Dr Sabrina Belbekhouche
email: belbekhouche@icmpe.cnrs.fr

Julie Oniszczuk

André Pawlak

Imane El Joukhar

Angélique Goffin

Gilles Varrault

Dil Sahali

Benjamin Carbonnier
email: carbonnier@icmpe.cnrs.fr

Cationic poly(cyclodextrin)/alginate nanocapsules: From design to application as efficient delivery vehicle of 4-hydroxy tamoxifen to podocyte in vitro

Keywords: particle-templated layer-by-layer assembly, poly(cyclodextrin), capsule, supramolecular chemistry, host-guest complexation, podocyte

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction.

Nowadays, the efficient delivery of hydrophobic drugs is still a key challenge in the field of biotechnology. [START_REF] Agarwal | Stable nanocolloids of poorly soluble drugs with high drug content prepared using the combination of sonication and layer-by-layer technology[END_REF][START_REF] Smith | Layer-by-Layer Platform Technology for Small-Molecule Delivery[END_REF] The limited clinical effectiveness may result from low solubility in aqueous media, toxicity profile, and/or other poor pharmaceutical characteristics. The development of drug vehicles has then been considered for encapsulating and delivering poorly water-soluble drugs for several therapies. [START_REF] Mura | Stimuli-responsive nanocarriers for drug delivery[END_REF]. For the design of polymer-based delivery systems various types of carrier-drug interactions, including electrostatic or hostguest ones, may be considered providing innovative solutions for carrying one or multiple drug payloads. Similarly to common host molecules such as pillararenes, cucurbiturils and crown ethers, low toxic cyclic oligosaccharides as cyclodextrins (CDs) can incorporate various guest molecules into their hydrophobic cavity, thereby enabling enhanced solubilization of high amounts of hydrophobic drugs and potentially transport though cell membranes. [START_REF] Hu | Cyclodextrin-Based Host-Guest Supramolecular Nanoparticles for Delivery: From Design to Applications[END_REF] In the literature, two strategies are presented to prepare CD-containing LbL assemblies. The first one involves the use of charged CDs, and the LbL assemblies are obtained through electrostatic interactions. [START_REF] Yang | Polyelectrolyte and molecular host ion self-assembly to multilayer thin films: An approach to thin film chemical sensors[END_REF][START_REF] Sato | Preparation of Polyelectrolyte-Layered Assemblies Containing Cyclodextrin and Their Binding Properties[END_REF][START_REF] Sato | Layered assemblies composed of sulfonated cyclodextrin and poly(allylamine)[END_REF] Nevertheless, molecular cyclodextrins did not provide highly stable entrapment of the guest molecules, leading to a rapid drug release. The second strategy for constructing CD-containing LbL assemblies relies on the use of host-guest complexation as the driving force for the assembly built up. [START_REF] Suzuki | Construction of positivelycharged layered assemblies assisted by cyclodextrin complexation[END_REF][START_REF] Van Der Heyden | Multilayer films based on hostguest interactions between biocompatible polymers[END_REF][START_REF] Kaftan | Probing Multivalent Host-Guest Interactions between Modified Polymer Layers by Direct Force Measurement[END_REF][START_REF] Dubacheva | Unlimited growth of host-guest multilayer films based on functionalized neutral polymers[END_REF] In this case, CD-containing polymers and guest molecule-appended polymers are built into LbL multilayers by forming host-guest complexes.

Herein we propose an alternative path based on supramolecular chemistry for both the selfassembly of multilayered nanocapsules and the encapsulation of 4-hydroxytamoxifen via electrostatic and hydrophobic effect driven inclusion complex formation, respectively. The asdesigned nanometer-sized hollow polymeric spheres (or capsules) are expected to exhibit high stability over a wide pH range, and enable efficient intracellular drug delivery. LbL-based hollow capsules are generally stable, explaining the interest of using them as drug carriers. [START_REF] Del Mercato | Biological applications of LbL multilayer capsules: from drug delivery to sensing[END_REF][START_REF] Belbekhouche | Promising sub-100 nm tailor made hollow chitosan/poly (acrylic acid) nanocapsules for antibiotic therapy[END_REF] Indeed, our approach involves the use of a charged polymeric carrier, namely a cationic polycyclodextrin, capable of complexation with a hydrophobic chemical in alternation with a degradable polyanion (the alginate). The synthesis of the polycation was previously reported [START_REF] Thuaud | Retention behavior and chiral recognition of β-cyclodextrinderivative polymer adsorbed on silica for warfarin, structurally related compounds and Dns-amino acids[END_REF] but no previous study explored its potential use for the elaboration of capsules with drug loading ability. Herein, the affinity of the cyclodextrin cavities has been investigated with a model hydrophobic probe and a drug, namely pyrene and 4-hydroxytamoxifen (OHT), respectively. [START_REF] Indra | Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases[END_REF] OHT is the major active metabolite of tamoxifen which binds selectively estrogen receptor (ER) and estrogen-related receptors (ERR) with estrogenic and anti-estrogenic effects.

Physiopathologic systems have been investigated through transgenic tools implying the targeted expression of the Cre-recombinase fused with a mutated form of the ER. This latter allows high affinity binding of OHT and a low affinity for endogenous estrogen [START_REF] Indra | Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases[END_REF][START_REF] Feil | Ligand-activated site-specific recombination in mice[END_REF]. The fusion protein translocates from the cytoplasm to the nucleus upon its binding with OHT. This involves the excision of genomic segments between loxP sequences.

In our study, we will use a conditionally immortalized mouse podocytes cell line which has been developed from transgenic mice Cmip conditional knock-out mice as described elsewhere [START_REF] Moktefi | Repression of CMIP transcription by WT1 is relevant to podocyte health[END_REF]. Indeed, these mice possess coding sequences for the chimeric Cre recombinase associated with the mutated domain of the estrogen receptor (CreERT2). Upon treated with binding OHT, the chimeric CreER protein translocation in the nucleus is induced where the recombinase can play its role and thus cause the deletion of the exon 8 of the second allele. It deserves noting that the formation the inclusion complex between tamoxifen and its derivatives and CD has already been reported [START_REF] Memisoglu-Bilensoy | Tamoxifen citrate loaded amphiphilic β-cyclodextrin nanoparticles: In vitro characterization and cytotoxicity[END_REF][START_REF] Daoud-Mahammed | Cyclodextrin and Polysaccharide-Based Nanogels: Entrapment of Two Hydrophobic Molecules, Benzophenone and Tamoxifen[END_REF] but to the best of our knowledge, there are no studies reporting the host-guest (polycationic CD-4hydroxytamoxifen) interaction-mediated Cmip exon 8 deletion in podocytes selected as an in vitro model. Thus, this contribution aims at providing evidences for (i) the efficient loading of 4-hydroxytamoxifen within polycationic CD-alginate multilayered nanocapsules through inclusion complex formation, (ii) the preservation of the biological activity of OHT through such delivery pathway.

Experimental section

Materials

The following chemicals were used as received: chitosan (chi + , Sigma-aldrich), alginate (alg -, Sigma-aldrich), sodium 2-mercaptoethanesulfonate (Sigma-aldrich, > 89.5%), pyrene (Sigma-Aldrich), 4-hydroxy-tamoxifen (OHT, Sigma-Aldrich), gold nanoparticles (60 nm, Sigma-Aldrich) and potassium cyanide (KCN, Sigma-Aldrich). The cationic cyclodextrin (P(CD + )) was synthesized according to the procedure developed by Thuaud et coll. [START_REF] Thuaud | Retention behavior and chiral recognition of β-cyclodextrinderivative polymer adsorbed on silica for warfarin, structurally related compounds and Dns-amino acids[END_REF] The cationic cyclodextrin polymer was synthesized by crosslinking the β-cyclodextrin (β-CD) with epichlorohydrin [START_REF] Renard | Preparation and characterization of water soluble high molecular weight β-cyclodextrin-epichlorohydrin polymers[END_REF] followed by a reaction with the 2,3 epoxypropyltrimethylammonium chloride. [START_REF] Thuaud | Retention behavior and chiral recognition of β-cyclodextrinderivative polymer adsorbed on silica for warfarin, structurally related compounds and Dns-amino acids[END_REF] Briefly, β-CD reacted in NaOH solution (33 %) with epichlorohydrin at 50 °C for 210 min. After neutralizing the pH, the polymer was concentrated by ultrafiltration to remove salt or low molecular compounds and recovered through freeze-drying process. The resulting β-CD polymer was chemically modified with quaternary ammonium side groups through reaction with 2,3-epoxypropyltrimethylammonium chloride in basic media. The resulting modified polymer was purified by a dialysis step using a cellulose dialysis membrane (1 kDa cut off). The product was isolated via freeze-drying to yield a white solid.

Average molar masses and molar masses distributions were determined by size exclusion chromatography (SEC) coupled online with multi-angle light scattering (MALS) and differential refractive index (DRI) detectors. The MALS apparatus is the DAWN Heleos-II from Wyatt Technology (Ca, USA) filled with a K5 cell and a Ga-As laser (λ= 690 nm). The DRI detector is a Shimadzu RID-10A (Japan). Columns [Shodex SB OHpak 804 and 806 HQ for alginate (dn/dc = 0.140 mL.g -1 [START_REF] Colinet | New amphiphilic modified polysaccharides with original solution behaviour in salt media[END_REF]) and PL aquagel OH 40+30 for the cationic polycyclodextrin] were eluted with 0.1mol.L -1 LiNO3 at 0.5 mL min -1 . The flow rate was fixed at 1 mL.min -1 . The samples were filtered on 0.45 μm unit filter before injection through a 100 μL full loop.

For the alginate, the value is 2.7 10 5 g•mol -1 , the value is 3.8 10 5 g•mol -1 . For the cationic poly(cyclodextrin), the value is 6.5 10 3 g•mol -1 , the value is 2.4 10 4 g•mol -1 and the degree of substitution is 0.40 ±0.05 (estimated from 1 H NMR [START_REF] Thuaud | Retention behavior and chiral recognition of β-cyclodextrinderivative polymer adsorbed on silica for warfarin, structurally related compounds and Dns-amino acids[END_REF]).

