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Biomedical tools based on magnetic nanoparticles

Anna R. Sabaa,b, Paula M. Castilloa, Elvira Fantechic, Claudio Sangregoriod, Alessandro Lascialfarie, Andrea Sbarbatib, Alberto Casuf, Andrea Falquia,f, and Maria F. Casulaa,*

a INSTM and Dipartimento di Scienze Chimiche e Geologiche, Università di Cagliari, I-09042 Monserrato (Ca), ITALY

b INSTM and Dipartimento di Scienze Neurologiche, Neuropsicologiche, Morfologiche e Motorie , Università di Verona, I-37134 Verona, ITALY

c INSTM and Department of Chemistry ‘U. Schiff’, Università di Firenze, I-50019 Sesto Fiorentino (Fi) ITALY

d CNR–ISTM Milano and INSTM via C. Golgi 19, I-20133 Milano, ITALY e CNR and Department of Physics, Università di Milano, I-20134 Milano, ITALY

f Nanochemistry, Istituto Italiano di Tecnologia, I.I.T. Via Morego 30, 16163 Genova, ITALY

ABSTRACT

Magnetic and superparamagnetic colloids represent a versatile platform for the design of functional nanostructures which may act as effective tools for biomedicine, being active in cancer therapy, tissue imaging and magnetic separation. The structural, morphological and hence magnetic features of the magnetic nanoparticles must be tuned for optimal perfomance in a given application. In this work, iron oxide nanocrystals have been prepared as prospective heat mediators in magnetic fluid hyperthermia therapy. A procedure based on the partial oxidation of iron (II) precursors in water based media has been adopted and the synthesis outcome has been investigated by X-Ray diffraction and Transmission electron microscopy. It was found that by adjusting the synthetic parameters (mainly the oxidation rate) magnetic iron oxide nanocrystals with cubic and cuboctahedral shape and average size 50 nm were obtained. The nanocrystals were tested as hyperthermic mediators through Specific Absorption Rate (SAR) measurements. The samples act as heat mediators, being able to increase the temperature from physiological temperature to the temperatures used for magnetic hyperthermia by short exposure to an alternative magnetic field and exhibit a reproducible temperature kinetic behavior.

Keywords: iron oxide, hyperthermia, ferrofluids

* [email protected]; phone | +39 070 675-4360; fax | +39 070 675-4388

Invited Paper

Colloidal Nanocrystals for Biomedical Applications VIII, edited by Wolfgang J. Parak, Marek Osinski, Kenji Yamamoto, Proc. of SPIE Vol. 8595, 85950Z · © 2013 SPIE

CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2003180

Proc. of SPIE Vol. 8595 85950Z-1

Downloaded From: http://proceedings.spiedigitallibrary.org/ on 10/22/2013 Terms of Use: http://spiedl.org/terms

1. INTRODUCTION

Magnetic nanoparticles are regarded as versatile tools for biomedicine due to their impact in diagnostics and therapeutics both for in vivo (Magnetic Resonance Imaging, drug-delivery, magnetic hyperthermia) and in vitro (magnetic separation, magnetic biosensing) use. [1-3] Among magnetic nanoparticles, iron oxides play a key role when in vivo applications are concerned, as their medical use has been already approved both the US Food and Drug Administration and the European Medicines Agency. In particular, at present superparamagnetic iron oxide nanoparticles (often referred to as SPIONS) coated by a suitable stabilizer and dispersed in water are being used in clinical trials in magnetic resonance imaging (MRI), as they have been demonstrated to act as effective contrast agents. [4-6]Recent studies, however, have pointed out that for other in vivo applications such as magnetic fluid hyperthermia based therapy, iron oxide nanoparticles with different morphology and magnetic properties may be desirable. Magnetic hyperthermia is based on the selective heating (up to 37-45°C) of the diseased tissue generated by magnetic nanoparticles exposed to an alternating magnetic field. [1] The heating efficiency of the nanoparticles depends both on the intrinsic features of the nanoparticles (composition, crystal structure, size, and shape) and on the experimental procedure (strength of the alternating field, temperature detection setup, heat insulation) and is usually estimated by the Specific Absorption Rate (SAR). Very high SAR values have been recently reported for magnetosomes, i.e. iron oxide cubes and cubooctahedra with size in the range 40-100 nm which are produced by Magnetotactic bacteria. [7,8] A very high activity as heat mediators has been observed for the extracted magnetosomes both as the original iron oxide nanocrystal chains coated by the phopsholipidic membrane and as individual magnetosomes. A limit of this procedure is that the magnetic particles require inactivation prior to use due to their bacterial derivation, and that nanocrystals with different features (hence magnetic behavior) are obtained depending on the used bacteria, culture media, and extraction procedure.

