Sara M A Pinto
email: smpinto@qui.uc.pt

Mário J F Calvete

Mariana E Ghica

Sérgio Soler

Iluminada Gallardo

Agnès Pallier

Mariana B Laranjo

Ana M S Cardoso

M Margarida

C A Castro

M A Brett

Mariette M Pereira

Éva Tóth

Carlos F G C Geraldes
email: geraldes@ci.uc.pt

A Biocompatible Redox MRI Probe Based on a Mn(II)/Mn(III) Porphyrin

For the development of redox responsive MRI probes based on the Mn III /Mn II couple, stable complexation of both reduced and oxidized forms of the metal ion and appropriate tuning of the redox potential in the biologically relevant range are key elements. The water soluble fluorinated Mn-porphyrin derivative Mn-3 satisfies both requirements. In aqueous solution, it can reversibly switch between Mn III /Mn II oxidation states. In the presence of ascorbic acid or β-mercaptoethanol, the Mn III form undergoes reduction, which is fully reversed in the presence of oxygen. A UV-Vis kinetic study of Mn III /Mn II reduction yielded second-order rate constants, k2, of 46.1 M -1 s -1 and 13.8 M -1 s -1 for the reaction with ascorbic acid and βmercaptoethanol, respectively. This could correspond to a half-life of a few minutes in blood plasma and a few seconds in circulating immune cells where ascorbic acid reaches 20-40 M and a few mM concentrations, respectively. Contrary to expectations based on the redox potential, reduction with glutathione or cysteine does not occur. It is prevented by the coordination of the glutathione carboxylate group(s) to Mn III in axial position, as it was evidenced by NMR data. Therefore, Mn III -3 acts as an ascorbate specific turn-on MRI probe, which in turn can be re-oxydized by oxygen. The relaxivity increase from the oxidized to the reduced form is considerably improved at medium frequencies (up to 80 MHz) with respect to the previously studied Mn-TPPS4 analogues; at 20 MHz, it amounts to 150%. No in vitro cytotoxicity is detectable for Mn-3 in the typical MRI imaging concentration range. Finally, 19 F NMR resonances of Mn III -3 are relatively sharp which could open further opportunities to exploit such complexes as paramagnetic 19 F NMR probes.

Introduction

In any living body, both extracellular and intracellular redox environments are tightly regulated [1][2][3] and their dysregulation may be associated with a wide range of pathophysiological conditions, including chronic inflammation, 4 neoplastic growth 5 and ischemia, 6 since several triggered biochemical cascade events can damage cellular or tissue components, promoting disease progression. [7][8][9] The redox environment in a biological fluid, organelle, cell, or tissue is maintained by a number of redox couples present which are linked to each other. Among these, glutathione (GSH) is the major thiol-disulfide redox buffer of the cell with an average concentration of 1-11 mM in the cytosol, much higher than most other redox active compounds. 10 Therefore, the redox state of the glutathione disulfide-glutathione couple (GSSG/2GSH) can serve as an indicator of the intracellular redox environment. Changes of the half-cell reduction potential (Ehc) of the GSSG/2GSH couple appear to correlate with the biological status of the cell: proliferation Ehc ~ -240 mV; differentiation Ehc ~ -200 mV; or apoptosis Ehc ~ -170 mV. 10 The extracellular space is typically more oxidized than the cytosol. In cell extracts, the total concentration of reduced (GSH) and oxidized (GSSG) glutathione present can be assessed by HPLC or fluorescent assays. 11,12 The development of imaging techniques capable of following in vivo tissue redox environment/activity is of utmost interest in clinical research and medical practice, particularly in diagnosis, prognosis or monitoring therapeutic response. 13,14 The fundamental goal of developing molecular probes capable of imaging redox activity has seen some clinical success. In particular, positron emission tomography (PET) probes capable of targeting hypoxic tissue, such as the radiotracers 64 Cu IIdiacetyl-bis(N4-methylthiosemi-carbazone) ( 64 Cu II -ATSM) and 18 F-fluoro-misonidazole ( 18 F-MISO), showed capacity to predict treatment outcome in patients undergoing radiotherapy. [15][16][17] The same hypoxia targeting mechanism of probe uptake (irreversible reaction and retention in oxygen-deprived tissue) has been extended to MRI contrast agents and fluorescent probes for optical imaging. 18,19 Redox-sensitive nitroxide derivative probes were also used in electron paramagnetic resonance (EPR) imaging and spectroscopy to detect reductive environment (thiols and other reducing species). 20,21 Magnetic resonance imaging (MRI) is an attractive modality to monitor redox dynamics thanks to its exceptional spatial resolution (<100 m using modern high-field equipment), lack of invasiveness and use of ionizing radiation, and its ability to simultaneously report physiological and anatomical information. Not surprisingly, the interest in the development of redox sensitive MRI probes has grown over the last years. 14,[22][23][24][25][26] These can be based on redox active ligands, undergoing a change in the ligand structure or the molecular rotation upon reduction/oxidation, while the oxidation state of the paramagnetic metal remains the same (e.g. merocyanine unit linked to a GdDO3A which isomerizes to the spirooxazine derivative by the redox stimulus of NADH). 27 Alternatively, complexes of redox active paramagnetic metals, like europium and manganese, can be also explored, in which the different oxidation states have different magnetic properties. 11,22 Utterly, both types of probes must fulfill several requirements: i) biocompatibility, ii) a redox half-cell potential which is compatible with the redox potential of biological reducing agents; iii) strong signal change, if possible signal increase upon activation ("turn-on" probe); and iv) rapid redox kinetics as compared to the imaging time scale. Importantly, the redox active metal ion needs to be complexed by a ligand that is capable of efficiently chelating both oxidation states such that reduction or oxidation does not result in the decomposition of the complex. Among redox active metal ions, manganese is particularly attractive for the development of redox responsive MRI probes, whose efficiency is measured by their proton relaxivity ri (i = 1,2) (paramagnetic enhancement of the water proton relaxation rates, Ri = Ti -1 , where T1 and T2 are, respectively, the longitudinal and transverse relaxation times, referred to 1 mM concentration of the paramagnetic ion). Manganese is a biogenic element and in its +2 oxidation state, Mn 2+ , is a very efficient paramagnetic relaxation agent. Mn 2+ (d 5 configuration, high spin S = 5/2), with long (in the 0.1 -1 ns range) longitudinal electronic relaxation times and labile water exchange, is an attractive alternative to Gd 3+ in MRI probes. 28,29 Indeed, in the last years, there has been intensive research to identify suitable ligand structures for Mn 2+ chelation. 30,31 Mn 3+ (d 4 configuration) is also paramagnetic, usually giving rise to high spin S = 2 compounds. While water exchange is also fast, 14 the longitudinal electronic relaxation times are shorter (≤ 10 ps), making them less efficient relaxation agents. 32 The nuclear relaxation mechanism of Mn 3+ is different from that of Mn 2+ and therefore their relaxation efficiency shows a very distinct dependence on the external magnetic field B0. While for the S = 5/2 Mn 2+ complexes, the classical Solomon-Bloembergen-Morgan (SBM) formalism describes well nuclear relaxation, leading to a decrease of r1 at high field, for the S = 2 Mn 3+ complexes the analysis of the water proton relaxation rates is much less straightforward. Water soluble and air stable Mn 3+porphyrins have been investigated as tumor targeted MRI agents. 33 For these compounds, the electron relaxation times are relatively long due to their higher symmetry. 32 For instance, the Mn III -TPPS4 (meso-tetra(4-sulfonatophenyl)porphine chloride) chelate 34 displays an anomalous r1 relaxivity, with a peak above approximately 2 MHz. It was shown that the NMR relaxation mechanism has several unique aspects, including the unusual role of large rhombic zero-field splitting (ZFS) interactions, which are of the order of the nuclear Zeeman energy. The effect of these interactions on the electron spin dynamics leads to those relaxivity anomalies at high field. 35,36 As a consequence of the different relaxation mechanisms, the proton relaxivity, and thus the MRI efficiency can be either higher or lower for the reduced Mn 2+ relative to the oxidized Mn 3+ form, depending on the external magnetic field and on the chelating ligand. Porphyrins, which are macrocyclic ligands per excellence and widely used in a multitude of applications, such as catalysis, 37-39 materials science [40][41][42][43] and biomedicine, including MRI, 30, [44][45][46][47][48] offer the possibility to obtain very stable chelates to host many metal ions, including paramagnetic ones. Such metalloporphyrins having a much reduced risk of transmetallation, may avoid potential in vivo metal release during the MRI examination which would cause toxicity, such as for instance gadolinium induced fibrosis disorders, 49 or manganese induced neurotoxicity. 50 50 50 50 50 50 50 50 50 50 50 Many studies have been reported of Mn III -porphyrin dimers, supramolecular and macromolecular conjugates as MRI contrast agents, 33,[51][52][53][54][55][56][57][58] including enzyme 55,56 and Zn 2+responsive 57, 58 agents. So far, the only studies on Mnporphyrins as redox MRI probes were reported by Aime and collaborators, 59 who explored a Mn II /Mn III -based water-soluble porphyrin (TPPS4) as an oxygen sensor. However, at clinically relevant fields ( 1 H frequencies of 60-120 MHz), and at 25 ºC, the r1 relaxivity values revealed to be very close for the Mn II and Mn III analogues. On the other hand, the formation of macromolecular conjugates, through the encapsulation of the Mn II -TPPS4 and Mn III -TPPS4 complexes in commercially available, water-soluble poly--cyclodextrin (CD), led to a marked difference in relaxivity at 20 MHz between the reduced and oxidized state.

Please do not adjust margins

Please do not adjust margins Caravan and collaborators 30 explored hydroxybenzylethylenediamine acetic acid derivatives for Mn II/III -chelation to create redox active contrast agents. The stability of the complexes was recently improved by using a "Janus" ligand that readily isomerizes between binding modes that favor either the Mn 3+ or Mn 2+ ion. 30 Rapid and reversible interconversion between the two oxidation modes was achieved by peroxidase activity (oxidation) and L-cysteine (reduction), which was translated into an unprecedented 9-fold relaxivity increase between the Mn 3+ and Mn 2+ analogue, respectively. One concern, nevertheless, about these openchain complexes is the lack of a sufficiently high kinetic inertness which is desirable for in vivo applications. Based on previous experience in porphyrin chemistry, our objective is to develop water soluble, biocompatible Mnporphyrin complexes with a redox potential that is adapted to biological requirements. The approach to the development of such specifically designed Mn II/III complexes consists of modulating the redox potential by using appropriate electron withdrawing substituents on the porphyrin structure. The finetuning of the redox potential would be highly important since most of the redox responsive MRI probes reported so far do not undergo redox chemistry in a biologically relevant range. 22 The capacity of porphyrin complexes to selectively localize in tumor cells is well known and will be a great advantage. Indeed, it can simplify probe design as no additional targeting and cell internalizing moieties are required, although they might be later added to the chemical structure to further improve these properties. Herein we report the synthesis and physical-chemical characterization of a new fluorinated Mn II/III porphyrin derivative which was conjugated with PEG chains for better solubility and biocompatibility. Its redox properties were studied by cyclic voltammetry. The Mn III reduction kinetics was followed in the presence of ascorbic acid and mercaptoethanol (BME) by UV-Vis. The relaxivity differences between the reduced and oxidized forms were investigated using 1 H Nuclear Magnetic Relaxation Dispersion (NMRD). Cytotoxicity evaluation of the system on epithelial HeLa cells is also reported.

Results and Discussion

Synthesis of the biocompatible porphyrin-based probes

The strategy adopted for the synthesis of the Mn III biocompatible probe 3 is depicted in Scheme 1. Scheme 1. Synthetic route for the preparation of the biocompatible porphyrin 3. Reagents and conditions: i) DMF, NaH, room temperature, 2h; ii) acetic acid/sodium acetate, Mn(II)acetate tetrahydrate, 80ºC, 2h.

