Mbolantenaina Rakotomalala

Mustapha Abdelmoula

Martine Mallet

Romain Coustel

The role of starch in nano-magnetite formation: a spectrometric and structural investigation

Keywords:

In the framework of pollutant sequestration by magnetic nano materials, this work focuses on the synthesis and the physico-chemical properties of starch functionalized nano magnetites. Nanoparticles were prepared by the Fe 2+ /Fe 3+ coprecipitation method with increasing amount of starch in the synthesis medium (starch to iron mass ratio (R) in the range 0-10). Obtained materials were characterized by Transmission Electron Microscopy (TEM), Powder X-Ray Diffraction (PXRD), Raman, Fourier Transform Infrared (FTIR) and 57 Fe Mössbauer spectroscopies, as well as by vibrating sample magnetometry. The presence of starch into the synthesis medium lowers magnetite crystallinity and increases the dispersion of the oxide particles. Both effects lead to fast relaxing superparamagnetic particles at RT for high R. The role of starch on nano-magnetite growth and stabilization is evidenced.

Introduction:

Magnetic nanoparticles (NP) have been attracting strong interest for the past decades due to their established as well as novel applications in the fields of data storage [START_REF] Dai | Magnetically-responsive self assembled composites[END_REF], medicine (contrast agents for biomedical imaging and drug or gene delivery) [START_REF] Hah | Mössbauer spectroscopy of superparamagnetic Fe3O4 nanoparticles[END_REF][START_REF] Hong | Facile synthesis of polymer-enveloped ultrasmall superparamagnetic iron oxide for magnetic resonance imaging[END_REF][START_REF] Miyamoto | Polysaccharide Functionalized Magnetic Nanoparticles for Cell Labeling and Tracking: A New Three-Dimensional Cell-Array System for Toxicity Testing[END_REF][START_REF] Uthaman | Polysaccharide-Coated Magnetic Nanoparticles for Imaging and Gene Therapy[END_REF][START_REF] Zhang | Long-Circulating Heparin-Functionalized Magnetic Nanoparticles for Potential Application as a Protein Drug Delivery Platform[END_REF], purification and extraction of biomacromolecules [START_REF] Eivazzadeh-Keihan | Functionalized magnetic nanoparticles for the separation and purification of proteins and peptides[END_REF], catalysis [START_REF] Iyengar | Fabrication of magnetite nanocrystals in alcohol/water mixed solvents: catalytic and colloid property evaluation[END_REF][START_REF] Zhang | Applications of Magnetic Nanomaterials in Heterogeneous Catalysis[END_REF], etc. Due to their high specific area, that provide high adsorption properties, NP are also considered for environmental processes [START_REF] Bée | Magnetic chitosan/clay beads: A magsorbent for the removal of cationic dye from water[END_REF][START_REF] Ren | Chromium (VI) adsorption from wastewater using porous magnetite nanoparticles prepared from titanium residue by a novel solid-phase reduction method[END_REF][START_REF] Yoon | Synthesis of magnetite/nonoxidative graphene composites and their application for arsenic removal[END_REF]. In this respect, the benefit of additional magnetic properties is that the adsorbent could be separated from contaminated water via a weak magnetic separator. To prevent particles agglomeration and, then, to preserve their specific area, organic functionalization appears as an efficient strategy. Thanks to their low-toxicity, abundance and cost-effectiveness, both polysaccharides and iron oxides are relevant building blocks for the design of ecofriendly materials for environmental [START_REF] Rakotomalala Robinson | As(V) and As(III) sequestration by starch functionalized magnetite nanoparticles: influence of the synthesis route onto the trapping efficiency[END_REF] but also medical [START_REF] Miyamoto | Polysaccharide Functionalized Magnetic Nanoparticles for Cell Labeling and Tracking: A New Three-Dimensional Cell-Array System for Toxicity Testing[END_REF] applications. From a fundamental point of view, the development of polysaccharide functionalized iron oxide NP raises, at least, two questions. Firstly, the nature of the molecular bonds between polysaccharide and iron oxide remains puzzling. Actually, the strong affinity between polysaccharide and iron oxide is well established and has long been used on an industrial scale, e.g. starch has been used as universal depressant in reverse flotation of iron oxide in the field of mineral processing [START_REF] Pattanaik | Investigation of Adsorption Mechanism of Reagents (Surfactants) System and its Applicability in Iron Ore Flotation -An Overview[END_REF]. Nevertheless, adsorption mechanisms are misunderstood: complexation with metal site [START_REF] Laskowski | Current understanding of the mechanism of polysaccharide adsorption at the mineral/aqueous solution interface[END_REF][START_REF] Weissenborn | Selective flocculation of ultrafine iron ore 2. Mechanism of selective flocculation[END_REF], hydrogen bonding [START_REF] Filippov | Mechanism of starch adsorption on Fe-Mg-Albearing amphiboles[END_REF][START_REF] Ma | Role of solvation energy in starch adsorption on oxide surfaces[END_REF] or hydrophobic interactions [START_REF] Laskowski | Current understanding of the mechanism of polysaccharide adsorption at the mineral/aqueous solution interface[END_REF] were proposed. A recent DFT calculation study [START_REF] Li | AFM and DFT study of depression of hematite in oleate-starch-hematite flotation system[END_REF] concludes that starch adsorbs on hematite through direct chemical bonds and hydrogen bonds involving water molecules. For a greater economic and environmental efficiency, it appeared relevant to synthesize NP and to functionalize them in a single synthesis step where iron oxide is precipitated into a polysaccharide solution. This raises the second question of how polysaccharides, and more widely polymers, affect NP formation. Two steps, nucleation and growth [START_REF] Jolivet | Metal oxide chemistry and synthesis: from solution to solid state[END_REF]LaMer and Dinegar, 1950;[START_REF] Mutaftschiev | The Atomistic Nature of Crystal Growth[END_REF], lead to crystal formation but the impact of polysaccharides is scarcely described in literature. Polymer chains can behave as nucleation point for oxide: carboxylate and amine functions are able to form complexes with aqueous Fe 2+/3+ ions or to bind NP oxide. So NP formation can take place into alginate [START_REF] Hernández | Structural Organization of Iron Oxide Nanoparticles Synthesized Inside Hybrid Polymer Gels Derived from Alginate Studied with Small-Angle X-ray Scattering[END_REF] or chitosan [START_REF] Wang | Chitosan-induced synthesis of magnetite nanoparticles via iron ions assembly[END_REF] gels as well as into an interpenetrating (carboxymethyl cellulose/chitosan) polymer network [START_REF] Kaihara | In situ synthesis of polysaccharide nanoparticles via polyion complex of carboxymethyl cellulose and chitosan[END_REF]. It has been demonstrated that iron complexation could be tapped to favor formation of NP into poly(acrylic acid) brushes [START_REF] Zhu | Spherical polyelectrolyte brushes as a nanoreactor for synthesis of ultrafine magnetic nanoparticles[END_REF] or along a DNA strand. [START_REF] Sarkar | Static and Dynamic Magnetic Characterization of DNA-Templated Chain-Like Magnetite Nanoparticles[END_REF] Several studies reported the synthesis of iron oxyhydroxide NP in presence of carrageenan. In these conditions, self-assembled polysaccharide behaves as nano-reactor providing localized area for iron hydrolysis and particle growth [START_REF] Jones | Iron oxyhydroxide colloids stabilized with polysaccharides[END_REF][START_REF] Rashid | Synthesis of magnetic nanostructures: Shape tuning by the addition of a polymer at low temperature[END_REF]. Carrageenan was claimed to stabilize iron oxide NP and to prevent their agglomeration. In spite of these examples, some authors pointed out that in-depth understanding of the mechanisms at play when oxide NP were prepared in presence of polymer remained unclear and challenging (Noqta et al., 2019). This work aims to get further insight on how polysaccharides affects iron oxide NP formation. As magnetite (Fe3O4) presents one of the highest saturation magnetizations among the iron oxides (92 emu g -1 at room temperature [START_REF] Han | Crystallite size effect on saturation magnetization of fine ferrimagnetic particles[END_REF]), we will focus on nano magnetite prepared by coprecipitation of Fe 2+ and Fe 3+ in alkaline condition with an increasing amount of starch. Actually, it should be note that the nucleation and growth of NP can be hardly tracked during the synthesis. In this paper, we restrict ourselves to characterize the obtained materials to infer the role of starch. NP were fully analysed by Transmission Electron Microscopy (TEM), Powder X-Ray Diffraction (PXRD), Raman, Fourier Transform Infrared (FTIR) and 57 Fe Mössbauer spectroscopies, as well as by vibrating sample magnetometry. Because of its unique ability to finely probe Fe-Fe interactions, Mössbauer spectroscopy allows to spot starch induced modification of the iron oxide NP. In particular, superparamagnetic behaviors of the NP were correlated to starch content. It comes that starch is able to disperse and stabilize iron oxide NP with reduced crystallinity.

