specific Radiofrequency-Induced Hyperthermia of Cancer. J. Magn. Magn. Mater. 1993, 122, 374−378. (2) Jordan, A. Inductive Heating of Ferrimagnetic Particles and Magnetic Fluids: Physical Evaluation of Their Potential for Hyperthermia. Int. J. Hyperthermia 1993, 9, 51−68. (3) Vergés, M. A.; Costo, R.; Roca, A. G.; Marco, J. F.; Goya, G. F.; Serna, C. J.; Morales, M. P. Uniform and Water Stable Magnetite Nanoparticles with Diameters Around the Monodomain−Multidomain Limit. J. Phys. D: Appl. Phys. 2008, 41, 134003. (4) Suto, M.; Hirota, Y.; Mamiya, H.; Fujita, A.; Kasuya, R.; Tohji, K.; Jeyadevan, B. Heat Dissipation Mechanism of Magnetite Nanoparticles in Magnetic Fluid Hyperthermia. J. Magn. Magn. Mater. 2009, 321, 1493−1496. (5) De la Presa, P.; Luengo, Y.; Multigner, M.; Costo, R.; Morales, M. P.; Rivero, G.; Hernando, A. Study of Heating Efficiency as a Function of Concentration, Size, and Applied Field in γ-Fe2O3 Nanoparticles. J. Phys. Chem. C 2012, 116, 25602−25610. (6) Hergt, R.; Hiergeist, R.; Hilger, I.; Kaiser, W. A.; Lapatnikov, Y.; Margel, S.; Richter, U. Maghemite Nanoparticles with Very High ACLosses for Application in RF-Magnetic Hyperthermia. J. Magn. Magn. Mater. 2004, 270, 345−357. (7) Mehdaoui, B.; Meffre, A.; Carrey, J.; Lachaize, S.; Lacroix, L. M.; Gougeon, M.; Chaudret, B.; Respaud, M. Optimal Size of Nanoparticles for Magnetic Hyperthermia: A Combined Theoretical and Experimental Study. Adv. Funct. Mater. 2011, 21, 4573−4581. (8) Zeisberger, M.; Dutz, S.; Müller, R.; Hergt, R.; Matoussevitch, N.; Bönnemann, H. Metallic Cobalt Nanoparticles for Heating Applications. J. Magn. Magn. Mater. 2007, 311, 224−227. (9) Lacroix, L. M.; Malaki, R. B.; Carrey, J.; Lachaize, S.; Respaud, M.; Goya, G. F.; Chaudret, B. Magnetic Hyperthermia in SingleDomain Monodisperse FeCo Nanoparticles: Evidences for Stoner− Wohlfarth Behavior and Large Losses. J. Appl. Phys. 2009, 105, 023911−023914. (10) Lee, S. W.; Bae, S.; Takemura, Y.; Shim, I.-B.; Kim, T. M.; Kim, J.; Lee, H. J.; Zurn, S.; Kim, C. S. Self-Heating Characteristics of Cobalt Ferrite Nanoparticles for Hyperthermia Application. J. Magn. Magn. Mater. 2007, 310, 2868−2870. (11) Skumiel, A. Suitability of Water Based Magnetic Fluid With CoFe2O4 Particles in Hyperthermia. J. Magn. Magn. Mater. 2006, 307, 85−90. (12) Kashevsky, B. E.; Agabekov, V. E.; Kashevsky, S. B.; Kekalo, K. A.; Manina, E. Y.; Prokhorov, I. V.; Ulashchik, V. S. Study of Cobalt Ferrite Nanosuspensions for Low-Frequency Ferromagnetic Hyperthermia. Particuology 2008, 6, 322−333. (13) Kim, D. H.; Nikles, D. E.; Johnson, D. T.; Brazel, C. S. Heat Generation of Aqueously Dispersed CoFe2O4 Nanoparticles as Heating Agents for Magnetically Activated Drug Delivery and Hyperthermia. J. Magn. Magn. Mater. 2008, 320, 2390−2396. (14) Seongtae, B.; Sang Won, L.; Hirukawa, A.; Takemura, Y.; Youn Haeng, J.; Sang Geun, L. AC Magnetic-Field-Induced Heating and Physical Properties of Ferrite Nanoparticles for a Hyperthermia Agent in Medicine. IEEE Trans. Nanotechnol. 2009, 8, 86−94. (15) Pradhan, P.; Giri, J.; Samanta, G.; Sarma, H. D.; Mishra, K. P.; Bellare, J.; Banerjee, R.; Bahadur, D. Comparative Evaluation of Heating Ability and Biocompatibility of Different Ferrite-Based Magnetic Fluids for Hyperthermia Application. J. Biomed. Mater. Res. B 2007, 81B, 12−22.

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CONCLUSIONS These NPs synthesized by electrochemical method are an ideal material that allow the systematic study of the influence of the size, magnetic field amplitude, and frequency on the particle heating efficiency. For NPs with crystal size of 28 nm, a maximum SAR value of 133 W/g was obtained at 101 kHz and with an amplitude of magnetic field of 51 mT. Numerical simulations are used for a quantitative analysis of hyperthermia experiments and seem to indicate that the larger NPs are multidomain. The H0 f product for the maximum temperature rise is below the biological and physiological safety range in biomedical applications (H0 f < 5 × 109 A/ms), which indicates that CoFe2O4 nanoparticles are suitable for hyperthermia application. In addition, cytotoxicity measurements of CoFe2O4 NPs in HeLa cells corroborated an easy penetration of these NPs into cells under our experimental conditions, as well as an absence of effects on cell survival. The results confirm the biocompatibility of both cobalt ferrite NPs at the applied doses, which were much higher than the currently approved dose (0.56 mg/kg) in humans.49

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AUTHOR INFORMATION

Corresponding Author

*(P.H.) E-mail: [email protected]. Tel: 0034914976496. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. F



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Magnetic Hyperthermia Properties of Electrosynthesized Cobalt Ferrite

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