Synthetic procedures

Elaboration of P(CD + )/alginate multilayers films onto flat gold substrate

A Q-Sense E1 device (Q-Sense, Sweden) was employed to monitor the in situ formation of the multilayer self-assembly of (P(CD + )/alg -) bilayers. The gold coated QCM crystals were first cleaned with water and ethanol, then treated in an UV/ozone chamber (BioForce UV/Ozone ProCleaner) for 20 min and pretreated with sulfonated thiol (5 g.L -1 , 10 mg sodium 2-mercaptoethanesulfonate in 2 mL of water). This sulfonated thiol derivative is used as an anchoring layer for enabling the further adsorption of the polycation layer. After a supplementary rinsing with ethanol, surfaces were dried under N2 flow. After mounting in the QCM-D flow chamber, the sensor was conditioned in water for 10 min prior to monitoring the self-assembly. All solutions were injected into the cell with a flow rate of 250 μL.min -1 at 25

°C. The self-assembly of P(CD) + / alg -was performed onto the pretreated negatively charged flat gold substrate via alternative contact with first the polycation followed by the polyanion solutions. Wetting angle measurements were performed at room temperature with a Krüss apparatus in the sessile drop configuration. A drop (5 μL) was laid upon a flat gold substrate bare or modified with the multilayer film, and then, an image was captured using a black and white CCD camera.

Fabrication of P(CD + )-based nanocapsules

In order to adsorb the cationic-poly(cyclodextrin) and the polyanionic alginate on the spherical surface, the colloidal gold nanoparticles were first pretreated with a thiol derivative (sulfonate groups on the surface). 500 µL of a filtered suspension of gold nanoparticles, 400 µL of aqueous solution and 100 µL of sodium 2-mercaptoethanesulfonate solution (5 g.L -1 , 10 mg in 2 mL of water) were mixed overnight in an Eppendorf tube. Free thiol derivative was removed from the supernatant after centrifugation (17968 g for 15 min). 100 µL of cationicpoly(cyclodextrin) (1 g.L -1 prepared in water) solution was added to a suspension of pretreated gold nanoparticles. After 20 min, the polyelectrolyte excess was removed from the supernatant fraction after centrifugation (17968 g for 15 min). This washing process was repeated two more times keeping the final volume equal to 1 ml. This adsorption process was further repeated with the addition of 100 µL of alginate solution (1 g.L -1 prepared in water) to the suspension of cationic-poly(cyclodextrin)-modified gold nanoparticles. This procedure describes the assembly of a single bilayer: (cationic-poly(cyclodextrin) /alginate)1

(P(CD + )/alg -)1. The LbL process was repeated to get the nanomaterials with a desired number of layers. This process was monitored with zeta potential measurement (Zetasizer Nano-ZS, Malvern Instrument).

Nanocapsules were obtained by dissolving the gold core using 100 µL of potassium cyanide solution (70 mg of KCN in 15 mL of water). This step was followed by a dialysis process to mol.L -1 , preparation described below) or a 4-hydroxy-tamoxifen solution (1 µM µmol.L -1 prepared in ethanol).

Preparation of a pyrene solution at 4.10 -7 mol.L -1 : A stock solution of pyrene (10 -3 mol L -1 ) was prepared in acetone. 200 µL aliquot of this solution was introduced into empty flask and the solvent was evaporated. After evaporation, the flask were filled with 500 mL of water and gently stirred for 24 h. The final pyrene concentration was 4.10 -7 mol.L -1 . All samples were excited at 332 nm and the emission spectra of pyrene showed five vibronic peaks notably at 372 nm and 382 nm. The ratios I372/I382 (error less than 5%) were then calculated leading to a measure of the polarity of the pyrene micro-environment. [START_REF] Kalyanasundaram | Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems[END_REF] Fluorescence measurements were performed with a spectrofluorimeter (Japan, Jasco) equipped with a xenon lamp. The amount of pyrene incorporated in the CD cavity was estimated by fluorescence intensity measurements after the extraction of pyrene from capsules with methanol. The suspension of capsules (100 μl, 950 million nanoparticles) was centrifuged, and the supernatant was removed. The experiment was performed with pyrene-loaded (P(CD + )/alg -)n-P(CD + )-coated (n = 2 and 4) particles. After the addition of methanol (500 µL), the suspension was again centrifuged, and the supernatant containing pyrene was recovered. This process was repeated two times to ensure the total extraction of pyrene from the capsules. Then, the fluorescence intensity of the solutions was measured.

In Vitro Biocompatibility Studies.

Cell Culture. Immortalized mouse podocytes were cultured under permissive conditions:

RPMI 1640 medium containing 10% fetal calf serum, penicillin (100 µg/ml), streptomycin (100 μg/ml), 1% of non-essential amino acids, 1% of HEPES, 1% of sodium pyruvate and interferon-γ (IFN-γ, 50 U/ml), at 33°C. At 60-80% confluence on 6-well plates, the 4hydroxy-tamoxifen-loaded nanomaterials (x 9.5 10 9 particles/ml) were immediately added to the cells. After exposure to nanoparticles for 48h, cells were harvested and genotyping was processed afterwards

Results and discussion

Polymeric nanoparticles/assemblies are promising candidates for oral drug delivery of hydrophobic drugs by virtue of their potential biocompatibility and biodegradability especially when using polysaccharides, ability to increase drug solubility and to shield the entrapped drug from external harsh conditions of chemical or enzymatic degradation, for instances. To elaborate capsules able to selectively entrapped hydrophobic molecules inside the particle shell, a cationic poly(cyclodextrin) was layer-by-layer assembled with alginate onto a sacrificial gold template as outlined in Figures 1 and2. Lastly, the template is selectively removed through gold hydrolysis and diffusion of the resulting water-soluble salts through the polyelectrolyte shell to ensure the capsules formation.

Elaboration of the cationic poly(β-cyclodextrin)

The cationic cyclodextrin polymer was synthesized by crosslinking the β-cyclodextrin (β-CD) with epichlorohydrin [START_REF] Renard | Preparation and characterization of water soluble high molecular weight β-cyclodextrin-epichlorohydrin polymers[END_REF] followed by a reaction with the 2,3 epoxypropyltrimethylammonium chloride. [START_REF] Thuaud | Retention behavior and chiral recognition of β-cyclodextrinderivative polymer adsorbed on silica for warfarin, structurally related compounds and Dns-amino acids[END_REF] Briefly, β-CD reacted in NaOH solution (33 %) with epichlorohydrin at 50 °C for 210 min. After neutralizing the pH, the polymer was concentrated by ultrafiltration to remove salt or low molecular compounds and recovered through freeze-drying process. This results in a β-CD polymer containing dihydroxypropyl groups as substituents and hydroxypropyl groups as bridges linking β -CD. This polymer was then chemically modified with quaternary ammonium side groups through reaction with 2,3epoxypropyltrimethylammonium chloride in basic media. The supposed structure is illustrated in Figure S1.The resulting modified polymer was purified by a dialysis step using a cellulose dialysis membrane (1 kDa cut off). The product was isolated via freeze-drying to yield a white solid.

For the cationic poly(cyclodextrin), the value is 6.5 10 3 g•mol -1 , the value is 2.4 10 4 g•mol -1 and the degree of substitution is 0.40 ±0.05 (estimated from 1 H NMR [START_REF] Thuaud | Retention behavior and chiral recognition of β-cyclodextrinderivative polymer adsorbed on silica for warfarin, structurally related compounds and Dns-amino acids[END_REF]).

Proof of concept: Elaboration of the multilayer films onto flat gold substrate.

To confirm the success of the multilayer film elaboration, static contact angle measurements are performed on flat gold surfaces before and after deposition of the polyelectrolytes (Figure 3). The contact angle decreases from ~50° for a gold substrate pre-treated with the sulfonated thiol derivative (data not shown) to ~20° upon the layer-by-layer deposition process, in agreement with the hydrophilic nature of the adsorbed polysaccharides. This variation in wettability after the multilayer build-up is found to be independent of the number of layers.

Figure 3: Water contact angle measurements on flat gold substrates A) after coating with (P(CD + )/alg -)4-P(CD + ) and B) after coating with pyrene-loaded(P(CD + )/alg -)4-P(CD + ) .

The assembly of P(CD) + and alg -in multilayer films was monitored by QCM-D measurements (Figure 4). A stable baseline is first measured prior to adsorption of the polyelectrolytes. After 10 minutes, a solution of P(CD + ) (1 mg.mL -1 ) is injected into the measurement chamber of the QCM-D. A decrease in frequency is then observed corresponding to the adsorption of the cationic P(CD + ) to the sulfonated anionic gold substrate. P(CD + ) is allowed to adsorb for 900 s before the rinsing step begins. It is assumed that this adsorption time enables sufficient surface coverage of sulfonated gold substrate with P(CD + ) to lead to charge reversal of the interfacial layer that is a prerequisite for further polyanion adsorption. Upon rinsing, a slight increase in the frequency shift assigned to the polyelectrolyte excess removal is observed. It must be kept in mind that for the studied films the frequency changes as observed during the rinsing process are much smaller than those measured during the adsorption steps. The polyanionic alginate (1 mg.mL -1 ) is then injected.

A decrease in the resonance frequency is observed due to the adsorption of the anionic alginate macromolecules to the cationic polycyclodextrin adsorbed layer. Once again, free polyelectrolyte chains are removed during the rinsing step before the multilayer assembly was continually built up with consecutive adsorption of P(CD + ) and alg -. Typical decrease in the frequency signal by further increasing the number of adsorption cycles is then observed, indicating that mass was being added onto the surface. This is in agreement with previously reported results indicating that the adsorption of polyelectrolytes onto oppositely charged surfaces essentially leads to irreversible adsorbed layers in a predetermined range of pH, temperature, ionic strength. [START_REF] Belbekhouche | Promising sub-100 nm tailor made hollow chitosan/poly (acrylic acid) nanocapsules for antibiotic therapy[END_REF] Further information may be obtained from dissipation monitoring about the viscoelastic properties of the polyelectrolyte multilayer films. The voltage imposed to the QCM is suspended and the decay of the amplitude of the crystal is measured as a function of time.

From this, the dissipation factor D, is defined according to the following equation:

= 2
Edissipated and Estored correspond to the dissipated energy and the stored energy respectively. A positive shift of D is typical assigned to less rigid (water-rich) structures.

Figure 4 shows the stepwise change in dissipation as the multilayer film is built up upon successive adsorption of P(CD + ) and alginate. The magnitude of the dissipation factor (dissipated energy) increases as a function of the bilayer number. For both polyelectrolytes, an initial rise in the dissipation is observed after flushing with polymer solution. The dissipation factor levels off rapidly yielding a plateau regime. Such behaviour suggests that upon adsorption of the polyelectrolyte layers, water molecules are coupled to the adsorbed polymer layers as an additional mass. The P(CD + )/alg -films can then be regarded as composed of polymer chains and entrapped water with viscoelastic characteristics. Figure 4: Monitoring of the LbL build-up of the (P(CD + )/alg -)n film on gold flat substrate by QCM-D experiment. The quartz crystal was initially placed in contact with solvent (noted "*"); after an equilibrium time of 10 min, cationic poly(cyclodextrin) (P(CD + )) and alginate (alg -) solutions (1 mg.mL -1 ) were alternatively added. A rinsing step was performed between each polymer deposition step.