Methods

Materials

All chemicals used in this work (obtained from multiple suppliers) were of analytical grade and used without further purification: ferrous sulphate heptahydrate (FeSO4.7H2O, Fisher Chemical), ferric chloride hexahydrate (FeCl3.6H2O, Sigma-Aldrich) potassium hydroxide (KOH, Riedel-de Haën), potassium nitrate (KNO3, Merck), ethanol 96% (VWR) and soluble starch (Sigma-Aldrich). Ultrapure water (18.2 MΩ cm -1 ) was used for the all experiments. Water was previously bubbled at least 1 h with N2 before use to prevent uncontrolled iron oxidation.

Synthesis of magnetic nano-particles

Magnetite nano-particles were prepared by coprecipitation of Fe 2+ and Fe 3+ with a base. Our protocol was established on the basis of the work of several authors [START_REF] Chang | Polysaccharides as stabilizers for the synthesis of magnetic nanoparticles[END_REF][START_REF] Culita | Superparamagnetic nanomagnetites modified with histidine and tyrosine[END_REF]Gnanaprakash et al., 2007bGnanaprakash et al., , 2007a;;[START_REF] Liang | Effects of Stabilizers and Water Chemistry on Arsenate Sorption by Polysaccharide-Stabilized Magnetite Nanoparticles[END_REF]. Typically, 70 mL of a solution containing Fe 2+ and Fe 3+ ions in a 2:1 molar ratio was prepared by dissolving 1130 mg of FeCl3.6H2O and 581 mg of FeSO4.7H2O in 0.1 M HCl solution. The dissolution was performed under nitrogen bubbling. The resulting mixture was heated to 80°C. Once the temperature was reached, 10 mL of a concentrated ammonia solution was added rapidly at one time to the reaction medium and the resulting suspension was heated for 10 minutes at the temperature of 80°C. Then, the suspension was cooled and aged overnight. Before washing, a neutralization step was necessary. For that purpose, concentrated hydrochloric acid (HCl) was added to the suspension to lower the pH to 7. The black precipitate was recovered by centrifugation, washed 5 times with ultrapure water and then 1 time with ethanol (96%) to remove the unreacted reagents. The resulting product was dried under vacuum. For the synthesis of starch functionalized NP, the synthesis method was slightly modified. Starch was gelatinized in the iron solution before the addition of the base. The starch to Fe mass ratio (R) ranged from 0 to 10 (R = 0 corresponds to starch free NP, R = 10 corresponds to 3500 mg of starch). The following steps remained the same as the ones for the starch free sample. The samples obtained by coprecipitation (CP) were labelled CP-RX where the number X corresponds to the starch to iron mass ratio, which constitutes the only varying parameter of this set of samples. An additional sample labelled CP-R10After was prepared adding gelatinized starch (R = 10) after Fe 2+ and Fe 3+ ions coprecitiation: 15 min after base introduction, starch was added to synthesis medium (the protocol for particle aging and particle recovery remained unchanged).

Materials characterization

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to ascertain the morphology of the as-prepared NP. The TEM micrographs were obtained on a JEOL 2100F and Philips CM200 microscopes both operating at an accelerating voltage of 200 kV. The samples were dispersed in ethanol (96%) under ultrasound. One drop of suspension was deposited on a metal grid covered with a carbon film. The SEM micrographs were obtained on a JEOL JSM-IT500HR operating at an accelerating voltage of 5.0 kV. Powder X-Ray Diffraction (PXRD) patterns were recorded with an X'Pert Pro diffractometer (PANalytical) equipped with a copper anode X-ray tube and a Ge(111) monochromator to exclude the Kα2 line. Then, diffractograms were recorded with the only Cu Kα1 line (λ(Kα1) = 1.5406 Å). Raman spectra were recorded on an inVia Qontor spectrometer (Renishaw) equipped with a Leica confocal microscope. The wavelength of the laser beam used was always 532 nm and the objective had a magnification ×50. The scattered light was collected by the same objective and then passed through a diffraction grating (2400 lines/mm). The scattered light was collected by a Peltier cooled CCD detector. A power of 0.05 mW at the sample level was used. The infrared spectra were acquired with a Vertex 70v spectrometer (Bruker) in transmission mode. The absorption bands of CO2 and water vapor were limited by the use of vacuum. The sample to be analyzed (0.5 mg) was ground with KBr (150 mg) in an agate mortar and compressed with a hydraulic press. For each sample, 100 spectra with a resolution of 4 cm -1 had been collected in the wavenumber range from 100 to 6000 cm -1 . 57 Fe Mössbauer spectra were collected using two conventional spectrometers in transmission geometry coupled with a cold head cryostat from Advances Research Systems (USA) or JANIS Research (USA), equipped with vibration isolation stand, developed in LCPME laboratory. The 50 mCi 57 Co in Rh matrix radioactive source was mounted in a constant acceleration velocity transducer. Measurements were taken at the wide range of velocity ±11 mm s -1 . The hyperfine interaction parameters were determined by fitting the experimental spectra by a least-squares method using the Recoil software (K. [START_REF] Lagarec | Recoil -Mössbauer Spectral Analysis Software for Windows[END_REF]. The center shifts were reported with respect to that of 25 μm-thick α-Fe foil at room temperature. A commercial MicroSense vibrating sample magnetometer was used to measure magnetic moment versus magnetic field amplitude at room temperature. The powder was place in quartz cup fixed onto a quartz sample rod.

Results

TEM

TEM micrographs evidenced that the coprecipation method provides finely dispersed iron oxide (see Fig. 2). In the absence of starch (CP-R0), round-shaped particles with diameter equal to 13 ± 3 nm were obtained (see Fig. S1). The orientation of lattice fringes probed by high resolution TEM allows to distinguish individual crystallites within aggregates. With starch to iron mass ratio equal to 10 in the synthesis medium, CP-R10 appears as flake of starch where particles seem incorporated (observed where the polymer matrix is thin enough to be transparent). Particles also present round shape when prepared with high amount of starch but show lower diameter (< 10 nm) than CP-R0. The corresponding selected area electron diffraction (SAED) patterns are displayed in Fig. 2. The CP-R0 material gives rings that can be indexed according to the inverse spinel iron oxide phase (JCPDS 19-0629). The spotty character of the rings is due to the limited number of probed crystallites. The SAED pattern of CP-R10 material can be indexed in the same way. Nevertheless, in the latter case both the extinction of several rings and the strengthening of the diffuse character of the rings should be attributed to poorer crystallinity of the oxide phase synthesized in presence of starch.

PXRD

To complete the microstructure analysis, the obtained products were further characterized by PXRD (see Fig. 3). In line with SAED results, CP-R0 obtained by the coprecipitation pathway shows PXRD pattern that can be indexed according to the magnetite structure (JCPDS 19-0629). The positions of the diffraction peaks remain unchanged when starch is introduced into the synthesis medium showing that the inverse spinel iron oxide structure is essentially preserved for CP-R0.31 to R10. For high value of R (R5, R10), starch gives a wide hump in the 10-25° region. The diffraction peaks of the iron oxide phase broaden with increasing value of R, evidencing the size reduction of the crystallites. The average crystallite dimensions <d> were evaluated applying the Scherrer's formula [START_REF] Scherrer | Bestimmung der Grosse und der Inneren Struktur von Kolloidteilchen Mittels Rontgenstrahlen[END_REF][START_REF] Suryanarayana | X-Ray Diffraction: A Practical Approach[END_REF] on the two most intense peaks ( 311) and ( 440) using X'Pert Highscore Plus v2.2b software. It appears that <d> tends to diminish with increasing starch amount: <d> equals 13.7 ± 0.4, 13.7 ± 0.6, 12.6 ± 0.4, 8.5 ± 0.4 and 8.8 ± 0.4 nm for R0, R0.31, R2, R5 and R10, respectively. One should note that magnetite and maghemite are both inverse spinel iron oxides with rather similar diffractogram [START_REF] Cornell | The iron oxides: structure, properties, reactions, occurrences, and uses, 2nd[END_REF] (see reference diffractograms for magnetite (JCPDS 19-0629) and maghemite (JCPDS 39-1346) in Fig. 4). Then, these phases are hardly distinguishable from PXRD measurements in particular when poorly crystallized or nanomaterials solids are considered. In here, definitive phase identification needs additional characterization by Raman and Mössbauer spectroscopies.

Raman spectroscopy

The Raman spectrum of the CP-R0 sample (Fig 4) presents bands at 670, 540 and 310 cm -1 attributed, respectively, to the A1g, T2g and Eg modes of magnetite [START_REF] De Faria | On the models for interparticle interactions in nanoparticle assemblies: comparison with experimental results[END_REF][START_REF] Shebanova | Raman spectroscopic study of magnetite (FeFe2O4): a new assignment for the vibrational spectrum[END_REF]. It should be noted that maghemite is expected to give Raman bands at 350, 500, 660 and 715 cm -1 as well as a broad band at ~1400 cm -1 [START_REF] Boucherit | Passivity of Iron and Iron Alloys Studied by Voltammetry and Raman Spectroscopy[END_REF][START_REF] De Faria | On the models for interparticle interactions in nanoparticle assemblies: comparison with experimental results[END_REF][START_REF] Hanesch | Raman spectroscopy of iron oxides and (oxy)hydroxides at low laser power and possible applications in environmental magnetic studies[END_REF][START_REF] Jacintho | Surface-enhanced Raman spectra of magnetic nanoparticles adsorbed on a silver electrode[END_REF][START_REF] Ohtsuka | Raman Spectroscopy of Thin Corrosion Films on Iron at 100 to 150 C in Air[END_REF]. The absence of these bands excludes maghemite phase in R0 samples at least in a minute amounts that could be detected by Raman spectroscopy. The bands originating from starch (see reference spectrum in Fig 4) arise for increasing value of R and they overlap with the weak T2g and Eg bands (similarly FTIR spectra present starch bands for increasing value of R confirming magnetite functionalization, see Fig S2). Concurrently, when R value is above 2, a shoulder at 710 cm -1 widens the main A1g magnetite band. As discussed bellow, this broadening may be attributed to starch induced defect into magnetite structure.

