Edouard Alphandéry
email: edouardalphandery@hotmail.com

François Guyot

Imène Chebbi

Preparation of chains of magnetosomes, isolated from AMB-1 magnetotactic bacteria, yielding efficient treatment of tumors using magnetic hyperthermia

Keywords: Magnetosomes, magnetotactic bacteria, cancer, tumor, chains of magnetosomes, individual magnetomes, alternating magnetic field, magnetic hyperthermia

Chains of magnetosomes isolated from AMB-1 magnetotactic bacteria by sonication at 30 W during 2 hours are tested for magnetic hyperthermia treatment of tumors. These chains are composed of magnetosomes, which are bound to each other by a filament made of proteins. When they are incubated in the presence of cancer cells and exposed to an alternating magnetic field of frequency 198 kHz and average magnetic field strength of 20 or 30 mT, they produce efficient inhibition of cancer cell proliferation. This behavior is explained by a high cellular internalization, a good stability in solution and a homogenous distribution of the magnetosome chains, which enables efficient heating. When the chains are heated during 5 hours at 90 C in the presence of 1 % SDS, the filament binding the magnetosomes together is denatured and individual magnetosomes are obtained. By contrast to the chains of magnetosomes, the individual magnetosomes are prone to aggregation, are not stable in solution and do not produce efficient inhibition of cancer cell proliferation under application of an alternating magnetic field.

INTRODUCTION

Magnetic hyperthermia is a technique by which magnetic nanoparticles are either introduced or sent within tumors and heated under the application of an alternating magnetic field (AMF). The heat produced locally by the nanoparticles induces anti-tumoral activity. Magnetic hyperthermia has been used both on animal models and on humans to treat a series of different cancers, including breast cancer, [START_REF] Denardo | Thermal Dosimetry Predictive of Efficacy of 111 In-ChL6 Nanoparticle AMF-Induced Thermoablative Therapy for Human Breast Cancer in Mice[END_REF][START_REF] Kikumori | Anti-cancer effect of hyperthermia on breast cancer by magnetite nanoparticle-loaded anti-HER2 immunoliposomes[END_REF], prostate cancer, [START_REF] Johannsen | Clinical hyperthermia od prostate cancer using magnetic nanoparticles: Presentation of a new interstitial technique[END_REF][START_REF] Johannsen | Thermotherapy of Prostate Cancer Using Magnetic Nanoparticles: Feasability, Imaging, and Three-Dimensional Temperature Distribution[END_REF][START_REF] Kawai | Anticancer Effect of Hyperthermia on Prostate Cancer Mediated by Mediated by Magnetite Cationic Liposomes and Immune-Response Induction in Transplanted Syngeneic Rats[END_REF][START_REF] Kawai | Effect of Heat Therapy Using Magnetic Nanoparticles Conjugated With Cationic Liposomes on Prostate Tumor in Bone[END_REF], glioblastoma, [START_REF] Maier-Hauff | Efficacy and safety of intratumoral thermotherapy using magnetic iron-oxide nanoparticles combined with external beam radiotherapy on patients with recurrent glioblastoma multiforme[END_REF], and head and neck cancer, [START_REF] Zhao | Magnetic Nanoparticle-Based Hyperthermia for Head and Neck Cancer in Mouse Models[END_REF]. Until now, most of the iron oxide nanoparticles tested were chemically synthesized with either a superparamagnetic or a weakly ferrimagnetic behavior at physiological temperature, [START_REF] Hergt | Magnetic hyperthermia: nanoparticle magnetism and material development for cancer therapy[END_REF][START_REF] Hergt | Magnetic particle hyperthermia-biophysical limitations of a visionary tumour therapy[END_REF][START_REF] Hergt | Effects of size distribution on hysteresis losses of magnetic nanoparticles for hyperthermia[END_REF][START_REF] Bordelan | Magnetic nanoparticle heating efficiency reveals magneto-structural differences when characterized with wide ranging and high amplitude alternative magnetic fields[END_REF]. For an applied magnetic field of frequency and strength kept below a toxicity threshold of  200 kHz and  100 mT respectively, [START_REF] Ivkov | Application of High Amplitude Alternating Magnetic Fields for Heat Induction of Nanoparticles Localized in Cancer[END_REF], the specific absorption rate (SAR) of the chemically synthesized nanoparticles is lower by a factor of 2 to 100 than that of the biologically synthesized nanoparticles, called magnetosomes, [START_REF] Hergt | Magnetic hyperthermia: nanoparticle magnetism and material development for cancer therapy[END_REF][START_REF] Hergt | Magnetic particle hyperthermia-biophysical limitations of a visionary tumour therapy[END_REF][START_REF] Hergt | Effects of size distribution on hysteresis losses of magnetic nanoparticles for hyperthermia[END_REF]. For this reason, there has been a surge of interest to carry out magnetic hyperthermia for cancer treatment using the magnetosomes, [START_REF] Hergt | Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools[END_REF][START_REF] Timko | Tomasovicova N, Magnetic properties and heating effect in bacterial magnetic nanoparticles[END_REF][START_REF] Alphandéry | Treatment of cancer or tumor induced by the release of heat generated by various chains of magnetosomes extracted from magnetotactic bacteria and submitted to an alternative magnetic field[END_REF][START_REF] Alphandéry | Chains of Cobalt Doped Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in Alternative Magnetic Field Cancer Therapy[END_REF][START_REF] Alphandéry | Chains of Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in Alternative Magnetic Field Cancer Therapy[END_REF]. In fact, the magnetosomes are monodomain, well-crystallized nanoparticles surrounded by a lipidic membrane with the unique property of being usually arranged in chains. They are synthesized by a species of bacteria, called magnetotactic bacteria, which use them as a compass to navigate in the direction of the earth magnetic field in search for an optimum environment. In a previous study, [START_REF] Alphandéry | Chains of Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in Alternative Magnetic Field Cancer Therapy[END_REF], 1 mg of a suspension of chains of magnetosomes extracted from AMB-1 magnetotactic bacteria has been administered within breast tumors xeno-grafted under the skin of mice. After three applications of an alternating magnetic field during 20 minutes, tumors were totally eradicated in several mice. These results suggest that chains of magnetosomes extracted from AMB-1 magnetotactic bacteria are highly efficient for solid tumor treatment using magnetic hyperthermia, [START_REF] Alphandéry | Chains of Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in Alternative Magnetic Field Cancer Therapy[END_REF].

In this article, we examine if the efficiency of chains of magnetosomes extracted from AMB-1 magnetotactic bacteria, designated as CM, is solely due to the high SAR of the magnetosomes or if it also arises from other factors such as a homogenous distribution of the magnetosome chains within the tissue or a faculty of the magnetosome chains to internalize within the cancer cells. To know the influence of the magnetosome chain arrangement on the efficiency of the therapy, we compare the properties of the chains of magnetosomes with those of individual magnetosomes, designated as IM, which are detached from the chains by heat and chemical treatments.

MATERIALS AND METHODS

Method used for the culture of the AMB-1 magnetotactic bacteria:

Magnetotactic bacteria belonging to the species Magnetospirillum magneticum, strain AMB-1, available at the ATCC under the reference 700264, were cultivated in a volume of 1 liter of bacterial growth medium. The cells were cultivated in micro-anaerobic conditions, i. e. in a culture medium that has not been degassed, but is closed and not in contact with oxygen. The culture of magnetotactic bacteria has been carried out in an incubator at 26 C in a slightly modified MSGM liquid culture medium, whose composition is described below.

For a volume of 1 liter, the standard culture medium contains 0.68 g of monobasic potassium phosphate, 0.85 g of sodium succinate, 0.57 g of sodium tartrate, 0.083 g of sodium acetate, 225 l of 0.2 % resazurine, 0.17 g of sodium nitrate, 0.04 g of L-ascorbic acid, 2 ml of a 10 mM solution of iron quinate, 10 mL of a solution of Woolf's vitamins and 5 mL of a solution of Woolf's minerals. The solution of iron quinate has been prepared by dissolving 0.19 g of quinic acid and 0.29 g of FeCl3.6H2O in 100 ml of distilled water. The solution of Woolf's minerals contains in 1 liter of distilled water, 0.5 g of nitrilotriacetic acid (NTA, C6H9O6), 1.5 g of magnesium sulfate heptahydrate (MgSO4.7H2O), 1 g of sodium chloride, 0. (vitamin B5), 0.4 mg of vitamin B12, 5.2 mg of amino-benzoic acid, 5.2 mg of thiotic acid and 900 mg of potassium phosphate. The pH of the culture medium was adjusted to 6.85 by using a 1 M solution of sodium hydroxide.