After elaboration of the (P(CD + )/alg -)4 P(CD + ) multilayer film, flushing the multilayer assembly with a solution of pyrene (4.10 -7 mol.L -1 prepared in water) leads to a decrease of the equilibrium frequency shift of 10 ± 2 Hz. This evidences an increase of the film weight and then suggests an entrapment of pyrene through the multilayer film. It is worth mentioning that no variation in the equilibrium frequency shift is seen for the (chi + /alg -)4 chi + multilayer film upon contact with the pyrene solution suggesting that the entrapment of pyrene within (P(CD + )/alg -)4 P(CD + ) results from inclusion complex formation. The water contact angle 3b) for the pyrene incubated with (P(CD + )/alginate)4-P(CD + ) multilayer film.

Note that after elaboration of the multilayer film ((P(CD + )/alg -)4 P(CD + )), the latter has been flushed with culture medium. This film is stable as evidenced by QCM measurement. (see Figure S2)

Once the feasibility of the LbL strategy is evidenced onto flat gold substrate, it was transposed to spherical templates, gold nanoparticles, having a well-defined size, dispersity and surface chemistry (sulfonated thiol stabilized gold nanoparticles) for the elaboration of capsules.

From flat to colloidal gold substrates: Elaboration of cationic poly(cyclodextrin)-based nanocapsules

Zeta potential measurement is used herein to follow each stage of the polyelectrolyte deposition onto the spherical substrate (Figure 5 A). [START_REF] Burke | pH-Responsive Properties of Multilayered Poly(llysine)/Hyaluronic Acid Surfaces[END_REF][START_REF] Belbekhouche | Latex nanoparticles surface modified via the layer-by-layer technique for two drugs loading[END_REF] The ζ-potential values for pretreated gold nanoparticles are -28 mV (± 2) due to the ionized sulfonate functions on the particle surface. As expected after the exposition of gold colloidal nanoparticles to P(CD) + and alginate solution, the value alternately switches to a positive one (~ +20 mV) and to a negative value (~ -20 mV), respectively. This clearly indicates the successful stepwise growth of polyelectrolytes multilayer films on colloids though robust adsorption of both polycationic and polyanionic polymers onto the colloidal material. [START_REF] Caruso | Investigation of Electrostatic Interactions in Polyelectrolyte Multilayer Films: Binding of Anionic Fluorescent Probes to Layers Assembled onto Colloids[END_REF][START_REF] Caruso | Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating[END_REF][START_REF] Caruso | Hollow capsule processing through colloidal templating and self-assembly[END_REF][START_REF] Sukhorukov | Intelligent micro-and nanocapsules[END_REF] We have evidenced the adsorption onto the gold nanoparticles surface by exploiting the sensitivity of the gold nanoparticle's surface Plasmon band. [START_REF] Belbekhouche | Promising sub-100 nm tailor made hollow chitosan/poly (acrylic acid) nanocapsules for antibiotic therapy[END_REF][START_REF] Gittins | Multilayered polymer nanocapsules derived from gold nanoparticle templates[END_REF] Towards this aim, UV-vis spectroscopy technique was used to follow each step of surface coating with the polyelectrolytes (Figure 5B). The absorption peak is correlated to the transverse surface Plasmon band of the bare colloidal gold nanoparticles. Compared to the bare gold nanoparticles, the coating of the gold nanoparticles with 2 and 4 (P(CD + )/alg -) leads to a redshift of 2 and 6 nm respectively in the Plasmon band maxima. All samples present stable electronic absorption spectra up to 4 weeks. The colloidal stability was also measured at 25°C and 37°C up to one month by DLS and zeta, a very good stability was observed, i.e. for ((P(CD + )/alg -)4 P(CD + ) around 60 nm and -20 mV. Moreover, the absence of a Plasmon band blue-shift clearly demonstrated the robust deposition of the polyelectrolytes on the gold nanoparticles that is a crucial point for the elaboration of stable hollow nanoparticles. A major extension of core-shell particles technology is the removal of the core, leading to hollow particles (nanocapsules) that are replicas of the template-core. The procedure lies on the inherent permeability of the multilayer shell enabling diffusion-controlled removal of the core decomposition components. Sufficient shell permeability is required to preserve the structural integrity of the hollow particles. Herein, the removal of the gold cores and consequently the formation of nanocapsules were induced through cyanide etching of the gold core. This leads to the formation of gold cyanide complexes (4 Au + 8 KCN + 2 H2O + O2 → 4 KOH + 4 KAu(CN)2) that can leach through the polymer capsule shell. This can be easily performed by repeated dialysis process. [START_REF] Gittins | Tailoring the polyelectrolyte coating of metal nanoparticles[END_REF] The dissolution process of the gold core was monitored by the change in the UV/Vis spectrum. As evidenced from figure 6, a gradual disappearance of the absorbance peak is observed due to KCN treatment. This result is in accordance with the dissolution of the gold core by cyanide (Figure 6). No presence of atomic gold was detected by inductively coupled plasma optical emission spectrometry analysis in the last dialysate solution and in the (P(CD + )/alg -)n nanocapsules. we successfully obtain capsules (Figure 7C). Pyrene is a fluorescent probe whose emission spectrum changes in response to changes in polarity of the surrounding solvent. [START_REF] Liu | Preparation of polyhedral oligomeric silsesquioxane-based hybrid monolith by ring-opening polymerization and postfunctionalization via thiol-ene click reaction[END_REF] The intensity ratio of the first peak (372 nm) and the third peak (382 nm) -I372/I382 -in its fluorescent spectrum is classically used to evidence of a subtle environment change in terms of polarity. [START_REF] Goddard | Fluorescence probes for critical micelle concentration determination[END_REF] For instance, the decrease in I372/I382 intensity indicates the formation of hydrophobic clusters expected to occur with the aggregation of amphiphilic polymers. [START_REF] Henni | Aggregation of hydrophobically modified polysaccharides in solution and at the air-water interface[END_REF] At the studied pyrene concentration of 4.10 -7

mol.L -1 , no excimer band due to the interaction of an excited-and ground-state pyrene species was observed. [START_REF] Amiji | Pyrene fluorescence study of chitosan self-association in aqueous solution[END_REF][START_REF] Fischer | Detection of Intramolecular Associations in Hydrophobically Modified Pectin Derivatives Using Fluorescent Probes[END_REF][START_REF] Belbekhouche | Association states of multisensitive smart polysaccharide-block-polyetheramine copolymers[END_REF] Such a low concentration was indeed chosen to minimize the influence of pyrene on the formation and/or the stability of hydrophobic domains. The initial I372/I382 value of the pyrene solution at 4.10 -7 mol.L -1 is found to be around 1.8 which is the classical value obtained for the pyrene in water at this concentration. After incubation with the P(CD + ), this value decreases to 0.8-0.9.

Of utmost importance, a similar I372/I382 value of 0.8-0.9 is measured for the nanocapsules composed by pyrene-loaded P(CD + ) and alginate, and the ratio value is found to be independent of the number of multilayers from 2 to 4. As a control experiment, we prepared nanocapsules using chitosan as polycation instead of the cationic polycyclodextrin. Chitosan is a linear copolymer of β-(1,4)-2-amino-2-deoxy-D-glucose and N-acetyl-D-glucosamine in variable ratios that bears does positive charges in acidic conditions but does not provide hydrophobic interaction domains. Chitosan is not able to be implied in host-guest inclusion complex formation in contrast to CD-based polymers. In the case of the chitosan/alginate nanocapsules, the I372/I382 value is measured around 1.8. This result can be fully explained by the fact that neither chitosan nor alginate can entrap pyrene through hydrophobic interaction and more importantly that the entrapment of pyrene within P(CD + )/alginate results from host-guest complex formation. We quantified the amount of pyrene entrapped in the capsules by fluorescence intensity measurements. After extraction of pyrene from the CD cavity with methanol, the effective concentration of pyrene in 100 μL of a capsule suspension was found to be 0.145 and 0.310 µmol.L -1 for pyrene-loaded particles coated with (P(CD + )/alg -)n-P(CD + ) with n= 2 and 4 respectively.

Then, a chemical presenting a biological activity, namely 4-hydroxy tamoxifen, has been loaded instead of pyrene. The ability of OHT-loaded (PCD + /alg -)2-PCD + nanomaterials to be incorporated inside immortalized podocytes was investigated and the impact of the presence gold core was also studied. The OHT-loaded nanomaterials induce Cmip exon 8 deletion, deletion highlighted by specific PCR reaction. This technique leads to a 976 bp exon 8 floxedarm band that is reduced or may fade out after deletion. In the conditions used for this experiment, exposure to the OHT-loaded nanovectors after 48h changes Cmip lox percentage.

The quantification of the PCR bands is shown on Figure 8. Note that the hydroxy-tamoxifenloaded nanomaterials have been dispersed in the culture medium. These nanoparticles could be used for cellular tests even after two months. The same biological results are obtained. This evidenced unambiguously the stability of the loaded nanoparticles in the culture medium. A significant Cmip lox exon 8 band decrease (figure 8) can be observed after exposure to nanomaterials for 48h, i.e. 70.1% for P(CD + )-coated nanoparticles with 2 layers. After removing the gold core, this value is 57.2 % P(CD+)-nanocapsules with 2 layers respectively.

Note that with the 1 µM µmol.L -1 4-hydroxdy-tamoxifen solution, the percentage falls below 20%. All these results are expressed in percentage of the negative control. This data suggests that the nanomaterials are able to enter cells and that the 4-hydroxy tamoxifen retains its biological activity despite encapsulation. While we expected that hollow nanocapsules would be more malleable, more flexible compared to the coated gold nanoparticles, thus allowing a better penetration into podocytes and, by extension, a greater Cmip extinction. Further experiments are required to understand the role of the structure and composition of nanoparticles as factors influencing the entry pathway and the molecule release.

In addition, a toxicity test (A morphological study by indirect immunofluorescence with Annexin V or cleaved caspase 3) could show higher mortality after exposure to gold nanoparticles (data not shown).