Mössbauer spectrometry

Mössbauer 57 Fe spectrometry measurements were performed to get a further insight into the impact of starch on both structural and magnetic properties of the CP materials. The Mössbauer spectra recorded from room temperature (RT) to 13 K on CP-R0, CP-R5 and CP-R10 are given in Fig. 5 while the corresponding hyperfine parameters are given in Table 1. In sake of clarity, let us compare these spectra to the ones of reference magnetite (labelled OP-R0) prepared according to the oxidation precipitation method (Schwertmann and Cornell, 2000) (see Fig. S4 and table 1). RT spectrum shows two sextets with hyperfine parameters equal to CS = 0.28 mm s -1 / Hhf = 49.0 T and CS = 0.66 mm s -1 / Hhf = 45.8 T. These sextets are assigned to Fe 3+ in tetrahedral site (SA) and Fe 2.5+ in octahedral site (SB), respectively according to results obtained on bulk magnetite or particles whose size is >30 nm [START_REF] Byrne | Controlled cobalt doping in biogenic magnetite nanoparticles[END_REF][START_REF] Daou | Hydrothermal Synthesis of Monodisperse Magnetite Nanoparticles[END_REF][START_REF] Vandenberghe | Mössbauer characterization of iron oxides and (oxy)hydroxides: the present state of the art[END_REF]. According to the recommendation of Zegeye et al. [START_REF] Zegeye | In situ monitoring of lepidocrocite bioreduction and magnetite formation by reflection Mossbauer spectroscopy[END_REF], the deviation (δ) from the ideal stoichiometry (Fe3-δO4) can be evaluated from the relative area of SA and SB. It comes δ = 0.01 indicating that OP-R0 is very close to pristine magnetite and confirming PXRD and Raman spectroscopy results. Below Verwey transition that occurs at 120 K [START_REF] Daniels | Mössbauer spectroscopy of stoichiometric and nonstoichiometric magnetite[END_REF][START_REF] Verwey | Electronic Conduction of Magnetite (Fe3O4) and its Transition Point at Low Temperatures[END_REF], the crystallite structure of magnetite undergoes a symmetry changes from cubic to monoclinic, while the electron hopping between Fe 3+ and Fe 2+ ions located in octahedral sites ceases freezing this process. As a consequence, the Mössbauer spectrum of magnetite at low temperature is relatively complex [START_REF] Sawatzky | Mössbauer Study of Electron Hopping in the Octahedral Sites of Fe 3 O 4[END_REF][START_REF] Srivastava | Mössbauer study of the low-temperature phase of magnetite[END_REF]. Nevertheless, it is commonly accepted that low temperature spectra can be fitted with five sextets assigned to Fe 3+ and Fe 2+ in tetrahedral and octahedral sites. Such an approach was successfully applied to the spectrum recorded on OP-R0 at 4 K (Fig. S4). A particular feature is a sextet that presents a low hyperfine field (34.1 T) with respect to the remaining four components and comparatively higher center shift (CS) and quadrupole shift (ε). This sextet represents 10 % of the relative area and it is attributed to Fe 2+ in octahedral site [START_REF] Daou | Phosphate Adsorption Properties of Magnetite-Based Nanoparticles[END_REF][START_REF] Srivastava | Mössbauer study of the low-temperature phase of magnetite[END_REF]. At RT, CP-R0 is composed of six broad lines suggesting hyperfine fields distributions (HFD). Satisfactory fit was obtained with two HFD sites (CS = 0.27 mm s -1 / <Hhf> = 44.0 T and CS = 0.54 mm s -1 / <Hhf> = 35.1 T, see Fig. 5) assigned to tetrahedral and octahedral ions, respectively. The deviation from ideal stoichiometry derived from the relative area of SA and SB [START_REF] Zegeye | In situ monitoring of lepidocrocite bioreduction and magnetite formation by reflection Mossbauer spectroscopy[END_REF] is low (δ = 0.03). This result supports Raman analysis that ruled out maghemite contribution. It is particularly noteworthy that CP-R0 shows Fe 2+ content close to the one of stoichiometric magnetite. Actually, significant oxidation is often reported small nano magnetite (particle size ~10 nm) with Fe 3+ /Fe 2.5+ Mössbauer ratio component above 2.5 [START_REF] Amir | Electrical properties and hyperfine interactions of boron doped Fe3O4 nanoparticles[END_REF][START_REF] Kotsikau | Structural, magnetic and hyperfine characterization of Zn x Fe 3-x O 4 nanoparticles prepared by sol-gel approach via inorganic precursors[END_REF] or even no Fe 2.5+ component [START_REF] Güner | Synthesis and characterization of oleylamine capped MnxFe1-xFe2O4 nanocomposite: Magneto-optical properties, cation distribution and hyperfine interactions[END_REF], which suggests the formation of maghemite. The Mössbauer spectrum of CP-R0 recorded at 20 K corresponds to low temperature magnetite footprint (Fig. 5) confirming the identification of the dispersed phase. It is noteworthy that the relative weight of S5 (high CS = 0.90 mm s -1 , high ε = 1.72 mm s -1 and low <Hhf> = 34.9 T) is only 3 % compared to 10 % for OP-R0. Although S5 is attributed to a fraction of Fe 2+ ions, its weakness does not match with low δ (poorly oxidized magnetite). When prepared with starch, the CP samples show substantially different Mössbauer profiles. CP-R5 spectrum at RT presents a broadening of measured spectral lines (superposition of broad sextets and central lines) (Fig. 5). For CP-R10 that corresponds to higher amount of starch, no more magnetic component was observed at RT but this component rises and gradually grows with decreasing temperature (Fig. 5), which is typical of superparamagnetic behavior. Following the procedure proposed by Tronc et al. for superparamagnetic nano-maghemite, CP-R5 and CP-R10 spectra were fairly reproduced with three components [START_REF] Tronc | Magnetic behaviour of γ-Fe2O3 nanoparticles by mössbauer spectroscopy and magnetic measurements[END_REF]. A quadrupole doublet, a magnetically split component, and a broad single line are attributed to particles in the superparamagnetic state, in the blocked state and with intermediate relaxation times, respectively. Below the Verwey transition, the remaining magnetic components correspond to magnetite.

Magnetization measurements

The magnetic properties of the hybrid materials were estimated from the specific magnetization curves as a function of magnetic field strength recorded at room temperature (Fig 6). All the samples show curves wich can be fitted with Langevin functions characteristic of superparamagnetic behaviour of nanoparticules. The saturation magnetizations (Ms) of the materials CP-R0, CP-R5 and CP-R10 are 63.5, 19.3 and 8.4 emu g -1 (as derived from Langevin function fits, see Fig 6). All CP materials present the typical superparamagnetic behavior, the absence of hysteresis loop is assigned to the small size of the NP having a single magnetic domain. The value obtained for the starch free material (CP-R0) is lower than the theoretical value of the bulk magnetite (92 emu g -1 [START_REF] Han | Crystallite size effect on saturation magnetization of fine ferrimagnetic particles[END_REF]). Previous reports on Fe3O4 NP [START_REF] Amir | Magneto-optical investigation and hyperfine interactions of copper substituted Fe3O4 nanoparticles[END_REF][START_REF] Byrne | Controlled cobalt doping in biogenic magnetite nanoparticles[END_REF][START_REF] Daou | Coupling Agent Effect on Magnetic Properties of Functionalized Magnetite-Based Nanoparticles[END_REF][START_REF] Daou | Hydrothermal Synthesis of Monodisperse Magnetite Nanoparticles[END_REF][START_REF] Kotsikau | Structural, magnetic and hyperfine characterization of Zn x Fe 3-x O 4 nanoparticles prepared by sol-gel approach via inorganic precursors[END_REF]Meite et al., 2022bMeite et al., , 2022a;;[START_REF] Zegeye | Effect of Sb on precipitation of biogenic minerals during the reduction of Sb-bearing ferrihydrites[END_REF] indicated that Ms decreases with particle size (see inset of Fig 6). Our results on CP-R0, as well as on OP-R0 (Ms = 89.1 emu g -1 for 67 nm particles), are fully consistent with the reported trend. Ms lowering is attributed to the surface contribution: spin canting, surface disorder, stoichiometry deviation, cation distribution and adsorbed water. [START_REF] Daou | Hydrothermal Synthesis of Monodisperse Magnetite Nanoparticles[END_REF] Hybrid materials (CP-R5 and CP-R10) show an even lower Ms than uncoated ferrite NP because of the reduced content of magnetic oxide [START_REF] Amir | Magneto-optical and catalytic properties of Fe3O4@HA@Ag magnetic nanocomposite[END_REF][START_REF] Baykal | Preparation and characterization of SPION functionalized via caffeic acid[END_REF][START_REF] Covaliu | Magnetic nanoparticles coated with polysaccharide polymers for potential biomedical applications[END_REF].

