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Optimal Parameters for Hyperthermia Treatment Using Biomineralized Magnetite Nanoparticles: A Theoretical and Experimental Approach Alicia Muela, David Muñoz, Rosa Martín-Rodríguez, Iñaki Orue, Eneko Garaio, Ana Abad Díaz de Cerio, Javier Alonso, Jose Angel Garcia, and Mª Luisa Fdez-Gubieda J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b07321 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016

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The Journal of Physical Chemistry

Optimal Parameters for Hyperthermia Treatment Using Biomineralized Magnetite Nanoparticles: a Theoretical and Experimental Approach Alicia Muela1,5, David Muñoz1, Rosa Martín-Rodríguez2, Iñaki Orue4, Eneko Garaio2, Ana Abad Díaz de Cerio1, Javier Alonso5, José Ángel García3,5, Mª Luisa Fdez-Gubieda2,5,* Depto. de Inmunología, Microbiología, y Parasitología1, Depto. de Electricidad y Electrónica2, Física Aplicada II3, Universidad del País Vasco (UPV/EHU), Leioa, Spain SGIker Medidas Magnéticas, Universidad del País Vasco (UPV/EHU), Leioa, Spain4 BCMaterials, Edificio No. 500, Parque Tecnológico de Zamudio, Derio, Spain5

*corresponding Author: [email protected] (+34 946012553)

ABSTRACT We hereby present an experimental and theoretical insight on the use of biomineralized magnetite nanoparticles, called magnetosomes, as heat nano-inductors in magnetic hyperthermia technique. The heating efficiency or Specific Absorption Rate of magnetosomes extracted from Magnetospirillum gryphiswaldense bacteria and immersed in water and agarose gel, was directly

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determined from the hysteresis loops obtained at different frequencies and magnetic field amplitudes. We demonstrate that heat production of magnetosomes can be predicted in the framework of the Stoner-Wolhfarth theory of uniaxial magnetic domains subjected to significant dipolar interactions which can be described in terms of an interaction anisotropy superimposed to that of each particle. Based on these findings, we propose optimal magnetic field amplitude and frequency values in order to maximize the heat production while keeping the undesired eddy current effects below safe and tolerable limits. The efficiency of magnetosomes as heat generators and their impact on cell viability has been checked in macrophage cells. Our results clearly indicate that the hyperthermia treatment causes both cell death and inhibition of cell proliferation. Specifically, only a 36% of the treated macrophages remained alive 2 hours after alternating magnetic field exposure and 24 hour later the percentage fell down to 22%.

INTRODUCTION Magnetic hyperthermia combines alternating magnetic field (AMF) and magnetic nanoparticles as a heating source for cancer cells treatment. Specifically, in hyperthermia therapy the magnetic nanoparticles are injected into the tumor cells and an AMF with frequency f and amplitude H is applied. Under the action of the AMF, the magnetic moment of nanoparticles describes a hysteresis loop, whose area A is proportional to the dissipated energy that increases the temperature of the tumor. By reaching temperatures around 40-45ºC in the tumor area, the cancer cells can be “deactivated” (dead or driven to apoptosis) without affecting the healthy ones1,2. The use of magnetic particles as hyperthermia mediators was first proposed in the 1950s3. However, the first phase I clinical trials were performed in the early 2000s on patients having prostate carcinoma4