Porphyrin 1 was synthesized by the NaY method, developed by some of us, 60 where NaY acts as a reusable catalyst. After workup 12% of the isolated porphyrin was obtained. In order to obtain free base porphyrin 2, nucleophilic aromatic substitution of 1 using mono methyl protected polyethylene glycol with Mw = ~500 g/mol (PEG500) was performed, in DMF as solvent and NaH as base, at room temperature for two hours. The product was obtained in 65% yield, after purification, and fully characterized by 1 H, 19 F NMR spectroscopy and ESI-FIA-TOF mass spectrometry (see Figs. 1, S1, S2 andS3, ESI). Its main characterization detailing feature was ascertained by observing the loss of the characteristic triplet (p-F) 19 F NMR signal at around -150 ppm (Fig. 1) for porphyrin 2, when compared with the 19 F NMR spectrum of 1, which presents its three typical (m-F), (p-F) and (o-F) signals at -160.2, -150.1 and -135.4 ppm, respectively (Fig. 1), confirming the tetrapegylation pattern obtained.

Please do not adjust margins

Please do not adjust margins The preparation of Mn III -3 proceeded by reacting 2 with excess manganese diacetate tetrahydrate in sodium acetate/acetic acid buffer solution at 80 ºC for two hours. After several extractions of the crude with dichloromethane/water, the organic layer was dried with anhydrous sodium sulfate and concentrated under vacuum giving 3 in 82% yield (Scheme 1). The Mn III -3 complex was characterized by ESI-FIA-TOF mass spectrometry (see Fig. S4,ESI), presenting the characteristic poly-dispersed m/z peaks from PEGylated 1121.3612, 1319.9802, 1386.0195, 1493.6057 [M] 2+ (polydisperse distribution). Additionally, Electron Paramagnetic Resonance (EPR) experiments were also performed, in order to confirm the absence of the Mn II salt used in the complexation reaction. The absence of the sharp sextet characteristic of the free aqueous Mn 2+ cation even at a very high amplification, led to the conclusion that the Mn III -3 sample was free of aqueous Mn 2+ (Figure S5, ESI). 47 The EPR spectrum of the Mn III -3 was also silent at room temperature, as expected from the fast electron relaxation characteristic of the integer-spin (non-Kramers) porphyrin compound. 61 Characteristic EPR resonances of Mn IIporphyrins have been observed at liquid nitrogen temperature (e.g. a strong sextet at g┴ ~ 5.9-5.2, a signal at g║ ~ 2.0-2.1, and sometimes three other weak signals at 1.23, 0.77 and 0.54). 61,62 If some Mn II -3 were present in the sample, a broad signal at giso ~2.0 would be observable at room temperature, 62 which was not the case.

Proton and 19 F NMR

The proton NMR spectrum of Mn III -3 in CDCl3 (Fig. S6a) shows a broad signal at -22.76 ppm corresponding to the -pyrrole protons, characteristic of Mn III high-spin meso-porphyrins. The large upfield shift and large broadening observed arise from the dominant contact mechanism, due to the small magnetic anisotropy of the coordinated Mn III ion and consequent dipolar shift induced. 63,64 The PEG chain CH2 protons at 3.66 ppm are unshifted and hardly broadened relative to the signal at 3.65 ppm for free PEG due to their long distance from the paramagnetic center. The 19 F NMR spectrum of Mn III -3 (Fig. S6b) consists of a broad signal centered at -126.2 ppm and a set of five upfield shifted (in the range of -149.7 to -157.

Please do not adjust margins

Please do not adjust margins

Cyclic Voltammetry

The electrochemical behaviour of the porphyrin TPFPP(PEG)4 (2) and the metalloporphyrins Mn III -TPFPP(PEG)4 (Mn III -3) and Zn II -TPFPP(PEG)4 (Zn II -3) was investigated in deoxygenated 0.1 M HEPES buffer solution, pH 7.4, by cyclic voltammetry at a glassy carbon electrode (GCE). The cyclic voltammograms obtained for the fluorinated porphyrins are presented in Fig. 2a. No adsorption of any porphyrin was observed in consecutive cycles.

-1.5 -1.2 -0.9 -0.6 -0.3 0.0 0.3 0.6 0.9 1.2 -2.5

I / nA (v / V s -1 ) 1/2 (b)
Figure 2. a) Cyclic voltammograms at GCE in 0.1 M HEPES buffer pH 7.4 for 2.2 mM of TPFPP(PEG)4 (black), Mn III -3 (red) and Zn II -3 (blue); b) Plot of the peak current versus square root of scan rate for the peaks IIIa/IIIc, corresponding to Mn II /Mn III for the CV of 2.2 mM Mn III -3 in 0.1 M HEPES buffer pH 7.4 at scan rates 10-100 mV s -1

In the potential range investigated, which is limited by water reduction and oxidation potentials, the following behavior was observed. The free porphyrin exhibited two redox couples IIa/IIc and Ia/Ic, with midpoint potential EpII m = -0.580 V and peak separation ΔEpII = 0.120 V and EpI m = -1.060 V and peak separation ΔEpI = 0.120 V, respectively. These couples were ascribed to the porphyrin ring two successive one-electron oxidation/reduction processes, the first involving a -radical anion and the second the porphyrin dianion formation, respectively, as suggested in the litterature. 66,67 The formal potential for these peaks shifted to more negative values for Mn III -3 (EpII´m= -0.900 V), as well as for Zn II -3 (EpII´m= -0.945 V) porphyrins, which is expected with the introduction of a metal ion in the porphyrin by its effect on the electronic structure of the macrocyclic ring. For Zn II -3 no additional peaks were observed in the potential range studied; those involving oxidation of the porphyrin ring should be higher than +1.20 V (see later). In the case of Mn III -3 two more redox couples IIIa/IIIc and IVa/IVc appeared. The IIIa/IIIc pair with EpIII m = +0.193 V and ΔEpIII = 0.150 V was assigned to the Mn II /Mn III redox process, while IVa/IVc with EpIV m = +0.642 V and ΔEpIV = 0.264 V was associated with the Mn III /Mn IV reaction. These assignments were confirmed by performing cyclic voltammetry under the same conditions in the presence of Mn III -acetate (Fig. S7a), where two redox couples also appeared. The pair IVa/IVc exhibited potential values EpIV´m= +0.575 V and ΔEpIV´ = 0.222 V for Mn IIIacetate, quite similar to those of Mn III -3 and of the cationic tetramethyl meso-substituted porphyrin, Mn III -PMPyP, at basic pH. 68- 70 The reversibility of this redox process was found not to depend on experimental conditions 71 and will not be further discussed here. However, for the pair IIIa/IIIc a large difference was observed; the peak separation was wider ΔEpIII´ = +0.778 V, whilst the formal potential was more negative, EpIII´m= -0.065 V for Mn III -acetate. An increase in peak current was also observed for the same redox couple, IIIa/IIIc, a factor of 2 for the oxidation and 3 for reduction process in the case of the Mn III -3 porphyrin compared with Mn IIIacetate. Thus, the introduction of manganese into the porphyrin has a positive impact, by greatly increasing its reversibility with regard to the Mn II /Mn III process. An improvement in the Mn II /Mn III redox reaction was also achieved when introducing manganese in a sulphonated porphyrin, Mn III -TPPS4, since this redox pair exhibited a peak separation ΔEpIII´´ = 0.252 V with midpoint potential EpIII´´m= -0.238 V (data not shown). However, the formal potential for Mn III -3 is more positive and the peaks are less separated, as well as exhibiting higher current peaks, compared with Mn III -TPPS4, which enables the former porphyrin to be elected as a better redox probe.

Although the Mn II /Mn III redox reaction in Mn III -3 was a quasireversible slow electron transfer, this is in agreement with what was found for non-fluorinated Mn-porphyrins, eg. Mn III -TPPS4, with EpIII m values of -0.24 V in DMF/0.1 M TBAP 72 and -0.22 V in water/0.3 M Na2SO4. 73 , as well as for manganese fluorinated porphyrin, Mn-TPFPP, in acetonitrile 61 , EpIII m value of +0.05 V (Table S1).

The cyclic voltammetric behaviour of the Mn III -3 porphyrin was investigated over a range of scan rates from 10 to 100 mV s -1 . The results showed a linear dependence of the anodic and cathodic peak currents of the redox couple Mn II /Mn III with the square root of the scan rate (ν) (Fig. 2b andS7b), indicating a diffusion-controlled electron transfer process, 74 in agreement with what was previously observed for the complex Mn III -HBET (HBET = N-(2-hydroxybenzyl)-N,N´N´-ethylenediaminetriacetic acid) 26 and other manganese-based porphyrins. 75 The stability of the Mn III -3 porphyrin was checked by performing cyclic voltammograms of the prepared solution over a period of one month. No change in peak current values was observed, indicating high stability of this compound in aqueous solution. In spite of the quasi-reversible process, the value of the redox potential achieved as well as its high stability ensures the ability of Mn III -3 to act as a redox probe, as proposed here.

The redox behaviour of the free porphyrin and the Zn II -3 and Mn III -3 derivatives was also investigated by cyclic voltammetry in DCE and 0.6 M Bu4NBF4 at a glassy carbon electrode (Table S1 and Fig. S7d,e).

The free porphyrin showed two redox couples at negative potentials with Ep m values at -0.452 V and -1.00 V, corresponding to the porphyrin ring anion and dianion formation. Zn II -3 showed two This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins reversible monoelectronic reduction peaks with Ep m values at -0.72 V and -1.10 V, also assigned to the reduction of the porphyrin ring to the -radical anion and dianion forms. The reversible monoelectronic reduction peak observed at -1.31 V and the irreversible peak at -1.64 V correspond to the PEG chains. Zn II -3 also exibited two irreversible monoelectronic anodic peaks at +1.61 V and +1.80 V, corresponding to the oxidation of the pyrrole groups to the radical cation and dication forms of the Zn porphyrin. This was followed by a multielectronic irreversible wave at + 2.80 V, corresponding to the degradation of the ether chains in the porphyrin. These data are in agreement with reported data for other perfluorinated Zn II porphyrins (Table S1). 65,66,76 The Mn III -3 porphyrin exhibited a similar behaviour to that observed in aqueous media: at negative potentials two redox couples with Ep m values at -0.828 V and -1.131 V, corresponding to porphyrin ring anion and dianion formation. These assignments were confirmed by comparison with the data obtained for the free porphyrin, which show a trend in the potentials involving the reduction of the porphyrin moiety to be less negative for the free porphyrin than in the Zn II -3 and Mn III -3 complexes, as also observed in HEPES buffer. The Mn II /Mn III core redox process in the Mn III -3 porphyrin was observed at Ep m = +0.335 V, with a peak separation ΔEp = 0.350 V, again in agreement with data for other perfluorinated Mn III porphyrins (Table S1). 65,66,76 All the redox potentials obtained simultaneously in the two solvents were found to be lower in H2O than in DCE, in agreement with their relative dielectric constants and complexing abilities.

Conversion between the Mn III -3 and Mn II -3 redox states

In order to evaluate the possibility of reduction of Mn III -3 to Mn II -3, two different biological reductants were used, ascorbic acid and BME. The reduction with ascorbic acid was followed by UV-Vis spectra, recorded in PBS (phosphate buffer solution) in a solution of the Mn III -3 porphyrin upon addition of increasing amounts of reductant (Fig. 3a). Addition of 0.28 equivalents of ascorbic acid caused a decrease in the absorption at 457 nm (typical wavelength for the charge transfer Soret band (band V) of Mn III porphyrins), with the concomitant appearance of a new band at 434 nm, which is a typical absorption wavelength for Mn II porphyrins. 77 Total reduction of Mn III -3 to Mn II -3 was achieved after the addition of 25.3 equivalents of ascorbic acid. Conversely, oxidation of Mn II -3 to Mn III -3 was evaluated by acquiring the UV-Vis spectra of a Mn II -3 PBS solution, following exposure to air for 10 min, 4 h and 24 h (Fig. 3b). The system displays complete reversibility after 24h. A similar study was performed with BME and complete reduction from Mn III -3 to Mn II -3 was achieved in the presence of equivalents of this reductant (Fig. 3c). In this case, total reoxidation by air exposure was achieved after 72h (Fig. 3d). Please do not adjust margins Please do not adjust margins exposure: (black line) -Mn II -3 before exposure; (red line) -after 30 min. exposure; (blue line) after 24 h exposure; (green line) complete conversion to Mn III -3 (after 72 h exposure).