Discussion

Coprecipitation vs oxidation precipitation

In a recent study [START_REF] Rakotomalala Robinson | Starch functionalized magnetite nanoparticles: New insight into the structural and magnetic properties[END_REF], we reported characterizations of starch functionalized spinel iron oxide NP obtained by the oxidation-precipitation method (Schwertmann and Cornell, 2000). In brief, this method consists of the partial oxidation of an Fe 2+ solution in alkaline medium by nitrate. The OP method was modified by introducing gelatinized starch in the Fe 2+ solution before the addition of the base. Then, starch functionalized NP were prepared with a starch to iron mass ratio varying in the range of 0 to 10 (labelled from OP-R0 to OP-R10). TEM measurements revealed that in the absence of starch OP-R0, the oxidation-precipitation path leads to cubic shape particles with much higher average size (80 ± 30 nm) than CP-R0 (13.7 ± 0.4 nm). As reported in this paper for CP method, increasing the amount of starch tends to reduce the size of the NP when prepared according to the OP method. But contrary to CP samples, OP-R10 also presents intriguing objects with rough surface (corrugation length of <10 nm) and shows lattice fringes, and then crystalline order, of several tens of nanometer. The PXRD patterns of the OP samples evidences the presence of spinel phase, whose crystallinity decreases when the amount of starch is increased: <d> drops from 67 ± 5 nm for OP-R0 to 44 ± 4 nm for OP-R0.31 and reaches 12 ± 4 nm for R10. Then, PXRD (as well as TEM and SAED) suggests that the functionalized CP particles are smaller than the ones obtained by the OP method and they also present narrower crystallite size distribution. Moreover, non-indexed diffraction peaks were reported for OP-R5 and OP-R10 while no extra crystallized phase can be suspected from PXRD records on CP products. Although CP-R0 (<d> = 13.7 ± 0.4 nm) presents poorer crystallinity than OP-R0 (<d> = 67 ± 5 nm), their Raman spectra present no difference (see Fig. 4 and (Rakotomalala Robinson et al., 2019)). This observation confirms that without starch the two synthesis routes lead to magnetite formation and highlights that Raman fingerprint of magnetite weakly depends on crystallite size (at least for <d> > 14 nm). Therefore, the asymmetry of the A1g band that was observed on OP-R2 (<d> = 30 ± 10 nm) cannot be attributed to size effect, but instead should be ascribed to defects due to partial oxidation and/or starch/oxide interactions [START_REF] Kumar | Theory, Production and Mechanism of Formation of Monodispersed Hydrosols[END_REF]. A similar cause may explain the shoulder (710 cm -1 ) of the A1g band for CP-R5 and CP-R10. While both CP and OP samples show similar PXRD patterns and Raman profiles, Mössbauer spectrometry clearly discloses additional consequences of the reduced crystallite size. Due to its selectivity on the element iron, the Mössbauer effect is very sensitive to hyperfine interactions and allows access to both magnetic properties and their relationship to the size-dependent effect of very small particles. This point will be discussed in the next section. 57 Fe Mössbauer fingerprint of starch functionalized nano magnetitesIn this study, magnetic properties of the nano materials were probed by Mössbauer spectrometry. The magnetic moment of nano-sized particle is subject to thermally driven fluctuation. The magnetic anisotropy of a single-domain particle is proportional to its volume (V) and its anisotropy energy is given by: 𝐸 = 𝐾. 𝑉. sin²(𝜃)

(1) where K is the magnetic anisotropy constant and θ is the angle between the (sublattice) magnetization vector and the easy axis. Therefore, if the volume is small, the energy barrier between two energy minima drops down to the range of thermal energy. In the high temperature (T) regime, the reversal of the magnetic moment among the easy direction of magnetization arises, which corresponds to superparamagnetic relaxation. At low temperature, the direction of the magnetic moment is retained. For isolated (non-interacting) particle, the Néel relaxation time τN of the process is given by [START_REF] Néel | Théorie du traînage magnétique des ferromagnétiques en grains fins avec application aux terres cuites[END_REF]:

𝜏 𝑁 = 𝜏 0 . exp ( 𝐾.𝑉 𝑘 𝐵 .𝑇 ) (2)
where τ0 is a constant in the range of 10 -9 -10 -12 s depending on the material, K the magnetic anisotropy constant, and kB the Boltzmann's constant.

If τN is larger than the Mössbauer measurement time (3 10 -8 s), the probed nucleus behaves as in steady magnetic surrounding giving rise to a magnetically split sextet. For low τN, fluctuation throughout the measurement time tends to reduce average magnetization and then, sextet collapses into one or two paramagnetic lines [START_REF] Fock | On the interpretation of Mössbauer spectra of magnetic nanoparticles[END_REF]. Nevertheless, the Néel's model is not sufficient to explain the asymmetric broadening of the Mössbauer lines observed for CP-R0 at RT. Actually, superparamagnetic behavior depends also on particle size distribution, particle morphology, surface effects as well as interparticle interaction (dipole-dipole interaction and exchange interaction) [START_REF] Fock | On the interpretation of Mössbauer spectra of magnetic nanoparticles[END_REF][START_REF] Tronc | Magnetic behaviour of γ-Fe2O3 nanoparticles by mössbauer spectroscopy and magnetic measurements[END_REF]. Theoretical description of inter-particle interaction is a complex issue whose theoretical description is controversial [START_REF] De Faria | On the models for interparticle interactions in nanoparticle assemblies: comparison with experimental results[END_REF][START_REF] Mørup | Magnetic interactions between nanoparticles[END_REF]. According to Mørup et al. interparticle interaction may result in a blocking or ordering of the magnetic moments of the particles. Then, the magnetic energy of a particle can be calculated using the superferromagnetism model in the framework of the mean-field model for interacting nanoparticles [START_REF] Fock | On the interpretation of Mössbauer spectra of magnetic nanoparticles[END_REF][START_REF] Mørup | A new interpretation of Mössbauer spectra of microcrystalline goethite: "Super-ferromagnetism" or "super-spinglass" behaviour?[END_REF]: 𝐸 ≈ 𝐾. 𝑉. sin 2 (𝜃) -𝐽 𝑒𝑓𝑓 . 𝑀 0 2 (𝑇). 𝑏(𝑇). cos (𝜃)

(3) where Jeff is an effective interaction constant describing the magnetic interaction between a particle with its neighbors, M0 is the magnetization of a particle and b(T) is an order parameter of the magnetic moments of the particles. b(T) is a parameter in the range 0-1 that decreases with increasing temperature as the direction of the magnetization fluctuate. When magnetic fluctuations are fast compared to the Mössbauer measurement time, the magnetic hyperfine splitting is proportional to b(T). Distribution of anisotropy energies and interaction energies will end to in a distribution of b, which results in sextets with asymmetrically broadened lines for CP-R0 at RT. At lower temperature, the increase of b leads to narrower hyperfine field distribution and, then, to narrower sextet lines as observed at 145 K (Fig 5). According to equation 3, the particle size reduction and the dispersion of the particles into a matrix cause the weakening of both anisotropy energy and interaction energy, which in turn favor the relaxation process. The results presented in this paper are consistent with this trend. At RT, CP materials present Mössbauer spectra that evolve from magnetic profile to central single line for increasing R. Actually, starch tends to reduce crystallite size and leads to an overall dispersion of the magnetic particles.