Preparation of the different suspensions containing the extracted chains of magnetosomes and individual magnetosomes:

Two different types of magnetosomes have been isolated from AMB-1 magnetotactic bacteria, those arranged in chains and those forming individual nanoparticles. To prepare them, the cells were first harvested by centrifugation at 4 000 g during 20 minutes. The supernate was then removed and the cells were re-suspended in deionized water. To extract the chains of magnetosomes, 1 ml of cell suspension obtained as previously described was centrifugated again and re-suspended in a 10 ml tris-HCl buffer of pH 7.4. The cellular suspension was then sonicated during 120 minutes at 30 W in order to lyse the cells and collect the CM. After sonication, the suspension containing the CM was separated from the cellular debris by positioning a strong neodynium magnet (0.1-1 T) next to the tube, and the magnetic material was then harvested. The supernate containing the cellular debris and other organic molecules was eliminated. CM were washed 10 to 20 times in deionized water at pH 7.4 and were then resuspended in deionized water. To prepare the suspensions containing the IM, the suspension containing the CM was heated during five hours at 90 C in the presence of 1 % SDS. IM were separated from the biological material remaining after the treatment by using a strong neodynium magnet (0.1-1T). The concentrations in maghemite of the various suspensions containing CM and IM were measured by absorption at 480 nm, [START_REF] Alphandéry | Chains of Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in Alternative Magnetic Field Cancer Therapy[END_REF].

Methods used for measuring the SAR and for heating the different suspensions of bacterial magnetosomes:

The SAR of suspensions of CM and IM were measured for bacterial magnetosomes, which were either able or unable to rotate under the application of an AMF, [START_REF] Alphandéry | Treatment of cancer or tumor induced by the release of heat generated by various chains of magnetosomes extracted from magnetotactic bacteria and submitted to an alternative magnetic field[END_REF]. The amount of heat produced in the gel by the CM and IM was low under the application of an AMF of frequency 108 kHz and magnetic field strength kept below 36 mT, [START_REF] Alphandéry | Treatment of cancer or tumor induced by the release of heat generated by various chains of magnetosomes extracted from magnetotactic bacteria and submitted to an alternative magnetic field[END_REF]. To enhance the magnetosome heating efficiency during the in-vitro and in-vivo heating experiments, the frequency of the AMF was therefore increased up to 198 kHz and the average magnetic field strength was kept below 30 mT to avoid the formation of eddy currents, which can induce toxicity. The average magnetic field strength was estimated from the variation with time of the magnetic field strength. It was measured using a 2D magnetic field probe designed by the company Fluxtrol. The value of the average magnetic field strength measured experimentally with this probe was 1.8 times lower than the theoretical value of the magnetic field, estimated using the relation B = 0NI/L, where 0 = 410 -7 is the vacuum permeability, N = 4 is the number of spires in the coil, I = 151, 300, 419 or 600 A is the amplitude of the alternating current flowing through the spires and L = 3.5 cm is the length of the coil (Suppl. table 1). The temperatures reached during the heating experiments were measured using a thermocouple microprobe (IT-18, Physitemp, Clifton, USA).

Percentage of inhibition and magnetization studies of HeLa and MDA-MB 231 cells incubated in the presence of the different suspensions of magnetosomes isolated from AMB-1 magnetotactic bacteria:

The HeLa and MDA-MB 231 cells were purchased from the ATCC. The cell lines were cultivated in Dulbecco's model modified Eagle's medium supplement, which contained 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 U/mL streptomycin (all purchased from Life Technologies Inc.). Cell viability was evaluated using the so-called MTT (microculture tetrazolium) assay, [START_REF] Mosmann | Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays[END_REF]. This technique measures the ability of mitochondrial enzyme to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (purchased from Sigma, St Louis, MO, USA) to purple formazan crystals.

MDA-MB-231 and HeLa cells were seeded at a density of 2.10 4 cells per well in 96-well flat-bottom plates (Falcon, Strasbourg, France) and incubated in completed culture medium for 24 hours. Then, the culture medium was removed and replaced by 10 % FCS-medium containing CM or IM with different concentrations in iron oxide (1 mg/mL, 500 µg/mL or 125 µg/mL). After 24 hours of incubation, the cells were exposed (or not) to an AMF of frequency 198 kHz and average magnetic field strength of  20 mT or  30 mT during 20 min. After having been exposed to the AMF, the cells were incubated at 37 °C in a 5 % CO2 humidified atmosphere during 48 hours. Following the incubation, cells were washed with phosphate buffered saline solution (PBS, Life Technologies) and incubated with 100 µl of MTT (2 mg/ml, Sigma-Aldrich) for an additional 4 hours at 37 °C. The insoluble product was then dissolved by addition of 100 ml of DMSO (Sigma-Aldrich). The absorbance of the solubilized formazan pellet, which reflects the relative viable cell number, was measured at 540 nm using a Labsystems Multiskan MS microplate reader. The measurements were carried out on DMSO solubilized formazan pellets using cells washed with PBS as a control, [START_REF] Yoshino | Magnetic Cell Separation Using Nano-Sized Bacterial Magnetic Particles With Reconstructed Magnetosome Membrane[END_REF]. The experiments were carried out in triplicates.

Internalization and magnetization studies of MDA-MB 231 cells incubated in the presence of various suspensions of magnetosomes:

5.10 5 MDA-MB-231 cells in suspension were mixed in 1 ml of a 10 % FCS-medium containing suspensions of CM or IM with a fixed concentration in iron oxide of 1 mg/ml. The cells were exposed from the reaction between Fe 2+ or Fe 3+ ions coming from the dissolved magnetosomes and the ferrocyanide ions. After having washed each Petri dish three times with PBS, Prussian blue staining was observed using an optical microscope. The experiments were carried out in triplicates.

Scanning electron microscopy, transmission electron microscopy and optical microscopy studies of MDA-MB 231 cells incubated in the presence of either chains of magnetosomes or individual magnetosomes:

Scanning electron microscopy (SEM) studies were carried out using a FEG-SEM ultra 55 from Zeiss equipped with an energy dispersive X-ray analysis system (EDX). The samples studied by SEM contained MDA-MB-231 cancer cells incubated during 24 hours in the presence of IM or CM. The cells were deposited on top of a carbon grid covered with a carbon film and fixed with NOVAprep, produced by Novacyt. Transmission electron microscopy (TEM) studies were also carried out using a JEOL 2100F-FEG operated at 200 kV. The samples studied by TEM contained 5 l of suspensions of IM or CM deposited on top of a carbon grid. Optical microscopy was also used to visualize precipitated magnetosomes within MDA-MB 231 cells. For that, the cells were incubated in the presence (or not) of suspensions containing either CM or IM. The cells were stained with Prussian blue, which precipitates in the presence of iron, producing a blue color in the micrographs, which reveals the presence of iron.

RESULTS AND DISCUSSION

The different types of bacterial magnetosomes chosen for magnetic hyperthermia treatment of tumors:

In whole magnetotactic bacteria, the chains of magnetosomes only occupy a small portion of the total volume of a bacterium ( 0.02 %). A TEM image of a typical AMB-1 magnetotactic bacterium is shown in Figure 1(a). Several chains of magnetosomes separated by more than ~ 100 nm are contained within this bacterium. Large regions of the bacterium are not occupied by chains of magnetosomes. Because of the low density of the chains of magnetosomes within whole magnetotactic bacteria, suspensions containing whole magnetotactic bacteria are expected to yield a low in vivo heating efficiency. In addition, the administration of whole inactive magnetotactic bacteria in humans is not recommended due to the presence of potentially toxic biologic materials such as bacterial DNA or endototoxins, [START_REF] Diem | Infrared Spectroscopy of Cells and Tissues: Shining Light onto a Novel Subject[END_REF]. For these two reasons, whole inactive magnetotactic bacteria were not tested in this study. In order to minimize the risks of toxicity and to enhance the heating efficiency, the bacterial magnetosomes were isolated from the bacteria.

The first type of bacterial magnetosomes tested for magnetic hyperthermia consisted of chains of magnetosomes extracted from AMB-1 magnetotactic bacteria, CM. phospholipids [START_REF] Song | Application of a magnetotactic bacterium, Stenotrophomas sp. to the removal of Au(III) from contaminated wastewater with a magnetic separator[END_REF][START_REF] Abramson | Study of Ionic Structures in Phospholipids by Infrared Spectra[END_REF], both located within the magnetosome membrane, [START_REF] Yoshino | Magnetic Cell Separation Using Nano-Sized Bacterial Magnetic Particles With Reconstructed Magnetosome Membrane[END_REF]. The peak at 580 cm -1 (peak 5, Figure 2(b)) is attributed to maghemite, [START_REF] Salazar | Characterization of natural microcosms of estuarine magnetotactic bacteria[END_REF]. These results suggest that both the filament binding the magnetosomes together, which is made of proteins such as MamJ or Mam K, [START_REF] Draper | Mam K, A bacterial actin, forms dynamic filament in vivo that are regulated by the acidic proteins MamJ and Lim[END_REF][START_REF] Scheffel | An acidic protein aligns magnetosomes along a filamentous structure in magnetotactic bacteria[END_REF][START_REF] Villanueva | The influence of surface functionalization on the enhanced internalization of magnetic nanoparticles in cancer cells[END_REF], and the bilayer surrounding the individual magnetosomes, which is composed of LPS and phospholipids, [START_REF] Xie | Production, Modification and Bio-Applications of Magnetic Nanoparticles Gestates by Magnetotactic Bacteria[END_REF], are contained within CM. After extraction, the magnetosome chains have kept the lipid bi-layer surrounding each magnetosome and the filament binding the magnetosomes together.