In contrast, reoxidation of Mn II -3 by addition of an increasing number of equivalents of hydrogen peroxide showed no reversibility to Mn III -3 (Fig. S8). Although the addition of up to the first equivalent gave mostly Mn III -3, further addition of a large excess led to formation of Mn IV and Mn V oxo derivatives (Soret bands in the 424-443 nm region), 69, 70 in agreement with the literature. 77,78 Besides the quite different wavelengths of the Soret band, the UV-Vis spectra of Mn III -3 and Mn II -3 show other distinctive differences (Fig. S9). In the UV region, Mn III -3 shows two other bands, (VI and Va), less intense than the Soret band, at 367 and 391 nm, respectively, which in Mn II -3 dramatically decreases intensity and shifts to 364 nm (VI), or disappears (Va). In the visible region, Mn III -3 displays a QIV band at 554 nm, which is shifted to 561 nm upon reduction, but the QIII band, usually observed in the 610-620 nm range, is not visible. 64,72,77 The absence of a decreased intensity of the Soret band and the absence of a band above 700 nm, show that the porphyrin macrocycle is not reduced. 79 In order to determine the empirical rate law for the reduction of Mn III -3 by ascorbic acid, a series of kinetic experiments were conducted at 25°C (pH=7.4) under pseudo-first order conditions, following the disappearance of the Soret band at 457 nm ( = 1.0110 4 M -1 s -1 ) over time in 50 ms intervals. Initial reaction rates were measured first at three different ascorbic acid concentrations (91, 191 and 390 M) keeping the initial Mn III -3 concentration constant at 8 M, and then at three different initial Mn III -3 concentrations (95, 150 and 330 M) in the presence of 9.8 mM ascorbic acid. The experimental data (Figs. S10 andS11) evidenced that the reduction is first-order in relation to both Mn III -3 and ascorbic acid concentrations, with an overall second-order rate constant k2 = 46.1 ± 5.6 M -1 s -1 . Similar experiments with BME as reductant yielded a k2 value of 13.8 ± 1.1 M -1 s -1 . These values are one to two orders of magnitude larger than the corresponding value of k2 = 0.38 M -1 s -1 reported for the reduction of Mn III -HBET by glutathione. 26 The normal blood plasma concentration of ascorbic acid is in the 20-40 M range, which can be somewhat reduced in some pathologies, such as systemic inflammation. 80,[START_REF] Levine | Proc. Natl. Acad. Sci[END_REF] For this ascorbic acid concentration range, the half-life of Mn III -3 is roughly 6-13 min. Ascorbic acid accumulates in circulating immune cells, such as neutrophiles, monocytes and lymphocytes, reaching up to 1.2 to 3.5 mM concentrations. [START_REF] Levine | Proc. Natl. Acad. Sci[END_REF] In this range of concentrations, the halflife of Mn III -3 would be expected to decrease to about 4 -12 s if it could enter those cells. Ascorbic acid, present in neutral solution predominantly in the monoanionic form (HA -), can be oxidized to dehydroascorbic acid, which appears in solution in the hydrate form Hy, with E o ´Hy/HA -= +0.184 V in 0.1 M HEPES buffer pH 7.4 (Fig. S7c), which is slightly lower than the value + 0.276 V reported in phosphate buffer. 82 This value is low enough to reduce the metal center of Mn III -3 (E o ´Mn(III)/Mn(II) = +0.193 V) (Keq 1 at 298 K) when present in excess, as in our experimental conditions. BME is a non-natural thiol (E o ´ = -0.26 V) 83 also capable of reducing Mn III -3. A similar study with an aqueous solution of Mn III -TPPS4 showed that no reduction was observed in the presence of ascorbic acid, which is in agreement with the reported redox potential of Mn III -TPPS4 in aqueous solution (E o ´Mn(III)/Mn(II) = -0.22 V). 73 Reduction of Mn III -3 to Mn II -3 was not observed in the presence of glutathione even in a large excess of this important biological reductant. This is thermodynamically unexpected on the basis of their redox potentials, as the E o ´ value (-0.240 V) 10 of the GSSG/2GSH couple is negative enough to reduce Mn III -3, as observed with BME. In order to rationalize this finding, we have compared the 13 C NMR spectrum of glutathione in the absence and in the presence of 0.1 equivalent of Mn III -3 in D2O (Fig. S12).

The addition of Mn III -3 causes broadening of specific glutathione 13 C resonances: the two carboxylate peaks at 173.52 and 172.30 ppm ( -O-C=O) and the signals of neighbouring carbons at 53.58 ppm ( -COO-C-NH2) and 41.46 ( -COO-C-NH-). This can be attributed to the binding of the terminal carboxylate groups of glutathione to the paramagnetic metal ion in the axial position of Mn III -3. Such binding mode is in agreement with X-ray crystal structures reported for complexes of Mn III -porphyrins with trichloroacetate. 64 On the other hand, we have shown above that electron transfer occurs with BME which has no carboxylate group. Altogether, this suggests that the coordination of glutathione to Mn III -3 to form a ternary complex blocks the electron transfer between the thiol group and the metal center and prevents the reduction. For the same reasons, reduction of Mn III -3 to Mn II -3 was also not observed, even in the presence of a large excess of cysteine (data not shown).

Relaxivity studies

The relaxation properties have been investigated for both the oxidized Mn III and the reduced Mn II form of the porphyrin complex 3. Mn II -3 was obtained by adding 30 equivalents of ascorbic acid to the Mn III analogue. Full conversion of Mn III -3 to Mn II -3 was evidenced by recording UV-Vis spectra before the relaxivity measurements. The NMRD profiles recorded at 25 °C for the reduced and for the oxidized form are presented in Fig. 3. The reduction of the d 4 Mn III -3 complex to the electronically more symmetric d 5 Mn II -3 analogue leads to a large increase in Please do not adjust margins Please do not adjust margins the proton relaxivities at low and medium magnetic fields. Although a similar trend has been previously found for the Mn II /Mn III -TPPS4 and Mn II /Mn III -TPPBr6S4 analogues, 59, 84 it was observable only in a much more restricted frequency range. Indeed, for those TPPS4 analogues, the two curves cross each other at ~10 MHz, 59 while for our system, even at 80 MHz, the relaxivity of the reduced form remains ~30% higher than that of the oxidized form. On MR images, this would correspond to a remarkable "turn-on" response to a reducing environment. As shown by Koenig 34 and later by Bryant, 84 attempts to fit the NMRD data for the oxidized Mn III -TPPS4 by using the Solomon-Bloembergen-Morgan theory do not yield satisfactory results for many of the fitted parameters. These include the distance between the Mn III ion and the protons of the coordinated water molecules, rMnH, that showed highly temperature-dependent values ranging from 2.13 Å (35 ºC) to 2.48 Å (5 ºC), which is way too short in the light of the structural data available from X-ray crystallographic studies. 85 They also used a value for the water exchange rate, kex 298 = 10 9 s -1 which is too large when compared with kex 298 values of 14 × 10 6 s -1 and 27.4 × 10 6 s -1 determined from the temperature dependence of 17 O NMR relaxation times. 86,87 It was indeed evidenced that the relaxation mechanism of Mn III porphyrin derivatives has unique aspects, the most important being the unusual role of ZFS interactions and the effect of these interactions on the electron spin dynamics. 35 Consequently, the analysis of the NMRD curves of Mn III -3 requires a complex mathematical treatment which, in the case of Mn III -TPPS4 gave a reasonable and temperatureindependent rMnH value of 2.80 Å. Values for other parameters were also calculated. At 20 ºC a value of the rotational correlation time, R = 900 ps and two different electron spin relaxation times S (±1) = 506 ps and S (±2) = 180 ps for the MS = ±1 and ±2 non-Kramers manifolds, respectively, were reported. 35 When compared with the corresponding calculated SBM parameters, 84 R = 275 ps and the parameters describing the electron spin relaxation, the correlation time for the modulation of the zero-field-splitting, v = 30 ps and the electron spin relaxation time at zero magnetic field, SO = 15 ps, one concludes that the SBM treatment underestimated R and SO and gave a too long v value. However, such a mathematical treatment for the analysis of the NMRD curves of Mn III -3 is beyond the scope of the present work. Despite this, our observation that the r1 values of Mn(III)-3 at low fields are systematically lower than those of Mn III -TPPS4 59, 84 can be qualitatively explained by their relative electronic relaxation parameters, dominate r1 at such fields. Thus, we can say that Mn(III)-3 has faster electronic relaxation than Mn III -TPPS4 as a result of the fluorinated phenyl substituents at the porphyrin meso positions.

Concerning the Mn II -3 complex, its NMRD curve has been analysed within the frame of the SBM theory (see ESI for equations). The outer sphere relaxation contribution was considered according to the translational diffusion model developed by Freed. 88 There are a large number of parameters that enter in the analysis and some of them have been fixed to reasonable values. These involve the distance between the Mn II electron spin and the proton of the coordinated water molecules (rMnH = 3.1 Å), the distance of closest approach of water protons diffusing around the complex which determines the outer sphere interactions (aMnH = 3.6 Å) and the relative translational diffusion coefficient (DMnH = 26 × 10 -10 m 2 s -1 ). The hydration number was also fixed to q =2, in analogy to the TPPS4 analogue. In the fit, we adjusted the rotational correlation time, R, the parameters describing electron spin relaxation, v, the correlation time for the modulation of the zero-field-splitting and 2 , the energy of the zero-field-splitting. The water exchange rate could not be measured for Mn II -3 by variable temperature 17 O NMR, as it is typically done for paramagnetic complexes, due to solubility problems at higher temperatures. Therefore, we also adjusted this parameter. The fit is rather satisfactory (Fig. 3) and the resulting parameters are given in Table 1, in comparison to some other Mn II complexes. We should note that the inclusion of a scalar relaxation mechanism into the fit by assuming a scalar coupling constant of Ah = 0.5 MHz did not improve the quality of the fit and did not have an important effect on the other fitted parameters. Bryant et al. reported some time ago on the analysis of the NMRD profile of Mn II TPPS4. 84 They concluded that it was impossible to describe the Mn II TPPS4 system by the SBM theory and with a reasonable set of parameters. In their best fit, they used a reasonable kex 298 = 10 7 s -1 , R 298 = 300 ps and a very short v 298 = 0.5 ps. The outer sphere contribution was parametrized with a very short distance for the closest approach of water protons to the manganese center (aMnH = 2.0 Å) and a very small value for the diffusion constant (6 × 10 -10 m 2 s -1 ). To explain these discrepancies, they proposed that the dipole-dipole approximation of the SBM theory might fail for these porphyrin complexes which can undergo an electron spin delocalization from the manganese ion to the porphyrin. This hypothesis seemed to be supported by the fact that a derivative, Mn II -TPPBr6S4, which was brominated on the pyrrole rings, had even higher proton relaxivities. In our analysis, the Mn II -proton distances and the diffusion constant have values typical of Mn II chelates. The water exchange rate is also reasonable. The rotational correlation time has a high value, but it is not surprising given the presence of four PEG chains attached to the porphyrin ring. The fact that the paramagnetic metal is in the barycenter of a large molecule implies that it takes full benefit from the slow rotation of the whole molecule. A porphyrin center bearing four Gd III complexes, with a similar molecular weight (Mw = 2.76 kDa), thus molecular size, as that of Mn II -3 (Mw = 2.99 kDa) has been recently reported. 92 Here the

Please do not adjust margins

Please do not adjust margins paramagnetic Gd III complexes are not in the barycenter of the molecule. However, the relaxivities have been analyzed with the Lipari-Szabo approach that allows calculating the global rotational correlation time, g, which can then be related to the rotational motion of the entire molecule. The global rotational correlation time for this tetra-Gd porphyrin was found to be g = 2.3 ns, similarly in the nanosecond range as the R calculated for Mn II -3. This gives confidence in the fit of the NMRD profile for Mn II -3. Even if the errors on the calculated parameters might be higher than usual given the lack of independent determination for the water exchange rate, their order of magnitude should be correct.