To get a further insight into the role of starch into the growth of the spinel phase, particles were prepared by the CP method: gelatinized starch was added 15 min after base introduction (ammonia) into the reactive medium (the protocol for particle aging and particle recovery was unchanged). The PXRD pattern of the obtained material (labelled CP-R10After) corresponds to iron oxide spinel with <d>CP-R10After = 11.8 ± 1 nm. Raman lines at 667, 540 and 310 cm -1 confirms magnetite formation while FTIR probes starch fraction (see Fig 7 andS3). Interestingly, the Mössbauer spectra of CP-R10After recorded at RT and 4 K show similar profile to that of CP-R0 (see Fig 7) with close hyperfine parameters (see Table 1). This result highlights the importance of starch during the spinel growth. Actually, starch addition after coprecipitation leads to the formation of iron oxide with structural properties close to the ones of CP-R0. This is probably due to the formation of crystallite aggregates whose size is sufficient to show similar magnetic properties as CP-R0. This assumption is supported by SEM picture that shows micrometer-sized aggregates of crystallites deposited on starch (see Fig 7). Then, starch addition after coprecipitation does not result into disperse oxide crystallites. One can infer that, when present during precipitation, starch prevents crystallites aggregation. This result is in accordance with adsorption ability of CP materials. As reported previously, the arsenate adsorption capacity of CP-R10 is more than 4 times stronger than for CP-R0 [START_REF] Rakotomalala Robinson | As(V) and As(III) sequestration by starch functionalized magnetite nanoparticles: influence of the synthesis route onto the trapping efficiency[END_REF]. Such difference should be mainly attributed to limited agglomeration that preserves the specific surface area (the particle size reduction could only explain an increase of the adsorption capacity by a factor close to 1.6). For CP pathway, rapid addition of ammonia favors oxide nucleation. Nevertheless, the presence of starch during precipitation tends to reduce the crystallite size, which can be attributed to an exacerbated nucleation. Actually, starch may alter particles formation in several ways: polymer chains may (i) limit the diffusion of molecular species toward growing crystallites, (ii) stabilize the surface of small particles, (iii) act as nucleation points. Both (ii) and (iii) would result in iron oxide intimately mixed with organic phase. It is worth noticing that the inclusion of polymer chains inside nano-magnetite particles was considered for synthesis in the presence of carboxymethylcellulose [START_REF] Maccarini | Interactions at the CMC/magnetite interface: Implications for the stability of aqueous dispersions and the magnetic properties of magnetite nanoparticles[END_REF]. In here, starch inclusion may contribute to the decay of spinel crystallinity and in turn to the weakening of Fe-Fe interactions and the strengthening of the relaxation process for high R. Then the magnetic behavior of the nano-materials obtained by the CP methods can be roughly rationalized considering that increasing value of R induces the reduction of the spinel iron oxide crystallites size (possibly by starch inclusion into the oxide structure), as well as the overall increasing of the inter particle distances. These factors would favor relaxation process. All these results lead to infer that during synthesis starch chains contribute to isolate NP from each other. Then starch would behave as nano-reactor providing localized area for iron hydrolysis and particle growth, which is role of carrageenan when iron oxyhydroxide NP are prepared [START_REF] Jones | Iron oxyhydroxide colloids stabilized with polysaccharides[END_REF][START_REF] Rashid | Synthesis of magnetic nanostructures: Shape tuning by the addition of a polymer at low temperature[END_REF]. Comparing both CP and OP materials bring some light on the role of starch upon the nanostructuration of the spinel iron oxide. In the absence of starch, the OP pathway gives smooth and sharp edge magnetite particles (67 ± 5 nm) showing magnetic properties close to bulk material. The increase of R value up to 10 strongly affects both microstructural and magnetic properties. Crystallite size declines with starch amount. For OP-R10, crystallite diameter drops down to 12 ± 4 nm [START_REF] Rakotomalala Robinson | Starch functionalized magnetite nanoparticles: New insight into the structural and magnetic properties[END_REF] and lies in between the ones of CP-R0 and CP-R10 in spite of quite dissimilar magnetic properties as probed by Mössbauer spectrometry. At RT, the spectrum of OP-R10 is the sum of a magnetic component and a doublet whose contribution vanishes at lower temperature. Such features have to be attributed to the peculiar structures obtained through the OP pathway. Actually, the HRTEM micrographs of OP-R10 evidence corrugated crystallites with serrated edge, which suggests that starch induces defects preventing even growth of the oxide crystals. It appears that starch decisively steers the growth and the structural properties of the nano iron oxide spinel phase.

Conclusions

In this study, we paid attention to how the properties of nano-magnetite prepared by coprecipatation pathway are altered when gelatinized starch is introduced into the synthesis medium (iron to starch mass ratio in the range 0-10). PXRD shows the spinel structure of the oxide is preserved when starch is used but with lower crystallinity (the crystallite size is reduced from 13.7 ± 0.4 nm for CP-R0 to 8.8 ± 0.4 for CP-R10). Raman and Mössbauer spectroscopies show that, in absence of starch, coprecipitation leads to the formation of nearly stoichiometric magnetite, while increasing R leads to material containing defective magnetite. All CP materials present superparamagnetic behavior but Mössbauer spectrometry allows to evidence distinct magnetic properties. Starch lowers magnetite crystallinity and increases crystallites dispersion, both effects contribute to exacerbate relaxation process [START_REF] Fock | On the interpretation of Mössbauer spectra of magnetic nanoparticles[END_REF], which leads to fast relaxing superparamagnetic particles embedded in starch matrix. When gelatinized starch is added in the synthesis medium after coprecipitation, the obtained oxide shows similar properties as starch free material. This result highlights the role of starch to steer the growth of nano-magnetite. One can infer that in the synthesis medium, starch chains could provide nucleation points and localized volume for iron hydrolysis and particle growth. Moreover, the presence of starch into the synthesis medium during Fe 2+ and Fe 3+ precipitation prevents particles agglomeration, which is of prime importance to elaborate competitive magnetic adsorbent. Finally, comparison with materials obtained in the presence of starch through the oxidation precipitation pathway illustrates the diversity of the structural and magnetic properties that can be reached. It appears that a wide range of easy-to-prepare and ecofriendly nanostructured materials is at hands by using polysaccharide to control oxide synthesis.

Fig. 1 .

 1 Fig. 1. Schematic presentation of synthesis of starch functionalized nano magnetites by coprecipitation method.

Fig. 2 .

 2 Fig. 2. TEM micrographs and SEAD patterns of CP-R0 and CP-R10.

Fig. 3 .

 3 Fig. 3. PXRD patterns of the as-synthetized CP products at different R values and corresponding crystallite size determined from Scherrer's formula.

Fig. 4 .

 4 Fig. 4. Raman spectra of CP products and native starch.

Fig. 5 .

 5 Fig. 5. Mössbauer spectra of CP-R0, CP-R5, CP-R10 between room temperature and 13 K.

Fig. 6 :

 6 Fig. 6: Magnetic hysteresis loops at room temperature of CP-R0 (black), CP-R5 (green) and CP-R10 (red) and Langevin function fits (dashed line). Inset: magnetization at saturation for Fe3O4 NP (open symbol this work, filled symbol see ref in text).

Fig. 7 .

 7 Fig. 7. Mössbauer spectra recorded at room temperature (a) and 4 K (b), PXRD pattern (c), Raman spectrum (d) and SEM image (e) of CP-R10After.

Table 1 .

 1 Mössbauer hyperfine parameters for the as-synthetized products (values obtained after fitting the experimental data).

	Sample	Site	CS	ε or Δ	Hhf or	RA
					<Hhf>
			(mm s -1 )	(mm s -1 )	(T)	(%)
	CP-R0@RT	SA	0.27	0.01	44.0	38
		SB	0.54	-0.03	35.1	62
	CP-R0@130K	SA	0.33	0.02	49.9	35
		SB	0.59	0.01	45.9	65
	CP-R0@20K	S1	0.41	0.1	51.9	14
		S2	0.45	-0.01	50.5	45
		S3	0.53	-0.03	53.2	25
		S4	0.73	-0.07	46.3	12
		S5	0.90	1.72	34.9	4
	CP-R5@RT	S1	0.30	-0.03	42.0	32
		D	0.31	0.69	-	13
		Sg	0.38	-	-	56
	CP-R5@146K	S1	0.35	0.10	46.4	41
		S2	0.51	-0.12	37.0	52
		D	0.32	0.73	-	7
	CP-R5@13K	S1	0.43	0.01	50.9	30
		S2	0.43	-0.02	48.7	21
		S3	0.50	-0.00	52.7	42
		S4	0.65	0.08	44.2	5
		S5	0.90	1.25	34.0	2
	CP-R10@RT	D	0.34	0.98	-	34
		Sg	0.50	-	-	66
	CP-R10@135	S1	0.49	0.03	44.7	24
		D	0.44	0.87	-	17
		Sg	0.43	-	-	59
	CP-R10@18K	S1	0.41	0	50.3	35
		S2	0.42	0	48.1	33
		S3	0.48	-0.04	52.1	22
		S4	0.92	0.27	48.4	7
		S5	1.21	0.87	33.1	3
	CP-R10After@RT	SA	0.28	0	43.0	36
		SB	0.62	-0.01	32.2	64
	CP-R10After@4K	S1	0.33	0.03	51.1	45
		S2	0.57	-0.08	53.8	16
		S3	0.69	-0.01	51.1	21
		S4	0.89	-0.14	46.9	14
		S5	1.05	1.50	34.2	4
	OP-R0@RT	SA	0.29	0.00	49.0	35
		SB	0.66	0.00	45.8	65
	OP-R0@4K	S1	0.44	0.03	50.4	46
		S2	0.47	0.03	54.1	12
		S3	0.71	-0.23	52.5	13
		S4	0.82	-0.22	47.4	19
		S5	1.03	1.37	34.1	10

Acknowledgements

The authors would like to thank Sylvie Migot (IJL-Nancy), Jaafar Ghanbaja (IJL-Nancy) and Liva Dzene (IS2M, Mulhouse) for TEM imaging. We thank the PMD2X X-ray diffraction facility and Pierrick Durand (CRM2-Nancy), for X-ray diffraction measurements, data processing and analysis, and providing of reports for publication: http://crm2.univ-lorraine.fr/lab/fr/services/pmd2x. FTIR, Raman and Mössbauer spectrometric measurements were performed at the spectroscopy and microscopy service facility of SMI LCPME (Université de Lorraine-CNRS-http://www.lcpme.cnrs-nancy.fr). The authors would like to thank Stephane Suire and CC MagCryo (IJL-Nancy) for help with magnetometry measurements.