To obtain suspensions of magnetosomes, which are similar to the chemically synthesized iron oxide nanoparticles, i. e. not arranged in chains, IM have been prepared by heating a suspension of CM at 90

C during 5 hours in the presence of 1 % SDS. TEM images of a suspension of IM deposited on top of a carbon grid are presented in Figures 1(c) and 1(d). It shows either assemblies of magnetosomes, which are much more aggregated than the chains of magnetosomes (Figure 1 suggest that the bi-layer surrounding each magnetosome has not been significantly removed during the heat treatment in the presence of a detergent.

SAR of the different suspensions of bacterial magnetosomes:

The SAR of suspensions containing IM and CM were measured in two situations in which the bacterial magnetosomes were either able or unable to rotate under the application of an AMF, [START_REF] Alphandéry | Treatment of cancer or tumor induced by the release of heat generated by various chains of magnetosomes extracted from magnetotactic bacteria and submitted to an alternative magnetic field[END_REF]. For the bacterial magnetosomes mixed in water and able to rotate, Figures 3(a) and 3(b) show that the amount of heat produced by the CM is larger than that produced by the IM. This behavior could be explained by the fact that CM are less prone to aggregation and therefore rotate more easily than IM under the application of an AMF. This analysis is further supported by the TEM image presented in Figure 1(b), which clearly shows that CM do not aggregate by contrast to IM (Figure 1(c)). For the bacterial magnetosomes mixed in water, the higher value of the SAR obtained for CM compared with IM could therefore be explained by a contribution of the rotation to the heat production, which is more significant for CM than for IM. By contrast, when CM and IM are fixed (either in a gel or on a solid substrate, [START_REF] Alphandéry | Treatment of cancer or tumor induced by the release of heat generated by various chains of magnetosomes extracted from magnetotactic bacteria and submitted to an alternative magnetic field[END_REF]), unable to rotate, mixed homogenously and exposed to the same AMF as above, IM produce a relatively similar amount of heat than CM (Figure 3), [START_REF] Alphandéry | Treatment of cancer or tumor induced by the release of heat generated by various chains of magnetosomes extracted from magnetotactic bacteria and submitted to an alternative magnetic field[END_REF]. These results suggest that when the rotation of the magnetosomes is not taking part in the mechanism of heat production, which is most probably the case in vivo, the quantity of heat generated by CM or IM is not only due to the magnetosome SAR but also to the spatial distribution of the magnetosomes.

Percentage of inhibition of HeLa and MDA-MB-231 cells incubated in the presence of CM or IM:

To compare the anti-tumoral activity of CM with that of IM, CM and IM were incubated in the presence of HeLa and MDA-MB-231 cells. The percentage of inhibition of cellular proliferation was then measured in the presence (or not) of an AMF. The results are presented for two suspensions of bacterial magnetosomes (CM or IM) with a concentration fixed at 0.125 mg/ml. In the absence of application of an AMF, the percentages of inhibition of MDA-MB-231 and HeLa cells incubated in the presence of CM or IM were as low as 2 % and 10 % (Figure 4(a)), respectively. When these cancer cells were incubated in the presence of the CM and exposed to an AMF of frequency 198 kHz and average magnetic field strength of 20 mT and 30 mT, the percentages of inhibition increased by factors of  3 to  5 for the HeLa cells and  7 to  16 for the MDA-MB-231 cells (Figure 4(a)). By contrast, when these cancer cells were incubated in the presence of IM and exposed to the same AMF as above, the percentage of inhibition of these cells remained almost identical (Figure 4(a)). A similar type of behavior was observed when MDA-MB6231 and HeLa cells were incubated in the presence of CM and IM at higher iron oxide concentrations of 1 mg/ml (Suppl. Fig. 1(a)) and 0.5 mg/ml (Suppl. Fig. 1(b)).

These results indicate that under the application of an AMF, CM yield a higher percentage of cell inhibition than IM.

Penetration of the chains of magnetosomes within the cancer cells under the application (or not) of an alternating magnetic field:

We examine if the different efficiencies observed between CM and IM are due to a different faculty of penetration within the cancer cells of these two types of bacterial magnetosomes. We first study if the application of an AMF induces the internalization of CM or IM within the cancer cells. To be able to estimate the magnetization of MDA-MB-231 cells, the latter had to be kept free and not fixed. The cells were incubated in the presence of the CM and IM and exposed to the same AMF as above. The cancer cells were then washed with water to remove CM and IM located at their surface and the percentage of magnetic cells was then measured. Following application of an AMF of frequency 198 kHz during 5 to 20 minutes, Figure 4(b) shows that the mass of maghemite per magnetic cell is always much higher for the cells incubated in the presence of CM than for those incubated in the presence of IM. This result suggests that under the application of an AMF, CM penetrate much more within the cancer cells than IM. Moreover, the high cellular magnetization, which can be reached in the presence of CM under the application of an AMF, could explain the high efficiency of CM for magnetic hyperthermia treatment of tumors.

We also study the internalization of CM and IM within the cancer cells as a function of the incubation time in the absence of an AMF. For that, the cells had to be fixed on top of a solid substrate. For a very short incubation time of less than 30 seconds, Figure 4 were detected (Suppl. Fig. 3). By contrast to the behavior observed with CM, IM were not observed within the MDA-MB-231 cell (Suppl. Fig. 2). The EDX measurements confirmed this behavior (Suppl.

Fig. 3). They showed that iron was not detected in the cellular regions with presumably no magnetosomes.

The internalization of CM within the cancer cells for a sufficiently long incubation time of 24 hours was further confirmed by optical microscopy.

Explanation for the high efficiency of the chains of magnetosomes:

Altogether, these results indicate that CM penetrate more easily within the cancer cells than IM. While the internalization within the cancer cells did not occur with IM, it was observed for CM in two conditions, either when an AMF of 198 kHz and average field strength of 20 mT was applied during 5 to 20 minutes and/or when the cells were incubated with CM for a sufficiently long time of 24 hours. The internalization of CM within the cancer cells could occur by a mechanism of endocytosis or by effective diffusion through the cell membrane. The mechanism of effective diffusion through the cell membrane could be favored by a temperature increase taking place during the application of the AMF, which would make the cell membrane permeable. This result agrees with previous studies, which showed that negatively charged maghemite nanoparticles penetrate within cancer cells, [START_REF] Wilhelm | Intracellular uptake of anionic superparamagnetic nanoparticles as a function of their surface coating[END_REF]. Since intra-cellular heating presumably efficiently damages cellular components, it may be a more efficient mechanism of cell destruction than extra-cellular heating. The higher efficiency of CM compared with that of IM could therefore be explained by the fact that CM internalize within the cancer cells by contrast to IM, which mostly remain located outside of the cells. Poorly efficient internalization of IM may be related to the more aggregated state of IM compared with that of CM as was observed in two cases, i. e. when suspensions of CM and IM were deposited on top of a carbon grid (Figures 1(b) and 1(c)) and in vivo when suspensions of CM and IM were administered within tumors xeno-grafted under the skin of mice and heated under the application of an AMF, [START_REF] Alphandéry | Chains of Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in Alternative Magnetic Field Cancer Therapy[END_REF]. A schematic summary showing the different distributions of CM and IM in the presence of cancer cells is proposed in Figures 9(a) and 9(b). The better internalization and more homogenous distribution of CM than IM could provide a reasonable explanation for the higher anti-tumoral efficiency observed for CM than for IM, [START_REF] Alphandéry | Chains of Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in Alternative Magnetic Field Cancer Therapy[END_REF].

Conclusion

In conclusion, we have studied two types of bacterial magnetosomes extracted from AMB-1 magnetotactic bacteria, the magnetosomes organized in chains and those forming individual magnetosomes detached from the chains by heat and chemical treatment. We have shown that chains of magnetosomes were more efficient than individual magnetosomes in inhibiting cancer cell proliferation under application of an alternating magnetic field. We have suggested that this increased efficiency was related to much less particle aggregation for the magnetosome chains than for the individual magnetosomes and as a consequence to a better faculty for the chains of magnetosomes than for the individual magnetosomes to internalize within the cancer cells. This paper clearly highlights the importance of nanoparticle distribution for efficient magnetic hyperthermia. It shows that the use of nanoparticles with high SAR is not sufficient to yield efficient magnetic hyperthermia.