In this work, we address the synthesis of iron oxide nanocrystals with structural and morphological features with close resemblance to magnetotactic crystals. We make use of a water-based partial oxidation procedure to obtain large amounts of well-faceted iron oxide nanocrystals with nearly cubic morphology. The synthesis was optimized based on X-Ray diffraction and Transmission Electron microscopy investigation and preliminary investigation on the ability of the nanoparticles to act as heat mediators for Magnetic fluid hyperthermia therapy was evaluated by specific absoprtion rate measurements.

2. METHODOLOGY

2.1 Materials Synthesis

Iron oxide samples were prepared according to synthetic protocols based on the partial oxidation of ferrous salts. The reaction is accompanied by a color variation, as the reaction mixture suddenly turns to black as soon as the base is added.Iron (II) sulfate eptahydrate (FeSO4•7H2O, > 99 %, Sigma Aldrich), Iron (II) chloride tetrahydrate (FeCl2•4H2O, > 99 %, Sigma Aldrich), Potassium nitrate (KNO3, plant cell culture tested, Sigma Aldrich, Potassium hydroxide (KOH, BioXtra, ≥85%, Sigma Aldrich) were used without further purification.Sample PO_SULF_1 was prepared according to the protocol proposed by Sugimoto et al [9] based on the partial oxidation of iron (II) sulfate. In particular, 17.7 g of FeSO4•7H2O are first dissolved in 200 mL of bi-distilled water; then a solution prepared by dissolving 10.11g KNO3 dissolved in 100 mL of bi-distilled water is added under vigorous stirring. Finally, a solution obtained by dissolving 13.81g of KOH in 50 mL of bi-distilled water is added to the mixture and kept under nitrogen at 90°C under reflux for two hours under vigorous stirring. The mixture is cooled down to room temperature (within about 1 hour) and the black precipitate is collected by magnetic separation and washed first with a



2L of a 2M solution of nitric acid and then with water until the pH of the supernatant was recorded to be 7. A suspension having a volume of 1L and a concentration of 4.5 mg/mL was obtained. The particles, however, tend to sedimentate within minutes.Sample PO_SULF_2 has been obtained by varying the procedure adopted for sample PO_SULF_1 as follows: a solution prepared by dissolving 30,02 g of FeSO4•7H2O, 2,89 g of KNO3, and 23,56 g of KOH in 300 mL of bidistilled water was heated under nitrogen under reflux at 80°C for 1 hour. [10 ] Once the mixture is cooled to room temperature, the black precipitate is collected by magnetic separation and washed repeatedly with water until neutral pH. A suspension having a volume of 300 mL and a concentration of 12 mg/mL was obtained. The particles, however, tend to separate from the suspension within minutes.Sample PO_SULF_3 has been obtained by preparing 100 mL of an aqueous solution containing 0.83 of FeSO4•7H2O, 0.56 g KOH, 4 g KNO3 which was kept under vigorous stirring for 5 minutes in nitrogen atmosphere and heated under reflux at 90°C for 5 minutes. The reaction flask is then sealed and kept in a pre-heated furnace at 90°C for 4 hours and manually shaken every 30 minutes.[11] Once the mixture is cooled to room temperature, the black precipitate is collected by magnetic separation and washed repeatedly with water until neutral pH. A suspension having a volume of 50 mL and a concentration of 5 mg/mL was obtained. The particles, however, tend to separate from the suspension within minutes.Sample PO_CHL_1 has been obtained by adopting the same procedure used to obtain PO_SULF_2, but in this case the iron(II) chloride was used instead of the sulfate. In particular, a solution prepared by dissolving 20 g of FeCl2•4H2O in 280 mL of bi-distilled water was heated to 90°C in nitrogen atmosphere under reflux under vigorous stirring. 120 mL of a water-based solution containing 3,23 g KNO3 and 22,45 g KOH was then added and the mixture is kept at 90°C for additional 40 minutes.[12] The mixture is aged at room temperature overnight and finally is washed through magnetic collection and washing with bi-distilled water until neutral pH is achieved. The precipitate is redispersed in 50 mL of water so that a concentration of 150 mg/mL is obtained. The suspension is stable for roughly 1 hour.Sample PO_CHL_2 has been obtained by a slight modification of the recipe adopted for sample PO_CHL_1. In this case, once the oxidizing solution is added at 90°C to the ferrous solution, it is kept under stirring only for 5 minutes, after which the mixture is rapidly quenched in water bath. The black precipitate is collected by magnetic separation and thoroughly washed with water. Finally, the powder was suspended in 250 mL of water so that a concentration of 30 mg/mL is obtained. The suspension is stable for roughly 4 hours.

