Sophie Tencé
email: sophie.tence@icmcb.cnrs.fr

Birgit Heying

Oliver Janka

Rainer Pöttgen
email: pottgen@uni-muenster.de

Bernard Chevalier
email: bernard.chevalier@icmcb.cnrs.fr

Hydrogenation-induced cerium valence change in CeNiZn

Keywords:

Polycrystalline CeNiZn (ZrNiAl type, P _ 62m, a = 715.6(4), c = 388.4(2) pm) forms a new hydride CeNiZnH1.2(1) under hydrogen exposure (10 bar) at 423 K. The resulting hydride crystallizes in a hexagonal structure (P6/mmm) with the unit cell parameters a = 433.19(2) and c = 424.37(2) pm. Thus, hydrogen atom insertion yields a structural transition from a ZrNiAl-to an AlB2-type structure with a high volume cell expansion of +20.1 %. The magnetic, thermal and transport properties of CeNiZnH1.2(1) were investigated. Susceptibility and specific heat measurements do not reveal magnetic order down to 1.8 K but most likely the presence of ferromagnetic correlations due to the proximity of a ferromagnetic order or a nonmagnetic atom disorder spin-glass state.

Moreover, the cerium valence is modified from an intermediate to a trivalent state, a change that results from the decrease of the hybridization strength between conduction and Ce(4f) electrons upon hydrogenation. Transport measurements also evidence a deviation from the typical resistivity behavior of metallic systems. Several scenarios are conceived to explain this change, in particular weak localization effect, knowing that it rarely occurs upon hydrogenation of intermetallics.

Introduction

The equiatomic cerium intermetallics CeTX (T = transition metal; X = element of the 3 rd , 4 th , or 5 th main group) cover more than 100 representatives with different crystal structures and broadly varying magnetic and electrical properties [1][2][3]. The physical properties of these materials depend on the valence electron concentration and the structure type. Several CeTX phases show pressure and / or temperature driven structural phase transitions along with distinct changes in the physical properties. These structure property relationships have recently been reviewed [1][2][3].

An interesting subgroup of the CeTX intermetallics concerns the CeTZn compounds.

Zinc occupies the position typical for a p element, underlining the covalent bonding within the two-or three-dimensional [TZn] networks. CeCuZn and CeAgZn [4] crystallize with the KHg2 type structure with random occupancy of the copper (silver) and zinc atoms on the mercury site. In contrast, CePtZn [5,6] and CeAuZn [7,8] show complete Pt (Au) / Zn ordering. Both compounds order antiferromagnetically below TN = 1.7 K [5][6][7][8][9]. CeNiZn [7,10] and CePdZn [10,11] crystallize with the hexagonal ZrNiAl type. The nickel compound shows intermediate cerium valence. CePdZn is dimorphic. The hightemperature modification adopts the orthorhombic TiNiSi type. This has consequences on the magnetic ground state. The low-temperature modification shows no magnetic ordering down to 2 K while the high-temperature modification orders antiferromagnetically at TN = 3.2 K [11]. Electronic structure calculations and the magnetic data point to larger Ce(4f)-Pd(4d) hybridization for the low-temperature type phase.

CeRhZn (orthorhombic LaNiAl type) [12] is one of the remarkable CeTZn compounds. Its crystal structure contains two crystallographically independent cerium sites and temperature dependent magnetic susceptibility measurements indicate Pauli paramagnetism, in line with an almost tetravalent state for both sites.

The magnetic ground state of CeTX intermetallics can be effectively influenced through hydrogenation. The studies on CeTXHx phases with X = p element of the 3 rd , 4 th or 5 th main group are summarized in [1][2][3]. We have now included the zinc compounds into these systematic studies. Herein we report on the hydrogenation behavior of intermediate valent CeNiZn and describe the structural, magnetic, thermal and transport properties of the CeNiZnHx phase.

Experimental

Synthesis

Starting materials for the preparation of the CeNiZn precursor were cerium pieces (Sigma-Aldrich, > 99.9 %), nickel powder (Merck, 99+) and zinc granules (Merck, > 99.9 %). Pieces of the cerium ingot were first arc-melted [13] to a small button under an atmosphere of ca. 700 mbar. The argon was purified over titanium sponge (900 K), silica gel and molecular sieves. The cerium button, nickel powder and pieces of the zinc granules (36 : 32 : 32 molar ratio) were then arc-welded in a tantalum ampoule. The latter was sealed in an evacuated quartz tube for oxidation protection, placed in a chamber furnace (Nabertherm P330) and heated to 1590 K within 6 h. This high temperature was kept for 10 min, followed by cooling to 920 K at a rate of 6 K min -1 . The temperature was kept for another 6 h followed by cooling to room temperature within 2 h. The polycrystalline sample could easily be separated from the container by mechanical fragmentation. CeNiZn is highly brittle and shows bright metallic lustre.

Hydrogen absorption experiments were performed by solid-gas reaction. An ingot of the CeNiZn precursor was crushed in small pieces prior to being introduced into a stainless steel container. The sample was activated under vacuum at 423 K for 2 hours and then exposed to a pressure of 10 bar of hydrogen gas at the same temperature for 1 day. The amount of hydrogen absorbed was determined volumetrically by monitoring pressure changes in a calibrated volume. After hydrogenation, we observe the decrepitation of the pieces into fine powder. The hydride is stable in air over months.

X-Ray diffraction

The polycrystalline CeNiZn precursor sample was checked by powder X-ray diffraction (XRD) (Enraf-Nonius FR552 Guinier camera) using CuK1 radiation and quartz (a = 491.30 and c = 540.46 pm) as an internal reference substance. The refined lattice parameters (a = 715.6(4), c = 388.4(2) pm) are in good agreement with earlier literature data [7,10]. The hydride CeNiZnHx was analysed by X-ray powder diffraction with a Philips1050-diffractometer using Bragg-Brentano geometry with CuKα radiation.

The crystal structure was refined by the Rietveld method using the FullProf program [14].

Physical property studies

Magnetic properties of the samples were investigated using a MPMS-XL Squid magnetometer from Quantum Design. Measurements were performed on free powder in the temperature range 1.8-300 K and under magnetic fields up to 7 T.

For the heat capacity measurements, one piece of the crushed pellet was fixed to a pre-calibrated heat capacity puck using Apiezon N grease to ensure thermal conductivity.

Measurements were realized with a PPMS from Quantum design in the temperature range of 2.0 to 300 K under applied external fields of 0, 0.1, 1 and 5 T.

Resistivity measurements were carried out with a PPMS from 2 to 325 K using a ac four probe method. Powder of CeNiZn and CeNiZnHx was cold pressed to form pellets on which electrical contacts were glued with silver paint.

Results and discussion

Structural aspects

At 423 K and exposed to 10 bars of H2 gas, CeNiZn uptakes hydrogen till reaching a pressure plateau in around 35 min corresponding to an absorbed H amount of 1.2(1) H/f.u.

(formula unit). Thus, this indicates the potential formation of the hydride CeNiZnH1.2(1).

The X-ray powder diffractograms of the precursor CeNiZn and the compound obtained after hydrogenation are shown in Fig. 1. The XRD pattern of CeNiZn is well indexed on the basis of the hexagonal ZrNiAl-type structure (space group P _ 62m), in agreement with data reported earlier in the literature [7,10]. In this structure, Ce atoms occupy the 3f (0.41576 0 0) site, Ni atoms 1a (0 0 0) and 2d (2/3 1/3 1/2) sites and Zn the 3g (0.76498 0 ½) site, as determined from single crystal data. After hydrogen exposure, the compound displays a completely different pattern, confirming the effective hydrogen uptake in CeNiZn without any sign of decomposition. The diffractogram of the resulting hydride CeNiZnH1.2 is perfectly indexed with the hexagonal space group P6/mmm, with the unit cell parameters a = 433.19 (2) and c = 424.37( 2) pm. The small extra peaks correspond to the presence of the Ce2Ni2Zn impurity already observed in the pristine compound as well as residual non-hydrogenated CeNiZn. A Rietveld refinement of the data was performed on the basis of the AlB2-type structure by analogy with the CeNiXHx hydrides (X = Al, Ga) that show similar X-ray patterns [15][16][17]. This Rietveld refinement (RBragg = 5.0 %) is depicted in Fig. 1. The Ce atoms are located in the 1a (0 0 0) site whereas the Ni and Zn atoms are statistically distributed on the 2d (1/3 2/3 1/2) site. However, it is noteworthy that the refinement of the structure considering an alternation of the Ni and Zn atoms within the (ab)-plane, i.e. with the LiBaSi-type structure (P _ 6m2), is only slightly worse since both elements have close x-ray scattering factors. The simple AlB2-type structure is built up of face-sharing trigonal prisms of Ce atoms [Ce6] filled either by Ni or Zn atoms in their centre. Thus, this structure contains [Ce3Ni] or [Ce3Zn] tetrahedra as potential interstitial sites for hydrogen atoms. It is noteworthy that [Ce3Ni] tetrahedra are also found in the ZrNiAl-type precursor CeNiZn. The positions of the hydrogen atoms could not be refined from X-ray data but we can assume that they are preferentially located in the 4h sites [Ce3Ni] (or [Ce3Zn]) tetrahedra. This hypothesis is based on neutron diffraction results obtained for the CeNiInD1.24 deuteride that report D atoms location in this position inducing short D-D interatomic distances of around 160 pm [18]. However, Shashikala et al. also mentioned the additional sites 6k and 6i for deuterium atoms in the CeNiAlD3.2 deuteride crystallizing in the AlB2 type structure [19]. These tetrahedral interstices are formed by 2 atoms of Ce and 2 atoms of Ni/Zn and are filled with a lower occupancy than the 4h one. Thus, we can assume that the 4h site is filled first, and then the 6k and 6i positions are occupied for higher H/D insertion rate. Neutron powder diffraction measurements will be needed to check this hypothesis for CeNiZnH1.2 and to determine more accurately the hydrogen composition within the structure.