Conclusion

We demonstrate an easy and efficient approach combining both templating method, selfassembly and host-guest complexation to obtain well-designed and stable cyclodextrincontaining capsules in aqueous system. This elegant strategy requires neither surfactants nor even organic solvents. The elaboration of these capsules relies on the ability of cationicpoly(cyclodextrin) to be layer-by-layer assembled with alginate onto sacrificial particles. The built-up process is driven by electrostatic interactions in aqueous solution as evidenced by zeta measurements. The results of TEM and Au plasmon absorbance evidence the successful formation of nanocapsules with spherical shape. We gained advantage of the presence of CD cavities to entrap 4-hydroxy tamoxifen within the nanocapsules' shell through inclusion complexion formation. It was further shown that 4-hydroxy tamoxifen can be efficiently delivered to podocytes in vitro using CD-containing nanocapsules as carriers.

2 . 3 .

 23 remove the gold complex (3.5 kDa cut off). Kinetic of the core removal was investigated through monitoring the spectroscopic signature of the gold core. Absorbance spectra were recorded with a UV-Visible spectrophotometer from Varian Cary 50 Bio. Non presence of gold complex was investigated in the last dialysat and in capsules by the Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) analyses (Au wt. %) (ICP-OES spectrometer, Simultaneous Varian Vista Axial). The capsules were viewed with transmission electron microscopy (TEM), a droplet of nanomaterial suspension and one of a solution of uranyle acetate (0.7 wt %) were deposited on a plasma pre-treated copper TEM grid and allowed to dry in air at room temperature prior analysing. Transmission Electron Microscopy (TEM) were conducted on a Tecnai F20 ST microscope (field-emission gun operated at 3.8 kV extraction voltage) operating at an acceleration voltage of 200 kV. Loading of hydrophobic chemicals inside CD cavity Hydrophobic chemicals (pyrene and 4-hydroxy-tamoxifen) were loaded inside the cyclodextrin cavity. The pyrene entrapment was achieved through simple diffusion process of this molecule from solution through P(CD + )-based multilayer films preassembled on pretreated gold substrates, namely flat surface (QCM sensor crystal) and nanoparticles. In the former case, gold coated QCM Sensor Crystal was initially modified with an anchoring sulfonate thiol layer and (P(CD + )/alg -)4-P(CD + ) LbL films under dynamic flowing of the corresponding solutions in the QCM cell. Polyelectrolytes-coated quartz slides were flushed with the pyrene solution (4.10 -7 mol.L -1 ) P(CD + )-based nanocapsules was overnight incubated with 50 µL pyrene solution (4.10 -7

  Genotyping. Podocytes were washed 3 times with 1X PBS solution and then suspended in lysis buffer (0.1 M Tris pH 8, 0.2 M NaCl, 0.2 % SDS, 5 mM EDTA, 150 μg/ml proteinase K). After incubation at 55°C and complete lysis of the sample, the DNA was purified by the phenol/chloroform extraction technique and then precipitated with ethanol. The resulting genomic DNA pellet was dissolved in water and the DNA concentration was measured by nanodrop. This solution was then diluted to a concentration of 150 ng/ml. 2 μl of this solution are used for the PCR reaction. The PCR conditions were as follows: step 1 : 95 °C for 5 minutes ; step2 : 95°C for 30 seconds ; step 3 : 60°C for 30 seconds ; step 4 : 72 °C for 90 seconds) and then repeat steps 2-4 for 30 cycles then finally keep at 72°C for 10 min. the PCR primer forward and reverse are respectively : Cmip lox Forward : , the samples migrate on a 1.2% agarose gel, in a migration buffer (Tris-base, boric acid, EDTA and H2O). The expected size of the fragments with the Cmip allele is 976 bp (Cmip lox).The following conditions have been tested on these podocytes with two controls. The first one consists in cells, untreated with nanoparticles (negative control), the second consists in cells treated with 2 layers 4-hydroxy-tamoxifen-loaded (P(CD + )/alg -)n-P(CD + ) particles with gold core. The third condition consists in cells treated 2 layers 4-hydroxy-tamoxifen-loaded (P(CD + )/alg -)n-P(CD + ) particles without gold core. 475 million nanoparticles (50 μl) are injected into each well. Finally the last condition consists in cells treated with 10 µl of a 4hydroxy-tamoxifen solution (1 µM µmol.L -1 prepared in ethanol).

Figure 1 :

 1 Figure 1: Schematic illustration of the polyelectrolyte adsorption process for the elaboration of cationic-poly(cyclodextrin) (P(CD + )) /alginate (alg -) capsules.

Figure 2 :

 2 Figure 2: Chemical structure of the cationic poly(β-cyclodextrin) (x" symbolises an integer number representing the degree of polymerization) and the polyanionic alginate.

Figure 5 :

 5 Figure 5: A) Zeta potential values measured upon alternating adsorption of P(CD + ) with alg - on pre-treated gold nanoparticles. B) UV-vis spectra of polyelectrolyte-coated gold nanoparticle as a function of the number of adsorbed polymer layers: curve a: uncoated gold nanoparticles; curve b: (P(CD + )/alg -)2; curve c: (P(CD + )/alg -)4; Curves are offset for clarity.

Figure 6 :

 6 Figure 6: UV-vis spectra of (P(CD + )/alg -)4 coated gold nanoparticle as a function of gold etching time: (a) 0 min, (b) 5 min, (c) 10 min, (d) 15 min, (e) 20 min, (f) 30 min, (g) 40 min, (h) 50 min and (i) 60 min.

Figure 7 :

 7 Figure 7: TEM micrographs of: A) bare gold nanoparticles, B) (P(CD + )/alg -)4-coated gold nanoparticles and C) (P(CD + )/alg -)4 nanocapsules.

Figure 8 :

 8 Figure 8: Quantification of Cmip lox PCR products in each condition: (P(CD + )/alg -)2-P(CD + )-coated gold nanoparticles, (P(CD + )/alg -)2-P(CD + ) nanocapsules, OHT (positive control) and negative control (cells, untreated with nanoparticles) characterizing the full Cmip lox expression.

Supporting information


Emmanuelle Reygagne
email: emmanuelle.reygagne@hotmail.fr

Foucaud Du Boisgueheneuc
email: foucaud.duboisgueheneuc@chu-poitiers.fr

Antoine Berger
email: antoine.berger@chu-poitiers.fr

Pierre Ingrand
email: pierre.ingrand@univ-poitiers.fr

Keywords: stereotactic, radiation therapy, brain metastases, cognitive test

Examining the Inter Hemispheric Transfer Time Test: A new computerized cognitive test to incorporate into therapeutic strategy for patients with brain metastases?

A pilot study

Introduction

Historically, whole brain radiotherapy (WBRT) was the standard treatment for multiple brain metastases. Its benefit in terms of improved tumor control and overall survival is well demonstrated [START_REF] Posner | Management of brain metastases[END_REF][START_REF] Tsao | Whole brain radiotherapy for the treatment of multiple brain metastases[END_REF][START_REF] Latorzeff | Radiotherapy for brain metastases[END_REF]. For one to four brain metastases, stereotactic radiotherapy has become a new standard for patients with a good prognostic score to preserve cognitive functions by sparing a part of the brain from brain irradiation [START_REF] Chang | Neurocognition in patients with brain metastases treated with radiosurgery or radiosurgery plus whole-brain irradiation: a randomised controlled trial[END_REF]. Stereotactic Radiotherapy (SRT) does not improve overall survival compared to WBRT [START_REF] Latorzeff | Radiotherapy for brain metastases[END_REF][START_REF] Kocher | Adjuvant whole-brain radiotherapy versus observation after radiosurgery or surgical resection of one to three cerebral metastases: results of the EORTC 22952-26001 study[END_REF][START_REF] Soffietti | A European Organisation for Research and Treatment of Cancer phase III trial of adjuvant whole-brain radiotherapy versus observation in patients with one to three brain metastases from solid tumors after surgical resection or radiosurgery: quality-of-life results[END_REF].

More and more studies have been conducted, including cognitive tests in patients with brain malignancies. In fact, neurocognitive status could have a predictive and prognostic value in this population and be of considerable help in choice of therapeutic strategy [START_REF] Sneed | A multiinstitutional review of radiosurgery alone vs. radiosurgery with whole brain radiotherapy as the initial management of brain metastases[END_REF].

Neurocognitive assessment must be brief and sensitive in this asthenic population. The most impaired brain-based cognitive skills after radiation therapy in patients with brain metastases are the executive functions [START_REF] Stokes | White matter changes in breast cancer brain metastases patients who undergo radiosurgery alone compared to whole brain radiation therapy plus radiosurgery[END_REF][START_REF] Meyers | Role and relevance of neurocognitive assessment in clinical trials of patients with CNS tumors[END_REF].

That is why we decided to choose the InterHemispheric Transfer Time Test (IHTTT) as a cognitive evaluation tool, which detects information processing speed impairment. It is short (10 minutes), repeatable, simple to administer but not standardized. In this prospective study, we share our initial experience using this computerized cognitive test for detection of information processing speed impairment in patients undergoing stereotactic radiotherapy for brain metastases.

The aim of this pilot observational study was to evaluate computerized IHTTT for the detection of information processing speed impairment in patients with radiation therapy for brain metastases.

Material and methods

Patients and study design

Eligibility criteria were as follows: patients (M/F) aged over 18 years, with one to four brain metastases treated by SRT with dose schedule: 33 Gy in 3 fractions, with a solid tumour, ≥70 neuropsychological tests [START_REF] Herman | Neurocognitive and functional assessment of patients with brain metastases: a pilot study[END_REF][START_REF] Meyers | The use of the mini-mental state examination to assess cognitive functioning in cancer trials: no ifs, ands, buts, or sensitivity[END_REF].

Frontal Assessment Battery (FAB) has been developed for the assessment of executive functions. It lasts 10 minutes. It consists of six subtests. Each subtest is scored on a maximum of 3 points, rendering a total maximum score of 18 [START_REF] Dubois | The FAB: a Frontal Assessment Battery at bedside[END_REF].

IHTT Test.

IHTT test is based on Poffenberger paradigm created to estimate the time required for the critical transfer of information from one hemisphere to other [START_REF] Poffenberger | Reaction time to retinal stimulation with special reference to time lost in conduction through nervous centers[END_REF].

During this test, the patient was seated in front of a computer with a joystick in his hands and instructed to look at the middle of the screen. Stimuli were arrows appearing tachitoscopically (250 ms) on the right or on the left and pointing to the right or to the left. The visual information was treated exclusively selectively by the hemisphere opposite to stimulation.