2.2 Materials Characterization

The crystal structure of the prepared materials was studied by X-ray diffraction (XRD) recorded in Bragg-Brentano geometry on a Panalytical Empyrean diffractometer equipped with Cu Kα radiation, a graphite monochromator on the diffracted beam and an X'Celerator linear detector. The samples were deposited by dropping the iron oxide suspension on a low-background silicon sample holder and by drying the sample under ambient conditions. The sample stage was spinned during data collection in order to average out potential contributions from preferential orientation effects. Phase identification was performed according to the Powder Diffraction File database and the average size of the nanocrystals was obtained by line profile analysis according to the Scherrer law [13 ]. Conventional Transmission Electron Microscopy (TEM) images were recorded on a HITACHI H-7000 microscope equipped with a thermionic W electron source operating at 125 KV and equipped with a 4 Mp ATM CCD camera from Deben Technologies. Prior to observations, the iron oxide suspension was sonicated and few drops were deposited on a carbon-coated copper grid and dried at ambient conditions.Nanoparticle suspension densities were determined by measuring the mass of the sample after drying 1 mL of each ferrofluid at 80 °C overnight. Inductively coupled plasma (ICP) atomic emission spectroscopy analyses were performed with a Varian Liberty 200 spectrophotometer to determine the Fe content. After sonication, 1 µL of the suspension was dissolved in concentrated nitric acid (5 mL) and the solution was diluted to 100 mL with bi-distilled water.The ability of the materials to act as mediators in magnetic fluid hyperthermia was tested by performing calorimetric measurements through a Fives Celes apparatus operating at an alternating field (AC) of 183 kHz. The field amplitude was evaluated with a AMF Life Systems ® high frequency magnetic field probe and was found equal to 17 kA ⋅m-1. Prior



to the experiments the suspension temperature was stabilized for at least 30 minutes at 37°C by thermal coupling with an ethylene glycol flow. [14] The Specific Absorption Rate (SAR), that is the adsorbed power per mass unit, was determined for all samples by collecting the temperature curves while the suspensions of given concentration were exposed to the AC magnetic field according to the following relationship:

dt

dTmc

mSAR

iii

metal

= ∑1

where i denotes every species involved in the heat exchange, ci is the specific heat, mi the mass and mmetal the total mass of metal. The dT/dt value was extrapolated by taking the initial slope of the temperature increase obtained from the linear term of a polynomial fit of the whole curve. The temperature kinetic curves were collected three times for each sample in order to determine the reproducibility of the measurements.

3. RESULTS AND DISCUSSION

X-Ray diffraction was used to gain information on the structure of the as-prepared materials. XRD patterns of all the samples are reported in Figure 1.a and show the occurrence of well-defined peaks, suggesting that the materials are highly crystalline. The XRD patterns of all the samples are quite similar and show typical reflections of the spinel structure associated to the magnetic iron oxides (magnetite Fe3O4 and maghemite γ-Fe2O3).The mixed valence (magnetite) or to the ferric (maghemite) spinel iron oxides have a slightly different cell parameter and are difficult to distinguish in their nanocrystalline by XRD.[15, 16] The black color of the precipitate, however, suggests the formation of magnetite.

10 20 30 40 50 60 70 80

Inte

nsity

(a

.u.)