The hydrogenation of CeNiZn induces a drastic increase of the molar volume from 0.0574 to 0.0690 nm 3 , i.e. an increase of +20.1 %. Additionally, it yields a structural transition from the ZrNiAl to the AlB2-type structure. Interestingly, similar high volume expansion and structural change upon hydrogenation (+19.7%) was also evidenced in the isotypic intermetallic uranium compound UNiZn [20]. In this case, the crystal structure of the resulting hydride UNiZnH2.3 was not unambiguously solved between the AlB2-type structure and a derivative of this structure with an ordering of the Ni and Zn atoms. But, the survival of the ZrNiAl-type structure was clearly ruled out the from x-ray diffraction analysis. In both U and Ce compounds, the observed structural modification is coherent with the high volume expansion. Indeed, the unit cell expansion exceeding 10-15% induces this type of structural transition as for CeNiGa (low-temperature form) or CeNiAl, whereas lower volume increase (<10-15%) keeps the initial ZrNiAl structure type as for CeNiIn [15][16][17].

The high volume expansion in the sequence CeNiZn → CeNiZnH1.2 results in increasing the interatomic distances in the structure, except for the shortest M-M distance of 250 pm, M being Ni or Zn (see Table 1). In the precursor CeNiZn, the shortest distances, found between the Ni and Zn atoms, were equal to 257 pm. However, in the hydride, these distances still remain slightly longer than the sum of the covalent radii (240 pm for Ni+Zn). A spectacular effect concerns the strong augmentation of the Ce-Ce distances from 371-389 to 424-433 pm. All these distances are much longer than those found in Ce metal (365 pm [START_REF] Teatum | Los Alamos Report[END_REF]) or in cerium hydride CeH2 (394 pm [START_REF] Knappe | [END_REF]). The Ce-M distances between Ce and M atoms also strongly increase upon hydrogenation, going from 289-322 pm in CeNiZn to 328 pm in the hydride. These distances variations suggest that f electrons could be more localized and the Ce valence state modified after hydrogen absorption.

Magnetic, thermal and transport properties

The temperature dependence of the magnetic susceptibility of CeNiZn and CeNiZnH1.2 measured in the field of 3 T is presented in Fig. 2. The curve of CeNiZn shows a rather temperature-independent behavior as reported earlier for this intermediatevalent cerium compound [7]. The small bump around 25 K, not observed in ref [7], could be due to the presence of a small amount of the Ce2Ni2Zn impurity observed in XRD. On the contrary, the hydride displays much higher values of the susceptibility and follows a Curie-Weiss behavior above 30 K (see inset of Fig. 2). The downward curvature in inverse susceptibility below 30 K might be due to crystal-field effects. The linear fit of data in the temperature range 25-200 K gives an effective moment µeff =2.49 µB/Ce atom and a paramagnetic Curie temperature θp = -15 K (Table 2). The effective moment close to that of the free Ce 3+ ion (2.54 µB) suggests that the cerium is trivalent and that Ni atoms do not carry magnetic moments. This means that hydrogenation induces a cerium valence change from intermediate-valent to a trivalent state, which is coherent with the strong increase of the Ce-Ni and Ce-Zn interatomic distances from CeNiZn to CeNiZnH1.2. For CeNiZnH1.2, the susceptibility measurement at 0.05 T (Fig. 3) shows an enhancement of χ(T) below 5 K but without clear evidence of any magnetic ordering down to 1.8 K. The small anomaly around 8 K may be attributed to some impurity since no peak is visible at this temperature on the specific heat measurement (Fig. 4). However, the negative curvature of the magnetic isotherm M(H) at 1.8 K (inset of Fig. 3) may indicate the existence of ferromagnetic correlations. It is noteworthy that this behavior cannot be accounted for the presence of a small quantity of a ferromagnetic impurity. The magnetization at 1.8 K reaches almost 1 µB/Ce atom at 7 T. At higher temperatures the negative curvature of the M(H) isotherms progressively disappears and at 15 K the curve is linear. These observations suggest that, at low temperature, CeNiZnH1.2 could be close to a long-range ferromagnetic order or to a nonmagnetic atom disorder spin glass state. A spin glass behavior could arise from the atomic disorder on the Ni/Zn site and on the H site that would yield a random exchange interaction between the Ce atoms. This was observed in the AlB2-type germanides Ce2NiGe3 and Ce2CuGe3 in which a random distribution of the Ge and Ni or Cu atoms on the 2d crystallographic site is also reported [23,24]. The M(H) isotherms at low temperatures for these two germanides are very similar to those measured on CeNiZnH1.2. However, the absence of a peak in the ZFC curve of the hydride at 20 Oe (not shown here) does not allow us defining a freezing temperature Tf as for Ce2NiGe3 (Tf = 3.5 K [23]) or Ce2CuGe3 (Tf = 3 K [24]).

The specific heat measurement in zero magnetic field shown in Fig. 4 clearly indicates the absence of a λ-peak characteristic of a magnetic order down to 2 K. However, the Cp(T) curve reveals a small upturn below 8 K which seems to reach a maximum around 2 K. This maximum becomes more visible with increasing fields, especially at 5 T. This behavior suggests the existence of a nonmagnetic atom disorder spin glass state or a ferromagnetic state. On the plot of the Cp/T versus T 2 , we observe a linear region above approximately 12 K. The fit of the data with the formula Cp/T = γ +βT 2 between 12 and 24 K at zero field gives the values γ = 44 mJ.mol -1 K -2 and β = 0.478 mJ.mol -1 K -4 (Table 2). The β coefficient, associated to the phonon contribution to the specific heat, gives an estimation of the Debye temperature θD of 230 K (considering only the 3 non-hydrogen atoms since hydrogen atoms are usually involved in higher energy optical phonons modes). The moderate γ value suggests that the Kondo effect is weak in the hydride, in agreement with the small paramagnetic Curie temperature θp. Nevertheless, below 12 K, the Cp/T curve strongly increases, reaching a value of 1120 mJ.mol -1 K -2 at 2 K. Such apparently large electronic coefficients γ are usually found in heavy-fermions or in spin glass systems [25] as for example in the Ce2NiGe3 and Ce2CuGe3 spin glasses in which Cp/T even attains a maximum [23,24]. It was also observed in the hydrides Ce2Ni2MgH7.7 [26] and CeNiGaH1.1 [17], the last one crystallizing also in the AlB2-type structure. The rather high γ value was associated to the existence of a strongly correlated system at low temperature in the former hydride and of a spin glass state in the latter one. However, in CeNiZnH1.2, this γ value depends on the magnetic field since it is well reduced to 800 mJ.mol -1 K -2 at 5 T (see inset of Fig 4). Therefore, it is reasonable to consider that this high γ value is not ascribed to a heavy-fermion character manifestation since, usually, γ is rather insensitive to the magnetic fields in heavy-fermion systems.

The resistivity measurements of the precursor CeNiZn and of the hydride are displayed on Fig. 5. The data were normalized to the value at 325 K because the use of compacted pellets for the measurements yields the presence of porosities and microcracks that hamper the accurate determination of ρ(T). The resistivity of CeNiZn behaves as a normal metal as typically expected for an intermediate-valent Ce compound with a high spin fluctuations temperature Tsf. On the contrary, the electrical resistivity of CeNiZnH1.2 presents different characteristics: ρ(T) rises continuously with decreasing temperature in the whole temperature range 1.8-325 K with a more pronounced augmentation below 25 K. This behavior is not typical to that of a classical metal but more similar to that of a disordered Kondo system or a Kondo insulator as CeNiSn for example [27]. However, by plotting ln(ρ) versus 1/T we do not clearly observe a linear region at low temperature allowing us to define a band gap Eg (maybe due to the lack of pellet densification). Besides, a Kondo insulator state implies the absence of structural disorder and of a long-range magnetic order ground state [28], ground state which is not ruled out in our hydride. Thus, it is more likely that the drastic change in the transport properties originates from structural disorder. This could arise from the random distribution on the Ni/Zn atoms on the 2d site and on the partial filling of the H site. This hypothesis includes the case of a disordered Kondo lattice in which disorder hampers the establishment of the low-temperature coherent regime, regime below the Kondo temperature TK where the resistivity drops with decreasing temperature. In contrast, in the incoherent regime, the magnetic part of the resistivity diverges logarithmically as the temperature decreases. A similar resistivity curve as that of CeNiZnH1.2 was observed in the hydride Ce2Ni2MgH7.7 [26]. But as such a behavior was also reported for the isotypic compound La2Ni2MgH8 [29], this suggests that the Kondo effect is not directly responsible for the non-metallic character of Ce2Ni2MgH7.7. Another possibility to consider is the weak localization of electron waves induced by atomic disorder. In this case, the electron motion experiences numerous random scatterings on scattering centers.

The resulting "circle-like manner" scattering paths give rise to interference phenomena which leads to an increase in the resistivity. This effect can be well reduced by the application of an external magnetic field. Therefore, further resistivity measurements under magnetic fields are planned in order to provide more evidences of weak localization effect in CeNiZnH1.2.

It is important to note that metal to non-metal transitions induced by hydrogenation occur rarely. Interestingly, such a transition was evidenced in the hydrides MAlSiH [30] and MGaXH [31] (M = Ca, Sr, Ba; X= Si, Ge, Sn) which crystallize in a structure closely related to the AlB2-type. In those systems, the calculated density of states (DOS) clearly revealed an indirect band gap opening upon hydrogenation with Eg values between 0.1 and 0.8 eV. It was ascribed to the removal of the partly filled antibonding π* band in the metal having a predominantly Ga or Al p orbital character that yields an energetically low Ga(Al)-H bonding band. Thus, it turns out that hydrogen captures electron density, leading to hydride formation with covalent Ga(Al)-H bonding. However, it is unlikely that a similar scenario takes place in CeNiZnH1.2 since the Sommerfeld coefficient suggests a significant density of state at the Fermi level and thus the conservation of a metallic state.