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and multiform glioblastoma5, showing that hyperthermia using magnetic nanoparticles was feasible, with the deposited nanoparticles being stable for several weeks, making sequential hyperthermia treatments possible. Magnetic hyperthermia treatment proceeded towards a phase II clinical trial as adjuvant therapy with conventional radiotherapy6, and it is authorized for cancer treatment since 2011. Current clinical trials carried out by MagForce AG company in Germany have shown a 7-8 months increase in the life expectancy of patients with Glioblastoma (www.magforce.de). The generated temperature depends on the magnetic properties of the nanoparticles, and it increases with magnetic field frequency and amplitude1,2,7,8. However, there are clinical upper limits for the magnetic field intensity and frequency values the human body can be exposed to, imposed by Eddy currents9,10. In addition, in order to minimize potential sideeffects, the dosage of nanoparticles administered during the hyperthermia treatment should be kept as low as possible11. Thus, the study of magnetic nanoparticles with high heating capability, namely large hysteresis loop area for a given magnetic field intensity and frequency, has generated wide interest. A promising new approach consists of using magnetic nanoparticles with either large saturation magnetization or enhanced magnetic anisotropy12–16. Magnetotactic bacteria have the ability to biomineralize magnetosomes, magnetic nanoparticles covered by a lipid bilayer membrane, which allow them to align and navigate along the geomagnetic field lines17–19. The magnetosomes hold great potential for hyperthermia applications, since they have already been proved to present large specific absorption rate (SAR) values20–23. The shape and size of the magnetosomes as well as the type of magnetic material depend on the species of magnetotactic bacteria. In particular, Magnetospirillum gryphiswaldense produces magnetite, Fe 3 O 4 , cuboctahedral shaped nanoparticles with an average size diameter of ≈ 45 nm. The fact that magnetosomes spontaneously self-assemble forming chains holds great potential to


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tailor their heating capabilities. Besides, the proteins present in the magnetosome membrane can be used to link bio-active molecules, making the magnetosomes highly biocompatible. Previous in vitro studies on magnetosomes toxicity indicate that they are compatible for biomedical applications24–26. In this paper we provide detailed and novel experimental and theoretical findings concerning the heat efficiency of magnetosomes in hyperthermia, including in vitro experiments. We make use of the powerful AC magnetometry to directly determine the Specific Absorption Rate in a wide range of excitation conditions. The main mechanism of the heat production is proved to be the intrinsic hysteresis losses which have been successfully modelled by a dynamical Stoner Wolhfart approach. In this framework, interparticle interactions are treated as an external interaction anisotropy and we deduce that they play an essential role to predict the SAR in this system for a given experimental conditions. We have also determined the optimal magnetic field frequency and amplitude values to get the maximum SAR of the magnetosomes within the clinical limits9,10. Finally, in vitro tests clearly indicate that the hyperthermia treatment with magnetosomes causes both cell death and inhibition of cell proliferation. These results pave the way for an efficient cancer treatment through magnetosomes mediated magnetic hyperthermia.

EXPERIMENTAL SECTION Magnetotactic bacteria culture and magnetosomes isolation: Magnetosomes were extracted from the Magnetospirillum gryphiswaldense strain MSR-1 (DMSZ 6631). The MSR-1 bacteria were cultured in a medium containing Fe(III)-citrate as we described elsewhere19. Magnetosomes were isolated according to the protocol described by Grünberg et al. with minor modifications30.


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Briefly, cultured magnetotactic bacteria were collected by centrifugation, suspended in 20mM HEPES-4mM EDTA (pH 7.4), and disrupted using French press at 1.4 kbar. The lysate was centrifuged at 600 g for 5 min to remove cell debris. Then, the magnetosomes were isolated from the supernatant using a magnetic rack and rinsed 10 times with 10 mM HEPES-200mM NaCl (pH 7.4). Finally, the isolated magnetosomes were dispersed in deionized water (pH 7.4), sterilized in autoclave (115ºC, 15 min), and stored at 4ºC. Transmission Electron Microscopy: Transmission electron microscopy (TEM) was performed on both unstained bacteria and isolated magnetosomes adsorbed onto 300 mesh carbon-coated copper grids. TEM images were obtained with a Philips CM120 Biofilter electron microscope at an accelerating voltage of 100 kV. The particle size distribution was analyzed using standard software for digital electron microscope image processing, ImageJ. Electron micro-diffraction was performed on a Philips CM200 electron microscope at an accelerating voltage of 200 kV and with an electron beam size of 40 nm. Infrared spectroscopy: FTIR spectroscopy was carried out in transmittance mode in a Jasco 4200 spectrometer. 30 μl of purified magnetosomes in aqueous solution (150 µg Fe 3 O 4 /ml) were airdried over a zinc selenide disk. Spectra were acquired from 4000 to 800 cm-1 with a spectral resolution of 4 cm-1 and accumulating 128 scans. The spectra were baseline corrected excluding the CO 2 bands and normalized to the amide I band at 1654 cm-1 to avoid the effect of different sample thickness. Zeta potential: Zeta potential was analyzed in a Zetasizer Nano (Malvern Instruments) at room temperature. Magnetosomes were suspended in deionized water at pH 7.0 to achieve a


