Clotilde Policar
email: clotilde.policar@ens.psl.eu

Jean Bouvet

Hélène C Bertrand

Nicolas Delsuc

SOD mimics: From the tool box of the chemists to cellular studies

Keywords: metallodrugs, X-fluorescence imaging or X-fluorescence microscopy XFM, Cellular studies nanoparticles, COVID, Coronavirus disease, cyt c, Ferricytochrome

Superoxide dismutases (SODs) are metalloproteins that protect cells against oxidative stress by controlling the concentration of superoxide (O 2 -) through catalysis of its dismutation.

The activity of superoxide dismutases can be mimicked by lowmolecular-weight complexes having potential therapeutic applications. This review presents recent strategies for designing efficient SOD mimics, from molecular metal complexes to nanomaterials. Studies of these systems in cells reveal that some SOD mimics, designed to react directly with superoxide, may also indirectly enhance the cellular antioxidant arsenal. Finally, a good understanding of the bioactivity requires information on the cell-penetration, speciation, and subcellular location of the SOD mimics: we will describe recent studies and new techniques that open opportunities for characterizing SOD mimics in biological environments.

Introduction

Superoxide dismutases (SODs) are metalloproteins involved in the cellular protection against superoxide, a reactive species derived from dioxygen by one-electron reduction. Superoxide (O 2 À [1]) is a metastable radical anion endowed with both signaling and deleterious properties [2,3]. SODs participate in the control of its cellular concentration (about 10 pM in mammalian cells [4]) by catalyzing its dismutation (1), resulting from two monoelectronic processes (2) and (3), with (1)

= (2) þ (3) 2 O 2 À þ 2H þ / O 2 þ H 2 O 2 (1) O 2 À / O 2 þ e À (2) O 2 À þ 2H þ þ e À / H 2 O 2 (3)
SODs are remarkably efficient proteins that catalyze superoxide dismutation with kinetics only limited by diffusion (ca. 10 9 mol À1 Ls À1 ). Four families of SODs use different metallic redox couples, Cu(II/I)eZn(II), Cu(II/ I), Fe(III/II), Mn(III/II) or Ni(III/II) [2,5], with a redox potential in a very narrow range (ca. 0.30 V/NHE) [2,6]. Optimal kinetics is expected for a potential at 0.36 V/ NHE [6,7], the mid-point potential [8,[START_REF] Barrette | Potentiometric titrations and oxidation-reduction potentials of several iron superoxide dismutases[END_REF] of the two couples involved in superoxide dismutation (E ' pH7, 25 C (O 2aq /O 2 À ) = À0.18 V/NHE and E ' pH7, 25 C (O 2 À / H 2 O 2 ) = 0.91 V/NHE, apparent redox potentials at pH7 and taking into account dioxygen low solubility in water [START_REF] Koppenol | Electrode potentials of partially reduced oxygen species, from dioxygen to water[END_REF]). This pinpoints the redox potential as a key parameter, but electrostatic attraction by a positively charged funnel described for all SODs, except Cu-only SOD in which the Cu active site is open to solvent [START_REF] Robinett | Eukaryotic copper-only superoxide dismutases (SODs): a new class of SOD enzymes and SOD-like protein domains[END_REF], also contributes to the fast, only diffusion-limited, reaction with superoxide [2]. Oxidative stress, described as an imbalance between the flow of reactive species and protective pathways, is associated with a range of physiopathological conditions. Chemists can take inspiration from nature to design complexes or materials reproducing SOD activity [6], and that could be used for therapeutic applications [12e14]. These aspects have been extensively reviewed [6, 13,15e19] along with the therapeutic applications of SOD mimics [START_REF] Batinic-Haberle | SOD therapeutics: latest insights into their structure-activity relationships and impact on the cellular redox-based signaling pathways[END_REF][START_REF] Bonetta | Potential therapeutic applications of MnSODs and SOD-mimetics[END_REF][START_REF] Batini C-Haberle | Spasojevi c I: Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential[END_REF][START_REF] Batinic-Haberle | H2O2-Driven anticancer activity of Mn porphyrins and the underlying molecular pathways[END_REF]. Note that the SOD mimics discussed here are presented in both the Scheme 1 (molecular structures and abbreviations) and Table 1 (physicochemical properties and references), Cp standing for compound and Pep for peptides.