The patient should click right when the arrow indicated right and left when the arrow indicated left. If the arrow appeared to the right and indicated the right or if the arrow appeared to the left and indicated the left, the situation was congruent. Visual information was perceived in level of the ipsilateral hemisphere, which controls motor control and therefore does not have to pass through the corpus callosum (which is the connective structure between the two cerebral hemispheres). Intra-hemispheric transfer was evaluated (additional material).

If the arrow appeared to the right and indicated the left or if the arrow appeared to the left and indicated the right, the situation was incongruent. The visual information is perceived in the hemisphere contralateral to the motor control and must therefore pass through the corpus callosum. Inter-hemispheric transfer was evaluated (additional material) [START_REF] Westerhausen | Interhemispheric transfer time and structural properties of the corpus callosum[END_REF]. Lesions of the white matter are indeed responsible for non-specific cognitive disorders (slowed processing speed, difficulties in multiple tasks) revealed by a deficit of the executive functions [START_REF] Stokes | White matter changes in breast cancer brain metastases patients who undergo radiosurgery alone compared to whole brain radiation therapy plus radiosurgery[END_REF][START_REF] Monaco | Leukoencephalopathy after whole-brain radiation therapy plus radiosurgery versus radiosurgery alone for metastatic lung cancer[END_REF].

The objective of this test was to sensitize evaluation of the executive functions by measurement of reaction time and comparison of intra-and interhemispheric transfer times during an inhibitor control task (Stroop test). Several variables were measured:

1-The reaction time (IHTT) between presentation of the visual stimulus and the response obtained by the joystick.

2-

The inter-hemispheric transfer index (IHTI), which corresponds to the delta of the response time between the incongruent and congruent situations because transfer of the information over the corpus callosum results in a time delay.

The hypothesis was that IHTT and IHTI are more sensitive measures than MMSE or FAB to evaluate executive functions in post-radiation lesions.

Quality of life assessment (QLQ C30)

EORTC QLQ-C30 is a specific tool assessing the quality of life of cancer patients is the most widely used test in clinical trials Four scores were calculated using the EORTC scoring procedures (rendering a score ranging from 0 to 100): global health status; functional scale; symptom scale; cognitive functioning [START_REF] Aaronson | The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-oflife instrument for use in international clinical trials in oncology[END_REF].

Statistical Analysis

IHTI measurement was the primary endpoint. Secondary endpoints included IHTT measurement, MMSE score, FAB score, quality of life scores, prognostic scores: recursive partitioning analysis (RPA) and graded prognostic assessment (GPA), progression-free survival (PFS, calculated from the date of the end of brain radiation therapy to the date of the first imaging showing cerebral progression) and overall survival (OS).

Descriptive statistics (mean and, standard deviation, or number and percent) were used to report data. Evolution of endpoints was analyzed by mixed linear models for repeated measures, taking into account the longitudinal design with incomplete observations over time.

Non-independences resulting from serial observations belonging to the same individual were accounted by a first-order autoregressive correlation structure. Deviations from distribution hypothesis were assessed on the residuals computed after fitting the models. Correlation analysis between IHTI, IHTT, GPA, MMSE, FAB, and QLQ-C30 scores was performed using the non-parametric Spearman correlation coefficient. PFS and OS analyses used the Kaplan-Meier method. The univariate survival analysis used the log-rank test. Multivariate survival analyses were performed using the Cox proportional hazards model with variables entering the model following a forward-stepwise selection procedure. For each test, a p-value ≤ 0.05 was considered statistically significant. Statistical analysis was carried out using SAS version 9.4 (SAS institute, Cary, NC).

Results

Thirty-three patients were eligible for stereotactic radiotherapy. Four patients were not included due to application of non-inclusion criteria. Twenty-nine patients were included from June 2014 through April 2015. At 6-month follow-up, 1 patient was excluded due to cerebral progression and two other patients at one-year follow-up. The cognitive test results of 26 patients in the stereotactic group were then analyzed (Figure 1). At baseline, 43% of patients received corticosteroids and 36% anti-epileptic drugs against 0% respectively at one year. The most frequent primary tumor site was lung (58%). Patient characteristics are represented in table 1.

Table 1 and Figure 1 approximately here

At baseline, IHTI and IHTT were respectively 131.0 ± 31.4 ms and 720.3 ± 26.5. IHTI and IHTT showed significant evolution over time after stereotactic radiotherapy (p=0.0001 and p=0.0010, respectively). IHTI and IHTT were stable at one month and then worsened over time (Figure 2).

Figure 2 approximately here

Preceding SRT, patients had no impairment regarding MMSE and FAB results with 29.5 ± 0.7 points and 17.4 ± 1.1 respectively. MMSE scores were significantly worse at one-year follow-up compared with baseline, one month and 6 month follow up (p=0.014). FAB showed a non-significant trend toward impairment at one-year follow-up (p=0.13). IHTT was correlated with the MMSE (Spearman rS=-0.56) and the FAB (rS=-0,68). The use of corticosteroids, RPA scores, brain metastasis surgery, sex, number of brain metastases and their volume were not associated with neurocognitive functioning (data not shown).

Compared with the general population, quality-of-life QLQ-C30 scores were similar in patients at baseline for global health status, global functional scales and cognitive functioning (76,2 ± 18,9 versus 78 for general population, 85,2 ± 15,4 versus 90, 86.2 ± 23.3 versus 92 respectively) [START_REF] Habets | Neurocognitive functioning and health-related quality of life in patients treated with stereotactic radiotherapy for brain metastases: a prospective study[END_REF]. Despite a decreasing quality of life of patients over time, this evolution was not statistically significant (Table 2). There was no correlation between IHTTT scores and QLQ-C30 scores (Table 3). The use of corticosteroids, RPA scores, brain metastasis surgery, number of brain metastases and their volume were not associated with quality of life (data not shown).

Tables 2 and3, and Figure 3

Discussion

Choice of cognitive tests

We chose IHTTT as a computerized cognitive evaluation test because of its speed of execution (10 minutes) and its ease to administrate. It evaluates psychomotor retardation, concentration and attention disorders. These functions are often impaired in brain radiotherapy patients [START_REF] Wefel | Neuropsychological dysfunction associated with cancer and cancer therapies: a conceptual review of an emerging target[END_REF]. This test seemed promising as a future evaluation tool, which could help us in therapeutic decision-making on the choice of radiotherapy modalities (WBRT versus stereotactic radiotherapy) or re-irradiation. But this test has yet to be standardized. That is why we added two other validated tests: the MMSE and the FAB.

The choice of MMSE as a cognitive test for irradiated patients for cerebral metastases can be criticized for its low sensitivity and specificity. Indeed, the MMSE is not a psychometric test but rather a standardized clinical examination that does not have the metrological qualities of the psychometric tests [START_REF] Meyers | The use of the mini-mental state examination to assess cognitive functioning in cancer trials: no ifs, ands, buts, or sensitivity[END_REF][START_REF] Murray | Importance of the mini-mental status examination in the treatment of patients with brain metastases: a report from the Radiation Therapy Oncology Group protocol 91-04[END_REF]. However, the MMSE is a good standardized tool because of its simple administration and deserves to be included, in addition to neuropsychological tests, in the systematic examination of patients with brain lesions. It is quick to administrate in tired patients. FAB appeared to be a good complement to the MMSE because it evaluates the executive functions, which are often impaired in patients irradiated for brain metastases, something that MMSE cannot do [START_REF] Meyers | Role and relevance of neurocognitive assessment in clinical trials of patients with CNS tumors[END_REF].

Results comments

Our results suggest that IHTTT could be a discriminant test measuring the cognitive impact of brain radiotherapy, especially stereotactic radiotherapy. IHTT as well as IHTI evolves over time significantly and conclusively. IHTT and IHTI are stable at one month and subsequently only worsen with time. We can suspect alteration of the white substance on the irradiated side, resulting in lengthening of the IHTT on that side only (transfer time for the information being longer) and also in IHTI lengthening. IHTTT results reflect attention disorders and psychomotor slowing induced by the rays. MMSE detects significant evolution of the cognitive functions contrary to FAB (respectively p = 0.014 and 0.13).

However, if we can detect cognitive toxicity in this population, this does not mean that radiotherapy is of no benefit to them. Indeed, the benefit of stereotactic radiotherapy in terms of its anti-tumor effect, loco-regional control and overall survival is well-established [START_REF] Aoyama | Stereotactic radiosurgery plus whole-brain radiation therapy vs stereotactic radiosurgery alone for treatment of brain metastases: a randomized controlled trial[END_REF][START_REF] Andrews | Whole brain radiation therapy with or without stereotactic radiosurgery boost for patients with one to three brain metastases: phase III results of the RTOG 9508 randomised trial[END_REF][START_REF] Kondziolka | Stereotactic radiosurgery plus whole brain radiotherapy versus radiotherapy alone for patients with multiple brain metastases[END_REF].

This is likewise reflected in our results through complete steroid anti-epileptic withdrawal and at one-year follow-up (36% of patients under anti-epileptics and 43% of patients under corticosteroids in stereotactic radiotherapy group baseline versus 0% at one-year follow up.

Quality of life tends to worsen over time but the results were statistically insignificant.

Concerning the correlations between the different cognitive tests and quality of life, IHTT was significantly correlated with the MMSE and the FAB, but not with patient quality of life.

Correlation between IHTTT and the FAB was not surprising because both of them evaluate the executive functions. Few studies have evaluated the changes in cognitive function following stereotactic radiotherapy for brain metastases. Concerning IHTTT, our results support the hypothesis of radiation-induced lesions of the white matter. Stokes' TB study supports our results [START_REF] Stokes | White matter changes in breast cancer brain metastases patients who undergo radiosurgery alone compared to whole brain radiation therapy plus radiosurgery[END_REF]. Indeed, the authors compared changes in white matter on T2weighted and FLAIR-weighted cerebral MRI in patients with brain-metastatic breast cancer.

One group received stereotaxy alone (n = 30) and the other received WBRT associated with cerebral stereotaxy (n = 35). Patients had cerebral MRI at baseline and then at one-year follow-up. At one-year follow-up, the stereotactic + WBRT group showed a high incidence of changes in white matter (71.5%, p <0.05) versus only 3.3% in the stereotactic group. The study by Monaco and al. confirms these findings in patients with brain metastatic lung cancer [START_REF] Monaco | Leukoencephalopathy after whole-brain radiation therapy plus radiosurgery versus radiosurgery alone for metastatic lung cancer[END_REF]. According to Hulst and al., lesions of the white matter are correlated with the deterioration of cognitive functions [START_REF] Hulst | Cognitive impairment in MS: impact of white matter integrity, gray matter volume, and lesions[END_REF]. Our study finds significant aggravation of the cognitive functions: +78 ms for the index and IHTT between baseline and one year follow-up.