Conclusions

The intermetallic CeNiZn forms the new hydride CeNiZnH1.2 upon exposure to 10 bar of H2 gas at 423 K. The hydrogenation of CeNiZn induces a structural transition from a ZrNiAl-type to an AlB2-type structure that goes along with a drastic unit cell volume expansion (+20.1 %) and a strong increase of the interatomic distances within the structure. As a result, the Ce(4f) electrons are more localized, the hybridization strength between conduction and f electrons decreases and the Ce valence changes from an intermediate to a trivalent state. According to magnetic and specific heat measurements, the hydride does not display magnetic order down to 2 K but is most likely close to a ferromagnetic order or to a spin-glass behavior as ground state. This second option could arise from the random distribution of the Ni and Zn atoms on the same Wyckoff position and on the partial filling of the H site inducing crystallographic disorder in the structure and thus the presence of competitive indirect RKKY (Rudermann-Kittel-Kasuya-Yosida) interactions between Ce atoms. Further magnetic and specific heat measurements at lower temperatures would be helpful to elucidate the ground state of CeNiZnH1.2.

Interestingly, hydrogen insertion also provokes a deviation from the classical metallic behaviour of the resistivity. In CeNiZnH1.2 it most likely results in atomic structural disorder leading to disordered Kondo lattice effect or weak localization of the electrons, as possible scenario. Further resistivity measurements under magnetic fields are planned in order to provide more evidences of weak localization effect in CeNiZnH1.2. Besides, it would be fruitful to better evidence the role of the structural disorder on the resistivity behavior notably. One possible way would be to modify the hydrogen amount in CeNiZnHx via different hydrogenation conditions. Such modifications of the transport properties, which are not often observed upon hydrogen insertion, can be interesting for applications as optical mirrors or hydrogen detector if they are reversible around room temperature.

Fig. 1 .Fig. 2 .

 12 Fig. 1. X-ray powder diffraction patterns of CeNiZn and CeNiZnH1.2. Indexation of the pattern on the basis of the AlB2-type structure is indicated for the hydride and Rietveld refinement is shown by a solid line. Stars symbols indicate the main visible peaks of the residual pristine compound.

Fig. 3 .

 3 Fig. 3. Temperature dependence of the susceptibility of CeNiZnH1.2 measured at 0.05 T. Inset: Magnetization isotherms of CeNiZnH1.2 at various temperatures.

Fig. 4 .

 4 Fig. 4. Temperature dependence of the specific heat of CeNiZnH1.2 measured at 0, 0.1, 1 and 5 T.The inset presents the Cp/T versus T 2 curve at 0 and 5 T with the linear fit Cp/T = γ+βT 2 in dotted line for zero magnetic field as mentioned in the text.

Fig. 5 .

 5 Fig. 5. Temperature dependence of the electrical resistivity of CeNiZn and CeNiZnH1.2, normalized to the value at 325 K.

Table 1 .

 1 Interatomic distances (pm) of CeNiZn and CeNiZnH1.2.

	For CeNiZn, Ni1 and Ni2

Table 2 .

 2 µeff, θP, γ and β fitting values of CeNiZn and CeNiZnH1.2 obtained as explained in the text from the Curie-Weiss fit of the susceptibility curve and from the low-temperature specific heat measurement. The data for CeNiZn are taken from ref[5].

		µeff	θp	γ	β
		(µB)	(K)	(mJ.mol -1 .K -2 )	(mJ.mol -1 .K -4 )
	CeNiZn	0.29	-2.8	-	-
	CeNiZnH1.2	2.49	-15	44	0.478

Acknowledgments

Authors gratefully acknowledge R. Decourt for his help for electrical measurements.


Edouard Alphandéry
email: edouardalphandery@hotmail.com

Ahmed Idbaih

Clovis Adam

Jean-Yves Delattre

Charlotte Schmitt

François Guyot

Imène Chebbi

Chains of magnetosomes with controlled endotoxin release and partial tumor occupation induce full destruction of intracranial U87-Luc glioma in mice under the application of an alternating magnetic field

Keywords: Magnetosomes, magnetotactic bacteria, magnetic hyperthermia, alternating magnetic field, glioblastoma

Previous studies have shown that magnetic hyperthermia could efficiently destroy tumors both preclinically and clinically, especially glioma. However, antitumor efficacy remained suboptimal and therefore required further improvements. Here, we introduce a new type of nanoparticle synthesized by magnetotactic bacteria called magnetosomes with improved properties compared with commonly used chemically synthesized nanoparticles. Indeed, mice bearing intracranial U87-Luc glioma tumors injected with 13 µg of nanoparticles per mm 3 of tumor followed by 12 to 15 of 30 minutes alternating magnetic field application displayed either full tumor disappearance (40% of mice) or no tumor regression using magnetosomes or chemically synthesized nanoparticles, respectively. Magnetosome superior antitumor activity could be explained both by the increased production of heat and by endotoxin release under the alternating magnetic field application. Most interestingly, this behavior was observed when magnetosomes occupied only 10% of the tumor volume, which suggests that an indirect mechanism, such as an immune activity, is involved in tumor regression. This is desired for the treatment of infiltrating tumors, such as glioma, for which whole tumor coverage by nanoparticles can hardly be achieved.

INTRODUCTION

A series of different nanotechnologies, such as lipid-based nanoformulations, polymer-drug conjugates, recombinant viruses, metallic nanoparticles, drug-targeted antibodies, and herbal nanoparticles, have been successfully developed for cancer treatment both at fundamental and industrial levels [START_REF] Alphandéry | Cancer therapy using nanoformulated substances: scientific, regulatory and financial aspects[END_REF]. Among them, technologies in which nanoparticle anti-tumor activity can be controlled by applying an external source of energy, such as a laser (2), [START_REF] El-Sayed | Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles[END_REF], radiation (4), [START_REF] Her | Gold nanoparticles for applications in cancer radiotherapy: Mechanisms and recent advancements[END_REF], ultrasound ( 6), [START_REF] You | Nanoparticle-enhanced synergistic HIFU ablation and transarterial chemoembolization for efficient cancer therapy[END_REF], or magnetic field [START_REF] Bañobre-López | Magnetic nanoparticle-based hyperthermia for cancer treatment[END_REF], [START_REF] Jordan | The effect of thermotherapy using magnetic nanoparticles on rat malignant glioma[END_REF] appear promising because they can potentially enhance both treatment safety and efficacy. Strategies to increase nanoparticle SAR by combining magnetic hyperthermia with photothermia using magnetoplasmonic nanohybrids have also been suggested [START_REF] Schwartzbaum | Epidemiology and molecular pathology of glioma[END_REF], [START_REF] Uk Cr | Types of primary brain tumors[END_REF]. There is interest in developing this type of nanotechnology for glioblastoma (GBM) treatment, especially for the most severe GBM cases, which are non-operable. With the current standard of care, i.e., maximal safe surgery, concurrent radiochemotherapy and adjuvant chemotherapy, GBM, which affects 25,000 patients per year in the United States and Europe [START_REF] Ostrom | The epidemiology of glioma in adults: a "state of the science[END_REF], [START_REF] Stupp | Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial[END_REF], remains incurable with a poor prognosis. The five-year survival time of patients with GBM is low at 10% [START_REF] Billecke | Polynuclear platinum anticancer drugs are more potent than cisplatin and induce cell cycle arrest in glioma[END_REF], [START_REF] Ross | Ethynyldeoxyuridine (EdU) suppresses in vitro population expansion and in vivo tumor progression of human glioblastoma cells[END_REF], demonstrating the need for new treatment options [START_REF] Yang | Treating glioblastoma multiforme with selective high-dose liposomal doxorubicin chemotherapy induced by repeated focused ultrasound[END_REF], [START_REF] Safdie | Fasting Enhances the Response of Glioma to Chemo-and Radiotherapy[END_REF], [START_REF] Bidwell | Thermally Targeted Delivery of a c-Myc Inhibitory Polypeptide Inhibits Tumor Progression and Extends Survival in a Rat Glioma Model[END_REF], [START_REF] Titsworth | Fighting Fire with Fire: The Revival of Thermotherapy for Gliomas[END_REF], [START_REF] Jiang | Combination Therapy with Antiangiogenic Treatment and Photodynamic Therapy for the Nude Mouse Bearing U87 Glioblastoma[END_REF]. Among the potential treatments for GBM are different types of nanothermotherapies [START_REF] Rabias | Rapid magnetic heating treatment by highly charged maghemite nanoparticles on Wistar rats exocranial glioma tumors at microliter volume[END_REF], such as photodynamic therapy [START_REF] Shinkai | Intracellular hyperthermia for cancer using magnetite cationic liposomes[END_REF] and magnetic hyperthermia, which have been tested to treat GBM [START_REF] Jordan | The effect of thermotherapy using magnetic nanoparticles on rat malignant glioma[END_REF]. Compared with other thermotherapies, magnetic hyperthermia [START_REF] Ito | Heat-inducible TNF-alpha gene therapy combined with hyperthermia using magnetic nanoparticles as a novel tumor-targeted therapy[END_REF], [START_REF] Ohno | Effective Solitary Hyperthermia Treatment of Malignant Glioma Using Stick Type CMC-magnetite. In vivo Study[END_REF], [START_REF] Le | Preparation of Tumor-Specific Magnetoliposomes and Their Application for Hyperthermia[END_REF], [START_REF] Maier-Hauff | Intracranial thermotherapy using magnetic nanoparticles combined with external beam radiotherapy: results of a feasibility study on patients with glioblastoma multiforme[END_REF], [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], in which tumors are typically heated to 43-50 °C by intratumor administration of iron oxide nanoparticles followed by alternating magnetic field (AMF) application, appears promising. This is due to more localized heat produced by the nanoparticles, which results in enhanced efficacy, and to tumor destruction achieved at lower temperatures, which strengthens treatment safety. In a clinical trial that enrolled 14 patients with GBM, magnetic hyperthermia treatment performed using small chemically synthesized iron oxide nanoparticles (IONP) was well-tolerated by patients, did not produce major side effects, and led to an increased survival time of 7 months compared with conventional treatments [START_REF] Alphandéry | Chains of Magnetosomes Extracted from AMB-1 Magnetotactic Bacteria for Application in Alternative Magnetic Field Cancer Therapy[END_REF], [START_REF] Alphandéry | Preparation of chains of magnetosomes, isolated from Magnetospirillum magneticum strain AMB-1 magnetotactic bacteria, yielding efficient treatment of tumors using magnetic hyperthermia[END_REF].