Dr Charlotte Lussey-Lepoutre
email: charlotte.lussey@inserm.fr

Alexandre Bellucci

Nelly Burnichon

Laurence Amar

Alexandre Buffet

Tom Drossart

Sébastien Fontaine

Olivier Clement

Paule Benit

Pierre Rustin

Lionel Groussin

Tchao Meatchi

Anne-Paule Gimenez-Roqueplo

Bertrand Tavitian

Judith Favier

Succinate detection by in vivo 1 H-MR spectroscopy identifies germline and somatic SDHx mutations in paragangliomas

Keywords: Magnetic resonance Spectroscopy, paraganglioma, Succinate, Succinate Dehydrogenase

Purpose

Germline mutations in genes encoding succinate dehydrogenase (SDH) are frequent in patients with pheochromocytoma and paraganglioma (PPGL). They lead to SDH inactivation, mediating a massive accumulation of succinate, which constitutes a highly specific biomarker of SDHx-mutated tumors when measured in vitro. In a recent pilot study, we showed that magnetic resonance spectroscopy ( 1 H-MRS) optimized for succinate detection (SUCCES) could detect succinate in vivo in both allografted mouse models and PPGL patients. The objective of this study was to prospectively assess the diagnostic performances of 1 H-MRS SUCCES sequence for the identification of SDH deficiency in PPGL patients.

Methods

Forty-Nine patients presenting with 50 PPGLs were prospectively enrolled in our referral center for 1 H-MRS SUCCES. Two observers blinded to the clinical characteristics and genetic status analyzed the presence of a succinate peak and confronted the results to a composite gold standard combining PPGL genetic testing and/or in vitro protein analyses in the tumor.

Results

A succinate peak was observed in 20 tumors, all of which had proven SDH deficiency using the gold standard (17 patients with a germline SDHx mutations, 2 with a somatic SDHD mutation and 1 with negative SDHB IHC and SDH loss of function). A false negative result was observed in 3 tumors. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of 1 H-MRS SUCCES were respectively 87%, 100%, 100%, 90% and 94%.

Conclusions

Detection of succinate by 1 H-MRS is a highly specific and sensitive hallmark of SDH-deficiency in PPGLs.

INTRODUCTION

Pheochromocytomas and paragangliomas (PPGL) are characterized by a very strong genetic determinism with almost 20 susceptibility genes identified so far, which mutations can impact the prognosis, clinical outcome [START_REF] Buffet | Positive Impact of Genetic Test on the Management and Outcome of Patients With Paraganglioma and/or Pheochromocytoma[END_REF] and response to treatment [START_REF] Favier | Paraganglioma and phaeochromocytoma: from genetics to personalized medicine[END_REF]. More than half of inherited PPGL involve mutations in genes encoding mitochondrial enzymes or transporters. Among them, SDHx genes (SDHA, SDHB, SDHC, SDHD, SDHAF2) encode the four subunits and the assembly factor of succinate dehydrogenase (SDH), which oxidizes succinate to fumarate in the tricarboxylic acid (TCA) cycle and constitutes the complex II of the electron transport chain. SDHx mutations predispose to multiple PPGL and SDHB mutated tumors are of strongly increased risk of metastatic progression [START_REF] Amar | Succinate dehydrogenase B gene mutations predict survival in patients with malignant pheochromocytomas or paragangliomas[END_REF][START_REF] Gimenez-Roqueplo | Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas[END_REF]. While SDHB mutations are found in around 10% of PPGL patients, it is estimated that more than one third of patients with metastatic PPGL carry an SDHB germline mutation. Similarly, it is estimated that approximately 15% of PPGL patients will ultimately develop a metastatic disease, versus 50% of SDHB-mutation carriers. Patients with hereditary SDHx-related PPGL carry a germline heterozygous mutation, which is associated with the somatic inactivation of the normal allele by somatic mutation or loss of heterozygosity according to the Knudson's 2-hit model for tumor suppressor genes. This bi-allelic inactivation induces a complete loss of activity SDH in the tumor, leading to the accumulation of succinate, which acts as an oncometabolite and is suspected to mediate most tumorigenic effects related to SDHx mutations [START_REF] Favier | The Warburg effect is genetically determined in inherited pheochromocytomas[END_REF][START_REF] Letouze | SDH mutations establish a hypermethylator phenotype in paraganglioma[END_REF][START_REF] Morin | Oncometabolites-driven tumorigenesis: From genetics to targeted therapy[END_REF][START_REF] Selak | Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase[END_REF].

In SDHx-mutated PPGL, succinate concentrations increase dramatically, up to 100-fold with respect to tumors without SDHx mutations [START_REF] Pollard | Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations[END_REF][START_REF] Richter | Krebs cycle metabolite profiling for identification and stratification of pheochromocytomas/paragangliomas due to succinate dehydrogenase deficiency[END_REF][START_REF] Rao | Genotype-specific abnormalities in mitochondrial function associate with distinct profiles of energy metabolism and catecholamine content in pheochromocytoma and paraganglioma. Clinical cancer research : an official journal of the[END_REF]. We recently adapted a magnetic resonance spectroscopy (MRS) method to detect succinate in tumors (SUCCinate Estimation by Spectroscopy (SUCCES)) relying on a monovoxel PRESS asymmetric « PROBE » sequence. This pilot study [START_REF] Lussey-Lepoutre | In Vivo Detection of Succinate by Magnetic Resonance Spectroscopy as a Hallmark of SDHx Mutations in Paraganglioma. Clinical cancer research : an official journal of the[END_REF] performed in 9 patients with PPGL (5 with SDHx mutations and 4 sporadic cases) demonstrated that succinate accumulation was indeed detectable in the tumors of all SDHx-mutated patients but not in non-SDHx tumors. An unexplained choline peak was also observed in SDHx-mutated cases. These results supported the proof-of-concept that noninvasive detection of succinate by in vivo 1 H-MRS may constitute a specific biomarker of SDHx mutations. Following this first-in-man study, we here report the results of a prospective study aiming at evaluating the clinical relevance of 1 H-MRS SUCCES in the routine care of PPGL patients.

MATERIALS AND METHODS

Patients

Patients explored for a PPGL in the hypertension unit of Hôpital Européen Georges Pompidou, Paris (France) were included prospectively if they met the following criteria: 1-age ≥ 18 years-old; 2-PPGL diagnosis in accordance with international clinical practice guidelines in a reference centre, relying on biochemistry, cross-sectional and functional imaging [START_REF] Lenders | Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline[END_REF]; 3-signed informed consent for PPGL genetic testing; 4-no contraindications to MRI (i.e metallic implants, claustrophobia,..).

Ethical approval for the study was obtained from the institutional review board (Comité de Protection des Personnes (CPP) Ile de France II). All patients provided written informed consent for participation to the study, collection of samples and subsequent analyses. When a surgical procedure was performed, fresh tumor samples were frozen immediately after surgical resection and subsequently stored at -80°C until processing following the COMETE collection procedures [START_REF] Plouin | COMETE, a network for the study and management of hypersecreting adrenal tumors[END_REF]. Histologic diagnosis was performed on paraffin-embedded formalin fixed samples.

Succinate detection by 1 H-MRS at 3 Tesla

Combined MR images and MR spectroscopic scans of patients were acquired in a 3 Tesla MRI clinical scanner (Discovery MR750w GEMSOW, GE Medical Systems, Milwaukee, WI), as previously described [START_REF] Lussey-Lepoutre | In Vivo Detection of Succinate by Magnetic Resonance Spectroscopy as a Hallmark of SDHx Mutations in Paraganglioma. Clinical cancer research : an official journal of the[END_REF]. 1 H-MRS spectra were acquired using the optimized SUCCES sequence: asymmetric Point REsolved SpectroScopy (PRESS) monovoxel acquisition based on the PROBE monovoxel sequence [START_REF] Webb | Automated single-voxel proton MRS: technical development and multisite verification[END_REF], with TR: 2500 ms; TE: 144 ms; Nex: 512 (22 min acquisition) or 1024 (44 min acquisition).

Detection of tumors and VOI (Volume of Interest) positioning were performed on thin-section highresolution T2-weighted fast spin-echo imaging in at least two orthogonal planes with the following parameters: TR: 2500 ms; TE: 85 ms; echo train length: 19; slice thickness: 2 mm; spacing: 0.3; field of view: 14x14cm for neck or 42x42 cm for whole body coil; matrix: 320×320 or on thin-section highresolution balanced steady-state gradient echo sequence (FIESTA) in at least two orthogonal planes with the following parameters: TR: 4.9 ms; TE: 1.4 ms; NEX:1; Flip angle : 50; slice thickness: 3 mm; spacing: 1; field of view: 14x14cm for neck or 42x42 cm for whole body coil; matrix: 256x256.