Evaluation of the cytotoxicity of Mn III -3 in a cell model

The in vitro cytotoxicity of Mn III -3 on the epithelial HeLa tumor cell line was evaluated using the Alamar Blue colorimetric assay. The cells were incubated with solutions containing different concentrations of Mn III -3 (0.25-5mM), prepared from a PBS stock solution and diluted in DMEM complete medium, during different time periods (2 h, 4 h and 24 h). The Mn III -3 compound was soluble in aqueous solution and the tested concentrations were in the meaningful range for the desired biomedical application.

As shown in Fig. 4, no statistically significant toxicity was observed after 2 and 4 h of incubation for all the concentrations used. After 24 h exposure to the compound, a statistically significant decrease in cell viability was observed for the highest concentrations tested (3 and 5 mM). In addition to its water solubility, the absence of statistically significant citotoxicity of Mn III -3 in the concentration and exposure time ranges normally used in in vivo MRI, are positive indications for its potential application as a redox-responsive agent.

Figure 5. Effect of increasing concentrations the Mn III -3 porphyrin compound on HeLa cells viability as a function of time. HeLa cells were plated in 96-well plates at a density of 2x10 4 cells per well. Twenty-four hours after plating, the cells were incubated with 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 5.0 mM solutions of Mn III -3. Cell viability was measured after 2, 4 or 24 h using the Alamar blue assay. Cell viability is presented relative to the percentage of untreated cells (control cells) considered as 100%. Data represent the mean ± SD of three independent experiments. Pairwise data comparisons were performed between cell viability values obtained with each Mn III -3 concentration, as compared with the immediately precedent concentration, for each incubation period (non-significant) and between cell viability values obtained for each incubation period with Mn III -3, as compared with the immediately precedent incubation period, for each concentration (**p<0.01, *p<0.05). The significance of the results was statistically analyzed by one-way analysis of variance (ANOVA) with Tukey's multiple pairwise comparison. Statistical significance was set at p < 0.05.

Conclusions

A new water-soluble fluorinated porphyrin derivative conjugated with PEG chains was synthesized. It is capable of stabilizing both Mn III and Mn II oxidation states in a biologically relevant range of redox potentials. In aqueous solution, Mn III/II -3 undergoes reversible reduction/oxidation reactions, promoted by the natural reducing agent ascorbic acid and oxygen, respectively. The reduction process with ascorbic acid has second-order kinetics, with a k2 value of 46.1 M -1 s -1 which is two orders of magnitude larger than the k2 of 0.38 M -1 s -1 reported for the reduction of Mn III -HBET by glutathione. 26 Reoxidation is slowly but fully achieved by air oxygen. Interestingly, 19 F NMR resonances of Mn III -3 are relatively sharp which could open further possibilities to exploit such complexes as paramagnetic 19 F NMR probes. 93 At medium frequencies (20-80 MHz), the relaxivity increase between the Mn III -3 and Mn II -3 complexes is considerably improved with respect to the previously studied Mn-TPPS4 analogue 59 (at 40 MHz, r1 ~100 %). Cytotoxicity evaluation on epithelial HeLa cells confirmed in vitro non-toxicity of the compound in the mM concentration range typically used for MR imaging.

In general, the development of MRI imaging probes to monitor redox environments in vivo faces several difficulties. Mn II/III chelates of open-chain hydroxybenzyl-ethylenediamine acetic acid derivatives have been reported to possess favorable in vitro properties as redox active MRI contrast agents, 30 but their kinetic stability might be insufficient. In this respect, Mn-porphyrin redox responsive probes can be advantageous. The present Mn II /Mn III -porphyrin couple satisfies many of the criteria required for a redox imaging probe: good water solubility, biocompatibility, non-toxicity, thermodynamic and kinetic stability in both metal oxidation states, a redox half-cell potential accessible to biologically relevant reducing agents with fast kinetics (adapted to the imaging time scale), and a strong "turn on" relaxivity response upon reduction. However, it is not reduced by glutathione or cysteine, the major thiol-disulfide redox buffers of the intracellular and extracellular spaces, respectively. In human plasma, cysteinecystine and glutathione reach levels of ~260 microM and ~7 microM, respectively, 94 while ascorbic acid has 20-40 microM concentration. 80,[START_REF] Levine | Proc. Natl. Acad. Sci[END_REF] Therefore, the role of ascorbic acid should not be ignored either in maintaining the extracellular redox state. Our redox sensitive MRI probe might have potential to report, with fast kinetics (few minutes half-life), on extracellular ascorbate without the interference of the cysteine-cystine buffer. In circulating immune cells for instance, ascorbic acid amounts to millimolar concentrations and controls the redox state. Provided the probe is modified for cell internalization to reach mM concentrations, it could be useful to assess intracellular ascorbic acid in such cells with very fast kinetics (few seconds half-life). Furthermore, MRI quantification of ascorbate could be also achieved by separating the effect of the in vivo concentration of the probe from the redox effect on the observed contrast. For this, a This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins ratiometric technique could be used, 95 based on injecting a cocktail of the 52 Mn-radiolabeled porphyrin and the normal one. Indeed, 52 Mn (t1/2 = 5.59 d, β + = 29.6%, Eβave = 0.24 MeV) is a promising positron emission tomography (PET) radioisotope, 96 which allows to directly combine PET and MRI detection. The advantage of this technique is the identical biodistribution of the PET and MRI probes.

As PET is a quantitative technique, it will allow assessing the concentration of the MRI probe as well, leading to quantification of the ascorbate concentration.

Experimental Section

Materials and methods

Commercially available reagents were bought from Aldrich, except PEG500 (mono methyl polyethyleneglycol) (Fluorochem) and used as received. All solvents were pre-dried according to standard laboratory techniques. For the cyclic voltammetry experiments, 1,2-dichloroethane (DCE) was purchased with the highest quality available, and stored in molecular sieves, under inert atmosphere. The other reagents were also purchased with the highest quality available and were used as received. 5,10,15,20-(pentafluorophenyl)porphyrin was synthesized according to the literature. 64 UV-visible absorption spectra were recorded on a Hitachi U-2010 using quartz cells. The molar absorption coefficients were determined using PBS as solvent. Free base 2 (100 mg, 0.045 mmol), sodium acetate (367 mg, 4.47 mmol) and manganese (II) acetate were dissolved in 3 mL of acetic acid. The reaction was stirred at 80 ºC and controlled by UV-Vis spectroscopy. After complete conversion to the manganese complex (2 hours), the reaction mixture was extracted several times with dichloromethane/water and then the organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. Porphyrin 3 was obtained in 85% yield. MS (ESI-FIA-TOF) m/z: 1121.3612, 1319.9802, 1386.0195, 1493.6057 [M] 2+ (polydisperse distribution). Free base 2 were dissolved in a mixture of chloroform/methanol (2:1) and zinc(II) acetate was added. The reaction was stirred at 50ºC and controlled by UV-Vis spectroscopy. After complete conversion (1h) the reaction mixtures was extracted several times with dichloromethane/water and the organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum. The zinc derivative was obtained in 89% yield. 1 Mn III -5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (Mn III TPPS4) TPPS4 was synthesized according to literature. 97 Mn III TPPS4 was obtained using the same procedure used for the porphyrin Mn III-3. Characterization is in accordance to literature. 67

Electron Paramagnetic Resonance (EPR)

X-band (0.34 T, 9.5 GHz) continuous wave (CW) EPR measurements were carried out on a Bruker EMX spectrometer, equipped with a continuous flow Oxford Instruments cryostat for low temperature measurements. The EPR experiments were performed on the Mn II salt MnCl2 and Mn III -porphyrin samples in aqueous solution at 25 ºC. (see supporting information). Simulation of the experimental spectra of aqueous Mn 2+ was performed using SimFonia v.1.2 (Bruker Instruments Inc. software). The EPR spectra showed that the residual Mn 2+ aqueous ion concentrations present in the Mn III -porphyrin solutions used for the NMRD measurements were lower than 10 M.

H Relaxometry

The longitudinal (T1) proton relaxation times of aqueous solutions of the Mn III and Mn II complexes were measured at 25 ºC and 37 ºC by nuclear magnetic resonance relaxometry on a Bruker Minispec mq20 relaxometer operating at a magnetic field of 0.47 T, corresponding to a proton Larmor frequency of 20 MHz, using an inversion recovery pulse sequence. All the experimental values were corrected taking into account the diamagnetic contribution of water. The corresponding relaxivity values (r1), were calculated through the least-squares curve fitting of the inverse relaxation time 1/T1 (s -1 ) vs. the Mn II/III Please do not adjust margins Please do not adjust margins concentration. 1 H NMRD (Nuclear Magnetic Relaxation Dispersion) profiles were recorded on a Stelar SMARtracer Fast Field Cycling NMR relaxometer (0.01-10 MHz) and a Bruker WP80 NMR electromagnet (20, 40, 60 and 80 MHz) adapted to variable field measurements and controlled by a SMARtracer PC-NMR console. The temperature was monitored by a VTC91 temperature control unit and maintained by a gas flow. The temperature was determined by previous calibration with a Pt resistance temperature probe. The longitudinal relaxation rates (1/T1) were determined in water at 25 ºC and 37 ºC. High field relaxivities were measured on a Bruker AVANCE NMR spectrometer at 400 MHz by placing the sample solution in an inner co-axial tube while the outer tube was filled with D2O.

Reduction kinetics

To stock 1.35×10 -5 M solutions of Mn III -3 in PBS (25ºC, pH = 7.4) were added appropriate volumes of 50 mM ascorbic acid or of 63 mM BME. For one set of experiments, the final concentration of Mn III -3 was kept at 0.008 mM and ascorbic acid was 0.390 mM, 0.191 mM and 0.091 mM. For the other set, the final concentration of ascorbic acid was constant at 9.7 mM and Mn III -3 was 0.230 mM, 0.150 mM and 0.095 mM. The same final concentrations were used for the BME experiments. The conversion of Mn III -3 to Mn II -3 was monitored by the disappearance of a strong UV-Vis absorbance at 457 nm ( = 1.0110 4 M -1 s -1 ) which is absent in the UV-vis spectrum of the Mn II -3 complex. The decrease of the absorbance at 457 nm of Mn III -3 in the presence of excess ascorbic acid or BME, with the concomitant appearance of a new band characteristic of the Mn II -3 spectrum, was monitored over time. The experimental setup used to monitor the kinetics of the reaction consisted of a fiber coupled Ocean Optics spectrometer (USB4000-UV-Vis) used in absorption mode together with a MiKropack DH-2000-BAL UV-Vis-NIR light source. The absorption spectra were recorded in intervals of 50 ms (for ascorbic acid) and 100 ms (for BME) and monitored up to the complete formation of the Mn II -3 species. The large excess of ascorbic acid and of BME used in the experiments ensured pseudo-first-order kinetics with respect to the Mn III -3 complex as expressed in equation (1): ln[Mn III -3]t = -kobs t +ln [Mn III -3]0

(1)

Here, kobs refers to the observed pseudo-first-order rate constant, which is the product of the actual rate constant (k) and [ascorbic acid]0, t is time and the subscripts "t" and "0" correspond to the calculated concentration at time "t" and the initial concentration, respectively.