Treatment efficacy could potentially be further improved by using chains of magnetosomes (CM), which are iron oxide nanoparticles synthesized by magnetotactic bacteria, instead of IONP. In previous studies, CMs have been shown to destroy MDA-MB-231 breast tumors subcutaneously xenografted under the skin of mice more efficiently than IONPs [START_REF] Alphandéry | The effect of iron-chelating agents on Magnetospirillum magneticum strain AMB-1: Stimulated growth and magnetosome production and improved magnetosome heating properties[END_REF], [START_REF] Alphandéry | Heat Production by Bacterial Magnetosomes Exposed to an Oscillating Magnetic Field[END_REF]. Magnetosome efficacy was attributed to the large magnetosome size, leading to stable ferrimagnetic properties and to a large amount of heat produced under AMF application, as well as to an arrangement of chains that prevented aggregation and promoted homogenous magnetosome distribution in the tumor [START_REF] Alphandéry | Use of bacterial magnetosomes in the magnetic hyperthermia treatment of tumours: A review[END_REF], [START_REF] Alphandéry | Applications of Magnetosomes Synthesized by Magnetotactic Bacteria in Medicine[END_REF], [START_REF] Bordelon | Magnetic nanoparticle heating efficiency reveals magneto-structural differences when characterized with wide ranging and high amplitude alternating magnetic fields[END_REF], [START_REF] Grüttner | Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy[END_REF].

In the present study, anti-tumor activity against U-87-Luc GBM was studied both in vitro and in vivo with a suspension containing CMs. We first demonstrated that the amount of endotoxins released from the CMs can be controlled under AMF applications. CM cytotoxicity and internalization properties in the presence of U87-Luc cells were also studied in vitro. Mice bearing intracranial U87-Luc tumors were then treated under conditions that could be followed clinically, i.e., using an AMF with 30 mT and 198 kHz (H.f = 8 10 8 A.m -1 .s -1 , below the threshold of 5.10 9 A.m -1 .s -1 , above which Eddy currents may occur) [START_REF] Maier-Hauff | Intracranial thermotherapy using magnetic nanoparticles combined with external beam radiotherapy: results of a feasibility study on patients with glioblastoma multiforme[END_REF], [START_REF] Cui | A quantitative study of factors affecting in vivo bioluminescence imaging[END_REF]. Treatment parameters, such as the number of magnetic sessions and quantity of CMs administered, that lead to complete disappearance of the intracranial U87-Luc tumors were determined and compared with IONP treatment, which has been previously used to perform preclinical magnetic hyperthermia tumor treatments [START_REF] Sun | Firefly luciferasebased dynamic bioluminescence imaging: a noninvasive technique to assess tumor angiogenesis[END_REF], [START_REF] Hashizume | A human brainstem glioma xenograft model enabled for bioluminescence imaging[END_REF]. During the various treatments, nanoparticle distribution in the tumors and the presence of different cell types in the brain were also examined by histology. This study also suggests a mechanism for tumor destruction and verified that the brain tumor disappeared fully following magnetic hyperthermia treatment with CMs.

MATERIALS AND METHODS

Preparation of the injectable CM suspension

Magnetospirillum magneticum AMB-1 magnetotactic bacteria at biosafety level 1 were purchased from ATCC (79024 strain). Five milliliters of a suspension containing 4.10 6 of these bacteria per milliliter were introduced into one liter of sterile 1653 ATCC culture medium containing nutrients and additives, such as minerals, vitamins, and an iron source, as listed in the 1653 ATCC medium, which is required for the proliferation of these bacteria and for magnetosome production. The bottles were completely filled with sterile medium to prevent contamination by other bacterial species and to avoid a high oxygen concentration in the growth medium, which would prevent magnetosome production. The media containing the bacteria were then placed in an incubator at 30 °C for 7 days to enable bacterial growth and magnetosome production. After 7 days, the media was centrifuged at 4000 g for 45 minutes. The bacterial pellet was washed using 1 ml of sterile water (Millipore®). Magnetotactic bacteria were concentrated using a strong Neodinium magnet (0.6 Tesla), re-suspended in 0.05 M TRIS and sonicated with a sonicating finger at 0 °C for 2 hours at 30 W. The suspension containing the chains of magnetosomes extracted from the magnetotactic bacteria was washed several times with sterile water using a magnet to isolate the CMs from the supernatant containing cellular debris and residual bacteria until the cellular debris disappeared from the supernatant. Sonication was performed at 30 W by a series of three 2-second pulses between each wash. The CMs were then re-suspended in 1 ml of sterile water.

For the intracranial injections, the CMs were re-suspended in a sterile injectable solution containing 5% glucose and exposed to ultraviolet lamp (UV) irradiation for 12 h for partial sterilization.

Preparation of IONPs:

IONPs, which are starch-coated iron oxide nanoparticles, were purchased from Micromod Partikeltechnologie GmbH company, Friedrich-Barnewitz-Str. 4, D-18119, Rostock, Germany (ref: 10-00-102).

Nanoparticle Characterization:

Nanoparticle sizes, shapes, and organization were determined using a transmission electron microscope (JEM-2100, JEOL, Japan). The iron concentrations of the different nanoparticle suspensions were measured by mixing these suspensions with hydrochloric acid and hydrogen peroxide to produce Fe 3+ ions complexed with potassium thiocyanate, and total iron was then determined via an absorbance measurement at 476 nm. The concentrations were expressed as mg of maghemite contained in the CMs and IONPs per ml of suspension or per mm 3 of tumor. The zeta potentials of the different nanoparticles in suspension were measured by dynamic light scattering (DLS, ZEN 3600, Malvern Instruments, UK).

The stability of the nanoparticle suspensions was estimated by measuring the changes in the optical densities of these suspensions, which were measured at 476 nm for 15 min. Nanoparticle FTIR spectra were recorded with a FTIR spectrometer (Vertex 70, Bruker, USA). The amount of organic material at the nanoparticle surface was measured using an elemental CHNS analyzer (Flash EA 1112, Thermo Fisher Scientific, USA). More details about each of these techniques are provided in the supplementary materials section.

Set-up for generating the alternating magnetic field and for measuring the temperature

We used an induction system operating at 198 kHz to generate the alternating magnetic field. Mice, Eppendorf tubes, petri dishes containing nanoparticles and possibly cells were inserted into a coil with a 7-cm diameter with four spires to generate a magnetic field with an average strength of 30 mT. We used an infrared camera (EasIR-2, Optophase, France) positioned 20 cm above the coil to measure the temperature both in vitro and in vivo. We verified the temperature with a thermocouple positioned at the tumor center and that the infrared camera measured the temperature at the tumor center. This was possible due to the shallowness of the tumor.

Amount of endotoxins released under AMF application

Suspensions containing 2 µl of CMs or IONPs at a concentration of 20 mg/ml in iron oxide were treated as follows. Nanoparticle suspensions were exposed to 0, 1, 2, 3, and 4 magnetic sessions, during which an AMF of 198 kHz and average strength of 30 mT was applied for 30 minutes. After AMF applications, 100 µl of nonpyrogenic water was added to the suspensions, and the suspensions were mixed for one minute and centrifuged at 13 000 rotations per minute for 15 minutes. The endotoxin concentration of the supernatant was measured using the Limulus amebocyte lysate (LAL) assay.

Materials and cells culture

U87-MG Luc human GBM cell lines, which were transduced with a luciferase gene, were obtained from Keith Ligon's lab and used in this study. After thawing, the U87-MG Luc adherent cells were cultivated at 37 °C in the presence of 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Once the cells reached confluence, the culture dishes were rinsed with Hank's Balanced Salt Solution (HBSS). The cells were then detached with trypsinization for 5 minutes at 37 °C in the presence of 5% CO2. The trypsin activity was stopped by the addition of medium containing FBS.

The cellular concentration was then determined using a Malassez cell.

U87-Luc cytotoxicity in the absence of AMF using an MTT assay

U87-Luc cytotoxicity studies were evaluated using an MTT assay. This technique measures the ability of mitochondrial enzymes to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to purple formazan crystals. Cells were seeded at a density of 10 4 cells per well in 96-well flat-bottom plates and incubated in completed culture medium for 24 h. Then, the U87-Luc cell medium was removed and replaced with 10% FCS-medium containing CMs or IONPs at varying concentrations ranging from 7.8 µg/ml to 1000 µg/ml in maghemite. After 24 h of incubation, the cells were washed with HBSS and incubated with 0.1 ml of MTT (2 mg/ml) for an additional 4 h at 37 °C. The insoluble product was then dissolved by the addition of 100 ml of isopropanol. The absorbance corresponding to the solubilized formazan pellets, which reflect the relative viable cell numbers, was measured at 540 nm using a microplate reader. We measured the absorbance at 540 nm for the cells with the nanoparticles without formazan blue and we have subtracted this signal from the signal for the cells with nanoparticles with formazan blue. The percent inhibition of cell proliferation, inhibition (%), was then estimated using the following formula: % inhibition = [1-(ODTC/ODUC)] × 100, where ODUC is the optical density of the suspension of untreated cell, i.e., cells that have been washed with PBS and have not been in the presence of nanoparticles, measured at 540 nm, and ODTC is the optical density of the suspension of treated cells, i.e., nanoparticle suspensions that have been added to cells, measured at 540 nm. The percent inhibition of cell proliferation was plotted as a function of the nanoparticle concentration. The experiments were performed in triplicate.