Immunochemistry

SDHA and SDHB protein expression were assessed on formalin-fixed paraffin embedded (FFPE) tumor samples by immunohistochemistry (IHC) as previously described [START_REF] Korpershoek | SDHA immunohistochemistry detects germline SDHA gene mutations in apparently sporadic paragangliomas and pheochromocytomas[END_REF][START_REF] Menara | SDHD immunohistochemistry: a new tool to validate SDHx mutations in pheochromocytoma/paraganglioma[END_REF][START_REF] Van Nederveen | An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC, or SDHD gene mutations: a retrospective and prospective analysis[END_REF] using the following antibodies and conditions: anti-SDHA (ab14715, Abcam; 1:1000) and anti-SDHB (HPA002868, Sigma-Aldrich Corp; 1:500) .

SDH activity

SDH activity was investigated, when necessary, on frozen tumor samples, homogenized using a 1 ml glass-glass Potter-Elvehjem, using a spectrophotometric assay, as previously described [START_REF] Benit | Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples[END_REF].

Genetic analysis

DNA extracted from leukocytes was genotyped in all included patients, as well as DNA extracted from frozen or FFPE-tumors when available. Genetic analyses were performed by Next Generation Sequencing (NGS) using the 'MASTR plus SDHv2' targeted panel (Multiplicom, Agilent technologies) as previously reported and in accordance with international guidelines [START_REF] Aim | Targeted next-generation sequencing detects rare genetic events in pheochromocytoma and paraganglioma[END_REF][START_REF] Ngsips | Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas[END_REF].

Data analysis

Two independent investigators (CLL and ABE), blinded from clinical observations and genetic test results at the time of analyses, examined all spectra in a qualitative manner and scored for the presence or absence of a succinate peak at 2.44ppm, lactate/lipid signal (0.9 to 1.3 ppm), choline peak (3.2 ppm). When spectra interpretation was different between the two investigators, discordance(s) have been resolved by consensus by a panel of experts (CLL, ABE, JF and BT). Tumor and VOI size as well as tumor heterogeneity were evaluated on the FIESTA or the T2-weighted fast spin-echo sequence. 1 H-MRS data were initially compared to the genetic gold standard of SDH deficiency assessed by germline genetic testing. In cases of discrepancies between germline genetic test and 1 H-MRS results, a composite gold standard was applied by considering the results of tumor tissue analyses (somatic NGS, SDHB IHC or SDH activity measurement in the tumor). Tumors were thus considered as SDH deficient when (i) an SDHx mutation was detected on germline and/or (ii) tumor DNAs, or (iii) when SDH inactivation was supported by both of the protein analyses of tissue samples (SDHB/A negative immunochemistry and loss of SDH activity). Sensitivity (Se), specificity (Sp), positive/ negative predictive values (PPV/NPV) and accuracy of the method were calculated using the SISA (Simple Interactive Statistical Analysis) procedure with continuity corrected Wilson 95% confidence intervals presentation.

RESULTS

Patients

Fifty-six patients, including the 9 from the original pilot study [START_REF] Lussey-Lepoutre | In Vivo Detection of Succinate by Magnetic Resonance Spectroscopy as a Hallmark of SDHx Mutations in Paraganglioma. Clinical cancer research : an official journal of the[END_REF], were prospectively enrolled between January 2015 and March 2019, leading to the exploration of 57 tumors with the 1 H-MRS SUCCES sequence (one abdominal PGL and one head and neck PGL were explored in patient 1 (previously reported in the pilot study, Table S1) carrying an SDHB mutation [START_REF] Lussey-Lepoutre | In Vivo Detection of Succinate by Magnetic Resonance Spectroscopy as a Hallmark of SDHx Mutations in Paraganglioma. Clinical cancer research : an official journal of the[END_REF]). Seven patients were excluded. One presented a surgical clip, one a non-evaluable highly necrotic tumor, one was diagnosed with a poorly differentiated neuroendocrine tumor instead of a PPGL following pathological analysis of the tumor and the "gold standard" data could not be obtained for four of them. Overall, 49 patients with 50 tumors were analyzed (Figure 1) and their main characteristics are presented in Table 1. Median age at the time of 1 H-MRS examination was 44.7 years-old .

The 50 tumors were located as follow: 24 head and neck PGL, 9 abdominal PGL and 17 pheochromocytomas (PCC).

H-MRS SUCCES performance and added value for management of patients

A succinate peak at 2.44ppm was observed in 20 tumors (Figure 2), while a choline peak was detected in 27 tumors, including 17 with an associated succinate peak. Genetic status was known at the time of 1 H-MRS for 9 patients (18%). For the remaining 40 patients, genetic status was available for clinician after genetic counseling and NGS analyses of germline and somatic DNA in average 12.2 months (± 8.6 months) after 1 H-MRS results. Among the 19 patients (20 tumors) showing a tumor succinate accumulation, 16 (17 tumors) were confirmed to carry a germline SDHx mutation: 1 SDHA, 4 SDHB, 2 SDHC and 9 SDHD.

In 3 patients, 1 H-MRS SUCCES suggested SDH deficiency despite a negative genetic test. In 2 of them, NGS analysis of tumor DNA identified an SDHD somatic mutation (patients 20 and 21, Table S1). The third patient (Patient 22, Table S1) was a forty-two year-old patient with a familial history PPGL (his father developed bilateral carotid body PGL diagnosed at 40 years-old). The patient suffered from a right vagal PGL operated ten years earlier and a left vagal PGL treated at the same period by external beam radiotherapy. 1 H-MRS SUCCES was performed in a metastatic relapse of the left vagal PGL and showed a succinate peak. PPGL genetic testing was negative both in germline and somatic DNA, but SDH deficiency was demonstrated by negative SDHB IHC and complete inhibition of SDH enzymatic activity in the tumor (Figure 4). Hence, there was no false positive finding.

Thirty tumors (30 patients) showed no succinate accumulation detectable by 1 H-MRS SUCCES. Gold standard confirmed the absence of SDH deficiency in 27 cases (Table S2). Two patients carried a germline mutation in the VHL gene, while germline genotyping was negative in 25 patients. In the latter cases, NGS identified 10 with somatic mutations (2 in EPAS1, 4 in HRAS, 3 in VHL and 1 in NF1 gene).

A false negative result (i.e. no succinate peak in a patient with an SDHx mutation) was observed in 3 tumors (Figure 3). The first patient (SDHB mutated, Patient 7) presented with a cervical PGL that included the carotid artery and the jugular vein, preventing the use of the 1 H-MRS SUCCES which had to be performed on a very small metastatic lymph node (voxel size 0.7 cm), which may have been a limiting factor. The second patient (Patient 9) carried a germline SDHD gene mutation and suffered from a right cervical PGL with numerous hemorrhagic and necrotic spots (voxel size 2.5 cm3). The third one was a 20 years old patient (Patient 6) followed in a familial context of germline SDHD gene mutation with a small left-sided PCC (voxel size 1.5 cm3), for which respiratory motion may have compromised the result.

Discordance between the 2 investigators (CLL and ABE) were observed for 2 cases (Patients 13 and 36, Table S1 andS2) and resolved by consensus by the panel of experts.

Altogether, at the lesion level, sensitivity, specificity, PPV, NPV and accuracy of 1

DISCUSSION

To our knowledge, this is the first prospective validation study demonstrating high sensitivity and specificity of non-invasive detection of succinate by in vivo 1 H-MRS. Our results qualify the presence of a succinate peak at 2.44 ppm as a specific bona fide biomarker of SDH deficiency in patients suffering from PPGL, whatever the SDHx gene mutated (SDHA, B, C, D).

We clearly show that 1 H-MRS SUCCES allows early suspicion of SDH deficiency in routine clinical practice. Indeed, for almost 80% of patients, results of 1 H-MRS SUCCES sequence were provided to clinicians before the results of genetic test. A recent multicentric retrospective study demonstrated that early knowledge of genetic status has a positive impact on the management and clinical outcome of patients with a germline SDHx mutation [START_REF] Buffet | Positive Impact of Genetic Test on the Management and Outcome of Patients With Paraganglioma and/or Pheochromocytoma[END_REF]. Identification of a succinate peak with 1 H-MRS SUCCES sequence should therefore accelerate genetic testing in these patients, focusing and facilitating the interpretation of genetic variations in SDHx genes provided by NGS. In a patient with initial PPGL diagnosis, early assessment of SDHx mutational status may impact patient's management, by taking into account the higher risk of metastasis of SDHB-related tumors, or the high rate of multiple cervical PGL for SDHD-mutated patients. Finally, early identification of patients with genetic predisposition will also accelerate the familial pre-symptomatic genetic screening and therefore benefit to relatives of index patients.