Cyclic Voltammetry

The electrochemical experiments in aqueous solution were carried out using a 0.1 M HEPES buffer pH 7.4, prepared from N-[2-hydroxyethyl]piperazine-N´-[2-ethanesulfonic acid]) hemi sodium salt (Sigma, St. Louis, USA), as supporting electrolyte.

The porphyrins stock solutions were prepared by dissolving the solid compounds in HEPES buffer, followed by further dilution in buffer when needed. Millipore Milli-Q nanopure water (resistivity ≥ 18 MΩ cm) was used for the preparation of all solutions. All experiments were performed at room temperature (25 ± 1 ºC). The pH measurements were carried out with a CRISON 2001 micro pH-meter (Crison, Spain). The cyclic voltammetry (CV) measurements were carried out with an Autolab PGSTAT-30 potentiostat/galvanostat (Eco Chemie, Utrecht, Netherlands), controlled by GPES 4.9 software. The electrochemical cell, with a 2 mL volume, contained a glassy carbon electrode (d = 1mm) as working electrode, a platinum wire as counter and Ag/AgCl (3 M KCl) as reference electrode.

Prior to the CV measurements, all solutions were degassed by N2 bubbling for 15 min and during potential cycling a flux of N2 was kept flowing on top of the cell solution. Some CV measurements were also carried out in DCE solutions in the presence of 0.6 M Bu4NBF4. These were performed using a homemade potentiostat. 98 A jacketed five neck conical cell was used. Three of the necks were used for the working, counter, and reference electrodes, while the other two necks were used for the argon inlet and outlet. A glassy carbon disk with a 1 mm diameter was used as working electrode, with the counter electrode being a 1 mm diameter Pt disk. The reference electrode was a saturated calomel electrode (SCE), separated from the working electrode compartment by a salt bridge with a ceramic frit. Experiments were performed at 10 °C, using a HAAKE F3 circulation system in order to avoid the DCE solvent evaporation. Electrochemical characterisation of ferrocene was used as an electrochemical standard in DCE. Ferrocene showed a one-electron transfer. The current values were standardised with respect to concentration and scan rate square-root, in order to determine the number of electrons associated with an unknown electronic transfer. All the potentials obtained were corrected to be reported referenced to the normal hydrogen electrode (NHE).

Cytotoxicity Studies

HeLa cells (human epithelial cervical carcinoma cell line) were maintained at 37 °C under an O2/CO2 (95%/5 %) atmosphere in Dulbecco's modified Eagle's medium (DMEM, Sigma, St. Louis, MO), supplemented with heat-inactivated fetal bovine serum (10 % (v/v), FBS, Sigma), penicillin (100 UmL -1 ) and streptomycin (100 μgmL -1 ). HeLa cells grown in monolayer were detached by treatment with trypsin solution (0.25 %, Sigma) and were seeded in medium (2x10 4 cells per mL (100 uL per well) in 96well culture plates. After 24 h, when the cells were at 50-70 % confluence, they were incubated with the compound to be tested. The cytotoxicity induced by the compound in HeLa cells was evaluated using the modified Alamar Blue colorimetric assay, as previously described. 99 Briefly, a stock solution of the compound under study was prepared in phosphate buffer saline (PBS), and successive dilutions in DMEM complete medium were prepared from this stock in order to achieve the concentration range of 0.25 mM to 5 mM. The diluted solutions were added to the cells 24 h after seeding, followed by incubation of the cultures at 37 °C for 2, 4, and 24 h. For each time point, 0.1 mL of a resazurin dye solution (1% (v/v), Sigma) in DMEM medium was added to each well. After 45 min of incubation at 37 °C, the absorbance was measured at 570 and This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins (Molecular Devices, Union City, CA). Although the compound under study is colored, its maximum absorbance is at   460 nm and at   440 nm, respectively for the oxidized and the reduced forms (Figures 3 andS6), thus not interfering with the results obtained from the Alamar Blue assay.

For each concentration of the compound, the percentage of cell viability was calculated according to equation (2):

% cell viability = [(A570 -A600) treated cells / (A570 -A600) control cells] x 100 (2)

where the control cells (control +) refers to cells not exposed to the compound, corresponding to 100% of cell viability. It was also considered a negative control (control -) of the resazurin dye solution and the respective absorbance was subtracted to the values measured in all the samples. Statistical analysis was carried out using one-way ANOVA with Tukey's multiple pairwise comparison. A value of p < 0.05 was considered as statistically significant

Figure 1 .

 1 Figure 1. 19 F NMR of porphyrin 1 (black spectrum) and porphyrin 2 (blue spectrum).

  7 ppm) sharper (linewidths around 190 Hz) signals of different intensities. This set of signals corresponds to the fluorine atoms at the meta positions of the meso-phenyl groups in the five possible combinations of the chain lengths of the four polydisperse PEG chains with n = 10-11. The broad signal around -126.2 ppm results from the overlap of the broader signals of the fluorine atoms at the ortho position, which are closer to the metal center.65,66 Upon reduction of Mn III -3 to Mn II -3, all the 19 F resonances are completely broadened out, in accordance with the efficient paramagnetic relaxation of Mn II (Fig.S6c).

Figure a )

 a Figure a) UV-Vis reduction titration of 0.041mM Mn III -3 with ascorbic acid, recorded in PBS (25 ºC, pH = 7.4). Number of equivalents of ascorbic acid added: (black line) -0; (red line) -0.28; (light blue line) -0.57; (light green line) -0.85; (pink line) -6.4; (dark green) -11.4; (dark blue) -25.3; b) UV-Vis spectra of the reoxidation of Mn II -3 upon air exposure: (black line) -Mn II -3 before exposure; (red line) -after 10 min. exposure; (blue line) after 4h exposure; (green line) complete conversion to Mn III -3 (after 24h exposure). c) UV-Vis reduction titration of 0.041mM Mn III -3 with , recorded in PBS (25 ºC, pH = 7.4). Number of equivalents of  added: (black line) -0; (red line) -0.43; (light blue line) -2.32; (light green line) -7.94; (pink line) -23.5; (dark green) -48; d) UV-Vis spectra of the reoxidation of Mn II -3 upon air

Figure 4 .

 4 Figure 4. Proton NMRD profiles recorded at 298 K for Mn III -3 (red ♦) and Mn II -2 (black ). The curve represents the least squares fit to the data points as explained in the text.

  / MHz This journal is © The Royal Society of Chemistry 20xx

A

  1 H-NMR and 19 F-NMR spectra were recorded on a 400 MHz Bruker Avance III NMR spectrometer (400.101 and 376.5MHz respectively). Proton chemical shifts are given in parts per million (ppm) relative to tetramethylsilane at δ 0.00 ppm and 19 F relative to TFA at δ -76.55 ppm. Mass spectra (ESI-FIA-TOF) were acquired using an Applied Biosystems Voyager DE-STR instrument equipped with a nitrogen laser (λ = 337 nm). mixture of 5,10,15,20-(pentafluorophenyl)porphyrin (140 mg, 0.14 mmol) and PEG500 (mono methyl polyethyleneglycol -Mw = ~500 Da) (0.359 mL, 0.718 mmol) was dissolved in 10 mL of dry DMF. After the addition of NaH (17 mg, 0.718 mmol) the reaction was left at room temperature, until complete consumption of 5,10,15,20-(pentafluorophenyl)porphyrin, checked by TLC (2 hours). The reaction mixture was extracted several times with dichloromethane/water and dichloromethane/aqueous 1M K2CO3 and the organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. Porphyrin 2 was obtained in 78% yield.1 H NMR (400 MHz, CDCl3) , ppm: 8.93 (s, 8 Hβ), 3.65-3.60 (m, 160 -196 (polydisperse distribution) HPEG), -2.90 (s, 2 HNH); 19 F NMR (376 MHz, CDCl3) , ppm: -138.86-(-138.04) (m, 4F), -157.97-(-157.03) (m, 4F). MS (ESI-FIA-TOF) m/z: 1028.3720, 1220.9980, 1287.0377, 1491.1818 [M] 2+ (polydisperse distribution). Mn III -5,10,15,20-(4-PEG500-2,3,5,6-fluorophenyl)porphyrin (Mn III -TPFPP(PEG)4 , Mn III -3)

Table 1 .

 1 Relaxivities and fitted parameters for Mn II -3 and other Mn II complexes from literature.

	Parameter	Mn II -3	[Mn(15-py N3O2)(H2O)2] 2+ 89 [Mn(H2O)6] 2+ 90
	r1 / mM -1 s -1 20 MHz/298 K	24.3	4.48	7.4 a
	kex 298 / 10 6 s -1	0.81±0.2	3.8	21
	rH 298 / ps	4800±1000	40.3	30 b
	v 298 / ps	5±1	3.3	3.3
	 2 / 10 18 s -2	0.50±0.2	6.6	5.6

a this work; b 308 K.

91

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 4

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

This journal is © The Royal Society of Chemistry 20xx

Acknowledgements

The authors thank the joint France (ANR) -Portugal (FCT) PESSOA 3750-37687RE project, FCT-Portugal (Portuguese Foundation for Science and Technology) and FEDER -European Regional Development Fund through the COMPETE Programme (Operational Programme for Competitiveness) for funding with PEst-OE/QUI/UI0313/2014 and POCI/H2020/27996. S.M.A.P., M.J.F.C., M.E.G. and A.M.S.C. are grateful for the SFRH/BPD/84619/2012, SFRH/BPD/99698/2014, SFRH/BPD/103103/2014 and SFRH/BPD/99613/2014 postdoctoral grants, respectively. E.T. is grateful to the Ligue contre le Cancer (France) for their financial support.

Notes and references

Conflicts of interest

There are no conflicts to declare.


Dr Jing Wen
email: jing.wen@neel.cnrs.fr

Patricia De Rango
email: patricia.derango@neel.cnrs.fr

Nathalie Allain

Laetitia Laversenne

Thierry Grosdidier

Improving hydrogen storage performance of Mg-based alloy through microstructure optimization

Keywords: Hydrogen storage, Fast forging, Hydrides, Microstructure, Texture, Magnesium

come   L'archive ouverte pluridisciplinaire

Introduction

Magnesium and its alloys have gained a great interest in the field of hydrogen storage owing to their light weight, high hydrogen-storage capacity, low-cost and abundance in Earth's crust [START_REF] Sakintuna | Metal hydride materials for solid hydrogen storage: a review[END_REF][START_REF] Jain | Hydrogen storage in Mg: a most promising material[END_REF].

In particular, Mg-based metal hydrides are still considered as one of the most promising solidstate hydrogen-storage media due to their higher energy density and safety advantages over the gas and liquid storage materials. However, the high thermodynamic stability of MgH ! as well as the slow desorption kinetics limit the application of Mg-based hydride in industry [START_REF] Zaluska | Nanocrystalline magnesium for hydrogen storage[END_REF][START_REF] Barkhordarian | Effect of Nb 2 O 5 content on hydrogen reaction kinetics of Mg[END_REF]. Efforts have been devoted to accelerate the absorption/desorption kinetics of Mg-based hydrides while lowering the temperature of reaction. This can be accomplished to some extent by modifying the microstructure of Mg-based hydrides. Fast sorption kinetics were achieved with nanosized MgH 2 powders produced by High Energy Ball Milling (HEBM) [START_REF] Huot | Structural study and hydrogen sortion kinetics of ball-milled magnesium hydride[END_REF][START_REF] Bobet | Hydrogen sorption of Mg-based mixtures elaborated by reactive mechanical grinding[END_REF]. Hydrogen diffusion is therefore accelerated owing to the increased density of grain boundaries, the larger reactive surface area and presumably a large amount of defects, although a recent review paper has outlined the limited understanding of the scientific community regarding the exact nature of structural defects and their effect [START_REF] Huot | Mechanochemistry of metal hydrides: recent advances[END_REF]. However, HEBM cannot be applied to large-scale production due to energy, safety and processing time constraints.