2 ϑ

PO_SULF_1

PO_SULF_2

PO_SULF_3

PO_CHL_1

PO_CHL_2

(a)

32 34 36 38

2 ϑ

<d> 33 nm

<d> 42 nm

<d> 54 nm

<d> 38 nm

<d> 98 nm

(b)

Figure 1. XRD patterns of the iron oxide samples obtained by a partial oxidation procedure using as iron (II) source either sulfate or chloride (a) and detail highlighting peak broadening of the main reflection of the spinel structure indicating differences in the average crystallite domain (b).

The peak broadening is different for the samples prepared according to different protocols, as pointed out by Figure 1.b which reports a detail of the XRD pattern related to the main peak (311 planes) together with the values of the average crystallite size as determined by the Scherer equation. In particular, samples obtained using the chloride salt as iron (II)



source exhibit broader diffraction peaks with respect to the samples obtained by sulfates, suggesting smaller average crystal size. Moreover, the average crystal size was found to decrease significantly from sample PO_SULF_1 to PO_SULF_3 and slightly from PO_CHL_1 to PO_CHL_2.The morphology of the prepared materials was investigated by TEM. Figure 2 shows representative TEM images collected under bright field mode of the iron oxide materials obtained by partial oxidation of iron (II) sulfate. All the materials are made out of faceted particles with well-defined morphology whose electron diffraction (data not shown) matches with the iron oxide spinel structure, in agreement with XRD data.Among these materials PO_SULF_1 shows larger particles, which are polydisperse in shape and in size (particle size ranges from 50 up to 600 nm). A better control in particle size and morphology has been achieved in PO_SULF_2, which has been obtained by a protocol which makes use of milder conditions with respect to PO_SULF_1: in particular the shorter crystal growth time and the lower reaction temperature, as well as the smaller amount of oxidizer favor the formation of less polydisperse particles (from 20 to 200 nm) with smaller average size. Finally, PO_SULF_3 shows a good homogeneity in particle size and morphology. In particular, the sample is mainly made out of cubes as cuboooctahedra, which are the desired product, with size ranging from 20 to 100 nm. This result can be ascribed to the combined fast oxidative hydrolysis of iron sulphate in alkaline media used for the PO_SULF_3 sample compared to samples PO_SULF_1 and 2, where substoichiometric amounts of oxidizer and stronger reaction conditions were adopted.

Figure 2. TEM images of the iron oxide samples obtained by a partial oxidation procedure using as iron (II) source the sulfate salt: PO_SULF_1 (a); PO_SULF_2 (b); and PO_SULF_3 (c).

Figure 3 shows TEM images of the materials obtained by partial oxidation of iron (II) chloride. Sample PO_CHL_1 shows a very broad distribution of particle size (in the range 50 to 300 nm) and of particle shape, with less defined morphology with respect to the PO_SULF materials. On the other hand, PO_CHL_2 shows faceted particles with well-defined shape and narrower size distribution (in the range 30 to 200 nm). This result indicates that by reducing the growth time a higher control on the quality of the iron oxide particles can be achieved.



42 -

Û 40-

38 -

(a)

0 100

40

39

ÛF

38

37

(b)

40 -

38 -

37 -

(c)

U

00 00 400

t (s)200

(s)

400 200

t (s)

400

Figure 3. TEM images of the iron oxide samples obtained by a partial oxidation procedure using as iron (II) source the chloride salt: PO_CHL_1 (a); and PO_CHL_2 (b).

The effect of particle morphology and size on their hyperthermic behavior was investigated by collecting the temperature kinetic curves upon exposure of the material to the alternating field, as detailed in the Methodology. In Figure 4 are reported the curves for the PO_SULF iron oxide particles: it is observed that all samples after the thermostatation step (≅30 minutes at 37°C) a temperature increase within few minutes occurs up to around 40°C. The measurement was repeated 3 times for each sample, observing a high reproducibility of the kinetic curves. The average SAR value calculated was 7.0±0.8W/g for PO_SULF_1, 33.8 ± 1.7W/g for PO_SULF_2, and 65.4 ± 7.6W/g for PO_SULF_3.

Figure 4. Temperature kinetic curves of the iron oxide samples PO_SULF_1 (a); PO_SULF_2 (b); and PO_SULF_3 (b) obtained by a partial oxidation procedure using as iron (II) source the sulfate salt when exposed to an AC field at 17 KA/m, 183 KHz.