The redox dependence mentioned earlier was not always experimentally observed for SOD mimics, suggesting that other parameters could be key players. Various studies on a range of SOD mimics, with Mn II (Cp22-23, Cp24-28), Cu II (Cp11, [START_REF] Bonetta | Potential therapeutic applications of MnSODs and SOD-mimetics[END_REF], or Ni II (Pep18), have shown the importance of a coordinated water molecule [8,22e25]. This can be rationalized either by a fast exchange with superoxide, in an inner-sphere electron transfer requiring that superoxide enters the coordination sphere of the metal ion [25] involving a M-OO intermediate, or through the effect of proton donation to the reduced peroxide to lead to H 2 O 2 release [26]. Second coordination sphere effects have also been described, revealing a possible fine modulation of the Mn-OO bond's strength upon variation of electronic effects on a Schiff-base ligand (Cp38-43) [START_REF] Palopoli | Insights into second-sphere effects on redox potentials, spectroscopic properties, and superoxide dismutase activity of manganese complexes with schiff-base ligands[END_REF]. The dimerization of monometallic Mn(II) complexes upon reaction with superoxide was also observed [START_REF] Palopoli | Insights into second-sphere effects on redox potentials, spectroscopic properties, and superoxide dismutase activity of manganese complexes with schiff-base ligands[END_REF], a known process leading to Mn(III)eMn(IV) m-oxo complexes [6,8] that are 'dead- end' for superoxide dismutation activity. This dimerization process was shown to be prevented by a sterically hindered metal ion environment [8]. Bulky substituents can also favor catalysis by modulating the stability of intermediates of the catalytic cycle (MÀOO). Indeed, isopropyl moieties (Cp9) were shown to preclude hydrogen bonds that stabilize a Cu-peroxo intermediate (with Cp10) [START_REF] Martínez-Camarena | Unveiling the reaction mechanism of novel copper N -alkylated tetra-azacyclophanes with outstanding superoxide dismutase activity[END_REF]. This was in favor of catalysis, as a too stable intermediate would disfavor the release of the product (H 2 O 2 ). Several studies of Cu(II) or Mn(II) complexes with cyclic polyamines also suggested structural effects that could finely modulate the SOD-like activity: flexibility of the ligand that would permit molecular rearrangements of the complex along the catalytic pathway [START_REF] Guijarro | Homo-and heterobinuclear Cu 2+ and Zn 2+ complexes of ditopic aza scorpiand ligands as superoxide dismutase mimics[END_REF], or that would favor Mn(II) oxidation to the Jahn-Teller distorted Mn(III) complex [START_REF] Maroz | Pulse radiolysis investigation on the mechanism of the catalytic action of Mn(II)-Pentaazamacrocycle compounds as superoxide dismutase mimetics[END_REF], in relation to redox tuning.

Below, we describe selected recent strategies for designing efficient SOD mimics and discuss the importance of cell studies to understand, and possibly improve, bioactivity, including the investigation of cellular pathways and of SOD mimics cellular fate with innovative imaging approaches.

New directions in the design of SOD mimics: from low molecular-weight complexes to nanomaterials Peptide-based complexes Peptides have been used as ligands to generate metal complexes mimicking the different SOD enzymes.

Linear short sequences (Pep4-17) engaged in complexes with Cu II [START_REF] Csire | Coordination, redox properties and SOD activity of Cu(II) complexes of multihistidine peptides[END_REF][START_REF] Dancs | Catechol oxidase and SOD mimicking by copper(II) complexes of multihistidine peptides[END_REF] or Ni II [START_REF] Lihi | The role of the cysteine fragments of the nickel binding loop in the activity of the Ni(II)-Containing SOD enzyme[END_REF] cations have shown promising intrinsic catalytic activities. To circumvent the high conformational flexibility of peptides that entropically disfavors coordination [START_REF]coordination reduces the conformational space of flexible ligands by imposing a specific conformation[END_REF], a cyclic peptide (Pep3) [START_REF] Kotynia | Bieli nska S, Kamysz W, Brasu n J: The analysis of Cu(II)/Zn(II) cyclopeptide system as potential Cu,ZnSOD mimic center[END_REF] or preorganized peptide scaffolds as de novo mini-proteins [START_REF] Mathieu | Rational de novo design of a Cu metalloenzyme for superoxide dismutation[END_REF] have been used. A strategy involving the generation of a library of peptides screened with metal ions through an activity-based assay has led to the characterization of a copper complex mimicking SOD (Pep1), efficient even in cellular models of oxidative stress [37]. This strategy has been successfully extended to the discovery of the first peptidyl dicopper(II) complex (Pep2) mimicking the catalase enzyme [38]. Interestingly, depending on the activity assay used for the screening, complexes possessing only the desired activity were selectededesired activity, namely, monoelectronic SOD activity with a monometallic peptide complex or bi-electronic catalase activity with a dicopper complex [37,38].

Redox-ligand based SOD mimics

So far, there are no known SODs using an organic redox cofactor, probably because monoelectronic redox processes could lead to reactive radical intermediates. However, Goldsmith et al. have prepared a Zn(II)complex with a di-hydroxybenzyl ligand (Cp34) Zn II -OPh-OH that could potentially lead to a Zn IIphenoxyl-radical ([Zn II -OPh-O À 4 Zn II -OPh-O ]) [39,40]. This Zn II -phenoxyl radical acted as a SOD mimic, cycling between a Zn II -para-quinone and a Zn II -phenoxyl radical. This reactivity is associated with the di-hydroxybenzyl moiety, with its rich monoelectronic chemistry involving radical-phenoxyl and quinone. Whereas Zn II analogs of some Mn II SOD mimics with a bound phenol (Cp29) have been shown to be redox inactive [41e44], the Zn II -OPh-OH complex (Cp34) was found electroactive, with a quasi-reversible wave at 0.35 V/NHE, very close to the optimal redox potential for superoxide dismutation (0.36 V/NHE). Zn II was suggested to play a double role, attracting the anion substrate [45] and facilitating the cycling between the phenoxyl radical and quinone. In 2021, the mechanism of superoxide dismutation was investigated for Mn(II) analogs (Cp35-36) using cryospray-ionization mass spectrometry (CSI-MS), and an intricate mechanism involving quinone intermediates, but also a quinone-Mn IV ]O, was beautifully characterized [46].