To overcome these drawbacks, Severe Plastic Deformation (SPD) such as Equal Channel Angular Pressing (ECAP) [START_REF] Jorge | An investigation of hydrogen storage in a magnesium-based alloy processed by equalchannel angular pressing[END_REF][START_REF] Popilevsky | Hydrogenation-induced microstructure evolution in as cast and severely deformed Mg-10 wt.% Ni alloy[END_REF][START_REF] Jorge | Correlation between hydrogen storage properties and textures induced in magnesium through ECAP and cold rolling[END_REF], Cold Rolling (CR) [START_REF] Popilevsky | Hydrogenation-induced microstructure evolution in as cast and severely deformed Mg-10 wt.% Ni alloy[END_REF][START_REF] Huot | Improvement of hydrogen properties of magnesium alloys by cold rolling and forging[END_REF] High Pressure Torsion (HPT) [START_REF] Revesz | Dehydrogenationhydrogenation characteristics of nanocrystalline Mg 2 Ni powders compacted by high-pressure torsion[END_REF][START_REF] Panda | Effect of initial powder type on the hydrogen storage properties of high-pressure torsion consolidated Mg[END_REF] as well as Surface Mechanical Attrition Treatments (SMAT) [START_REF] Edalati | Effect of gradient-structure versus uniform nanostructure on hydrogen storage of Ti-V-Cr alloys: Investigation using ultrasonic SMAT and HPT processes[END_REF][START_REF] Novelli | Microstructural details of hydrogen diffusion and storage in Ti-V-Cr alloys activated through surface and bulk severe plastic deformation[END_REF] have been recently employed to improve the H-storage properties through microstructure modifications.

Additionally, a preferential texture is developed upon SPD, which has been reported to improve hydrogen absorption kinetics in AZ31 samples produced by ECAP and CR [START_REF] Jorge | Correlation between hydrogen storage properties and textures induced in magnesium through ECAP and cold rolling[END_REF]. Forging is an alternative SPD route, which has been widely used for centuries in metallurgy. Compared to ECAP, HPT and CR, forging is a simple, safe and cost-efficient approach to produce heavily deformed materials. According to the works of J. Huot [START_REF] Huot | Improvement of hydrogen properties of magnesium alloys by cold rolling and forging[END_REF] and A.C. Asselli [START_REF] Asselli | Hydrogen storage properties of MgH 2 processed by cold forging[END_REF], the hydrogen sorption kinetics of forged MgH ! is similar to that observed in the samples produced by CR or HPT. However, large amounts of MgO formed as the number of forging passes increased, with a detrimental effect on the hydrogen absorption kinetics [START_REF] Asselli | Hydrogen storage properties of MgH 2 processed by cold forging[END_REF]. To avoid the formation of MgO upon multi-passes forging, single-pass fast forging (high deformation rate forging) under Ar atmosphere has been developed. Fast forging has also been shown to produce microstructures containing large amounts of defects as well as a preferential texture [START_REF] Popa | High-speed forged NdFe12-XVX compounds for bonded magnets[END_REF][START_REF] De Rango | Fast forging: a new method to synthesize Mg-based alloys for hydrogen storage[END_REF], both leading to significantly modified sorption kinetics.

Among Mg-based alloys, Mg ! Ni has been considered as one of the most promising hydrogen storage media owing to its favorable thermodynamics and relatively high hydrogen capacity [START_REF] Eisenberg | The effect of surface nickel on the hydriding-dehydriding kinetics of MgH 2[END_REF][START_REF] Reilly | Reaction of hydrogen with alloys of magnesium and nickel and the formation of Mg 2 NiH 4[END_REF]. Upon hydrogenation at moderate temperature, Mg ! Ni transforms into a ternary hydride Mg ! NiH ! containing a hydrogen capacity of 3.6 wt.% [START_REF] Reilly | Reaction of hydrogen with alloys of magnesium and nickel and the formation of Mg 2 NiH 4[END_REF]. In the present work, with the aim of enhancing the hydrogen storage performance of a Mg-based alloy (Mg-22 wt.% Ni), microstructure optimization through fast forging was investigated either at room or elevated temperatures. Moreover, annealing followed by fast cold forging was carried out to produce a specific microstructure characterized by fragmented Mg ! Ni lamellae embedded in a textured Mgmatrix. The microstructure developed through fast forging was examined in the present work using a combined analysis of X-ray diffraction (XRD) and field emission scanning electron microscopy (SEM) coupled with electron backscatter diffraction (EBSD).

Experimental

The hypoeutectic composition Mg-22 wt.% Ni (equivalent to 78 wt.% Mg -22 wt.% Ni) was selected to produce composites consisting of Mg ! Ni domains embedded in a Mg matrix, by refering to the Mg-Ni phase diagram. This composition is close to the Mg-rich eutectic at Mg-11.3 at.% Ni, but contains lower amount of Ni to avoid the formation of higher amount of MgNi ! phase. Mg powder (atomized spherical particles of about 5 µm, 99.8% purity from SFM (Société pour la fabrication du magnésium; Switzerland) and nickel powder (30-40 µm, 99.5% purity from Neyco, France) were used as raw materials. Prior to fast forging, samples were prepared using the following procedures: firstly, a Mg-Ni mixture was fabricated by manual grinding of the Mg and Ni powders in a glove box under argon atmosphere; subsequently, the Mg-Ni pellets were compacted in a die using a 1 T/cm 2 press; finally, each sample containing 3 pellets (13 mm in diameter) was inserted into a thin cylindrical stainless steel sheath (22 mm in height) to avoid a burst effect of matter at shock [START_REF] De Rango | Fast forging: a new method to synthesize Mg-based alloys for hydrogen storage[END_REF]. The compacted powders in the form of pellets are referred as starting materials in the present work.

Plane-strain fast forging was performed at the laboratory scale using a device initially designed for hard magnets [START_REF] Popa | High-speed forged NdFe12-XVX compounds for bonded magnets[END_REF]. A 150 kg hammer is dropped from a height of 1.5 meters at a rate of 4.7 m/s onto a piston. The piston is in direct contact with the sample which is positioned on the anvil of a specially designed chamber. Three series of samples were prepared under argon atmosphere:

1) forged at room temperature (subsequently referred to as Cold forged, CF); 2) forged at the temperature of 450°C (referred to as Hot forged, HF); and 3) forged at room temperature after a preliminary annealing (referred to as Annealed + Cold forged, A + CF). Regarding the preliminary annealing, three pellets (inserted into a thin cylindrical stainless sheath) were heated by means of a retractable high frequency coil to 530 °C and maintaining for 30 minutes under Ar atmosphere. It should be noted that the temperature of the sheath gradually decreased as the annealing proceeded due to the increase of the electric conductivity of the sample; the final temperature was recorded as 455 °C. After fast forging, the height of the sample was reduced from 75 % to 93 % (Table 2) depending on the forging conditions. The sample geometry before and after fast forging is shown in Fig. 1a.

Following fast forging, pieces were extracted from the center of the samples for microstructure characterizations, as illustrated in Fig. 1a. The sectioned faces were mechanical polished using SiC papers with grits from 1000 to 4000 followed by electrolytic polishing in an electrolyte of 62% phosphoric acid and 38% ethanol at 2V for 3 min, at -15 °C. Phase identifications were conducted on the horizontal polished surface using X-Ray Diffraction (XRD). The weight fraction of each phase component and the corresponding lattice strain were determined by Rietveld refinement using the FullProf suite [START_REF] Rodriguez-Carvajal | Recent Developments of the Program FULLPROF[END_REF]. Energy-dispersive X-ray (EDX) spectroscopy was employed to determine the chemical composition of the annealed sample. To collect the high-quality Kikuchi patterns, the samples were Ar ion polished with a PECS-II GATAN using 3.0 keV beam energy under dual beam condition, with gun angle of 2° and for about 3 hours.

Then EBSD characterization was performed using a JEOL-6500F SEM equipped with the AZtec acquisition software package at a step size of 0.15-0.30 µm. In addition to the EBSD microtexture measurements, XRD macro-texture measurements were carried out to have a better representation of the crystallographic texture of the fast forged samples. Totally, five pole figures (0002) !" , (1010) !" , (1011) !" , (1012) !" and (1120) !" have been measured and the texture components were recalculated using the ATEX software [START_REF] Beausir | Analysis Tools for Electron and X-ray diffraction[END_REF]. The hydrogen storage properties of the fast forged samples were examined using a Sievert device from HERA. Hydrogen absorption and desorption kinetics were measured at temperature of 310 °C under a hydrogen pressure of 20 bars and 1.5 bars, respectively. In total, 12 cycles of absorption/desorption were conducted for each forged sample. Ueda et al. [START_REF] Ueda | Preparation and hydrogen storage properties of Mg-Ni-Mg 2 Ni laminate composites[END_REF], cannot be ruled out. XRD patterns recorded on the as-forged samples are shown in Fig. 1c. For the CF sample, only the initial phases Mg and Ni were detected, indicating that no phase transformation occurs under the impact loading. Nevertheless, the relative high intensity of the (0002) !" peak reveals that a basal texture is generated in the Mg phase upon the cold deformation. Regarding the HF sample, a Mg ! Ni phase formed during the forging process. According to the Mg-Ni phase diagram, this intermetallic compound is expected to form at the eutectic temperature of 506˚C. Obviously, during fast forging, under the impact loading a part of the kinetic energy is converted into heat which is dissipated in the sample. This stored energy might facilitate the formation of Mg ! Ni below the eutectic temperature through an accelerated metal diffusion at the Mg/Ni interface.

Results and discussion

According to numerical simulation performed in Ref. [START_REF] De Rango | Fast forging: a new method to synthesize Mg-based alloys for hydrogen storage[END_REF], only one shock of forging resulted in an increase of temperature within the sample of 150-200 ˚C. Therefore, for the HF sample, with the assistance of the heat transformed from the kinetic energy, the real temperature of the sample overpassed the eutectic one, which enabled the formation of Mg ! Ni at a local scale via fast alloying. For the A + CF sample, in addition to the relative high intensity of the (0002) !" peak, broadening of the diffraction peaks is observed for both the Mg phase and the Mg ! Ni intermetallic compound due to the generation of lattice strain upon cold forging.

The weight fraction of each phase and the corresponding lattice strain, extracted from Rietveld refinements are presented in Table 1 andTable 2, respectively. The estimated weight fractions of Mg and Ni in the CF sample are consistent with the ones we designed. Nevertheless, a higher extent of lattice strain is generated in the CF samples. For the HF sample, a very low fraction (~ 2.22 wt. %) of Ni retained in the sample implies that Ni is not completely consumed upon the formation of Mg ! Ni. This could be due to either the inhomogeneous distribution of heat within the sample or the size effect of the Ni particle [START_REF] Skryabina | Effect of temperature on fast forging process of Mg-Ni samples for fast formation of Mg 2 Ni for hydrogen storage[END_REF]. After the first hydrogenation, the forged samples are based on three major phases: MgH ! (tetragonal), Mg ! NiH ! (monoclinic) and metallic Mg indicating that the hydrogenation is incomplete (Fig. 1d). Also the observation of Mg ! NiH !.! reveals that some of the hydrogen atoms are dissolved as a solid solution within the Mg ! Ni lattice.