(

  or not) to an AMF of frequency 198 kHz and average field strength of  20 mT during 5, 10, 15 and 20 minutes. Following the application of the AMF, the MDA-MB-231 cells were collected with a strong neodynium magnet (0.1-1 T). For each sample, 10 4 cells were used to quantify the mass of iron oxide per cell. Solutions containing various concentrations of FeCl3 mixed with potassium thiocyanate (2 M) were prepared for calibration. Solutions containing one volume of HCl (6 N) mixed with 20 volumes of H2O2 were added to the cells to dissolve the iron oxide into Fe 3+ ions and to denature all biological material. The cells were further lysed by sonication during 30 minutes at 30 W (0 °C) and mixed with a solution of potassium thiocyanate (2 M), which induces the formation of a complex composed of Fe 3+

Figure 1 (Figure 2 (

 12 Figure 2(b)) and 1530 cm -1 (peak 2, Figure 2(b)), which arise from the Amide I and Amide II bands and

1 and 2 ,

 12 (c)), or loops (Figure1(d)). At pH = 7, Figure2(a) shows that suspensions of IM possess very different values of their zeta potential depending on whether or not they are mixed homogeneously by sonication. When IM are mixed homogenously in solution, they possess a negative zeta potential of  -35 mV at pH  7,[START_REF] Sunderland | Targeted nanoparticles for detecting and treating cancer[END_REF]. When they are not sonicated, the IM zeta potential is 10 mV at pH  7. The different values of the Zeta potential suggest that IM easily form aggregates in solution and are therefore unstable in the absence of sonication,[START_REF] Sunderland | Targeted nanoparticles for detecting and treating cancer[END_REF]. The infra-red absorption spectrum of a powder containing a lyophilized suspension of IM integrated in a KBr matrix is presented in Figure2(b). It shows Amide I and Amide II peaks (peaks Figure2(b)), suggesting that most of the proteins contained in the filament binding the magnetosomes together have been removed. The relatively strong peaks at 1050 cm -1 and 1240 cm-1

  (b) shows that neither CM nor IM penetrate within MDA-MB-231 cells. Suspensions containing CM and IM were then incubated in the presence of MDA-MB-231 cells for a longer incubation time of 24 hours. The cells were deposited on top of a carbon grid substrate and imaged under scanning electron microscopy. Figure 5(a) shows a portion of a typical MDA-MB-231 cell. Enlargements of two regions of the cell in which CM are observed are presented in Figures 5(b) and 5(c). Since CM are observed within intracellular vesicles, designated as V in Figure 5(b), which can be lysosomes or endosomes, CM are internalized within the cancer cell. The chain of magnetosome surrounded by a green box in Figures 5(c) and 5(d) contains two large magnetosomes at its end and several small magnetosomes at the middle. This type of magnetosome arrangement is clearly different from that observed for the magnetosome chains, which are not incubated in the presence of cancer cells (Figure 1(b)). It could be due to the degradation of the chains of magnetosomes by the cells following their internalization. Another reason why CM are internalized within the cells and not bound at the cell surface comes from the fact that the cells were washed prior to the observation by scanning electron microscopy. The presence of CM within the cells was further confirmed by EDX measurements, which revealed the presence of iron oxide in the region where CM

Figure 8

 8 shows optical microscopy observations of MDA-MB-231 cells incubated in the absence of magnetosomes (Figure 8(a)), in the presence of CM during 24 hours (Figure 8(b)) or in the presence of IM during 24 hours (Figure 8(c)). For MDA-MB-231 cells incubated in the absence of magnetosomes or in the presence of IM, no Prussian blue coloration was observed within the cells. By contrast, for the MDA-MB-231 cells incubated in the presence of the CM, Prussian blue coloration was observed within the cells. These results agree with those presented above (Figures 5) and suggest that CM penetrate within the cells by contrast to IM, which remain located outside of the cells.

Figure 1 :

 1 Figure 1: (a) Transmission electron microscopy image of a typical whole inactive magnetotactic

Figure 2 :

 2 Figure 2: (a) Zeta potential of suspensions of chains of magnetosomes and individual magnetosomes as

Figure 3 :

 3 Figure 3: (a) Specific absorption rate (SAR) of suspensions of chains of magnetosomes, which are able

Figure 4 :

 4 Figure 4: (a) Percentage of inhibition of HeLa and MDA-MB-231 cells incubated in the presence of CM

Figure 5 :

 5 Figure 5: Scanning electron microscopy image of a MDA-MB-231 cell incubated in the presence of CM.

Figure 6 :

 6 Figure 6: Confocal optical microscopic images of cancer cells stained with Prussian blue and incubated

Figure 7 :

 7 Figure 7: Conceptual summary describing the effect of aggregation on the penetration of magnetosomes

  MDA-MB-231 cells were seeded on Petri dishes (Ø 30 mm, density  5. 10 5 cells per Petri dish), grown during 24 hours and incubated (or not) during 24 hours in the presence of suspensions of CM or IM at a concentration of 142 µg/mL. Cells were washed twice in PBS and fixed in 4 % paraformaldehyde (PFA) (Sigma) during 15 minutes at 4 °C. MDA-MB-231 cells were washed twice more time in PBS and were then stained with Perl's Prussian blue solution during 20 minutes at 37 °C in an atmosphere containing 5 % CO2. Perl's Prussian blue solution was prepared by mixing two solutions of equal volumes containing 4 % potassium ferrocyanide and 4 % HCl. Staining (bright blue pigment: Fe III [Fe III Fe II (CN)6]3) resulted

and SCN -. Quantification of iron oxide was estimated by absorption measurements at 480 nm (UVICON 923, Bio-Tek, Kontron Instrument, Italy). The untreated MDAMB-231 cells were used as reference. The experiments were carried out in triplicates.

2.6 In vitro Prussian blue assay:

ACKNOWLEDGMENTS

François Guyot does not claim any inventive contribution in this work. We thank the company NOVAcyt for giving us the NOVAprep product free of charge.


E Alphandéry

C Carvallo +

N Menguy

I Chebbi

Chains of Cobalt doped magnetosomes synthesized by AMB-1 magnetotactic bacteria for application in AMF hyperthermia

Chains of Cobalt Doped Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in AMF hyperthermia

23/06/17

New treatments of cancer use nanoparticles as therapeutic agents. Among the different approaches tested, it has been suggested to send iron oxide nanoparticles within cancer cells and then kill these cells by heating them under the application of an alternative magnetic field (AMF). This technique has been tested on animals by several different groups (1)(2)(3) and is now at the stage of clinical trials for several different cancers, such as prostate, esophageal and pancreatic cancers (4). This technology can be improved further by increasing the specific absorption rate (SAR) of the nanoparticles currently tested.

One way to achieve this aim is to dope the iron oxide nanoparticles with cobalt (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Indeed, the insertion of cobalt within the iron oxide nanoparticles results in increasing hysteresis losses and nanoparticle SAR (17). Although encouraging, this property has been obtained for a rather large percentage of cobalt doping. An increase by a factor of two of the magnetocrystalline anisotropy constant is reached for a percentage of Co-doping larger than 10 % (18), (19). For such a large percentage of cobalt doping, the medical applications of these chemically synthesized iron oxide nanoparticles are uncertain due to the risks of toxicity.

In order to lower the percentage of cobalt doping while still retaining the desired magnetic properties, cobalt doped iron oxide nanoparticles can be synthesized biologically by using magnetotactic bacteria (20). Indeed, when they are grown in the presence of cobalt quinate, AMB-1 magnetotactic bacteria synthesize iron oxide nanoparticles, called magnetosomes, which contain a percentage of cobalt lying between 0.2 and 1.4 % (20). It has been shown that the magnetic properties of these cobalt doped magnetosomes are considerably improved even for such a low percentage of cobalt doping (20).

In this letter, we provide an explanation for the change of the magnetic properties of cobalt doped magnetosomes. We show that this behavior is essentially due to two factors, i. e. the increase of the magnetosome interactions in the presence of cobalt and the arrangement of the magnetosomes in chains (21). We also demonstrate that chains of magnetosomes extracted from magnetotactic bacteria, which 3 23/06/17 are currently tested for cancer thermotherapy (22), produce a larger SAR when they are doped with cobalt than when they are undoped.

Magnetospirillum magneticum strain AMB-1 was purchased from the ATCC (ATCC 700274). Cells were grown microanaerobically at room temperature (~25°C) in liquid culture in MSGM medium (ATCC Medium 1653). Undoped magnetosomes were obtained by introducing only iron ions (Fe 2+ or Fe 3+ ) in the growth medium using an iron quinate solution (21, 23). Co-doped magnetosomes were synthesized by introducing a 20 M cobalt quinate solution in addition to the iron source. Different types of samples were then prepared, containing either whole magnetotactic bacteria, chains of magnetosomes extracted from magnetotactic bacteria and individual magnetosomes unbound from the chains, which were obtained by heat and SDS treatments of the extracted chains of magnetosomes. Details about the sample preparations are given in the supplementary information section.

Two electron microscopes were used to characterize the magnetosomes. A JEOL (100 kV) model JEM 1011 transmission electron micrograph (TEM) was used to obtain low-magnification micrographs of the different samples mentioned above. For that, 10 l of suspensions containing either whole bacteria, extracted chains of magnetosomes, or individual magnetosomes with 2 10 -4 % by weight of magnetosomes were deposited on top of a carbon grid. After it had dried, the carbon grids containing the different suspensions were placed inside the electron microscope for analysis. A JEOL 2100F was used to carry out energy electron loss spectroscopy (EELS) on single magnetosomes and to verify that the magnetosomes synthesized in the presence of cobalt quinate contained cobalt. As shown in the supplementary information section, the EELS spectrum of the magnetosomes synthesized in the presence of cobalt quinate (Suppl. Fig. 1(b)) shows a peak arising from cobalt. This confirms the presence of cobalt within the magnetosomes synthesized in these conditions (20).