The temperature kinetic curves for sample PO_CHL_1 and PO_CHL_2 were also collected and are reported in Figure 5. Also in this case in few minutes a significant temperature increase was observed (up to around 45 °C). Sequential measurements show a high reproducibility of the kinetic curves, from which an average SAR value of 3.48±0.02W/g and of 17.1±0.8W/g was determined for the PO_CHL_1 and PO_CHL_2 samples, respectively.



ÛF

60

56

52

44

40

36

46

44

42

ÛI- 40

100 200

t (S)

300 400

38

36

266

t (s)

466

Figure 5. Temperature kinetic curves of the iron oxide sample PO_CHL_1 (a); and PO_CHL_2 (b), obtained by a partial oxidation procedure using as iron (II) source the chloride salt when exposed to an AC field at 17 KA/m, 183 KHz.

Although the obtained SAR values may appear low, if compared to what can be found in the literature, actually they do not preclude to a possible applications as heat mediators in magnetic fluid hyperthermia, Indeed, it should be pointed out that the hyperthermic behavior characterization which is reported in the literature is performed by a variety of different experimental setups, and the direct comparison among the observed behaviors is hampered by the lack of a clear definition of a protocol for SAR evaluation, which actually requires the control of many environmental parameters. For example, experiments are performed with a large variety of different experimental setups, which differ in alternate magnetic field and amplitude. In particular, by increasing the strength and frequency of the magnetic field, a higher hyperthermal effect is recorded, but, there is an upper limit (called physiological limit) beyond which deleterious response of the living tissues are observed. A reasonable estimation of this limit is represented by the product H∙ν<4.85∙108 A m-1s-1 (or 5∙109 if only small portions and not the whole body are exposed). [17] Unfortunately, the physiological limit is often overcome in the experimental studies reported in the literature. In this work, however, we have investigated the response of the materials under external magnetic field amplitude and frequency compatibles with the in vivo applications. The limited temperature increase observed can then be related to the moderate amplitude of the magnetic field adopted. In addition, the whole system is thermostatized at 37°C throughout all the experiment, so as to better simulate the cellular environment within a living being, while the most of measurements are performed using adiabatic set-ups. In such a non-adiabatic system, the sample is subjected to heat dissipation by interaction with the surroundings and consequently the temperature increase is limited.

4. CONCLUSIONS

Iron oxide magnetic nanoparticles are a versatile tool for biomedical use since its properties can be tuned for targeted applications. In this work, we made use of a water-based procedure based on the controlled oxidation of ferrous salts in order to obtain iron oxide magnetic particles for prospective use in alternative magnetic field cancer therapy.The procedure was effective in producing large amounts of magnetic iron oxide with high crystallinity without any further heat treatment, as shown by XRD and TEM investigation. It was found that the size and morphology of the iron oxide nanoparticles is strongly affected by the synthetic parameters which affect the rate of partial oxidation and nanoparticle growth. In particular, when chloride precursors are used as iron (II) source stronger conditions are required with respect to the sulfate precursor. The best synthetic control was achieved by using sulfates as precursors and by



adopting fast oxidation rates followed by rapid quenching and finally digestion of the particles (sample PO_SULF_3): cubic and cubooctahedra magnetic iron oxide with average size around 50 nm were obtained by this route.The samples act as heat mediators, being able to increase the temperature from physiological temperature to the temperatures used for magnetic hyperthermia under an alternative magnetic field and exhibiting a reproducible temperature kinetic behavior. The heating efficiency of the materials could be tuned over one order of magnitude by changing the morphology of the iron oxide nanocrystals. The highest SAR value obtained for PO_SULF_3 was of 65 W/g, which is fairly high taking into account the features of the experimental setup.

ACKNOWLEDGEMENTS

The "Associazione Italiana per la Ricerca sul Cancro" is gratefully acknowledged for financial support through the IG 11993/ AIRC Grant. P.M.C. thanks the Junta de Andalucia through the "Fundación Progreso y Salud" for a mobility fellowship. Additional financial support from the Italian MIUR through project “FIRB RBAP114AMK Riname” is also acknowledged. Dr. D. Meloni is gratefully acknowledged for ICP analyses.