In vitro efficacy of hyperthermia

A total of 500,000 U87-Luc cells were seeded in a Petri dish (35 mm) for 24 hours. Then, 2 ml of IONP and CM suspensions were added to the cells at different concentrations varying between 1 mg/ml, which is sufficient to induce an increase in temperature increase, and 40 µg/ml, which corresponds to the maximum magnetosome concentration in the tumor, i.e., 40 µg of magnetosomes per mm 3 of tumor.

When the same amount of nanoparticles used, we observed an increase in the temperature in solution but not in vitro, possibly due to the lower concentrations in vitro (2 ml at 20 µg/ml) than in solution (2 µl at 20 mg/ml). Next, these assemblies were exposed (or not) to a 30 mT and 198 kHz AMF for 30 minutes. The cells were then incubated with the nanoparticles for 24 h. The nanoparticle media was removed, and the cells were washed twice with cold PBS. We verified that there were no observable nanoparticle aggregates at the cell surface and that the nanoparticles were mostly internalized in cells by optical microcopy. It is possible that nanoparticles that were not observed with the optical microscope remained at cell surface, meaning that the measurement provides an estimate of the maximum quantity of internalized nanoparticles or the amount of nanoparticles internalized in cells or at the cell surface.

Next, 250 µl of trypsin were added to detach the cells, and then the culture media was mixed with cells.

Following this in vitro treatment, the mixtures were centrifuged at 800 rpm for 7 minutes. The supernatant was removed and replaced with 100 µl of annexin-binding buffer, 5 µl of Alexa Fluor 488 Annexin V for early apoptosis detection, and 1 µl of propidium iodide at 100 µg/ml for late apoptosis and necrosis detection. The mixtures were incubated at room temperature for 15 minutes. Then, 400 µl of annexin-binding buffer was added to this cell suspension and mixed gently. Ten microliters of this suspension were loaded into the sample slide and were inserted completely into a Countess™ II FL Automated Cell Counter (Thermo Fisher scientific), which was able to detect the Annexin and Propidium Iodide emissions. The percentages of necrotic and apoptotic cells were thus obtained.

Internalization of nanoparticles

Following the in vitro treatments described in 2.7, the different mixtures were loaded into the sample slide and completely inserted into a Countess™ II FL Automated Cell Counter to count the cells. The mixtures were then centrifuged, and the supernatant was removed and replaced with 286 µl of HNO3 (70%); the resulting treated mixtures were maintained at 4 °C for 24 h to lyse cells and dissolve the nanoparticles into free iron. Finally, 10 ml of filtered water was added to all the treated mixtures and the iron concentration was then determined using ICP-AES measurements. We deduced the average quantity of iron coming from the nanoparticles, which was internalized in each cell.

In vivo experiments

In vivo experiments were performed following ethical guidelines and surgery was performed following the guidelines of the Institutional Animal Care and Use Committee ("Ethic committee Charles Darwin N°5", project ethic number: 00764.03). Mice were fed and watered according to these guidelines and euthanized by cervical dislocation when their weights had decreased by more than 20% or when signs of pain, unusual posture or prostration were observed. At day 0 (D0), a cell suspension containing 10 5 U87-Luc cells per microliter was first inoculated into the brains of 7-week-old CD-1 female nude mice with a mean weight of 20 g from Charles River. To achieve the surgical procedure leading to cell implantation, the mice were first anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (8 mg/kg). The mouse head was then fixed in a stereotactic frame, a craniotomy was performed at the fixed coordinates (0.2.0) and 2 µl of a suspension containing 2.10 5 U87-Luc cells were injected at the coordinates (0.2.2).

Tumors grew for 8 days between D0 and D8. At D8, 7 different groups containing 10 mice each were treated as follows (Table S1(a)):

-Group 1 received a 30 mm 3 GBM at coordinates (0.2.2) mm and 2 µl of an isotonic solution containing 5% glucose without any further treatment; -Group 2 received a 5 mm 3 GBM at coordinates (0.2.2) mm and the same glucose solution as For the different groups of mice, the bioluminescence intensity emitted by the living tumor cells was measured on D7, D14, D21, D28, D35, D42, D49, D56 and D150.

During each magnetic session (S1 to S15), the mice were exposed to an AMF at an average strength of 30 mT and a frequency of 198 kHz that was applied for 30 minutes.

Bioluminescence intensity and tumor volume measurements

The mice were first anesthetized using isoflurane. The bioluminescence intensity was then measured using an IVIS Spectrum "In Vivo Imaging System" (PerkinElmer, Inc.) 10 minutes after intraperitoneal injection of 100 mg/kg of beetle luciferin potassium salt (Promega: E1605) in PBS, which reacts with luciferase to produce luminescent oxo-luciferin, (39), ( 40), [START_REF] Zhou | Dynamic Near-Infrared Optical Imaging of 2-Deoxyglucose Uptake by Intracranial Glioma of Athymic Mice[END_REF]. Images were analyzed with the living Image 4.0 software from Caliper, Life Sciences. The bioluminescence intensity at the tumor site was divided by the bioluminescence intensity measured in the flanks to remove the mouse autoluminescence signal. Then, the relationship between tumor bioluminescence intensity and tumor volume was determined. For that, bioluminescence intensities were measured at D7, D14, D21, D28 and D35 in a series of living mice. The tumor volumes were deduced from surface measurements of several horizontal histological sections positioned at various tumor heights, which were collected from mice euthanized on the same day at the BLI measurements. A linear relationship was observed between the logarithm of tumor bioluminescence intensity (BLI), log(BLI), and the logarithm of the tumor volume expressed in mm 3 , log (volume(mm 3 ))(Fig. S3). A linear coefficient of 1.4 was deduced by fitting the plot in Fig. S3 with a linear function, a value that agrees with previously reported values (40), ( 42), [START_REF] Kim | Macitentan, a Dual Endothelin Receptor Antagonist, in Combination with Temozolomide Leads to Glioblastoma Regression and Longterm Survival in Mice[END_REF], [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].

Representation of tumor volume variations and estimates of survival time

The variations in tumor volume in time were represented in Tukey diagrams using the Kaleida Graph 4.03 software. Mouse survival times are plotted using the Kaplan-Meier model method [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF], [START_REF] Branquinho | Effect of magnetic dipolar interactions on nanoparticle heating efficiency: Implications for cancer hyperthermia[END_REF].

The statistical significance of the survival time between the different groups was evaluated using the log rank test. Parameters are expressed as the median with p-values (9).

Temperature measurements

Intratumor temperatures were measured as a function of time during the various treatments in living mice using an infrared camera (EasIRTM -2 , Optophase) placed 20 cm above the coil.

Histological analysis

Histological studies were performed on extracted brains that were fixed with a 4% solution of formaldehyde for 24 hours, cut into 2-mm thickness transverse slices, washed in an ethanol (70%) bath for 12 hours and included in paraffin. Sections of paraffin blocks with a thickness of 4 µm were deposited on glass slides and stained with hematoxylin-eosin (H&E) and/or Prussian blue to distinguish between healthy and tumor areas and to determine the location of the magnetosomes, polynuclear neutrophils (PNN), and tumor and healthy cells. Histological analysis was also performed on the mice that received glucose on day 8 and were euthanized 6 or 24 hours following glucose injection (Figs.

RESULTS AND DISCUSSION

Properties of CMs and IONPs used for the tumor magnetic hyperthermia treatments

CMs were extracted from AMB-1 whole magnetotactic bacteria, a species of bacteria that produces chains of nanoparticles called magnetosomes, as shown in the TEM image of a typical magnetotactic bacterium presented in Figure 1 Magnetosomes are composed of a core, which is made of iron oxide, as revealed by the FT-IR Fe-O peak at 580 cm -1 (table 1) and further determined to be maghemite by saturating isothermal remnant magnetic measurements [START_REF] Grüttner | Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy[END_REF]. The core is surrounded by a coating of organic material, including phospholipids and lipopolysaccharides, as revealed by a series of FT-IR peaks [C=O at 1740 cm -1 , amide at 1650 cm -1 , amine at 1530 cm -1 , P=O at 1250 cm -1 , and PO at 1050 cm -1 (Table 1)]. The coating, whose function is to bind magnetosomes together in chains [START_REF] Casella | Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant[END_REF], is estimated to be 1 to 5 nm thick (Figure 1(b)) and to contain 13.9% carbon, as estimated by CHNS analysis. LAL measurements further indicated that the organic material at the magnetosome surface is partially derived from endotoxins whose concentration in CM suspensions is estimated as 1400-8400 EU per ml per mg of iron oxide. Moreover, CM suspensions appear sufficiently stable to enable their in vivo administration, as their absorption measured at 480 nm does not decrease by more than 30% within 20 minutes (Table 1). Compared with CMs, IONPs are also ferromagnetic iron oxide nanoparticles but characterized by the following distinct features. Their geometric structure is parallelepipedic, they are not organized in chains and tend to form small aggregates as observed in the TEM image in Figure 1(c). INOPs are made of a core measuring ~19 nm by TEM [START_REF] Astiz | Pretreatment of normal humans with monophosphoryl lipid A induces tolerance to endotoxin: a prospective, double-blind, randomized, controlled trial[END_REF], and whose composition is iron oxide as determined by FT-IR measurements that display an Fe-O peak at 610 cm -1 (Table 1). The IONP core is surrounded by synthetic hydroxyethyl starch, as suggested by FT-IR and CHNS measurements performed on the IONPs, which shows FT-IR peaks at 1150 cm -1 and 1050 cm -1 that are attributed to hydroxyethyl starch ether and alcohol functional groups, and has a carbon percent of 8.7%, revealing the presence of an organic coating surrounding the inorganic iron oxide core (Table 1). Compared with CMs, the IONP surface contains a much lower endotoxin concentration of 50 EU per ml per mg of iron oxide, as measured with the LAL assay. Furthermore, IONPs are characterized by ferrimagnetic properties at physiological temperatures but at the following lower values: (1) Hc  120 Oe and (2) Mr/Ms  0.15 [START_REF] Astiz | Pretreatment of normal humans with monophosphoryl lipid A induces tolerance to endotoxin: a prospective, double-blind, randomized, controlled trial[END_REF], (Fig. S6 and Table 1), which should yield lower SAR values. Furthermore, given that they are not less stable than CMs (Table 1) and that their anti-tumor efficacy has been demonstrated on tumorbearing mice using magnetic hyperthermia [START_REF] Liu | Effects of DMSA-Coated Fe3O4 Nanoparticles on the Transcription of Genes Related to Iron and Osmosis Homeostasis[END_REF], IONPs appear to be good standard for comparison to assess CM efficacy.