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To characterize the magnetic properties of the different samples, a vibrating sample magnetometer (VSM) from Quantum Design and an alternative gradient magnetometer (AGM) from Princeton Measurements Corporation were used. For that, 25 l of suspensions of whole bacteria, extracted chains of magnetosomes, or individual magnetosomes containing 4 10 -4 % by weight of magnetosomes were deposited on top of a silica substrate (50 x 50 mm 2 ). After it had dried, the silica substrates covered by the different suspensions were placed inside a VSM or AGM for magnetic measurements. We measured hysteresis loops of the different samples at 20 K and 300 K using a VSM and room temperature First Order Reversal Curve (FORC) diagrams using an AGM. Details about the principles of the FORC diagrams can be found in the supplementary information section. From the FORC diagrams, we deduced the distributions of mean interacting field, Hu, of the local interacting field, Hu, and of the microcoercivity, Hc, which is similar to the coercivity estimated by hysteresis loop measurements except that in this case the samples are subjected to a series of magnetic fields of different strengths.

The focus of this letter is the study of chains of magnetosomes extracted from the bacteria, which have been shown to be efficient for cancer thermotherapy (22). Fig. 1(a) shows a TEM micrograph of typical chains of magnetosomes extracted from magnetotactic bacteria. This sample is characterized by the presence of chains of magnetosomes containing more than 10 magnetosomes in average. To characterize the magnetic properties of these chains, hysteresis loops of the extracted chains of magnetosomes were measured at 20 K. Figure 1(b) shows that between the undoped and Co-doped magnetosomes, the coercivity increases from Hc ~ 360 Oe up to Hc ~ 3130 Oe. Assuming that for such a low level of Co-doping (< 2 %, (20)), the saturation magnetization of the assembly of magnetosomes is that of maghemite and using the relation Keff ~ MsHc/2, where Keff reflects the average magnetocrystalline anisotropy constant associated to each magnetosome, we estimate that Keff increases from Keff ~ 12 KJ/m 3 in the absence of cobalt up to Keff ~ 104 KJ/m 3 in the presence of cobalt.

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To understand if the origin of this behavior is due to increasing interactions between the magnetosomes in the presence of cobalt, we have carried out FORC measurements at 300 K. These measurements enable an estimate of the mean and local interacting fields, which reflect the strength of the interactions between the magnetosomes. The FORC diagrams of the extracted chains of magnetosomes are shown in Figs. 2 for the magnetosomes synthesized either in the absence (Fig. 2(a)) or in the presence (Fig. 2(b)) of cobalt. The FORC distribution is displaced below the Hu=0 axis, showing that the mean interacting field, Hu, is weak but non-zero and negative, indicating the presence of weak interactions between the chains of magnetosomes (23). Moreover, the mean interacting field increases in magnitude from Hu ~ -1 mT in the absence of cobalt (Fig. 2 To discern between the role played by these interactions and by the arrangement of the magnetosomes in chains, we have studied the properties of two other samples. The first one contains whole magnetotactic bacteria in which the chains of magnetosomes are well separated and noninteracting (Suppl. Fig. 2(c)). The coercivity increases by ~ 40 % in the presence of cobalt (Suppl. Figs. The heating efficiency of the co-doped magnetosomes arranged in chains and extracted from the bacteria has also been studied. For that, 100 l of a suspension containing extracted chains of magnetosomes with ~ 1.5 mg of maghemite per milliliter is submitted to an alternative magnetic field of frequency 183 KHz and field amplitude 80 mT. As can be seen in Figure 3, the application of the alternative magnetic field produces a larger temperature increase for the co-doped magnetosomes (34 C in 15 minutes) than for the undoped magnetosomes (27 C in 15 minutes). We also estimated a higher SAR of ~ 500 W/gFe for the cobalt doped magnetosomes than for the undoped magnetosomes (SAR ~ 400 W/gFe), (24). This indicates an enhancement of the heating capability of the chains of magnetosomes extracted from bacteria, which have been cultivated in the presence of cobalt quinate.

In conclusion, we have shown in this letter that the change of the magnetic properties of Co-doped magnetosomes is due to their arrangement in chains. For this reason, the change of the magnetic properties induced by the presence of cobalt is observed for a percentage of cobalt within the magnetosomes, which is smaller than that usually obtained with chemically synthesized nanoparticles, which are not arranged in chains. Furthermore, we have shown that the cobalt doped magnetosomes 23/06/17 arranged in chains produce a larger amount of heat than the undoped magnetosomes arranged in chains when they are exposed to an alternative magnetic field.

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Sample preparation:

Details about the growth conditions of AMB-1 magnetotactic bacteria including their growth curves can be found elsewhere (Yang. et al, Enz. Microb. Techno. 29, 13-19 (2001), ATCC protocol to grow strain AMB-1/700264, ATCC growth medium 1653 available on the website of the ATCC). Cells were harvested at stationary phase. Stationary phase occurred when the medium became completely reduced as indicated by a change in the coloration of the growth medium, from pink to colorless.

Three different types of samples were prepared. The living bacteria were first centrifuged at 8000 rpm for 15 minutes. The solution was then placed against a magnet and the supernatant containing the growth medium was removed and replaced by 2 ml of deionized water. Hence we obtained 2 ml of a solution of whole bacteria dispersed in water, which we redispersed in a 10 mM Tris buffer and sonicated during 60 minutes at 30 W to extract the chains of magnetosomes from the whole bacteria.

After sonication the solution containing the extracted chains of magnetosomes was placed against a magnet and the supernatant was removed to get rid of most of the biogenic material. The solution was washed 10 times in this way. 1 ml of this solution was transferred into an ependorf tube and was not treated further. It contained the extracted chains of magnetosomes. 1 ml of the same solution was transferred in another ependorf tube and heated for one hour at 90 0 C in the presence of 1 % sodium dodecyl sulfate (SDS). The solution was heated by placing the ependorph inside boiling water. The second solution contained the individual magnetosomes, which were both extracted from the whole bacteria and detached from the chains.

Electron energy loss spectroscopy measurements:

In order to determine that the magnetosomes synthesized in the presence of cobalt quinate were doped with cobalt, electron energy loss spectroscopy measurements were carried out on a series of individual magnetosomes. For the magnetosomes produced in the absence of quinate cobalt, Suppl. Fig. 23/06/17 1(A) only shows two peaks at 710 eV and 720 eV, which are attributed to the L2 and L3 peaks of iron. This indicates that the magnetosomes are pure iron oxide in this case. For the magnetosomes produced in the presence of a 20 M cobalt quinate solution, the L2 and L3 peaks of iron are still present but in addition, we observe the L2 peak of cobalt at 780 eV in the EELS spectrum (Suppl. Fig. 1(B). , where Cwater is the specific heat capacity of water (Cwater = 4.184 J/g.K), xm is the concentration of iron in g per mL of solvent (water) and (T/T) is the variation with time of the temperature measured at 25°C.

  (a)) up to Hu ~ -2.3 mT (Fig. 2(b)) in the presence of cobalt. The local interacting field, Hu, estimated from the FWHM of the distribution of interacting fields across the maximum Hc value, also increases from ~ 7 mT in the absence of cobalt up to ~ 11 mT in the presence of cobalt (Figs. 2(a) and 2(b)). These results indicate that there is an enhancement of the interactions between the magnetosomes in the presence of cobalt and that this enhancement could cause the change of the magnetic properties observed between Figs. 2(a) and 2(b).