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concentration of 30 μg/ml. Before measurement, the suspensions were sonicated for 10 min to reduce aggregates. Magnetic characterization: The room temperature hysteresis loops were measured uing a homemade vibrating sample magnetometer (VSM) up to a maximum applied field of 1 T. The thermal dependence of the magnetization was carried out in lyophilized magnetosomes under a zero-fieldcooling/field-cooling (ZFC/FC) protocol using a SQUID magnetometer (Quantum Design, MPMS-7). In the ZFC/FC measurements, the sample was cooled from room temperature down to 5 K, without applied magnetic field (zero-field-cooling), and then a magnetic field of 5 mT was applied, and the magnetization was measured while the sample was heated up to room temperature. Afterwards, the process was repeated but during the cooling phase, a magnetic field of 5mT was applied (field-cooling), and then the magnetization was measured while heating. SAR measurements: The SAR was measured in a home-made AMF magnetometer previously described by Garaio et al.31. Briefly, it consists of an air-core inductor part of a resonant circuit fed by a power amplifier. The dynamic magnetization, M t , is obtained by a pick-up coil system composed of two coils wound in opposite direction. The signal is filtered using a low-pass filter with the cutoff frequency at 3 MHz. For the SAR measurements the magnetosomes were dispersed in two different media, deionized water and 2% (w/v) agarose gel. In both cases, the magnetite mass concentration was c ≈ 0.2 mg·ml-1, as determined from saturation magnetization, measured using the VSM, and considering as reference the saturation magnetization value of 92.3 A·m2kg1

, corresponding to pure magnetite. The dynamic hysteresis loops were measured at room

temperature (25ºC) at selected frequencies 75 kHz, 149 kHz, 302 kHz and 532 kHz, and with an applied magnetic field ranging from 0 to 45 kA·m-1.


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In-vitro hyperthermia experiments: The experiments were carried out on two pair of cell samples simultaneously. Each pair consisted of a batch of “natural” cells (A) and another batch of magnetosome-loaded cells (B). One of the pairs was exposed to an AMF (A+, B+) and the other remained untreated (A, B). Cell viability and number of cells were measured several times during the experiment. By comparing the results between different batches, the effect of hyperthermia treatment (B+ vs. A), the cytotoxicity of the magnetosomes (B vs. A) and the effect of AMF exposure (A+ vs. A; B+ vs. B) was assessed. The experiment was performed six times and data are represented as mean ± standard deviation (M±SD). Statistical analysis was performed using Student’s t-test to compare mean values between groups. Differences were considered statistically significant at probability P90% of cells, which reflect the presence of magnetosomes both inside the cytoplasm and on the cytoplasmic membrane.

Figure 7. Observation of magnetosomes internalized in macrophages using bright field and fluorescence microscopy. Magnetosomes were labelled with FITC and cells were stained with DAPI and PI. a) Filter settings for DAPI show cellular nucleus in intense blue. b) Filter settings for FITC show internalized magnetosomes in green and nucleus of the dead cells in red. c) Macrophage-loaded cells stained with Prussian blue after 24 hour of internalization. Blue inclusions correspond to the internalized magnetosomes.


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Subsequently, the macrophages loaded with magnetosomes were exposed to an AMF for 30 minutes, applying the optimal conditions to achieve the maximum SAR, as explained above: H = 24 kA/m and f = 149 kHz. It must be noted that the applied frequency is slightly smaller than the optimum one we calculated before, 200 kHz, but this is due to restrictions on the frequencies available in our hyperthermia system. Cell phenotype was analyzed by flow cytometry. Figure 8 shows the forward-scattered light (FS) and side-scattered light (SS) resulting from a representative experiment. Events in red correspond to dead cells. AMF application did not induce changes in size, as estimated from FS, or internal complexity of the cells, as inferred from SS. On the contrary, the presence of magnetosomes inside the cells changed the SS distribution drastically. The increase of internal complexity of the magnetosome-loaded cells was reflected in the higher SS values. Interestingly, the uptake of nanoparticles by cells can be easily and quickly confirmed by FS/SS analysis, complementarily to the more subjective microscope observations. In addition, a clear reduction of the number of cells present in the culture was detected for macrophages loaded with magnetosomes and exposed to an AMF (Figure 8d).