Under AMF applications, CM can release heat and an immune-stimulating substance more efficiently than IONPs

In an attempt to mimic the conditions in the in vivo treatments and to study the release of endotoxins from the CM and IONP surfaces in these conditions, we introduced 2 µl of suspensions containing 40 µg of these nanoparticles in iron oxide at the bottom of a tube that we exposed to 1, 2, 3, and 4 magnetic sessions during which an AMF of 198 kHz and average strength 30 mT was applied for 30 minutes.

During each of these 4 magnetic sessions, CM and IONP suspensions led to maximal temperatures (Tmax) of 57 C and 28 C, respectively, after 3 minutes of AMF application (Fig. S2(a)). Specific absorption rates (SAR) were deduced from the formula SAR=Cwater(T/t)/Cm, where T/t are the initial slopes of the plot in Fig. S2(a) [T/t = 0.27 °C/sec and 0.05 °C/sec for CM and IONP, respectively], Cwater= 4.2 J/g.K is the specific heat capacity of water, and Cm = 0.02 g/mL is the concentration in iron oxide of the different nanoparticle suspensions. The SARs were estimated as 57 ± 6 W/gFe and 10 ± 3 W/gFe for CMs and IONPs, respectively. Therefore, the heating properties of CMs appear far superior to those measured for IONPs. Moreover, the percentage of endotoxin release, which remains relatively similar at 1 to 3% during the first 2 magnetic sessions, strongly increased to 11% and 32% following the third and fourth magnetic sessions, respectively (Fig. 2(a)). This indicates that endotoxins, which are initially at the magnetosome surface, are progressively released from CMs under AMF application. Using CMs exposed to AMF, it therefore appears possible to simultaneously produce heat and release an immune-stimulating substance such as an endotoxin. Although endotoxins cannot be used in medicinal preparations, they may be replaced by a nontoxic equivalent, such as M-PLA, (51), [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. Combining the production of localized heat with the release of such a substance may possibly enhance antitumor efficacy. Compared with CMs, IONPs released a lower quantity of endotoxins and their percentage of endotoxin release did not exceed 5% between 1 and 4 S (Fig. 2(a)). Taken together, these heating and endotoxin release behaviors suggest that IONPs are less promising than CMs for the magnetic hyperthermia and endotoxin treatment in tumors.

CMs induce enhanced U87-Luc early apoptotic cell death and internalization in vitro

In the absence of the AMF, the cytotoxicity of CMs towards U87-Luc cells was compared with that of IONPs using nanoparticle suspensions that were added to the cells and incubated for 24 and 72 hours at concentrations between 40 µg/mL and 1 mg/mL. At 24 hours, the percent inhibition of cell proliferation increased with increasing nanoparticle concentrations from 0 to 40% for CM and remained at 0% for the IONPs (Figure 2(c)). At 72 hours, the percent inhibition increased more significantly for CMs, i.e., from 0 to 60%, than for IONPs, i.e., from 0 to 100% (Figure 2(d)). While CMs were observed to be more cytotoxic than IONPs at 24 hours, the opposite behavior was observed at 72 hours.

U-87-Luc cells were also incubated for 24 hours with CMs and IONPs at three different concentrations of 40, 650, and 1000 µg/ml and then exposed to an AMF of 198 kHz and strength 47 mT for the first 30 seconds and 30 mT for the remaining 29.5 minutes in an attempt to reach 41 C, a temperature above which mild hyperthermia conditions are expected to occur [START_REF] Alphandéry | The effect of iron-chelating agents on Magnetospirillum magneticum strain AMB-1: Stimulated growth and magnetosome production and improved magnetosome heating properties[END_REF]. The mechanisms of cellular death induced by CMs and IONPs were further examined. CMs appear to induce cellular death mainly through early apoptosis, with a percentage of early apoptotic cell death that increased with increasing CM concentrations from 15% at 40 µg/ml to 50% at 1 mg/ml. Similar percentages were observed for CMs with/without AMF (Fig. 2(e)); this cell death mechanism was not observed with IONPs (Fig. 2(f)).

The differences in cell death mechanisms observed for IONPs and CMs as well as the more pronounced decrease in heating efficacy for CMs than IONPs observed between the in vitro and in suspension heating conditions may be explained by the different internalization properties. Indeed, the quantity of iron released from the nanoparticles, which are internalized in each cell, increased from 36.8 and 5.

CMs promote enhanced antitumor efficacy towards intracranial U87-Luc glioblastoma

In the in vivo studies, mice receive the following treatment at the center of intracranial U87-Luc tumors:

(1) glucose followed by treatment or not with magnetic sessions (groups 1 and 2), (2) 40 µg of CMs or IONPs in maghemite without any further treatment (groups 3 and 6), (3) 40 µg of CMs in maghemite followed by 15 magnetic sessions (groups 4 and 5), or (4) 40 µg of IONPs in maghemite followed by 12 magnetic sessions (group 7). The average size of the treated tumors was 3-5 mm 3 , except in group 5

where it was 25 mm 3 . Following injection, the mice were exposed to 12 to 15 magnetic sessions, during which an AMF of 198 kHz and strength 30 mT was applied for 30 minutes (see Table S1(a) for a summary of the treatment conditions). In this study, U87 cells were labeled with the firefly luciferase gene to evaluate the changes in tumor volumes following the treatments via tumor bioluminescence intensity (BLI) measurements using the relationship between BLI and tumor volumes that we established (Fig. S3) (40), ( 42), ( 43), [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]. For the mice belonging to the different groups without any measurable production of heat (groups 1, 2, 3, 6, and 7), Figures 3(a) to 3(e) show that the tumor volumes increased following tumor cell implantation without any signs of antitumor activity. Mice belonging to these groups were rapidly euthanized between days 28 and 42, leading to a rather low median survival time of 42 to 46 days. In contrast, in the mice belonging to groups with heated tumors (groups 4 and 5), antitumor activity is clearly observed and is more pronounced in the small (group 4) than in the large (group 5) treated tumors. On day 150, 40% of the mice from this group were still alive without any unusual behaviors. Then, the mice were euthanized and slides of their brains from the tumor cell implantation region were imaged by optical microscopy, revealing the absence of tumors or tumor lesions and a brain neuronal structure identical to that of a healthy brain (Figure 6). These results suggest that the tumors had fully disappeared in these mice. Further analysis of mice from group 4 suggests that treatment does not result in any healthy tissue damage. Indeed, it can be deduced from the optical microscopy images of the brain Thus, the treatment of the mice in group 4 appears safe and fully efficient. In contrast with CMs, IONPs do not produce any heat in vivo under the same excitation conditions. This together with rather low SAR may undermine the IONPs' heating power, as was observed in vivo.

CM exposed to AMF activate PNN, which are possibly involved in antitumor activities

Previous studies have reported that immune cells, such as dendritic cells, natural killers, and B and T cells, may participate in tumor destruction following hyperthermia treatment [START_REF] Asín | Induced cell toxicity originates dendritic cell death following magnetic hyperthermia treatment[END_REF]. Here, we examined potential involvement of other types of immune cells, i.e., polynuclear neutrophils (PNN), which have only rarely been associated with antitumor activities (57), [START_REF] Liang | Neutrophils Promote the Malignant Glioma Phenotype through S100A4[END_REF]. While PNNs were not detected after glucose (Fig. S5) or IONP (Fig. S4) administration with/without magnetic sessions, we observed that these cells were localized at the same sites as the magnetosomes, forming a complex designated M-PNN in the mouse euthanized 4 hours after CM administration with/without magnetic sessions (Figs. S2). Because PNNs are known to be recruited by endotoxins [START_REF] Segal | How Neutrophils Kill Microbes[END_REF], this behavior could be attributed to the endotoxins surrounding the magnetosome mineral core that attract PNN. Seventy-two hours later, the M-PNNs had disappeared in the absence of AMF (Figs. 9(b) and 9(d) and Table S2) but were detected in large quantities of 27,500 per mm 2 (Table S2) after 3 magnetic sessions either within the tumor periphery These interesting behaviors might suggest that in the absence of AMF, endotoxins are progressively deactivated, perhaps due to magnetosome internalization followed by magnetosome and endotoxin decomposition. However, in the presence of AMF, the endotoxins are re-activated, possibly through the release of endotoxins from the magnetosome surface, a behavior that perfectly fits with the observations in solution (Fig. 2(a)) and/or via the destruction of cells in which magnetosomes are initially internalized before AMF application. M-PNN complexes may have two main functions. First, they may be involved in tumor destruction, as they are observed in the tumor region and Toll-like receptor (TLR) from the treated mice could be activated and trigger antitumor activity; this was previously reported for mice bearing B16 tumors [START_REF] Silva | Application of hyperthermia induced by superparamagnetic iron oxide nanoparticles in glioma treatment[END_REF]. Second, they may also be involved in magnetosome capture and redistribution. Indeed, after one and three magnetic sessions, magnetosomes and PNNs alone are rarely observed in the entire brain, including the tumor regions (Figs. vessels. This behavior further agrees with the absence of heat production after D17, which suggests that magnetosomes have left the tumor by this date and/or have been dissolved into free iron. This idea is also in line with the well-known function of PNNs, which is to remove bacterial residues (61), [START_REF] Chu | Thermal ablation of tumours: biological mechanisms and advances in therapy[END_REF].