2Fig. 3 ,

 3 Fig. 3, 21). In this sample, the local interacting field and coercivity remain relatively unaffected by the

Figures: Figure 1 :

 1 Figures:

Figure 2 :

 2 Figure 2:

Figure 3 :

 3 Figure 3:

First

  order reversal curve measurements: FORC diagrams, which were measured at the Laboratoire des Sciences du Climat et de l'Environnement (LSCE), Gif-sur-Yvette, France, are constructed by measuring partial magnetic hysteresis curves known as first-order reversal curves or FORCs (Pike et al, J. Appl. Phys. 85, 6660-6667 (1999), Roberts et al, J. Geophys. Res. 105, 28,461-28,475 (2000)). Starting at positive saturation, the applied field is decreased until a specified reversal field (Hr) is reached. A FORC is the magnetization curve measured at regular field steps from Hr back up to positive saturation. A large number of FORCs is measured, so that the FORCs fill the interior of a major hysteresis loop. The magnetization (M) on the FORC with reversal field Hr is denoted by M(Hr, H). The FORC distribution is defined as the mixed second derivative: which is well defined for H > Hr. When plotting FORC distributions on FORC diagrams, it is customary to change coordinates from {Hr, H} to Hc = (H -Hr)/2 and Hu = (H + Hr)/2 (Chen et al, J. Geophys. Res. 112, B08S90, doi:10.1029/2006JB004575 (2007). FORC diagrams provide an alternative method of measuring Preisach distributions, which gives information about the spectrum of coercivity and interaction field within a sample (26, 27). In the case of ideal single-domain (SD) grains and under the assumption that Hc and Hu are fixed for each grain but different from grain to grain, the FORC function is equivalent 23/06/17 to a Preisach function. According to Néel's interpretation (Néel, L.; Appl. Sci. Res. Sect B 4, 13-24 (1954)), the distribution along the Hc axis is equivalent in a first approximation to the distribution of particle microcoercivities, and a cross-section through the peak of the FORC distribution parallel to the Hu axis left of the central peak is equivalent to the distribution of magnetostatic interaction fields. However, the exact theory developed by Egli (Egli, Geophys. Res., 111, B12S17, doi: 10.1029/2006JB004567 (2006)) shows that the accurate representation of the coercivity distribution of SD particles is the marginal distribution (Winklhofer et al; J. Appl. Phys. 99, 08E710, doi:10.1063/1.2176598 (2006)). Measurements of FORC diagrams on natural and synthetic samples have provided the following interpretative framework (Roberts et al; J. Geophys. Res. 105, 28,461-28,475 (2000), Winklhofer et al; J. Appl. Phys. 99, 08E710, doi:10.1063/1.2176598 (2006), Muxworthy et al; J. Geophys. Res. 108(B11), 2517, doi: 10.1029/2003JB002588 (2003). SD particles are characterized by closed concentric contours about a central peak, while FORC diagram of multidomain particles go from contours that diverge away from the horizontal axis close to the origin, to nearly vertical contours with the peak close to Hc=0. Two sorts of FORC diagram features can be caused by interactions. Firstly, a shift of the FORC diagram maximum above or below the Hc=0 axis is caused by negative or positive mean interaction field, respectively (Stancu et al, J. Appl. Phys. 93, 6620-6622 (2003), Pike et al; J. Appl. Phys. 85, 6660-6667 (1999). Secondly, the spreading of the FORC contours parallel to the Hu axis indicates the presence of a local interaction field (Pike et al; J. Appl. Phys. 85, 6660-6667 (1999). One hundred FORCs were measured to construct each FORC diagram. The smoothing factor (Roberts et al; J. Geophys. Res. 105, 28,461-28,475 (2000)) was set at 3 for all the FORC diagrams.23/06/17 16. Yan, C., H.; Xu, Z. G.; Cheng, F. X.; Wang, Z. M.; Sun, L. D.; Liao, C. S.; Jia, J. T.; Solid State Commun. 111, 287 (1999). 17. Veverka, M.; Veverka, P.; Kaman, O.; Lanëok, A.; Závèvta, K.; Pollet, E.; Knízek, K.; Báhaèek, J.; Benes, M.; Kaspar, P.; Duguet, E.; Vasseur, S.; Nanotechnology 18, 345704 (2007). 18. Franco, A. Jr.; Zapf, V. J. Mag. Mag. Mat. 320, 709 (2008). 19. Tackett, R.; Sudakar, C.; Naik, R.; Lawes, G.; Rablau, C.; Vaishnava, P., P; J. Magn. Magn. Mater., 320, 2755 (2008). 20. Staniland, S.; Williams, W.; Telling, N.; Van Der Laan, G., Harrison, A.; Ward, B.; Nature Nanotechnology 3, 158 (2008). 21. Alphandéry, E., Ding, Y., Ngo, A.T., Wu, L.F., Pileni, M.P.; ACS Nano 3, 1539 (2009).22. We have injected a suspension of extracted chains of magnetosomes within tumor mice, which we have then heated by applying an alternative magnetic field and we have shown that this yielded anti-tumoral activity (the disappearance of the tumor 30 days after the treatment). For suspensions containing individual magnetosomes or whole bacteria injected within the tumor mice, we have not observed any anti-tumoral activity. 23. Alphandéry, E.; Ngo, A., T.; Lefevre, C.; Lisiecki, I.; Wu, L., F.; Pileni, M., P.; J. Phys. Chem. C 112, 12304 (2008).24. To measure the SAR, we use the formula

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Acknowledgment:

We thank Fériel Skouri-Panet for her help with the culture of the bacteria. We thank Eric Larquet and Jean-Michel Guigner for their help with TEM measurements.

Supplementary Figure 1:

EELS spectra of the extracted chains of magnetosomes synthesized in the absence of cobalt quinate, (a), or in the presence of cobalt quinate, (b).

Supplementary Figure 2:

FORC diagrams measured at 300 K of the whole magnetotactic bacteria synthesized in the absence of


Edouard Alphandéry
email: edouardalphandery@hotmail.com

Matthieu Amor

François Guyot

Imène Chebbi

The effect of iron chelating agents on Magnetospirillum magneticum strain AMB-1: stimulation of growth, magnetosome production and improvement of magnetosome heating properties

Keywords: Magnetosomes, magnetotactic bacteria, iron chelating agents, siderophore, magnetic hyperthermia, alternating magnetic field

The effect of iron-chelating agents on Magnetospirillum

magneticum strain AMB-1: stimulated growth and magnetosome production and improved magnetosome heating properties

INTRODUCTION

For a number of applications, it is advantageous to use large, single magnetic domain and wellcrystallized magnetic nanoparticles, which possess a ferrimagnetic behavior at room temperature and are not prone to aggregation [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field[END_REF]. Indeed, these types of nanoparticles possess a magnetic moment, which is stronger and more stable, than that of the smaller superparamagnetic magnetic nanoparticles, usually tested in biotechnological applications. Whereas monodomain nanoparticles, which are ferrimagnetic at room temperature, can hardly be produced chemically (Borderon et al 2011), certain species of bacteria, called magnetotactic bacteria (MTB), synthesize them.

The organelles made of vesicles embedding magnetic crystals are called magnetosomes. They are usually composed of magnetite in the bacteria but they can oxidize into maghemite after their extraction from the bacteria due to their contact with oxygen [START_REF] Alphandéry | Difference between the Magnetic Properties of the Magnetotactic Bacteria and Those of the Extracted Magnetosomes: Influence of the Distance between the Chains of Magnetosomes[END_REF]. In addition, the magnetosomes isolated from the bacteria are not prone to aggregation due to their chain arrangement and the lipidic bi-layered membrane surrounding the magnetic crystal [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field[END_REF][START_REF] Komeili | Cell biology of magnetosome formation[END_REF]. MTB use the magnetosomes as a compass to navigate in the direction of the Earth magnetic field and to presumably find with increased efficiency the optimum environment for their growth and survival [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. The magnetosomes have already been shown to be useful for a number of applications in the commercial, scientific or medical fields. For example, they can be used to extract DNA, to magnetically detect biomolecular interactions or to separate cells (Arakaki et al 2005). The anti-tumoral activity of a complex formed by bacterial magnetosomes and doxorubicin has also been demonstrated experimentally [START_REF] Sun | In vitro and in vivo antitumor effects of doxorubicin loaded with bacterial magnetosomes (DBMS) on H22 cells: The magnetic bionanoparticles as drug carriers[END_REF]. Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria have also been shown to be efficient to eradicate tumors. For that, they have been administered within tumors xeno-grafted under the skin of mice and heated under the application of an alternating magnetic field. In several mice, this treatment produced the disappearance of the tumor one month following the treatment [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field[END_REF]. To consider a commercial application of the magnetosomes, the production yield of the MTB still needs to be increased. It has recently been improved in several species of magnetotactic bacteria including Magnetospirillum magneticum strain 4 AMB-1 [START_REF] Matsunaga | Mass culture of magnetic bacteria and their application to flow type immunoassays[END_REF], Matsunaga et al 1996a, Matsunaga et al 1996b[START_REF] Matsunaga | Production of Luciferase-Magnetic Particle Complex by Recombinant Magnetospirillum sp[END_REF], Yang et al 2001a, Yang et al 2001b, Yang et al 2001b), Magnetospirillum magnetotacticum strain MS-1 [START_REF] Kundu | Enhancement of magnetotactic bacterial yield in a modified MSGM medium without alteration of magnetosomes properties Indian[END_REF] and Magnetospirillum gryphiswaldense strain MSR-1 [START_REF] Guo | A novel rapid and continuous procedure for large-scale purification of magnetosomes from Magnetospirillum gryphiswaldense[END_REF][START_REF] Heyen | Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor[END_REF][START_REF] Lang | Biogenic nanoparticles: production, characterization, and application of bacterial magnetosomes[END_REF][START_REF] Liu | Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density[END_REF][START_REF] Sun | High-yield growth and magnetosome formation by Magnetospirillum gryphiswaldense MSR-1 in an oxygen-controlled fermentor supplied solely with air[END_REF]. To date, the highest production yield, which has been achieved, is 55 mg of magnetosomes synthesized per liter of growth medium and per day [START_REF] Liu | Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density[END_REF].

In this article, we present a method, which can be used to stimulate the growth of MTB and to improve their production yield. This method uses iron chelating agents, which are introduced to the bacterial growth medium of AMB-1 magnetotactic bacteria. A series of different iron chelating agents are tested.