Dane and al [START_REF] Dane | Sex and handedness differences in eye-hand visual reaction times in handball players[END_REF] demonstrated that reaction times were longer in women than in men and that left handed players have probably an intrinsic neurological advantage. Controlling theses variables should not change our results because our patients had a right hand preference and groups were comparable.

Moreover, patient quality of life after neuro-radio-induced toxicity requires close evaluation and analysis. Indeed, in patients with cerebral metastases, neurocognitive functions and activities of daily life (QoL) could be correlated. A decline in neurocognitive functions would be predictive of a decline in the QoL score [START_REF] Li | Relationship between neurocognitive function and quality of life after whole-brain radiotherapy in patients with brain metastasis[END_REF]. Our study does not find this correlation between cognitive functions and quality of life because IHTTT investigate a relatively narrow aspect of cognitive performance .

The median OS of 15 months for RPA Class I and II was longer than the value predicted in the original paper by Gaspar et al. (7.1 vs. 4.2 for Class I and II respectively) [START_REF] Gaspar | Recursive partitioning analysis (RPA) of prognostic factors in three Radiation Therapy Oncology Group (RTOG) brain metastases trials[END_REF]. This can be explained by the fact that patients have fewer than 5 brain metastases and that 30% of patients underwent surgery for their brain metastases. Median OS after resection of brain metastasis is differently reported in literature in a range between 6 and 17 months [START_REF] Weidle | The Blood-Brain Barrier Challenge for the Treatment of Brain Cancer, Secondary Brain Metastases, and Neurological Diseases[END_REF]. The most frequently used scoring systems to predict the outcome are RPA and GPA [START_REF] Sperduto | A validation study of a new prognostic index for patients with brain metastases: the Graded Prognostic Assessment[END_REF]. In our study, IHTI seems to be an independent factor for prediction of brain PFS. Being more accurate than RPA and GPA in our study, it could be incorporated into therapeutic decisionmaking.

Study limits

The study was not optimally designed. Indeed, our study was not a true longitudinal study.

We were unable to monitor each patient over time because many of them died prematurely or rapidly deteriorated, preventing any cognitive test. Other patients left the study at their request because of displacement-related fatigue when they lived far away. To compensate for the large number of patients who left the study early, we chose to include patients crosssectionally at 6 months and at one-year follow-up to increase our enrollment. Our study was therefore both longitudinal and transversal. We consequently evaluated evolution of the cognitive functions over time in patients who were not always the same, thereby exposing to selection bias. But group characteristics were comparable.

Perspectives

To preserve cognitive functions in patients irradiated on the brain, drugs could be efficient. A phase III study (RTOG 0614) studied memantine for the prevention of radio-induced cognitive toxicity. Memantine is an a drug blocking anti-methyl aspartate receptor that has been shown to be effective in Alzheimer disease vascular dementia. In this study 508 patients with cerebral metastases were randomized between complete brain irradiation with memantine for 6 months, and complete brain irradiation with a placebo for 6 months. Patients in the memantine group had a probability of cognitive decline at 6 months of 54% versus 65% in the placebo group (HR = 0.78, p = 0.01 [START_REF] Brown | Memantine for the prevention of cognitive dysfunction in patients receiving whole-brain radiotherapy: a randomized, double-blind, placebo-controlled trial[END_REF]. At present, an on-going randomized phase III trial is comparing memantine hydrochloride and whole-brain radiotherapy with or without hippocampal avoidance as a means of reducing neurocognitive decline in patients with cancer that has spread from the primary site to the brain (NCT02360215)

Moreover, the impairment of cognitive functions in patients irradiated on the brain raises questions on the possible efficacy of cognitive rehabilitation. The study of Gehring et al. Finally, a treatment adapted to each patient is necessary. We should neither over-treat patients with negative prognosis nor treat those with favorable prognosis (long-term adverse effects).

Taking a rapid cognitive test (which is feasible, for example, during consultation with a radiotherapist) before deciding on cerebral therapeutic strategy could be a better way of selecting patients. The test seems predictive of brain progression-free survival and could be incorporated into therapeutic decision-making.

CONCLUSION

This prospective pilot study suggests the interest of IHTTT in therapeutic management.

The IHT test would be a discriminatory test to assess cognitive function in evaluation of patients with brain metastases irradiated by SRT. The IHTTT could become an assessment tool to be considered as a new element in a therapeutic strategy.

  studied the cognitive impact of cognitive rehabilitation programs in brain tumor patients. A group of patients with brain tumor utilized from a computerized program of attention training and learning of cognitive impairment rehabilitation techniques for 6 weeks. Another group of control patients did not yet benefit from the cognitive Rehabilitation Program (waiting list). Patients who had utilized the cognitive rehabilitation program presented significantly better results in neuropsychological tests at 6 months compared with the control group [30]. Kesler et al. found a beneficial effect of a computerized cognitive re-education program on executive functions, verbal fluency and information-processing speed [31]. On account of brain plasticity, cognitive rehabilitation should be interesting in this population. A clinical trial is currently recruiting participants to assess the Impact of Cognitive Rehabilitation and Physical Activity on Cognition in Patients with Metastatic Brain Tumors Undergoing RT (NCT03096431).

4 not included: - 1

 1 refusal to participate -1 history of focal radiotherapy -1 blind patient -1 hemiplegic patient 33 patients eligible for stereotactic radiotherapy. 29 patients included in our center 14 patients in baseline sub group 11 patients in 1 month follow-up: -longitudinally: 10 patients -cross sectionally: 1patient 3 patients excluded due to brain progression: -1 in 6 month follow-up -2 in 1 year follow-up 26 patients analyzed 11 patients in 6 month follow-up: -longitudinally: 5 patients -cross sectionally: 6 patients 10 patients in 1 year followup: -longitudinally: 5 patients -cross sectionally: 5 patients

Figure

  Figure I : Flow chart

Figure 2 .

 2 Figure 2. Evolution of IHTT and IHTI over time (mean and sd, milliseconds).

Figure 3 .

 3 Figure 3. Overall survival (OS) and cerebral progression-free survival (PFS, months)


M Saidi

S Walha

K Nouri

A Kabadou

Lotfi Bessais
email: bessais@icmpe.cnrs.fr

M Jemmali

An investigation of the Gd-Fe-Cr phase diagram: Phase equilibria at 800 °C

Keywords:

diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

I. INTRODUCTION

In the last decades, the magnetic refrigeration (MR) based on the magnetocaloric effect (MCE), became the best alternative of the conventional gas-compression refrigeration due to its high thermodynamic efficiency and environmental safety [1][2][3][4].

The properties of ternary rare earth iron-rich compounds of the type RFe 12-x M x (R: rare earth metal, M: transition-metal or p-block metal) for (R = Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, Y and Lu; M= Si, Ti, V, Cr, Mn, Zr, Nb, Mo and W) have been studied intensively [5][6][7][8][9][10][11][12][13]. This research has been stimulated by the possibility of using some of these materials for high performance applications such as magnetic refrigeration.

Additionally, investigation on the phase diagrams of R-Fe-M systems is still a great concern for researchers in the interest of providing a basis for searching new magnetic materials and improving their performance.

In the R-Fe-M ternary systems the phase equilibrium has already been established for (R = Nd, Sm, Gd, Er and Y; M = Al, Co, Ni, Ge and Ga) [14][15][16][17][18], and in the R-Fe-Cr ternary systems the phase relationships have already been determined for (R = Tb, Dy, Ho, Er and Y) [19][20][21][22][23]. So far, no investigation of the Gd-Fe-Cr ternary system have ever been made. Thus, the purpose of the present paper is to provide basic information on compound formation and crystal structures of the binary and ternary compounds in the complete isothermal section of the ternary system Gd-Fe-Cr at 800 • C.

A detail description of the binary Gd-Fe, Gd-Cr and Fe-Cr phase diagrams was given in references [START_REF] Zhang | [END_REF][START_REF] Elliott | Constitution of Binary Alloys[END_REF][START_REF] Massalski | Binary Alloy Phase Diagrams[END_REF], with the available crystallographic data and stability temperature range of all the intermediate phases. The Gd-Fe system was investigated in the early 60s by Novy et al. [24] who showed the existence of seven binary compounds which have a peritectoid decomposition: Gd 2 Fe 17 , GdFe 5 , GdFe 4 , Gd 6 Fe 23 , GdFe 3 , GdFe 2 and Gd 2 Fe 3 . This binary system was later revised by Copeland et al. [START_REF] Copeland | Proceedings of the Conference on Rare Earth Research[END_REF] and Savitskii et al. [START_REF] Savitskii | Handbook on the Physics and Chemistry of Rare-Earths[END_REF]. In 1998, the study of Zhang et al. [START_REF] Zhang | [END_REF] showed that this system comprised four equilibrium intermetallic compounds, namely Gd 2 Fe 17 (Th 2 Zn 17 -type structure), Gd 6 Fe 23 (Th 6 Mn 23 -type structure), GdFe 3 (PuNi 3 -type structure) and GdFe 2 (MgCu 2 -type structure). The Gd-Cr binary phase diagram has been studied by Elliott [START_REF] Elliott | Constitution of Binary Alloys[END_REF] in 1965 which was based on Copeland and Kato [START_REF] Copeland | Proceedings of the Conference on Rare Earth Research[END_REF]. No binary compounds was found in this phase diagram at 800 • C. As for the Fe-Cr binary system, Massalski et al. [START_REF] Massalski | Binary Alloy Phase Diagrams[END_REF] reported that it has only one intermetallic compound namely σ(FeCr).

The phase diagrams for the three binary systems Gd-Fe [START_REF] Zhang | [END_REF], Gd-Cr [START_REF] Elliott | Constitution of Binary Alloys[END_REF],and Fe-Cr [START_REF] Massalski | Binary Alloy Phase Diagrams[END_REF] adopted in this study are shown in Figure 1.

The ternary compound GdFe 10 Cr 2 was previously identified by Bara et al. [11]. This later crystallizes with ThMn 12 -type of the I4/mmm space group.

In this experimental study, the complete isothermal section of the Gd-Fe-Cr ternary system at 800 • C was determined.

II. EXPERIMENTAL DETAILS

Bulk metals of gadolinium, iron and chromium, all with purities of 99.99 wt% were respectively used as the starting materials in this work. A total of 75 alloy buttons with weight of 0.5 g each were made in an electric arc furnace under high purity argon using a nonconsumable tungsten electrode and a water-cooled cooper crucible. A pure zirconium button was used as an oxygen-getter during the melting process. The alloys were re-melted three to four times, and each time the buttons were turned around in order to attempt complete fusion and ensure homogeneous composition. After alloying, the total mass of each sample was checked. The weight loss was less than 1% of the initial mass. After melting, each ingot the well-homogenized samples were quenched into ice-water mixture to preserve the high temperature state.