REFERENCES

[1] Colombo, M, Carregal-Romero, S., Casula, M.F., Gutiérrez, L., Morales, M.P., Böhm, I.B.J., Heverhagen, J.T., Prosperi, D., Parak, W. J. "Biological Applications of Magnetic Nanoparticles" Chem. Soc. Rev. 41, 4306-4334 (2012). [2] Roca, A.G., Costo, R. A., Rebolledo, F., Veintemillas-Verdaguer, S., Tartaj, P. , González-Carreno, T., Morales, M.P., Serna, C.J. "Progress in the preparation of magnetic nanoparticles for applications in biomedicine" J. Phys. D: Appl. Phys. 42, article n. 224002 (2009)[3] Laurent, S., Forge, D., Port, M., Roch, A., Robic, C., Vander Elst, L., Muller, R. N. "Magnetic iron oxide nanoparticles: Synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications" Chem. Rev. 108, 2064-2110 (2008)[4] Jun, Y-w., Huh, Y-M., Choi, J-s, Lee, J-H, Song, H-T., Kim, S., Yoon, S., Kim, K-S., Shin, J-S., Suh, J-S., Cheon, J. "Nanoscale Size Effect of Magnetic Nanocrystals and Their Utilization for Cancer Diagnosis via Magnetic Resonance Imaging" J. Am. Chem. Soc. 127, 5732-5733 (2005)[5] Casula, M.F., Corrias, A., Arosio, P., Lascialfari, A., Sen, T., Floris, P., Bruce, I.J. "Design of Water-based Ferrofluids as contrast Agents for Magnetic Resonance Imaging" J. Coll. Int. Sci. 357, 50-55 (2011). [6] Casula, M.F., Floris, P., Innocenti, C., Lascialfari, A., Marinone, M., Corti, M., Sperling, R., Parak, W.J., Sangregorio, C. "Magnetic Resonance Imaging Contrast Agents based on Iron Oxide Superparamagnetic Ferrofluids" Chem. Mater. 22, 1739-1748 (2010).[7] Alphandery, E., Faure, S., Sekses, O., Guyot, F., Chebbi, I., "Chains of Magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy" ACS Nano 5, 6279-6296 (2011).[8] Devouard, B., Posfai, M., Hua, X., Bazylinski, D.A., Frankel, R.B., Buseck, P.R. "Magnetite from magnetotactic bacteria: size distributions and twinning" American Mineralogist 83, 1387-1398 (1998)[9] Bruce, I.J. Taylor, J., Todd, M., Davies, M.J., Borioni, E., Sangregorio, C., Sen, T. "Synthesis, characterization and application of silica-magnetite nanocomposites", J. Mag. Mag. Mater. 284, 145-160 (2004). [10] Nyirő-Kósa, I., Csákberényi Nagy, D., Pósfai, M. "Size and shape control of precipitated magnetite nanoparticles", Eur. J. Mineral. 21, 293-302 (2009)[11] Arakaki, A., Masuda, F., Amemiya, Y., Tanaka, T., Matsunaga, T. "Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria" J. Coll. Int. Sci. 343, 65-70 (2010)



[12] Schwertmann, U., Cornell, R. M., [Iron Oxides in the Laboratory. Preparation and Characterization. Second, Completely Revised and Extended Edition], WILEY-VCH, (2000) [13] Klug, H.P., Alexander, L.E., [X-ray Diffraction Procedures], Wiley, New York, (1974)[14] E. Fantechi, PhD Thesis, university of Florence (2013)[15] PDF Card 25-1042 [γ-Fe2O3] and 19-629 [Fe3O4]; PDF-2 File, ICDD International Centre for Diffraction Data, 1601 Park Lane, Swarthmore, USA.[16] Corrias, A., Mountjoy, G., Loche, D., Puntes, V., Falqui, A., Zanella, M., Parak, W.J., Casula, M.F. "Identifying spinel phases in nearly monodisperse iron oxide colloidal nanocrystals" J. Phys. Chem. C 113, 18667-18675 (2009)[17] Hergt, R., Dutz, S. "Magnetic particle hyperthermia—biophysical limitations of a visionary tumour therapy" J. Magn. Magn. Mater. 311, 187-192 (2007)



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