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Figure 8. Dot-plots of FS and SS light obtained by flow cytometry in a typical experiment. a) Control macrophages. b) Magnetosome-loaded macrophages after 24 h of internalization. c) Macrophages 2 h after AMF exposure. d) Magnetosome-loaded macrophages after 2 h of AMF exposure. The evolution of cell size and internal complexity can be followed from the distribution of FS and SS light. The values shown inside the plots correspond to the number of cells recorded in 5 min. Events in red correspond to dead cells according to the PI staining.


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In addition, to get a better depiction about the state of the macrophage cells after the hyperthermia treatment, the cells were also stained with Annexin V-FITC/Propidium Iodide to discriminate alive, early apoptotic and dead cells. Annexin, a phospholipid binding protein, links cells in the early phases of the apoptotic death during which the cell membrane still remains intact. On the contrary, Propidium Iodide (PI), as we commented before, only enters dead cells, once membrane integrity has been lost. The results of a representative experiment are shown in Fig. 9. The events in Q1, annexin(-)/PI(-), are unstained cells and were considered alive. The events in Q2, annexin(+)/PI(-) bind only annexin thus correspond to early apoptotic cells. The events in Q3, annexin(+)/PI(+), do not exclude Propidium Iodide therefore they were considered dead cells. And the events in Q4 corresponds to the necrotic cells. After the hyperthermia treatment it can be noticed a significant drop in living cells concomitant with an increase in dead cells even though no more cells were observed in the early phase of the apoptotic process.


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Figure 9. Fluorescence dot-plots of Annexin/PI stained cells obtained by flow cytometry in a representative experiment. a) Control cells. b) Magnetosome-loaded cells after 2 h of AMF exposure.

Considering these data, the results obtained were analyzed under three different views: i) The global effect of the hyperthermia treatment, which includes the uptake of magnetosomes and application of an AMF, ii) the cytotoxic effect due to the presence of magnetosomes inside the cells, and iii) the possible side-effect of an AMF on the cells. Figure 10 summarizes the response of the macrophages to the hyperthermia treatment, as deduced from the flow cytometry data. Living cells population is estimated as the product of the viability (%V) by the total number of cells (N), %V·N, and normalized to the un-treated “natural” populations, which is considered to be 100% and serves as control. It must be noted that the treated population exhibits a strong decrease of living cells as compared to the control. Specifically, only a 36% of the treated macrophages remained alive 2 hours after the AMF exposure, and 24 hour later, the percentage fell down to 22%. This highly promising result confirms the potential of magnetosomes exposed to an AMF for cancer therapy. Magnetic hyperthermia based on magnetosomes has not been extensively analyzed, but our results agree with previous studies23. Alphandery et al., working with tumor cells and applying an AMF similar to us (183 kHz, 16 – 47 kA/m), found that living cells were reduced to between 10% and 60%, depending on the concentration of magnetosomes (0,125 mg/ml to 1 mg/ml) incubated with the cells24,51. Liu et al. obtained cell inhibitory rates around 80% in human breast cancer cells applying a much higher dose of 10 mg/ml of magnetosomes and an AMF of 300 KHz and 110 Oe (8.75 kA/m)52. Interestingly, both studies needed a concentration of magnetosomes around fifteen and three


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hundred times higher than us (30 µg/ml) respectively, to obtain an anti-tumoral efficiency similar to the one reported in this work. Such a huge difference could rely on the particular type of cells employed in the assays, which determines the effective cellular internalization of magnetosomes. Liu et al. used a magnetic field amplitude which is below the threshold limit of 10 kA/m.

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Figure 10. Effect of hyperthermia treatment on macrophage populations. Magnetosome-loaded macrophages were exposed to an AMF (H=24 kA/m, f=149 kHz) for 30 min and the effect was evaluated 2 and 24 hours after the exposure. Natural untreated macrophages were used as control. Data represent the mean ± standard deviation, n=6. * P

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