All samples were broken into two parts, one part for X-ray powder diffraction, the other for metallographic investigation. The first part of the samples, was crushed to powders and sieved for XRD analysis while the second part was ground, polished and cleansed in an ultrasonic bath before examination. The X-ray diffraction (XRD) data were collected on a Bruker D8 diffractometer (Bruker AXS, Karlsruhe, Germany) using CuKα radiation (λ Kα1 = 1.540562 Å). Data for Rietveld refinement were collected in the 2θ range from 20

•
to 80 • with a step size of 0.015 • and a counting time of 13.5 s per step. The experimental diffraction patterns were compared to those calculated from known structure types using the PowderCell [START_REF] Kraus | [END_REF] and FullProf [START_REF] Rodriguez-Carvajal | FULLPROF: A program for Rietveld Refinement and Pattern Matching analysis[END_REF] softwares.

The metallographic examination was carried out to check the results of X-ray diffraction analysis. Phase identification and composition analysis were investigated by Scanning Electron Microscopy (SEM) with a Silicon Drift Detector (SDD)-X-Max 50 equipped with energy dispersive X-ray spectroscopy (EDS).

III. RESULTS AND DISCUSSION

All the nominal compositions synthesized for the isothermal section Gd-Fe-Cr at 800 • C are plotted in Figure 2.

A. Binary systems

Taking into consideration the literature of the previous work [START_REF] Zhang | [END_REF], four binary compounds, namely Gd 2 Fe 17 , GdFe 3 , GdFe 2 and Gd 6 Fe 23 , were found in the binary system Gd-Fe. In the same binary system studied at 800 reference [START_REF] Saidi | [END_REF].

For the sake of confirming this result, the X-ray diffraction patterns of all the samples prepared in the two-phase regions 5 of the isothermal section Gd-Fe-Cr, showed two-phase equilibrium between Gd 2 Fe 17 and GdFe The Rietveld analysis of the binary compound GdFe 2 is represented in Figure 5. The lattice parameter for this phase was: a = 7.380(5) Å.

The Gd 2 Fe 17 compound displays two polymorphic forms: hexagonal with P 6 3 /mmc space group (Th 2 Ni 17 -type structure) and rhombohedral with R 3m space group (Th 2 Zn 17 -type structure) [33]. In this study, the peaks of the X-ray diffraction pattern of this sample were indexed with the rhombohedral unit-cell. Figure 6 shows the Rietveld refinement for the binary Gd 2 Fe 17 . This analysis proves that this intermetallic compound adopts the A series of samples near the σ(FeCr)-phase region were prepared in the Fe-Cr binary system, in order to confirm the existence of the σ(FeCr) phase. The results of X-ray diffraction show that these samples had patterns of σ(FeCr). Therefore, it was confirmed that the σ(FeCr) phase exists in the Fe-Cr binary system at 800 • C with a narrow homogeneity range of 45.3-49 at. % Cr.

B. Ternary system

The phase RFe 12 is based on the tetragonal ThMn 12 -type structure, with the I4/mmm space group [34]. In this structure, the rare earth Th atoms occupied the 2a (0,0,0) site and the Mn atoms occupy three crystallographic non-equivalent sites, denoted as Mn (8i), Mn (8j) and Mn (8f): 8i (x, 0, 0), 8j (x, 1/2, 0) and 8f (1/4, 1/4, 1/4) [35]. Figure 7 shows the crystal structure of ThMn 12 .

FIG. 7: Tetragonal crystal structure of ThMn 12 [35].

It is well known [5][6][7][8][9][10][11][12][13] that the pure phase RFe 12 is not stable and the addition of C. Solid solubility

According to the results obtained from SEM/EDS combined with the X-ray diffraction technique, the extended regions of solid solubility of each single phase in the 800 • C isothermal section were clearly identified. The Gd-Fe-Cr phase diagram was characterized by a large homogeneity ranges covering the iron-rich region which is summarized in Table II.

In order to identify the Gd(Fe,Cr) 2 solid solution at 800 • C, several samples on this binary extension and in the regions either side were synthesized. The existence of the GdFe 2-x Cr x solid solution was proven by the two samples with nominal compositions Gd44-Fe37-Cr19 and Gd18-Fe44-Cr38 prepared respectively in the three phase-region A and the two-phase region 1 of the Gd-Fe-Cr ternary phase diagram. The XRD pattern (Figure 9) of the Gd44-Fe37-Cr19 composition (N • 31 in Figure 1) showed a three-phase equilibrium between the upper limit of the solid solution GdFe 2-x Cr x (x = 0.35), (Gd) and (Cr). This result was confirmed by the microstructure in Figure 10. Similarly, the X-ray powder pattern and the microstructure of the Gd18-Fe44-Cr38 composition (N • 52 in Figure 1), which was melted and heat-treated at 800 • C, revealed a solid state equilibrium between the upper limit of the solid solution GdFe 2-x Cr x (x = 0.35) and

(Cr). The powder pattern of the Gd18-Fe44-Cr38 composition is given in Figure 11. In the SEM image (Figure 12), the light grey area had an atomic composition in agreement with II). Figure 13 presents the Rietveld refinement of the GdFe The stability of the Gd 2 Fe 17-x Cr x solid solution was confirmed using the SEM-EDS and the X-ray difraction. The synthesis of the Gd25-Fe64-Cr11 nominal composition (N • 12 in Figure 2) in the three phase-region G of the Gd-Fe-Cr ternary phase diagram, revealed the limit of this solid solution. The XRD pattern (Figure 14) and the SEM micrograph (Figure 15) of the Gd25-Fe64-Cr11 sample clearly shows a three-phase equilibrium between the upper limit of the solid solution Gd 2 Fe 17-x Cr x (x = 2.5), the upper limit of the solid solution GdFe 2-x Cr x (x = 0.35) and the phase GdFe 3 . In Figure 16, the dark area was The maximum solid solubility of Cr in Gd 2 Fe 17-x Cr x was determined to be around 13.2 at. % Cr (Table II). The Rietveld analysis of the Gd 2

Å) (Table

14

The experimental X-ray diffractogram (Figure 18) of the Gd11-Fe61-Cr28 nominal composition (N • 38 in Figure 2) located in the three-phase region C of the Gd-Fe-Cr ternary system, was indexed to three type structures, and shows the three-phase equilibrium between the following phases: GdFe 2-x Cr x (x = 0.35) (MgCu 2 -type structure) + GdFe 12-x Cr x (x = 3) (ThMn 12 -type structure) + σ(FeCr) (W-type structure). The SEM image cofirmed this result (Figure 19). From the combination of XRD analysis and SEM-EDS results obtained from 75 binary and ternary samples, the phase equilibria in the Gd-Fe-Cr ternary system were identified and the 800 • C isothermal section was constructed (Figure 20). A total of four binary compounds, namely GdFe 2 , GdFe 3 , Gd 2 Fe 17 and σ(FeCr) were characterized.

This study has enabled the determination of three solid solutions, and the characterization of divers equilibrium areas. All binary phases of the Gd-Fe binary system extended into the ternary, except GdFe 3 . The GdFe 2-x Cr x and Gd 2 Fe 17-x Cr x solid solutions showed maximum extensions up to x = 0.35 and x = 2.5 respectively. Also, the Gd-Fe-Cr system contained one ternary solid solution GdFe 12-x Cr x , having small homogeneity domain which extended from the GdFe 12-x Cr x (x = 2) composition to the GdFe 12-x Cr x (x = 3) composition.

The determined phase relationship indicates that the isothermal section of the Gd-Fe-Cr ternary system at 800 • C consists of eight single-phase regions, eight two-phase regions and seven three-phase regions. In order to further identify these phase relationships, Table III and Table IV present respectively the details of the measured compositions by EDS with their numbers as shown in Figure 2 in the two-phase regions and three-phase regions of the Gd-Fe-Cr ternary system. In this work, by comparing and analyzing the XRD experiments and SEM/EDS examinations of all the samples and identifying the phases in each composition, the isothermal section of the Gd-Fe-Cr ternary system at 800 • C was constructed (Figure 20). The complete investigation of the Gd-Fe-Cr phase diagram confirms the literature data and provides information relating to the solubility limit and phase relations in this ternary system.

IV. CONCLUSIONS

The isothermal section of the phase diagram of Gd-Fe-Cr system was determined at 800 • C. It consists of eight single-phase regions, eight two-phase regions and seven threephase regions. Four binary compounds, namely GdFe 2 , GdFe 3 , Gd 2 Fe 17 and σ(FeCr) and one ternary compound, GdFe 10 Cr 2 , were confirmed to exist in this section. Conversely, the

FIG. 1 :

 1 FIG. 1: Gd-Fe, Gd-Cr and Fe-Cr binary phase diagrams.

FIG. 2 :

 2 FIG. 2: Prepared compositions of the Gd-Fe-Cr samples.

3 .

 3 No diffraction reflections which belonged to the phase Gd 6 Fe 23 were observed for these samples, showing no evidence to support the existence of Gd 6 Fe 23 at 800 • C. Figure 3 presents the Rietveld refinement of the Gd13-Fe82-Cr5 nominal composition (N • 4 in Figure 2), located in the two-phase region 5 of the ternary phase diagram, consisting of the two phases Gd 2 Fe 17 and GdFe 3 . The microstructure was examined by SEM and clearly showed the existence of Gd 2 Fe 17 and GdFe 3 , as presented in Figure 4. The darker grey areas show the phase Gd 2 Fe 17 and the bright grey areas show the phase GdFe 3 .

Phase 1: GdFe 3 Phase 2 :

 32 FIG.3: Rietveld analysis for X-ray diffraction pattern of the Gd13-Fe82-Cr5 nominal composition.

FIG. 5 :

 5 FIG.5: Rietveld analysis for X-ray diffraction pattern of GdFe 2 binary compound.

  a transition metal or a p-block metal M (M = Si, Ti, V, Cr, Mn, Zr, Nb, Mo and W) is necessary to stabilize the ThMn 12 structure and form the RFe 12-x M x compounds. In the current research, and by analyzing and comparing the X-ray diffraction patterns and micrographs, the existence of the ternary compound GdFe 10 Cr 2 was confirmed. The Rietveld refinement for the XRD pattern of the compound GdFe 10 Cr 2 with cell parameters a = 8.480(6) Å and c = 4.755(3) Å, is shown in Figure 8. Crystal structure and lattice parameter data of the binary and ternary phases are given in Table I with references.