In addition, we also show that the introduction of iron chelating agents to the bacterial growth medium yields magnetosomes with increased magnetosome sizes and magnetosome chain lengths and results in improved magnetosome heating properties under the application of an alternating magnetic field.

MATERIALS AND METHODS

MTB belonging to the species Magnetospirillum magneticum, strain AMB-1, available at the ATCC under the reference 700264, have been cultivated in a volume of either 10 ml or 500 ml. A suspension containing either 100 l of  5.10 8 bacteria or 5 ml of  2.10 9 bacteria was first inoculated in 10 ml or 500 ml of ATCC bacterial growth medium 1653 respectively. The cells were cultivated in microanaerobic conditions, i. e. in a culture medium that had not been degased, but that was closed and thus not in contact with air. The culture of MTB was carried out in an incubator at 26 C using a slightly modified ATCC medium 1653, whose composition is given by the ATCC. The pH of the culture medium was adjusted to 6.85 by using a 5 M solution of sodium hydroxide. The suspensions containing the various chelating agents (EDTA, ethylenediaminetetracetic acid, rhodamine B, ascorbic acid, erythrosine, anthranilic acid, citric acid, 3-(N-morpholino)propanesulfonic acid, 3-(cyclohexylamino)-1propanesulfonic acid, calcein, dextran, alendronic acid, neridronic acid, nicotinamide) mixed in water were then inserted within the bacterial growth medium. The concentration of iron chelating agents contained within the bacterial growth medium was varied between 0.4 M and 400 M.

After incubation, two different types of suspensions containing either whole inactive MTB or extracted chains of magnetosomes mixed in water were prepared. To prepare the suspensions containing the whole MTB, the cells were harvested by centrifugation at 4 000 g during 20 minutes at room temperature. The supernate was removed and the cells were resuspended in milli-Q water. To extract the chains of magnetosomes, 2 ml of cell suspension obtained as previously described was centrifugated again and resuspended in a 10 ml tris-HCl buffer of pH 7.4. The cellular suspension was then sonicated during 120 minutes at 30 W in order to lyse the cells and collect the chains of magnetosomes. After sonication, the suspension containing the chains of magnetosomes was separated from the cellular debris by positioning a strong neodymium magnet (0.1-1 T) next to the tube, and the magnetic material was then harvested. The supernate containing the cellular debris and other organic molecules was eliminated.

The chains of magnetosomes were washed 10 to 20 times in deionized water at pH 7.4 and were then resuspended in 500 L of milli-Q water.

In order to obtain a sufficiently large amount of chains of magnetosomes, the MTB were first cultivated in a volume of 500 ml either in the absence or in the presence of various chelating agents. They were centrifugated (4000 g, 20 min) after 7 days of growth. The supernate was then removed and the bacteria were resuspended in 2 ml of mili-Q-water. For the various suspensions of MTB, the absorption was measured at 565 nm [START_REF] Heyen | Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor[END_REF] and the number of bacteria was estimated using a Beckman Coulter Z1 DT. The concentrations in maghemite of the various suspensions of extracted chains of magnetosomes were measured by absorption at 480 nm [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field[END_REF].

The growth curves of the MTB grown either in the absence or in the presence of the various iron chelating agents were measured for the MTB cultivated in 10 ml elongated tubes allowing the presence of an oxygen gradient similar to that encountered in natural environments. The bacteria were centrifugated 1 day, 2 days, 3 days, 4 days, 7 days, 8 days and 9 days following the inoculation of the MTB. The supernate was removed and the bacteria were resuspended in 1 ml of mili-Q-water. The growth curves of the various suspensions of whole MTB were then measured during the ten days following the inoculation by measuring both the absorption at 565 nm [START_REF] Heyen | Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor[END_REF] and the number of bacteria using a Beckman Coulter Z1 DT. In order to evaluate the presence (or not) of the magnetosomes within the bacterial growth medium, the magnetic moments of the different suspensions of magnetotactic bacteria were measured. For that, 600 l of the various suspensions of MTB were deposited on top of non magnetic absorbing paper, which was then inserted within a capsule of gelatin.

This capsule was positioned in a SQUID. The magnetic moment of the MTB was estimated under the application of a magnetic field of 1000 Oe.

Transmission electron microscopy (TEM) studies were carried out using a JEOL 2100F with field emission gun. For the TEM measurements, 5 l of a suspension of whole MTB was deposited on top of a copper grid covered with a thin amorphous carbon film. TEM measurements were used to measure the sizes of the magnetosomes and to estimate the lengths of the chains of magnetosomes in bacteria cultivated either in the absence or in the presence of iron chelating agents.

The maghemite composition of the magnetosomes was determined by measuring the saturation isothermal remanent magnetization curves of the magnetotactic bacteria cultivated in various conditions following the same method as that described previously [START_REF] Alphandéry | Difference between the Magnetic Properties of the Magnetotactic Bacteria and Those of the Extracted Magnetosomes: Influence of the Distance between the Chains of Magnetosomes[END_REF]. The absence of the Verwey transition indicates that the magnetosomes are oxidized into maghemite.

The heating curves of suspensions containing 300 l of extracted chains of magnetosomes mixed in water with a concentration in maghemite of 478 g/ml were measured. The maghemite composition of the chains of magnetosomes extracted from AMB-1 magnetotactic bacteria was estimated following a method previously described [START_REF] Alphandéry | Difference between the Magnetic Properties of the Magnetotactic Bacteria and Those of the Extracted Magnetosomes: Influence of the Distance between the Chains of Magnetosomes[END_REF]. The concentration in maghemite of the different suspensions of extracted chains of magnetosomes was measured at 480 nm. To generate heat, the suspensions of extracted chains of magnetosomes were exposed to an alternating magnetic field of frequency 198 kHz and average field amplitude of 20 mT using a 10 kW EasyHeat power supply from Ambrell, Soultz, France. The average field strength within the coil was measured using a 2 D probe (Suppl. Fig. 1). The temperature of the suspensions was measured using a thermocouple microprobe (IT-18, Physitemp, Clifton, USA).

RESULTS

Figure 1 shows the optical absorption, measured at 565 nm, of suspensions of MTB cultivated either in the absence or in the presence of various iron chelating agents and harvested after 7 days of growth.

About 70 % of the iron chelating agents tested produces an increase of the absorption of the suspension of MTB. This indicates that in most cases the presence of iron chelating agents within the bacterial growth medium stimulates the growth of the MTB. The growth stimulation is observed at low concentrations of iron chelating agents, which lie below 40 M (Figure 1). The most pronounced growth stimulations are observed for hemoglobin at concentration 40 M, citric acid at concentration 4 M, and hemoglobin at concentration 4 M for which the optical absorptions of the suspension of MTB is 1.5 to 2.5 times larger than that of the bacteria cultivated in the absence of iron chelating agents. Figure 2 shows the concentration in maghemite of the suspensions containing chains of magnetosomes extracted from whole MTB harvested after seven days of growth and cultivated in the same conditions as those presented in Figure 1. Since the magnetosomes studied here are oxidized into maghemite [START_REF] Alphandéry | Difference between the Magnetic Properties of the Magnetotactic Bacteria and Those of the Extracted Magnetosomes: Influence of the Distance between the Chains of Magnetosomes[END_REF], the concentration in maghemite, which is measured, is equal to the concentration in magnetosomes. We therefore deduce from Figure 2 that the production of magnetosomes is increased for a majority of iron chelating agents tested ( 65 %) and for concentrations in iron chelating agents, which lie below 40 M. The strongest enhancement of the magnetosome production is observed when 0.4 M or 4 M of hemoglobin is introduced within the bacterial growth medium. In this case, enhancements of the magnetosome production by factors of 6 are achieved. It is also worth mentioning that the concentration in iron chelating agent, which produces the largest growth stimulation, for example 40 M for hemoglobin (Figure 1), is not always the same as that, which yields the largest magnetosome production (for example 4 M for hemoglobin).

The influence of introducing iron chelating agents to the bacteria growth medium on several MTB and magnetosome properties is studied next. We describe the results obtained with an iron chelating agent, rhodamine B, which shows a typical behavior, i. e. which stimulates well both the growth of the MTB and the production of the magnetosomes. Figure 3(a) shows the growth curves of MTB cultivated either in the absence of rhodamine B or in the presence of different concentrations of rhodamine B. In the presence of 0.4 M, 4 M or 40 M of rhodamine B introduced to the bacterial growth medium, the growth of MTB is more rapid than in the absence of rhodamine B. The concentration of 400 M of rhodamine B does not stimulate the growth of MTB. The growth stimulation induced by the presence of rhodamine B is significant during the 3 or 4 first days of growth, i. e. during the exponential phase. After that, the bacteria reach the stationary phase and then the absorption of the different suspensions of bacteria decreases most probably due to the death of the bacteria (Suppl. Fig. 2). In order to determine the rate at which the magnetosomes grow, the magnetic moments of the suspensions of MTB cultivated in the absence or in the presence of various concentrations of rhodamine B were measured for an applied magnetic field of 1000 Oe. Before the fourth day of growth, the magnetic moments of the various suspensions of bacteria were not detected indicating the absence or undetectable concentrations of magnetosomes in these suspensions. At days 4, 7 and 8, the magnetic moments of the suspensions containing MTB cultivated in the presence of 0.4 M, 4 M or 40 M of rhodamine B were detected.