Importantly, we demonstrate that, in case of negative germline genotyping, the finding of a positive succinate peak using 1 H-MRS SUCCES should definitively prompt to search for SDHx somatic mutations in tumor DNA. In our series, this approach led to the identification of 2 tumors with somatic SDHD variants, which have only been very rarely reported in the past [START_REF] Aim | Targeted next-generation sequencing detects rare genetic events in pheochromocytoma and paraganglioma[END_REF][START_REF] Curras-Freixes | Recommendations for somatic and germline genetic testing of single pheochromocytoma and paraganglioma based on findings from a series of 329 patients[END_REF][START_REF] Van Nederveen | Somatic SDHB mutation in an extraadrenal pheochromocytoma[END_REF]. It could also prompt to go deeper in the exploration of some patients like in patient 22, familial case presenting with an SDH deficiency despite the absence of germline and somatic SDHx variants.

Several hypotheses such as an intronic mutation on germline DNA not detected by NGS analyses, epigenetic mutations in SDHx promoters or mutation in a regulator gene of SDHx genes expression, are under evaluation to go further in this case by performing a whole genome sequencing.

Moreover, the identification of a succinate peak in a tumor is a major argument to support the pathogenicity of a variant of unknown significance (VUS) in an SDHx gene, which is essential to assess the risk of recurrence, to adapt the follow-up and to manage the genetic counseling to relatives [START_REF] Aim | Targeted next-generation sequencing detects rare genetic events in pheochromocytoma and paraganglioma[END_REF][START_REF] Favier | Paraganglioma and phaeochromocytoma: from genetics to personalized medicine[END_REF]. In our cohort, a VUS was detected in 3 patients and classified as deleterious or not (a likely benign VUS in SDHA was ruled out, and 2 VUS, 1 on SDHB (Patient 1) and 1 on SDHD (Patient 14) genes, were eventually classified as pathogenic), based on in silico predictions and SDHB IHC. In these 3 patients, 1 H-MRS results were concordant with IHC results, suggesting that 1 H-MRS SUCCES is a of useful contribution for patient management, especially whenever the absence of tumor tissue precludes immunohistochemical analyses. Assessing this tumor hallmark in vivo in patients with SDHx-related tumors will present benefits in other aspects. In a patient carrying a germline SDHx variant it could help: i) to classify a suspicious lesion detected during follow-up and ii) to confirm the metastatic development of the disease after the identification of one (or several) dubious lesion(s) in extra-paraganglionic sites on imaging follow-up. SDHx mutations also predispose to other types of tumors such as GIST (GastroIntestinal Stromal Tumors), rare cases of renal cell carcinomas and of pituitary adenomas, which are still debated [START_REF] Boikos | Molecular Subtypes of KIT/PDGFRA Wild-Type Gastrointestinal Stromal Tumors: A Report From the National Institutes of Health Gastrointestinal Stromal Tumor Clinic[END_REF][START_REF] Evenepoel | Toward an improved definition of the genetic and tumor spectrum associated with SDH germ-line mutations[END_REF][START_REF] Mason | Conventional Risk Stratification Fails to Predict Progression of Succinate Dehydrogenase-deficient Gastrointestinal Stromal Tumors: A Clinicopathologic Study of 76 Cases[END_REF][START_REF] Ricketts | Germline SDHB mutations and familial renal cell carcinoma[END_REF]. Interestingly, in a recent study, the application of 1 H-MRS was successfully extended to metastasis of PPGL and GIST and permitted to exclude SDH deficiency in a pituitary adenomas developed in an SDHB mutation carrier [START_REF] Casey | Translating in vivo metabolomic analysis of succinate dehydrogenase deficient tumors into clinical utility[END_REF]. However, it is important to note that in our study, only 2 patients with an SDHx mutation were explored for an abdominal PGL. Additional studies should be performed in the future to evaluate 1 H-MRS SUCCES accuracy for this particular location.

Regarding clinical relevance, we suggest to perform 1 H-MRS SUCCES in all patients with a newly discovered PPGL, in patients with negative germline genetic testing in case of multiple tumors and/or a metastatic development of the disease to tailor therapeutic choices, and in patients already identified to carry a germline SDHx mutation in case of dubious finding on imaging follow-up. By contrast, 1 H-MRS SUCCES would not present any relevance in patients with a known mutation on a non-SDHx susceptibility gene.

Imaging of patients with PPGL is crucial in every step of their management and functional imaging using PET tracers provide important information on tumor biology, highly connected with genetic status [START_REF] Taieb | Molecular imaging and radionuclide therapy of paraganglioma and pheochromocytoma[END_REF]. Recent development of PET/MR devices would allow to combine functional imaging and 1 H-MRS, providing simultaneously and non-invasively coregistered anatomical, metabolic (i.e. FDG uptake) and metabolomics (succinate accumulation) information on the tumor. H-MRS SUCCES permitted in this study to assess for the presence (or absence) of a succinate peak in a qualitative manner. However, quantification of succinate in vivo with 1 H-MRS is feasible and development of a specific post-processing method allowing assessment of succinate as a quantitative biomarker over the time course of the disease would be of great interest especially for the evaluation of response to treatment. It should also be stressed that SUCCES has the same limitations as any 1 H-MRS approach, in particular sensitivity in the low millimolar range. Accordingly, its capacity to detect succinate in PPGL lesions is dependent on: (i) the size of the tumor, which is directly related to detection sensitivity: a voxel size smaller than 1 cm 3 is associated with a high risk of failure (Table 2); (ii) the presence of hemorrhagic or necrotic spots, which is a source of technical flaws: a large necrotic area or inclusion of blood vessel within a tumor may discourage exploration by 1 H-MRS SUCCES (Figure 3). Nevertheless, there is still room for progress and in this respect, several adjustments may improve sensitivity and decrease the sequence duration. These include increasing MRI field using the new research-dedicated 7-tesla devices, post-processing of the spectra or respiratory gating for abdominal tumors. Indeed, respiratory gating would be a possible adjustment for abdominal tumors and in particular for PCC but with an increase of total duration of the sequence, a risk of patient discomfort and then motion. Nevertheless, one solution could be to analyse respiratory-gated acquisition combined with an increased MRI field (7 tesla) that could allow reducing averages number and thus sequence duration.

In conclusion, using 1 H-MRS to detect specific tumor phenotypes induced by genetic mutations is innocuous (no tissue sampling, no injection of radiopharmaceutical or contrast agent are needed) and precise. Following the initial pilot studies [START_REF] Lussey-Lepoutre | In Vivo Detection of Succinate by Magnetic Resonance Spectroscopy as a Hallmark of SDHx Mutations in Paraganglioma. Clinical cancer research : an official journal of the[END_REF][START_REF] Varoquaux | Magnetic resonance spectroscopy of paragangliomas: new insights into in vivo metabolomics[END_REF][START_REF] Casey | Translating in vivo metabolomic analysis of succinate dehydrogenase deficient tumors into clinical utility[END_REF], this study crosses the second translational gap and demonstrates that 1 H-MRS SUCCES can be performed in a radiology department during routine clinical practice.

TABLES AND FIGURES

1

 1

  1 H-MRS improves genetic diagnosis, providing VUS characterization and guiding geneticists to extend investigations to somatic NGS. 1 H-MRS SUCCES is ideal for assessing the presence of succinate repeatedly over the time course of the disease, for clinical surveillance, postoperative follow-up, and evaluation of treatment efficacy. AKNOWLEGMENT We thank Catherine Tritscher for administrative assistance, Estelle Robidel, Anne-Laure Gauthier and Benjamin Banting for technical assistance. This work has received funding from the Paradifference Foundation, the European Union's Horizon 2020 research and innovation program under grant agreement No 633983 and by the Institut National du Cancer and the Direction Générale de l'Offre de Soins (PRT-K 2014, COMETE-TACTIC, INCa-DGOS_8663) and the Cancer Research for Personalized Medicine -CARPEM project (Site de Recherche Intégré sur le Cancer -SIRIC). Our team is supported by the Ligue Nationale contre le Cancer (Equipe Labellisée). The genetical and phenotyping parts of this study were granted by the Institut National du Cancer and the Direction Générale de l'Offre de Soins (PRT-K 2014, COMETE-TACTIC, INCa-DGOS_8663).

Figure 1 :Figure 2 : 1 H

 121 Figure 1: Flowchart

Figure 3 :

 3 Figure 3: 1 H-MRS SUCCES failures in SDH-deficient tumors. A) Left panel shows both the cervical

Figure 4 :

 4 Figure 4: Patient 22 with SDH deficiency demonstrated by functional studies. A) Axial T2-weighted

Table 1 : Baseline characteristics of patients (N=49 patients, 50 tumors)Table 2 : Comparison between successes and failures of 1 H-MRS SUCCES in patients with an SDH deficiency Table S1: Characteristics of the 22 patients with an SDH deficiency Table S2 : Analyses performed to rule out SDH deficiency in 27 patients

 12

statement No funding was received for this work

Age (years, Median [range])

44.7

Gender

Female (%) [START_REF] Evenepoel | Toward an improved definition of the genetic and tumor spectrum associated with SDH germ-line mutations[END_REF]

Click here to access/download Supplementary Material Lussey-Lepoutre-Table S1.docx Table S2 Click here to access/download Supplementary Material Lussey-Lepoutre-Table S2.docx