Only a small amount of the Mg ! NiH ! hydride is observed in the CF sample and most of the nickel remains as metallic Ni particles. In contrast, the quantity of Mg ! NiH ! hydride is quite high for the forged samples containing the Mg ! Ni phase (A + CF and HF). Moreover, we noticed that a peak appears between the doublet of Mg ! NiH ! in the XRD pattern at about 23-24º. These reflections can be assigned to stacking faults in Mg ! NiH ! [START_REF] Blomqvist | Mechanically reversible conductor-insulator transition in Mg 2 NiH 4[END_REF][START_REF] Tran | In-situ investigation of the hydrogen release mechanism in bulk Mg 2 NiH 4[END_REF], which can act as quick pathways for the transportation of hydrogen [START_REF] Hongo | Significance of grain boundaries and stacking faults on hydrogen storage properties of Mg 2 Ni intermetallics processed by high-pressure torsion[END_REF].

Texture development upon fast forging

Since plastic deformation is dominated by magnesium for the powder mixture of Mg-22 wt.% Ni, the texture development of Mg upon fast forging was examined using XRD, as shown in Fig. 2, where the recalculated (0002) and (10-10) pole figures are presented. For the CF sample (Fig. 2a), a fiber texture characterized by a majority of c-axes approximately aligned with the forging direction is developed under forging. The formation of this {0001} basal fiber texture is related to the slip systems {0001}< 1120 > operating on the basal planes [START_REF] Wang | Texture analysis in hexagonal materials[END_REF]. Regarding the HF sample (Fig. 2b), the {0001} texture intensity has been weakened due to the occurrence of dynamic recrystallization upon hot deformation. With respect to the A + CF sample (Fig. 2c), the texture intensity is comparatively lower than that for the CF sample. Keeping in mind that the intensity of texture is proportional to the volume fraction of grains having a preferential crystallographic orientation, this lower fraction can be explained by differences in the fine scale deformation mechanisms due to the composite nature of the deformed materials containing hard Mg ! Ni particles.

Microstructure analysis of the cold forged (CF) sample

EBSD technique was used to investigate the microstructure evolution upon cold forging of the CF sample (Fig. 3). As illustrated in the schematic drawing in Fig. 1a, EBSD measurements have been carried out on a cross-section along the forging direction (FD). In the inverse pole figure

(IPF) map of Fig. 3a, the Mg grains are colored while the Ni phase remains in white. The majority red color of the Mg grains indicates that they globally exhibit a preferential orientation (texture). Indeed, in agreement with the macro-texture determined by XRD (Fig. 2a), the (0002) pole figure in Fig. 3b, which corresponds to the IPF map, confirms the presence of a fiber texture with a majority of grains in the CF sample having <0001> directions approximately parallel to the forging direction.

Fig. 3c shows an IPF map (corresponding to the selected zone in Fig. 3a) of the Mg grains superimposed with their grain boundaries taken at a smaller step size of 0.10 µm, in which the high and low angle boundaries are outlined by black and grey colors, respectively. According to

Ref. [START_REF] Zherebtsov | Evolution of grain and subgrain structure during cold rolling of commercial-purity titanium[END_REF], high-angle boundaries (HABs) are identified with a minimum of 15° misorientation angle across the grain boundaries. Low-angle boundaries (LABs) are defined with misorientation angle below 15°. Due to the experimental error in evaluating orientations by EBSD [START_REF] Humphreys | Quantitative metallography by electron backscattered diffraction[END_REF], grain boundary misorientations below 2° are excluded from the data analysis. As seen, the grain boundaries of magnesium are decorated with high angle boundaries, whereas the interior of that is filled with low angle boundaries. Fig. 3d shows the IPF map of Ni-phase corresponding to the selected zone in Fig. 3a. The Ni grains, with size less than 1 µm, are distributed within the Mg matrix in the form of agglomerations. (Fig. 4a). As for the Mg-phase misorientation angle distribution in the CF sample, the black line in Fig. 4b indicates the random distribution of misorientation angle expected for hexagonal structure without any texture [START_REF] Mason | The generalized Mackenzie distribution: Disorientation angle distributions for arbitrary textures[END_REF], often referred as Mackenzie distribution. Because of the presence of a fiber texture in which many grains have a common crystallographic axis parallel to the forging direction, the 2D misorientations between adjacent grains are generally of lower magnitude in the forged samples than the expected ones in a material having a random orientation of grains (no texture).

For comparison, the grain size distributions for HF and A + CF samples are also given in Fig. 4.

The HF specimen contains mostly fine Mg grains ranging within the 1 to 6 µm size range with an average size of ~2.7 µm, as shown in Fig. 4c. The newly formed Mg ! Ni intermetallic compound is composed of fine to coarse grains ranging from ~1 to 10 µm in size (Fig. 4d). For the A + CF sample, because of some grain growth during pre-annealing, there is a more pronounced heterogeneity in Mg-grain size, as depicted in Fig. 4e. Due to the "fragmentation" of the brittle Mg ! Ni domains upon cold forging, the Mg ! Ni grains are slightly finer: 2.6 µm for the A + CF condition (Fig. 4f) compared to 3.7 µm for the HF sample (Fig. 4d). recrystallization is a mechanism operating by a nucleation and growth process. While it is well established that the nuclei are regions that already exist in the deformed microstructure, the growth stage requires a sufficient misorientation between these nuclei and the deformed grains that they are consuming to develop [START_REF] Wang | Texture analysis in hexagonal materials[END_REF][START_REF] Wagner | Evolution of recrystallisation texture and microstructure in low alloyed titanium sheets[END_REF]. This inevitably generates a weakening of the deformation texture [START_REF] Wagner | Evolution of recrystallisation texture and microstructure in low alloyed titanium sheets[END_REF]. [START_REF] Zaluska | Nanocrystalline magnesium for hydrogen storage[END_REF][START_REF] Chen | The activation mechanism of Mg-based hydrogen storage alloys[END_REF]. In this study, hydrogenation of all samples was carried out at 310°C under a hydrogen pressure of 20 bars. A comparison of the hydrogen uptakes recorded for fast forged samples as well as for compacted powders (starting materials) is shown in Fig. 8a. The beneficial effect of the peculiar microstructure developed upon cold forging is noticeable with fastest hydrogenations -without any incubation time -and highest uptakes recorded for both CF and A + CF samples.

The basal fiber texture as well as a amount of cracks induced upon cold forging may facilitate hydrogen diffusion through the samples leading to faster absorption rates than the one observed for the starting materials. Our results are in line with the work of Jorge Jr et al. [START_REF] Jorge | Correlation between hydrogen storage properties and textures induced in magnesium through ECAP and cold rolling[END_REF],

who showed that the fiber texture is the best orientation for hydrogen absorption. Structural defects like dislocations, grain boundaries, twins and stacking faults produced by the severe plastic deformation are likely to create diffusion paths and increase the surface reactivity, thereby improving hydrogen sorption kinetics of magnesium-based alloys [START_REF] Jorge | Correlation between hydrogen storage properties and textures induced in magnesium through ECAP and cold rolling[END_REF][START_REF] Huot | Improvement of hydrogen properties of magnesium alloys by cold rolling and forging[END_REF][START_REF] Revesz | Dehydrogenationhydrogenation characteristics of nanocrystalline Mg 2 Ni powders compacted by high-pressure torsion[END_REF][START_REF] Panda | Effect of initial powder type on the hydrogen storage properties of high-pressure torsion consolidated Mg[END_REF][START_REF] Edalati | Effect of gradient-structure versus uniform nanostructure on hydrogen storage of Ti-V-Cr alloys: Investigation using ultrasonic SMAT and HPT processes[END_REF][START_REF] Novelli | Microstructural details of hydrogen diffusion and storage in Ti-V-Cr alloys activated through surface and bulk severe plastic deformation[END_REF][START_REF] Asselli | Hydrogen storage properties of MgH 2 processed by cold forging[END_REF][START_REF] Hongo | Significance of grain boundaries and stacking faults on hydrogen storage properties of Mg 2 Ni intermetallics processed by high-pressure torsion[END_REF]. On the other hand, the hot forged sample exhibited the slowest absorption kinetics and the lowest hydrogen uptake capacity (~1 wt.% of hydrogen) after 2 hours of hydrogenation due to the weaker basal fiber texture, the lower amount of structural defects and the absence of any porosity and cracks within the sample. The compacted powder displays a relatively fast absorption owing to the facile hydrogen diffusion between the less compacted particles.

Interestingly, a two-stage hydrogen activation is observed for the CF sample: a fast absorption occurs at the very beginning of the reaction followed by a gradual stabilization and a hydrogen uptake of about 1.2 wt.%. After 50 minutes the uptake absorption kinetics goes again through a strong increase leading to an uptake of about 6 wt.% after 6.5 hours (curve not shown). The apparent limited maximum uptake is due to a deliberate choice to stop the experiment. As it has been checked also for the A + CF sample, a full hydrogenation of the samples is achieved for longer time. Regardless of the proportion of Mg alloyed with nickel, the theoretical hydrogen gravimetric capacities for the HF, CF and A + CF samples are 5.93 wt.%. Thus no significant loss of hydrogen capacity is detected. Moreover, the phenomenon of hydrogen trapping by lattice defects, such as grain/phase boundaries and dislocations [START_REF] Orimo | Materials science of Mg-Ni-based new hydrides[END_REF][START_REF] Hou | Hydrogenation thermodynamics of meltspun magnesium rich Mg-Ni nanocrystalline alloys with the addition of multiwalled carbon nanotubes and TiF 3[END_REF] cannot be evidenced due to the experimental resolution. To understand the kinetics mechanism behind this two-stage hydrogen absorption, two general kinetic models, Jonhson-Mehl-Avrami-Kolomogorov (JMAK) model [START_REF] Johnson | Reaction kinetics in processes of nucleation and growth[END_REF] and Jander model [START_REF] Jander | Reaktionen im festen Zustande bei höheren Temperaturen. Reaktionsgeschwindigkeiten endotherm verlaufender Umsetzungen[END_REF], have been used to fit the isothermal data. However, the obtained data cannot be fitted with any of these models, even the improved classical nucleation-growthimpingement mode (C-JMAK) recently proposed by Pang et al. [START_REF] Pang | A review on kinetic models and corresponding analysis methods for hydrogen storage materials[END_REF]. This means that the kinetic mechanisms responsible for this two-stage hydrogen absorption cannot be solely determined by kinetic models. One possible explanation may arise from the phase composition. XRD patterns (Fig. 1c Nevertheless, it is worth to mention that hydrogen was not fully released from the hydrogenated samples after the first desorption, as revealed in Fig. 8b, which is associated with the remaining hydrogen atoms present in solid solution Mg ! NiH !.! .

Hydrogen sorption kinetics after activation

Successive absorption/desorption kinetics were measured under conditions similar to the one used for activation. Fig. 8c shows the hydrogen absorption kinetics recorded at the second cycle for the forged samples. Since these samples were partly activated after the first cycle, the second absorption kinetics was remarkably improved for all the forged samples (Fig. 8c). Nevertheless, the hot forged sample still displays the slowest absorption kinetics due to the lack of any porosity and/or cracks for hydrogen penetration. Fig. 8d displays the hydrogen desorption kinetics recorded at the second cycle. The hydrogen desorption kinetics has improved to some extent for all the forged samples, despite the CF sample which still exhibits the slowest desorption behavior.

Although the sorption cycles can be treated as annealing processes to homogenize the microstructure, the sluggish desorption observed in the CF sample reveals that the amount of Ni alloyed with Mg remains low and Mg ! Ni content is poor after two cycles of sorption.