FIG. 8 :

 8 FIG. 8: Rietveld analysis of GdFe 10 Cr 2 annealed at 800 • C. The ticks refer to the main phase and (αFe).

FIG. 9 :

 9 FIG. 9: Experimental XRD pattern of the Gd44-Fe37-Cr19 composition showing the equilibrium between GdFe 1.65 Cr 0.35 , (Gd) and (Cr) phases.

FIG. 10 :

 10 FIG. 10: Backscattered electron SEM image of the Gd44-Fe37-Cr19 sample showing the equilibrium between GdFe 1.65 Cr 0.35 , (Gd) and (Cr) phases.

Phase 1 :

 1 FIG. 11: Rietveld analysis for X-ray diffraction pattern of the Gd18-Fe44-Cr38 composition. All the diffraction peaks were indexed with both the cubic structures of GdFe 1.65 Cr 0.35 and (Cr).

35 FIG. 12 :

 3512 FIG. 12: Backscattered electron SEM image of the microstructure of the sample with nominal composition Gd18-Fe44-Cr38 showing the equilibrium between GdFe 1.65 Cr 0.35 and (Cr).

  2-x Cr x x = 0.1 and x = 0.27 series. During the study of this homogeneity domain, the same type of structure of MgCu 2 was retained.

FIG. 13 :

 13 FIG. 13: Rietveld analysis of GdFe 2-x Cr x compounds: a) x = 0.1, and b) x = 0.27.

FIG. 14 : 3 Gd 2 5 FIG. 15 :

 1432515 FIG. 14: Experimental XRD pattern of the Gd25-Fe64-Cr11 composition showing the equilibrium between Gd 2 Fe 17-x Cr x (x = 2.5), GdFe 2-x Cr x (x = 0.35) and GdFe 3 .

  Fe 17-x Cr x x = 1 and x = 2 series are represented in Figure 16. The lattice parameters for Gd 2 Fe 16 Cr 1 were: a = 8.544(2) Å and c = 12.443(9) Å and the lattice parameters for Gd 2 Fe 15 Cr 2 were: a = 8.549(2) Å and c = 12.452(3) Å. The existence of the ternary line compound GdFe 12-x Cr x was revealed by annealed samples of several compositions. The analysis of the results of the scanning electron microscope coupled to the X-ray diffractograms of each sample allowed the delimitation of the GdFe 12-x Cr x ternary solid solution. The homogeneity range of the GdFe 12-x Cr x solid solution, which crystallizes in the tetragonal ThMn 12 type-structure with the I4/mmm space group, extended from about 15.4 at. % Cr to 23.1 at. % Cr at 800 • C (TableII). The SEM image (Figure17) for the Gd8-Fe81-Cr11 nominal composition (N • 17 in Figure2) located in the three-phase region F of the ternary phase diagram, shows the thermodynamic equilibrium between the lower limit of the GdFe 12-x Cr x solid solution which is GdFe 10 Cr 2 , the upper limit of the Gd 2 Fe 17-x Cr x (x = 2.5) solid solution and (αFe).

FIG. 17 :

 17 FIG. 17: Backscattered electron SEM image of the Gd8-Fe81-Cr11 sample showing the equilibrium between GdFe 10 Cr 2 , Gd 2 Fe 14.5 Cr 2.5 and (αFe) phases.

FIG. 18 :FIG. 19 :

 1819 FIG. 18: X-ray diffraction pattern of the Gd11-Fe61-Cr28 composition.

FIG. 20 :

 20 FIG. 20: Isothermal section at 800 • C of the Gd-Fe-Cr phase diagram. The areas of the threephase regions are identified with letters and the areas of the two-phase regions are identified with numbers.

  • C, only the three binary compounds Gd 2 Fe 17 , GdFe 3 and GdFe 2 were confirmed, but the compound Gd 6 Fe 23 prepared under the same circumstances was unstable, because there were two-phase mixtures containing Gd 2 Fe 17 and GdFe 3 . As a result, Gd 6 Fe 23 is not stable at 800

• C and should not existed in the isothermal section Gd-Fe-Cr. This result is consistent with the findings of our previous work mentioned in

TABLE I :

 I

	Phase	Structure	Space	Lattice parameters ( Å)	References
	Composition	type	group	a	c
	GdFe 10 Cr 2	ThZn 12	I4/mmm	8.507	4.769	[11]
	GdFe 10 Cr 2	ThZn 12	I4/mmm	8.480(6)	4.755(3)	This work
	Gd 2 Fe 17	Th 2 Zn 17	R 3m	8.510	12.510	[36]
	Gd 2 Fe 17	Th 2 Zn 17	R 3m	8.513(6)	12.453(2)	This work
	GdFe 3	PuNi 3	R 3m	5.148	24.62	[37]
	GdFe 3	PuNi 3	R 3m	5.166(4)	24.705(1)	This work
	GdFe 2	MgCu 2	F d 3m	7.390	-	[38]
	GdFe 2	MgCu 2	F d 3m	7.380(5)	-	This work
	σ(FeCr)	W	Im 3m	2.863	-	[39]
	Gd	Mg	P 6 3 /mmc	3.634	5.785	[40]
	(αFe)	W	Im 3m	2.868	-	[41]
	Cr	Cr	Im 3m	2.899	-	[42]

Crystallographic data of intermetallic compounds in Gd-Fe-Cr ternary system at 800 • C.

TABLE II :

 II Lattice parameters and homogeneity ranges of the identified solid solutions in the Gd-Fe-Cr system at 800 • C.

	Phase	Homogeneity	Lattice parameters ( Å)
		ranges	a	c
	GdFe 2-x Cr x	(0 ≤ x ≤ 0.35)
	x = 0		7.380(5)	-
	x = 0.1		7.385(8)	-
	x = 0.2		7.388(3)	-
	x = 0.27		7.397(5)	-
	x = 0.35		7.398(3)	-
	Gd 2 Fe 17-x Cr x	(0 ≤ x ≤ 2.5)
	x = 0		8.513(6)	12.453(2)
	x = 0.5		8.543(2)	12.436(8)
	x = 1		8.544(2)	12.443(9)
	x = 2		8.549(2)	12.452(3)
	x = 2.5		8.556(5)	12.466(4)
	GdFe 12-x Cr x	(2 ≤ x ≤ 3)
	x = 2		8.480(6)	4.755(3)
	x = 2.5		8.485(1)	4.756(7)
	x = 3		8.487(8)	4.757(3)
	while Fe was found to extend up to 43.5 at. % Cr. The σ(FeCr) compound has a narrow
	homogeneity range of 45.3-49 at. % Cr in the binary Fe-Cr.

D. Phase equilibrium at 800 • C

TABLE III :

 III Phase compositions of the two-phase regions in the Gd-Fe-Cr ternary system at 800 • C.

					EDS results (at.%)
	Phase regions	No	Nominal compositions	Phases
					Gd	Fe	Cr
				(Cr)	0.0	40	60
	1	52	Gd18-Fe44-Cr38
				GdFe 1.65 Cr 0.35	33.1	55.2	11.7
				σ(FeCr)	0.0	52.2	47.8
	2	59	Gd2-Fe52-Cr42
				GdFe 9 Cr 3	7.7	69.2	23.1
				(αFe)	0.0	59.8	40.2
	3	33	Gd4-Fe73-Cr23
				GdFe 10 Cr 2	7.7	76.9	15.4
				Gd 2 Fe 14.5 Cr 2.5	10.5	76.3	13.2
	4	1	Gd7.2-Fe88.7-Cr3.7
				(αFe)	0.0	99.7	0.3
				Gd 2 Fe 14.5 Cr 2.5	10.5	76.4	13.1
	5	7	Gd19-Fe77-Cr4
				GdFe 3	25	75	0.0
				GdFe 1.65 Cr 0.35	33.1	55.3	11.6
	6	25	Gd24-Fe62-Cr14
				GdFe 9.5 Cr 2.5	7.7	73.1	19.2
				GdFe 1.65 Cr 0.35	33.2	55.2	11.6
	7	14	Gd30-Fe66-Cr4
				GdFe 3	24.8	75.2	0.0
				GdFe 1.65 Cr 0.35	33.1	55.1	11.8
	8	16	Gd54-Fe43-Cr3
				(Gd)	99.9	0.1	0.0

TABLE IV :

 IV Phase compositions of the three-phase regions in the isothermal section of Gd-Fe-Cr ternary system at 800 • C.

					EDS results (at.%)
	Phase regions	No	Nominal compositions	Phases
					Gd	Fe	Cr
				(Gd)	99.8	0.0	0.2
	A	31	Gd44-Fe37-Cr19	GdFe 1.65 Cr 0.35	33.2	55.1	11.7
				(Cr)	0.3	0.0	99.7
				σ(FeCr)	0.0	51	49
	B	51	Gd10-Fe51-Cr39	GdFe 1.65 Cr 0.35	33.2	55.1	11.7
				(Cr)	0.0	39.8	60.2
				GdFe 9 Cr 3	7.7	69.4	22.9
	C	38	Gd11-Fe61-Cr28	GdFe 1.65 Cr 0.35	33.3	55.3	11.4
				σ(FeCr)	0.0	51.8	48.2
				(αFe)	0.0	60	40
	D	47	Gd2-Fe60-Cr38	σ(FeCr)	0.0	54.7	45.3
				GdFe 9 Cr 3	7.7	69.3	23
				GdFe 10 Cr 2	7.7	77	15.4
	E	21	Gd10-Fe75-Cr15	Gd 2 Fe 14.5 Cr 2.5	10.5	76.2	13.3
				GdFe 1.65 Cr 0.35	33.3	55.2	11.5
				GdFe 10 Cr 2	7.6	77	15.4
	F	17	Gd8-Fe81Cr11	Gd 2 Fe 14.5 Cr 2.5	10.6	76.2	13.2
				(αFe)	0.0	99.8	0.2
				GdFe 3	24.9	75.1	0.0
	G	12	Gd25-Fe64-Cr11	Gd 2 Fe 14.5 Cr 2.5	10.5	76.3	13.2
				GdFe 1.65 Cr 0.35	33.2	55	11.8

This work was mainly supported by the CNRS, the Tunisian Ministry of Higher Education and Scientific Research and Technology (LAB MESLAB) (Tunisia-Sfax) and the Higher Education, Scientific of French (PHC MAGHREB project 15MAG07).

VI. REFERENCES