The magnetic moments of the suspensions containing MTB cultivated in the absence of rhodamine B were detected at a later stage during the ninth day of growth, indicating that the production of the magnetosomes is more rapid for the MTB cultivated in the presence of rhodamine B than for those cultivated in the absence of rhodamine B. The stimulations of the growth of the bacteria and of the production of the magnetosomes were observed for other iron chelating agents introduced within the bacterial growth medium, such as ascorbic acid (Suppl. Fig. 3).

The magnetosome sizes and magnetosome chain lengths of the extracted chains of magnetosomes were measured for the MTB cultivated in the absence of rhodamine B and for those cultivated in the presence of 4 M of rhodamine B (Figure 4). The distributions in magnetosome sizes and magnetosome chain lengths are bimodal. For the magnetosomes of small sizes, the magnetosome sizes and magnetosome chain lengths increase from an average of  27.5 nm and  150 nm in the absence of iron chelating agent up to an average of  35 nm and  200 nm in the presence of 4 M rhodamine B, respectively (Figures 4(a)-(d)). The largest magnetosomes of the longest chains increase from an average of  42.5 nm and  750 nm in the absence of rhodamine B up to an average of  65 nm and  800 in the presence of rhodamine B, respectively (Figures 4(a)-(d)). The increase of the magnetosome sizes and magnetosome chain lengths was also observed in the presence of other iron chelating agents such as EDTA (Suppl. Fig. 4).

Three suspensions containing 300 l of extracted chains of magnetosomes with a concentration in maghemite of 478 g/ml were then heated under the application of an alternating magnetic field of frequency 198 kHz and average magnetic field strength of 20 mT. These suspensions contain chains of magnetosomes synthesized either in the absence or in the presence of 4 M Rhodamine B or 4 M EDTA. Figure 5

DISCUSSION

We have shown that the presence of iron chelating agents within the bacterial growth medium stimulates the production of the magnetosomes. The function of the iron chelating agents is to chelate Fe 3+ iron ions and to enhance the bioavailability of iron by forming Fe 3+ -ligand complexes. The iron chelating agents could be involved in one or several of the following steps of the magnetosome formation, (i) the formation of the vesicle in which the magnetosomes are formed, (ii) the iron uptake by the cells, (iii) the iron transport within the cells and (iv) the control of the biomineralization of the magnetosome formation in Fe3O4 or Fe2O3 [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. They could therefore favor the magnetosome formation by making each or several of these steps more efficient.

We examine why the presence of iron chelating agents stimulates the growth of the bacteria. The mechanisms, which enhance the production of the magnetosomes and those, which stimulate the growth of the bacteria, are expected to be different. The MTB are believed to synthesize siderophores, which help to incorporate iron within the bacteria (Calugay 2006). In the absence of such complexing agents, Fe 3+ iron ions precipitate mostly as ferrihydrite and their bioavailability is likely strongly decreased.

When iron chelating agents are introduced to the bacterial growth medium, the MTB have less need to synthesize siderophores. It is thus likely that the energy allocated for siderophore synthesis can then be used for bacterial growth. This could explain the more efficient growth observed in the presence than in the absence of iron chelating agents.

We also observe that the highest stimulation of the bacteria is observed for large iron chelating agents, such as hemoglobin. This result suggests that iron is chelated in a more efficient way using high molecular weight than low molecular weight iron chelating agents. This is in agreement with previous study [START_REF] Reichard | Dissolution mechanisms of goethite in the presence of siderophores and organic acids[END_REF]. There are several possible explanations for this behavior. The stability formed by a large iron complex could be higher than that formed by a smaller iron complex. A large iron complex may also more easily enter within the periplasm of the cells. It is also possible that a large iron complex would more easily release iron within the bacterial cells or that it would favor oxidoreduction reactions involved in the magnetosome production or crystallization process.

We have shown that above a concentration in iron chelating agent lying between 40 M and 400 M, the stimulation of the growth of the MTB and of the production of the magnetosomes is not observed. This result agrees with previous reports, which showed that for a ratio of 100 to 500 between the iron chelating agent and iron concentrations, no growth stimulation was observed for the species MSR-1 [START_REF] Schüler | Iron-limited growth and kinetics of iron uptake in Magnetospirillum gryphiswaldense[END_REF]. This indicates that the iron chelating agents are toxic above concentrations lying between 40 M and 400 M. We propose two hypotheses to explain this result. Either a high iron chelating agent concentration yields a high iron concentration within the bacterial growth medium, which is toxic for the magnetotactic bacteria. Or the reactive oxygen species, which are formed because of the alcohol functional groups getting unbound from iron, for example in rhodamine B, damage the membrane of the bacteria.

The kinetics of the magnetosome formation is reported for the AMB-1 species. For this species, the magnetosomes are probably mainly formed after the magnetotactic bacteria have reached the stationary phase [START_REF] Yang | Synthesis of Bacterial Magnetic Particles During Cell Cycle of Magnetospirillum magneticum AMB-1[END_REF]. We have shown that the introduction of iron chelating agents within the bacterial growth medium of AMB-1 magnetotactic bacteria yields faster growth of magnetosomes. This should therefore enable a significant increase in the rate of production of the magnetosomes by the AMB-1 magnetotactic bacteria. In strain MSR-1, the magnetosomes are produced earlier, before the bacteria have reached the stationary phase [START_REF] Heyen | Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor[END_REF]. This behavior could be explained by a different mechanism of iron uptake or of magnetosome formation between these two species of magnetotactic bacteria. Therefore, the influence of introducing iron chelating agents within the bacterial growth medium on growth rate of magnetosome production may be less pronounced for the MSR-1 than for the AMB-1 species.

The introduction of iron chelating agents to the bacterial growth medium also increases the magnetosome sizes and magnetosome chain lengths, which results in an improvement of the magnetosome heating properties. The specific absorption rate of suspensions of extracted chains of magnetosomes increases by a factor of 3 between the MTB cultivated in the absence of iron chelating agent and those cultivated in the presence of 4 M EDTA. High values of SAR are advantageous to carry out magnetic hyperthermia treatments of cancers since they enable the use of a small quantity of magnetosomes and/or the application of an alternating magnetic field of low strength, hence decreasing the risks of toxicity.

Further studies should explore separately the effect of iron on either the bacterial growth or the production of magnetosomes.

In conclusion, the introduction of iron chelating agents within the bacterial growth medium enables the stimulation of the growth of the MTB, the enhancement of the production yield of the magnetosomes, the increase of the magnetosome sizes and magnetosome chain lengths as well as the enhancement of the magnetosome heating properties under the application of an alternating magnetic field.

FIGURES

  (a) shows that the absorption curves of the three suspensions are the same, indicating that their concentration is identical. The variations of temperature as a function of time of the three suspensions are shown in Figure 5(b). The increase in temperature is more important in the presence of 4 M rhodamine B or 4 M EDTA than in the absence of iron chelating agent. The slopes, measured at 25 C, of the variations of temperature with time shown in Figure 5(b), designated as T/t, increase from T/t  0. 005 C/sec. in the absence of iron chelating agent up to T/t  0.011 C/sec in the presence of 4 M Rhodamine and 0.017 C/sec in the presence of 4 M EDTA. Using the relation between the specific absorption rate (SAR) and (T/t), which is given by SAR = Cv(T/t), where Cv = 4.2 J/(g.K) is the specific heat capacity of water, we find that the SAR increases from  222 W/gFe in the absence of iron chelating agent up to  444 W/gFe in the presence of 4 M rhodamine B and  723 W/gFe in the presence of 4 M EDTA.

Figure 1 :

 1 Figure 1: Absorption measured at 565 nm and number of magnetotactic bacteria per ml of suspensions

Figure 2 :

 2 Figure 2: The concentration in maghemite of suspensions containing chains of magnetosomes extracted

Figure 3 :

 3 Figure 3: (a) The growth curves of magnetotactic bacteria cultivated either in the absence of rhodamine

Figure 4 :

 4 Figure 4: (a), (b) The magnetosome size distribution of the magnetosomes contained within the

Figure 5 :

 5 Figure 5: (a) The absorption curves of three suspensions of extracted chains of magnetosomes. The

ACKNOWLEDGMENTS

F. Guyot and M. Amor do not claim any inventive contribution in this work. They only analyzed experimental results. Yang CD, Takeyama H, Tanaka T, Matsunaga T (2001a) Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1 Enz. Microb. Tech. 29:13-19. Yang C, Takeyama H, Matsunaga T (2001b) Iron Feeding Optimization and Plasmid Stability in Production of Recombinant Bacterial Magnetic Particles by Magnetospirillum magneticum AMB-1 in Fed-Batch Culture J. Bioscience Bioeng. 2:231-216. 19 20