After 11 sorption cycles, all the samples display similar absorption curves, with a markedly fast kinetics during the first 5 minutes followed by a strong slowdown, as depicted in Fig. 8e. The observations may indicate that the absorption/desorption cycles -and the associated volume changes and consequent lattice strain -have modified the initial microstructures to reach a "normalized" structure rather identical to all the 3 samples. Several modifications can take place under cycling such as recovery / recrystallization, grain growth, fragmentation of the samplesassociated to the volume changes under hydrogenation -to form fine powder particles, reannealing of these powder particles, etc. [START_REF] Popilevsky | Hydrogenation-induced microstructure evolution in as cast and severely deformed Mg-10 wt.% Ni alloy[END_REF][START_REF] Revesz | Dehydrogenationhydrogenation characteristics of nanocrystalline Mg 2 Ni powders compacted by high-pressure torsion[END_REF]. This generates a microstructure of cycled materials that depends less on the initial processing, which is however essential to improve the first hydrogenation. Whereas the unified microstructure leads to fast hydrogen absorptions for all three samples, the desorption kinetics of the CF sample remains low compared to the HF and A + CF samples even after 11 cycles (see Fig. 8f). The dehydrogenation curve of CF sample remains quasi identical from the first to the 12 following desorptions suggesting that the catalytic phase Mg 2 NiH 4 is not in sufficient quantity or in adequate distribution in the cold forged sample despite several cycles.

It is interesting to note that a two-stage hydrogen desorption is even observed for the three samples as depicted by the insert in Fig. 8f. The fast desorption at the first stage can be attributed to a rapid decomposition of the Mg ! NiH ! hydride. The slower desorption kinetics at second stage can be then ascribed to the slow release of H from the stable MgH ! hydride. The further analysis of the kinetic mechanisms involved in absorption/desorption investigated by in-situ neutron diffraction is in progress and will be reported later. [START_REF] Gajdics | Characterization of a nanocrystalline Mg-Ni alloy processed by high-pressure torsion during hydrogenation and dehydrogenation[END_REF][START_REF] Revesz | Hydrogen storage of nanocrystalline Mg-Ni alloy processed by equal-channel angular pressing and cold rolling[END_REF]. Without considering the effect of absorption pressure, the hydrogen storage capacity is significantly increased for the sample prepared by annealing and fast cold forging, as compared to the maximum hydrogen uptake achieved for the sample prepared by combination of HEBM and HPT, which remains lower than expected (3.0 wt.% instead of 4.2 wt.%). For the starting material made by HEBM, the maximum hydrogen uptake is evidently promoted by HPT, whereas it is decreased by 35% through 6 passes of ECAP or one pass of CR [START_REF] Revesz | Hydrogen storage of nanocrystalline Mg-Ni alloy processed by equal-channel angular pressing and cold rolling[END_REF]. Interestingly, after 10 passes of CR, the maximum hydrogen uptake is identical to the initial value (2.4 wt.%) recorded for the HEBM sample. These differences could result from the distinct extents of shear strain imposed by the different SPD modes [START_REF] Revesz | Hydrogen storage of nanocrystalline Mg-Ni alloy processed by equal-channel angular pressing and cold rolling[END_REF]. The multi-passes SPD often generates a preferential texture that promotes hydrogen absorption, however, the absence of any porosity retards hydrogen penetration from surface into bulk [START_REF] Jorge | An investigation of hydrogen storage in a magnesium-based alloy processed by equalchannel angular pressing[END_REF]. The enhanced hydrogen storage performance achieved in this work is due to the coexistence of a preferential fiber texture and amounts of the cracks, the interfaces and the structural defects. The primary advantage associated the fast cold forged sample subjected to preannealing is the generation of considerable amounts of cracks accompanied with the fragmentation of the Mg ! Ni lamellae, which act as hydrogen diffusion paths from surface through bulk.

Conclusions

The aim of the present work was to improve our understanding of microstructure modifications (including nature of phases and structural defects, grain sizes and texture) on the H-storage behavior in Mg-based composites (Mg -22 wt.% Ni). In terms of activation (i.e. first absorption), it appears that a stronger {0001} Mg fiber texture formed during cold deformation as well as the presence of structural defects and micro-cracks in the processed samples are beneficial since they create easier propagation paths for H diffusion within the bulk of the sample. Regarding the desorption behavior, the best desorption kinetics were obtained in the presence of the Mg 2 Ni phase due to the weaker stability of the Mg 2 NiH 4 hydride phase which favors the H release upon desorption. A consequence of these observations in terms of optimal processing to create a material having both an improved absorption as well as an improved desorption is the A + CF processing route that generates the formation of a cracked microstructure combining fine grains of Mg ! Ni phase (favorable for desorption) and textured Mg grains (favorable for absorption).

3. 1

 1 Structural and microstructural analysis 3.1.1 Phase analysis Phase composition was studied by XRD. Fig. 1b shows a comparison of the XRD patterns of the starting material Mg-22 wt.% Ni collected before and after annealing performed at 530°C under inert atmosphere for 30 min. A Mg ! Ni phase formed by diffusion of Mg and Ni atoms during annealing. The weight fraction of Mg ! Ni is determined to be 41 wt.% by Rietveld refinement, in agreement with the starting composition. Additionally, some weak diffraction peaks which correspond to the formation of small amounts of the MgNi ! compound are observed. While Mg ! Ni have formed by solid state diffusion of Ni within the Mg particles, the possibility of the formation of MgNi ! by phase separation during local solidification, as previously reported by

Fig. 1 .

 1 Fig. 1. Phase identification using XRD measurement. (a) Schematic of samples geometry and analyzed surfaces. Comparison of XRD patterns of (b) Starting material before and after annealing, (c) Cold forged, hot forged and annealed + cold forged samples, and (d) Cold forged, hot forged and annealed + cold forged samples after the first hydrogenation.

Fig. 2 .

 2 Fig. 2. Pole figures of (0002) and (10-10) for Mg-phase measured using XRD: (a) CF, (b) HF and (c) A+ CF. (RD: radial direction).

Fig. 3 .

 3 Fig. 3. EBSD characterization taken from the cross-section of CF sample: (a) IPF map of Mgphase, Ni indicated by white color, (b) (0002) pole figure, (c) IPF map superimposed with grain boundary corresponding to the selected zone in (a) taken at a smaller step size and (d) IPF map of Ni corresponding the selected zone in (a). High and low angle grain boundaries are indicated by black and grey, respectively.

Fig. 4 .

 4 Fig. 4. Grain size distributions of Mg-phase for CF (a), HF (c) and A + CF (e) samples. Misorientation angle distribution of Mg-phase for CF sample (b). Grain size distributions of Mg 2 Ni for HF (d) and A + CF (f) samples.

Fig. 5 .

 5 Fig. 5. SEM-EBSD characterization taken from the cross-section of HF sample: (a) EBSD phase map superimposed with band contrast, in which the phases Mg, Mg ! Ni and Ni are indicated by red, blue and yellow, respectively; IPF maps of Mg (b), Mg ! Ni (c) and Ni (d), respectively; and (e) SEM-SEI image corresponding to the selected area in (a).

Fig. 6 .

 6 Fig. 6. Microstructure of the annealed Mg-22 wt. % Ni, (a) SEI image; (b) EDX spectrum taken from three different areas 1, 2 and 3, as indicated by the rectangles in (a); (c) zone 3 at higher magnification and (d) superimposed map of the elements distribution (Mg and Mg 2 Ni indicated by red and blue, respectively.)

Fig. 7 .

 7 Fig. 7. SEM-EBSD characterization of A + CF sample. SEM-SEI images taken from the crosssection (a) and zoom of the selected area (b); SEM-BSE image (c); phase map superimposed band contrast in which Mg and Mg 2 Ni are indicated by red and blue, respectively (d). IPF maps of Mg (e) and Mg 2 Ni (f), respectively.

  ) have shown that the CF sample is only composed of elemental Mg and Ni while additional Mg 2 Ni alloy is visible in the A + CF sample. This difference in phase distribution may explain the unlike absorption profiles between CF and A + CF samples. In the A + CF sample, the already formed Mg 2 Ni contributes to the fast kinetics of the first hydrogenation, while only interfaces between Mg and Ni particles can be part of the kinetics enhancement in the CF sample. After completion of the first hydrogenation, Ni is partly alloyed with Mg (as shown in XRD pattern Fig.1d and the two stage behavior is no more visible (Fig. 8c). The present experimental observations are in line with previous reports that describe sorption mechanisms in Mg-Ni alloys. According to Refs [52,53], hydrogenation starts with the dissolution of hydrogen atoms into Mg 2 Ni towards the formation of the Mg 2 NiH 0.3 solid solution, and, in a second stage, the magnesium hydride appears. The ternary hydride Mg 2 NiH 4 crystallizes as soon as the hydrogen content in Mg 2 NiH 0.3 reaches saturation.

Fig. 8 .

 8 Fig. 8. Hydrogen sorption curves for different cycles. Absorption was performed at 310 ˚C, 20 bars and desorption at 310 ˚C, 1.5 bars. First (a), second (c) and 12 th (e) absorption steps; first (b), second (d) and 12 th (f) desorption steps. Absorption measurements were intentionally stopped after 80 min, 110 min or 140 min to limit the acquisition duration.

Table 1 :

 1 Weight fraction of each phase component in annealed and as-forged samples.

		Forging	Weight fraction (wt. %)
	Sample series	temperature (°C)	Mg	Ni	Mg 2 Ni
	CF	25	78.22 ± 2.07	21.78 ± 2.06	-
	HF	450	62.36 ± 1.50	2.22 ± 0.78	35.42 ± 2.02
	Annealed (530°C)	-	56.85 ± 2.30	1.50 ± 0.16	41.65 ± 1.95
	A + CF	25	57.75 ± 2.89	1.40 ± 0.17	40.85 ± 1.95

Table 2 :

 2 Thickness reduction ratios and lattice strain of each phase in annealed and as-forged samples.

	Sample series	Reduction		Lattice strain (%)
		ratio (%)	Mg	Ni	Mg 2 Ni
	CF	79.0	0.14 ± 0.01	0.18 ± 0.01	-
	HF	93.0	0.09 ± 0.01	0.08 ± 0.01	0.05 ± 0.01
	Annealed (530°C)	-	0.08 ± 0.01	0.05 ± 0.01	0.06 ± 0.02
	A + CF	75.0	0.16 ± 0.01	0.13 ± 0.01	0.08 ± 0.02

Table 3

 3 Comparison of hydrogen sorption properties of Mg-Ni samples obtained in the presentwork and from literature[START_REF] Gajdics | Characterization of a nanocrystalline Mg-Ni alloy processed by high-pressure torsion during hydrogenation and dehydrogenation[END_REF][START_REF] Revesz | Hydrogen storage of nanocrystalline Mg-Ni alloy processed by equal-channel angular pressing and cold rolling[END_REF].

	Sample	Techniques	Sorption	Absorption	Desorption	Max uptake	H retained
			temperature	pressure	pressure	after 1 st	after 1 st
			(°C)	(bars)	(bars)	absorption	desorption
						(wt.%)	(wt.%)
	Mg-22 wt.% Ni	CF				5.8	0.80
	(this work)	HF	310	20	1.5	2.0	0.25
		A + CF				6.0	0.70
	Mg 75 Ni 25	HEBM + HPT	300	10	0.1	3.0	0.45
	Mg-45 wt.% Ni
	[41]
	Mg 75 Ni 25	HEBM	300	10	-	2.4	-
	Mg-45 wt.% Ni	HEBM + ECAP				1.5	-
	[43]	HEBM + CR				2.4	-

Table 3

 3 summarizes the H-storage properties of the Mg-Ni powders obtained by fast forging in the present work and by different SPD routes from the literatures [41, 43]. Regarding the chemical composition of Mg-Ni powder, a lower amount of Ni contained in the current samples (Mg-22 wt.% Ni) leads to a theoretical hydrogen capacity of 5.97 wt. %, as compared to 4.2 wt. % for the (Mg-45 wt.% Ni) one employed in Refs.

Acknowledgement

The authors appreciate the financial support from the IDEX-CDP Eco-SESA program, from the University Grenoble Alpes, France. This work was also supported by the French State through the program "Investment in the future" operated by the National Research Agency (ANR) and referenced by ANR-11-LABX-0008-01 (Labex DAMAS).