Magnetosome progressive departure from the tumor, possibly followed by magnetosome redistribution within the mouse, could be appealing for the treatment by preventing crystallized magnetosomes from remain in the brain for too long a period of time. Optimum treatment conditions correspond to those where magnetosomes remain in the brain long enough to perform the treatment, but not for too long, to avoid unnecessary long-term magnetosome interactions with brain tissues. These conditions may be identified by controlling PNN activation and M-PNN complex formation under AMF application.

CMs appear to destroy glioblastoma tumors more efficiently than chemically synthesized nanoparticles under AMF applications

Using the same quantity of nanoparticles administered, we have shown in this study that CMs lead to 40% of mice with full tumor disappearance, while IONPs do not yield any observable antitumor activities. This result agrees with previous animal studies performed on mice and rats bearing GBM using intratumor administration of chemically synthesized nanoparticles exposed to AMF that did not improve survival [START_REF] Wang | Hyperthermia Promotes Apoptosis and Suppresses Invasion in C6 Rat Glioma Cells[END_REF] or increased survival by 15 days without resulting in a full cure [START_REF] Morlé | Hyperthermia restores apoptosis induced by death receptors through aggregation-induced c-FLIP cytosolic depletion[END_REF], [START_REF] Maier-Hauff | Intracranial thermotherapy using magnetic nanoparticles combined with external beam radiotherapy: results of a feasibility study on patients with glioblastoma multiforme[END_REF]. In addition to being more efficient, magnetosomes could also be used in a lower quantity, thus strengthening treatment safety. Indeed, the quantity of magnetosomes necessary to eradicate U87-Luc GBM in this study, which was 1 to 13 µg in maghemite per mm 3 of tumor volume, is much less than 60 µg per mm 3 of tumor volume, which produced less-efficient anti-tumor activities with chemically synthesized nanoparticles [START_REF] Jordan | The effect of thermotherapy using magnetic nanoparticles on rat malignant glioma[END_REF].

CONCLUSION

We demonstrated that 40% of mice bearing intracranial U87-Luc tumors displayed full tumor disappearance after intratumor administration of 40 µg of CMs followed by 12 to 15 magnetic sessions, during which an AMF of average strength 30 mT and frequency 198 kHz was applied for 30 minutes. In contrast, similar antitumor efficacy was not observed when mice were treated with the same conditions and administered chemically synthesized nanoparticles instead of magnetosomes. Furthermore, the absence of mouse brain damage during and following the various treatments appears to indicate that a safe treatment could be performed with magnetosomes, provided that the endotoxins are replaced with an equivalently active but nonpyrogenic substance such as M-PLA. Antitumor activity does not appear to be dominated by nanoparticle cytotoxicity, as the CMs, which are more efficient than IONPs in vivo, do not enhance cytotoxicity towards U87-Luc cells compared with IONPs. Moreover, nanoparticle administration without AMF does not produce any observable anti-tumor effects. Instead, tumor destruction appears to be due to the heat produced by the CMs under AMF application, as the only mice that displayed clear anti-tumor effects had their tumors heated (groups 4 and 5). Under heating conditions, tumor tissue destruction may be caused by irreversible cell damage; cytolysis; actin filament, microtubule or mitochondrial dysfunction; DNA replication or RNA synthesis inhibition; or DNA polymerase denaturation [START_REF] Nikfarjam | Mechanisms of Focal Heat Destruction of Liver Tumors[END_REF], but these mechanisms seem insufficient to fully explain the observed antitumor activities. Indeed, tumor destruction does occur when the magnetosomes occupy a small percentage of the total tumor volume (~10%), suggesting that magnetosomes occupying the tumor can induce the destruction of the entire tumor. We have identified the following three possible mechanisms that could be responsible for full tumor destruction:

i) Early apoptosis, as apoptotic cell death is observed to be dominant in vitro for CMs and heat was previously shown to favor apoptotic cell death (65), (66), (67);

ii) Microvascular damage due to the presence of magnetosomes in the ventricles that possibly leads to the destruction of the blood vessels supplying the tumor with oxygen, which was previously reported to occur during hyperthermia, (68), and could possibly occur because CMs were observed in the ventricles near the blood vessel; and

iii) the involvement of PNN, which were previously reported to be involved in antitumor activity, [START_REF] Liang | Neutrophils Promote the Malignant Glioma Phenotype through S100A4[END_REF] and were attracted by the magnetosomes in the tumor region due to the presence of endotoxins at magnetosome surface.

Finally, our study also suggests a method to control magnetosome distribution by attracting PNNs to the magnetosome region, which promotes the formation of magnetosome/PNN complexes that may progressively remove magnetosomes from the tumor through the ventricle blood vessels and enable their elimination. This process appears to be governed by the application (or not) of an AMF.

UV

Group 1 ,

 1 followed by magnetic treatment consisting in 12 magnetic sessions (S) on D8 (S1), D9 (S2), D10 (S3), D15 (S4), D16 (S5), D17 (S6), D22 (S7), D23 (S8), D24 (S9), D29 (S10), D30 (S11), and D31 (S12); -Group 3 received a 3 mm 3 GBM at coordinates (0.2.2) mm and a suspension containing 40 µg of magnetosome chains in maghemite without any further treatment; -Group 4 received a 3 mm 3 GBM at coordinates (0.2.2) mm and the same suspension as Group 3, followed by magnetic treatment consisting in 15 magnetic sessions on D8 (S1), D9 (S2), D10 (S3), D15 (S4), D16 (S5), D17 (S6), D22 (S7), D23 (S8), D24 (S9), D29 (S10), D30 (S11), D31 (S12), D36 (S13), D37 (S14), and D38 (S15); -Group 5 received a 25 mm 3 GBM at coordinates (0.2.2) mm and the same suspension as group 3, followed by 15 magnetic sessions on D8 (S1), D9 (S2), D10 (S3), D15 (S4), D16 (S5), D17 (S6), D22 (S7), D23 (S8), D24 (S9), D29 (S10), D30 (S11), D31 (S12), D36 (S13), D37 (S14), and D38 (S15); -Group 6 received a 5 mm 3 GBM at coordinates (0.2.2) mm and 40 µg of IONPs in maghemite without any further treatment; and -Group 7 received in a 3 mm 3 GBM at coordinates (0.2.2) mm and the same suspension as Group 6, followed by 12 magnetic sessions on D8 (S1), D9 (S2), D10 (S3), D15 (S4), D16 (S5), D17 (S6), D22 (S7), D23 (S8), D24 (S9), D29 (S10), D30 (S11), and D31 (S12).

  S5(a) and S5(c)). Six mice were analyzed at various times, including after 1 magnetic session (Figs. S5(b)) or 24 hours after 3 magnetic sessions (Fig. S5(d)), in which they received CM on day 8 and were euthanized 4 hours (Figs. 9(a) and 9(c)) or 72 hours (Figs. 9(b) and 9(d)) after the CM injection; just after or 4 hours after 1 magnetic session (Figs. 8(a), 8(b) and 8(c)); and 24 hours after 3 magnetic sessions (=72 hours, Figs. 8(d) to 8(g)), in which they received IONPs on day 8 and were euthanized 6 or 72 hours after IONP administration (Figs. S4(a) and S4(c)) or 24 hours after 3 magnetic sessions (72 hours (Figs. S4(b) and S4(d)).

  (a). After extraction from the magnetotactic bacteria, the magnetosomes maintain their organization in chains (Figure1(b)), a type of organization that is advantageous for the treatment because it prevents aggregation and promotes uniform distribution and homogenous heat production. Magnetosomes appear to possess a cubooctahedric geometry, more uniform sizes and shapes than IONPs (Figs.S7(a) and S7(b)), a size that is larger than 20 nm for the majority of them and a mean size of 45 nm (Fig.S1(a)), which leads to a coercivity Hc of 200-300 Oe and a ratio between remnants and saturating magnetization of Mr/Ms ~ 0.35 at physiological temperature (Fig.S6),[START_REF] Grüttner | Synthesis and antibody conjugation of magnetic nanoparticles with improved specific power absorption rates for alternating magnetic field cancer therapy[END_REF].

Figure 1 :

 1 Figure 1: Transmission electron microscopy images of (a) an AMB-1 Magnetospirillum magnetotacticum magnetotactic bacterium, (b), chains of magnetosomes extracted from magnetotactic bacteria and (c) an IONP. Seven microliter suspensions containing magnetotactic bacteria, chains of magnetosomes, and IONPs were deposited and dried on top of a carbon grid before TEM observation.

Figure 2 :

 2 Figure 2: (a) Percentage of endotoxin release from 2 µl suspensions containing 40 µg of CM or IONP in iron oxide that were exposed to 1, 2, 3 or 4 S with an AMF of 198 kHz and average strength of 30 mT applied for 30 minutes. (b) The quantity of iron released from MCs and IONPs, measured in pg and internalized per cell from U87-Luc cells incubated for 24 hours with 1 mg/mL of CM or IONP in maghemite with(+H)/without(-H) AMF, where the AMF is the same as in (a). (c and d) The percentage inhibition of cell proliferation as a function of nanoparticle concentration for CMs and IONPs in maghemite in U87-Luc cells incubated for (c) 24 hours or (d) 72 hours with nanoparticle concentrations varied between 8 µg/mL and 1 mg/mL. (e and f) The percentages of late apoptotic (+ necrotic) and early apoptotic cell death as a function of nanoparticle concentration with(H)/without(-H) AMF where the AMF is the same as in (a) in U87-Luc cells incubated for 24 hours with (e) CM and (f) IONP at concentrations of 40 µg/mL, 650 µg/mL, and 1 mg/mL.

  The 40 and 650 µg/ml concentrations led to temperature increases of less than 2 C, which were insufficient to reach the hyperthermia conditions. These concentrations resulted in necrosis and late apoptosis percentages that remained below ~ 4% with/without AFM for the CMs and IONPs and early apoptosis percentages that were similar with/without AFM at ~ 15% and ~ 40% for CMs at 40 and 650 µg/ml in iron oxide, respectively, and at 0% for IONPs (Figs. 2(e) and 2(f)). In contrast, at a concentration of 1 mg/ml, both CMs and IONPs yielded a more significant temperature increase of 7 C up to 41 C (Fig.S2(b)), which resulted in an increase in the necrotic and late apoptotic cell death percentages by 10%. These in vitro heating behaviors are In contrast, with those observed in solution, where CMs were observed to heat much more than IONPs.

Figure 3 :

 3 Figure 3: Variations in tumor volumes as a function of time during the days following U87-Luc tumor cell implantation at D0 for (a) untreated mice (group 1), (b) mice exposed to 15 magnetic sessions (group 2, +H), mice receiving a suspension containing either (c) 40 µg of CMs in iron oxide at the site of tumor cell implantation (group 3) or 40 µg of IONPs in iron oxide (group 6, IONP), or (e) 40 µg of IONPs in iron oxide and exposed to 15 magnetic sessions. These variations are plotted using Tukey diagrams, where error bars designate the maximum and minimum tumor volume values, circles represent single tumor values, lower and upper horizontal rectangular sides designate lower and upper quartiles, respectively, and horizontal lines within each box plot represents median tumor volumes. The number of living mice used to draw each box plot (n) is also indicated above each box plot.

Figures 4 (

 4 Figures 4(a) shows that tumor growth is delayed in group 5 with a median tumor volume that first

Figure 4 :

 4 Figure 4: (a) Variations in tumor volumes as a function of time during the days following U87-Luc tumor cell implantation at D0 for mice receiving a suspension containing 40 µg of CMs in iron oxide. The tumors reach an average volume of 25 mm 3 at D7 and were treated with 15 magnetic sessions (group 5, CM+H). (b) Variations in tumor volume and bioluminescence intensity as a function of time during the days following tumor cell implantation in a mouse in group 5 with a tumor volume at D7 of 7.5 mm 3 that received 40 µg of a suspension of CMs in iron oxide on D7 followed by 15 magnetic sessions (S1 to S15).

Figure 5 (

 5 Figure 5(a) shows more significant antitumor activity in group 4 with full tumor disappearance at day 35

Figure 5 :

 5 Figure 5: (a) Variations in tumor volumes as a function of time during the days following U87-Luc tumor cell implantation at D0 for mice receiving a suspension containing 40 µg of CMs in iron oxide when the tumors reach an average volume of 3 mm 3 at D7 followed by 15 magnetic sessions (group 4, CM+H). (b) Variations in tumor volume and bioluminescence intensity as a function of time during the days following tumor cell implantation for a mouse belonging to group 4 with a tumor volume at D7 of 3 mm 3 that receiving 40 µg of CMs in iron oxide on D7 followed by 15 magnetic sessions (S1 to S15).

  sections from mice treated with CM with/without AMF collected at different times ranging from 4 hours to 150 days after CM administration (Figs. S4(a) to S4(d), S5(a) to S5(d) and Figs. 8(a) to 8(g), 9(a) to 9(d)) that healthy tissues in the hippocampus and brain do not contain lesions or edema. Such observations can be explained by an LPS-activated neuro-protective response by the microglia (55).

Figure 6 : 3 . 5 .

 635 Figure 6: Optical microscopy image of a 4-µm-thick brain section stained with hematoeosin from a mouse in group 4 that received 40 µg of CMs in iron oxide followed by 15 magnetic sessions. This mouse was euthanized 7 months after magnetosome administration. No tumor was observed in this brain section.

Figure 7 :

 7 Figure 7: (a), Temperature increases above 28.5 °C observed during each magnetic session (T) on D8, D9, D10, D15, D16 and D17 in the mice from groups 4 and 5 treated with CMs. (b) The same as in (a) for the mice in group 7 treated with IONPs. After D17, tumor temperature increases were no longer observed. (c)Variations in mouse survival times as a function of time during the days following U87-Luc tumor cell implantation for mice with the following treatments: (1) untreated (group 1), (2) exposed to 15 magnetic sessions (group 2), (3) treated with suspensions containing 40 µg of CMs in iron oxide without magnetic sessions (group 3), (4) treated with 40 µg of CMs in iron oxide in ~3 mm 3 tumors followed by 15 magnetic sessions (group 4), (5) administered 40 µg of CMs in iron oxide in ~25 mm 3 tumors followed by 15 magnetic sessions (group 5), (6) administered 40 µg of IONPs in iron oxide (group 6), and administered 40 µg of IONPs in iron oxide followed by 15 magnetic sessions (group 7).

Figure 8 :

 8 Figure 8: Optical microscopy images of 4 µm thick brain sections stained with hematoeosin from mice receiving 40 µg of CMs in iron oxide and exposed to (a-c) one or (d-g) three magnetic sessions. The mice were euthanized (a) 0 hours (0 h), (b and c) 4 hours (4 h), or (d-g) 72 hours (72 h) after MC administration with either (a-c) one or (d-g) three magnetic sessions. (c) An enlargement of the region in (b). The red arrows in (c) designate polynuclear neutrophils. (e), (f), and (g) are enlargements of three different regions from (d). PNN, M-PNN, U87-Luc, M-PNN & U87-Luc, and U87-Luc & magnetosomes designate polynuclear neutrophils, magnetosomes colocalized with polynuclear neutrophils, U87-Luc cells, magnetosomes colocalized with polynuclear neutrophils and U87-Luc cells, and U87-Luc cells colocalized with magnetosomes, respectively. The regions containing these different cells are surrounded in (d), while individual cells are indicated by arrows in (c), (e) and (f).

  behavior can be explained by their lower concentration in the tumor under AMF applications, i.e., they are observed at the tumor surface as well as part of the tumor periphery at 6 hours (Figs.S4(a) and S4(c)); their more rapid diffusion in the ventricles, i.e., IONPs and CMs localize in the ventricles at 6 and 72 hours, respectively, (Figs. S4(a) and S4(b), Figs. 8(d)); and their earlier internalization in tumor U87-Luc and healthy BETZ brain cells, i.e., IONPs and CMs are internalized at 6 and 72 hours, respectively, (Figs. S4(c) and S4(d), Fig. 8(e)). The combination of a low concentration in the tumor

  8(b), 8(c), 8(d), 8(f), 8(g), 9(a) and 9(c)). More precisely, M-PNNs designate assemblies of magnetosomes and PNNs where the magnetosomes are less than 1 µm away from the PNNs. The number of M-PNNs was estimated as 14000-19000 per mm 2 4 hours after CM administration (Table

(

  Fig. 8(d) and 8(f)) or in the tumor (Figs. 8(d) and 8(e)).

Figure 9 :

 9 Figure 9: Optical microscopy images of 4 µm thick brain sections stained with hematoeosin from mice receiving 40 µg of CMs in iron oxide without magnetic sessions and euthanized (a) 4 hours (4 h) or (b) 72 hours (72 h) after magnetosome administration. (c) and (d) are enlargements of two regions in (a) and (b), respectively. PNN are observed in (c) and indicated by red arrows, but not in (d).

  7(b) and 7(d)), and M-PNNs appear to be located either in the tumor, near the tumor periphery or in the ventricles (Figs. 7(b) and 7(c)). These results suggest that PNNs progressively migrate in the region where the magnetosomes are located between 0 and 4 hours after CM administration to form M-PNN complexes and that these complexes carry the magnetosomes from the inside to the outside of the tumor, possibly through the ventricle blood

Table 1 :

 1 TheEstimates of the coating thickness measured by transmission electron microscopy and percentage of carbon (% C) measured with CHNS and assignments from the different FT-IR peaks measured by FT-IR for the CM and IONP dried suspensions.Estimates of the specific absorption rates (SAR) in W per gram in iron for 2 µl containing 40 µg of CMs or IONPs in iron oxide mixed in water or introduced inside the mouse brains.

endotoxin content estimated in Endotoxin units (EU per mg in iron per ml), percent decreased absorption measured at 480 nm in suspensions containing CM and IONP at a concentration of 1 mg in iron per ml after twenty minutes, and estimates of the isoelectric point and zeta potential as a function of pH for suspensions containing CMs or IONPs.

  enhanced by the presence of the AMF for both types of nanoparticles. This enhancement is more pronounced for CMs than for IONP by factors of 7 and 4, respectively (Figure2(b)). The different internalization properties for the CMs and IONPs may be explained by differences in their charges, i.e., CM and IONP are negatively and positively charged at physiological pH, respectively (Table1)); differences in organization, i.e., CM and IONP are well-dispersed and tend to aggregate, respectively; and to larger values of coercivity for CMs (200-300 Oe) compared to IONPs (120 Oe) that may enhance CM magnetic field coupling. Furthermore, CM organization in chains may favor internalization by groups containing several magnetosomes[START_REF] Alphandéry | The effect of iron-chelating agents on Magnetospirillum magneticum strain AMB-1: Stimulated growth and magnetosome production and improved magnetosome heating properties[END_REF], i.e., potentially 6 for chains averaging 250 nm in length (Fig.S1(b), while IONPs may result in nanoparticles being internalized one by one or at a lower rate. The enhanced internalization of CMs may favor early apoptosis, which was

5 pg per cell without AMF application to 255.7 and 19.8 pg per cell with AMF application for the CMs and IONPs, respectively. It therefore appears that CMs are internalized better than IONPs and that the internalization is previously observed with iron oxide nanoparticles, (53), and reduce its heating properties, possibly by preventing nanoparticle Brownian heating mechanisms (54).

ACKNOWLEDGMENTS

We would like to thank the Eurostars Program (Nanoneck-2, E9309), subvention AIR from the region of Paris (A1401025Q), the ANRT, the ANR (Méfisto project), the French Research Tax Credit Program, Paris Biotech Santé, BPI, the University Paris 6, the University Paris XI, Marianne Labussière and Elvis Dervishi, as well as all other people and structures in France and Europe that have helped us and been supportive and enthusiastic about our research activities.

Abbreviations


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