Nanoparticles

Nanoparticles have been developed, either through the incorporation of known discrete SOD mimics in nonactive materials (silica, alumina) or by the use of metal-based materials, including metallic nanoparticles or metaleorganic frameworks, in which the basic structural unit can be responsible for antioxidant Cu II /Cu II 2 /Cu II Zn II cyclic(HKHGPG) 2 Pep3 -MCF NBT (7.4) = 6.67/6.71/6.92 (T)

[35] Ac-HAAHVH-NH 2 [Cu II H -1 L5] + Pep4 +202 MCF NBT (7.4) = 7.59 (P) [31] Ac-HAAHGH-NH 2 [Cu II H -1 L6] + Pep5 +214 MCF NBT (7.4) = 7.79 (P) [31] Ac-HGGHGH-NH 2 [Cu II H -1 L7] + Pep6 +230 - [31] Ac-HGGGHGH-NH 2 [Cu II H -1 L8] + [Cu II H -2 L8] Pep7 +247 MCF NBT (7.4) = 7.56 (P) [31] Ac-HHGH-NH 2 [Cu II L9] 2+ [Cu II H -1 L9] + Pep8 +313/+264 - [31] Ac-S2H3-NH 2 [Cu II L10] 2+ [Cu II H -1 L10] + Pep9 +293 MCF NBT (7.4) = 7.30 (P) [31] Ac-S3H4-NH 2 [Cu II L11] 2+ Pep10 - MCF NBT (7.4) = 7.81 (P) [31] Cu II Ac-HKHKH-NH 2 Pep11 - - [32] Cu II Ac-HHKHKH-NH 2 Pep12 - - [32] Cu II Ac-KAHEFGHSLGLDHSK-NH 2 Pep13 - - [32] Cu II HADHDHKK-NH 2 Pep14 - - [32] Cu II Ac-RHQAGPPHSHR-NH 2 Pep15 - - [32] Ni II HHDLPCGVY-NH 2 Pep16 - MCF NBT (7.8) = 4.85 (P) [33] Ni II HCDLPHGVY-NH 2 Pep17 - MCF NBT (7.8) = 3.89 (P) [33] [Ni II L 3S (OH 2 )] - Pep18 Epa = +724 (100 mV/s) SF(7.75) = 5.26(H) [24] Nanoparticles CeO 2 NP - - MCF cyt c (7.2) = 9.56 (T) a [51] MnO NP (8 nm size) - - MCF cyt c (7.4) = 9.38 (P) a [54] Mn 3 O 4 NP - - 20 mg/mL eq. 1U/mL SOD a,b [56] Cu-TBAP nanodots - - 12.5% of SOD activity a,c [62]
Half-wave redox potentials (E 1/2 ) have been converted and expressed in mV vs. NHE (Normal Hydrogen Electrode) for comparison, with E 1/2 = (E anodic peak + E cathodic peak )/2. The intrinsic activity of the SOD mimics logk cat (O 2 .-); hence, the kinetics of reaction with superoxide out of any cellular context [6], is displayed under the form method detector (pH) = X (Buffer) indicating method used (MCF, SF, PR, see below), buffer, pH. When several buffers are mentioned: P corresponds to a phosphate buffer whereas H is for HEPES, T is for TRIS buffer, and M for MOPS buffer, concentrations are not shown. 'MCF' refers to the rate of the constant back-calculated from the McCord and Fridovich assay [START_REF] Beauchamp | Superoxide dismutase: improved assays and an assay applicable to acrylamide gels[END_REF]. This assay is a kinetic competitive assay between the SOD mimic studied and a detector. In this assay, superoxide is produced by xanthine oxidase as a slow flow. Several UV-visible detectors have been used in the literature, including nitro blue tetrazolium 'NBT', ferricytochrome c 'cyt c', 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxianilide 'XTT' as a detector. The IC 50 is the concentration of the SOD mimic that reduces the reaction rate of the detector with superoxide by a factor of 2. Then k cat = k detector x[detector]/IC 50 [6,8,[START_REF] Pasternack | Superoxide dismutase activities of an iron porphyrin and other iron complexes[END_REF]. Back-calculating the k cat was only possible when the authors explicitly gave the concentration used for the detector. 'PR' stands for pulsed radiolysis and 'SF' for Stopped-Flow (see Ref. [START_REF] Friedel | Comparative studies on manganese-based SOD mimetics, including the phosphate effect, by using global spectral analysis[END_REF] for a comprehensive description of the methodology). Both in PR and SF, a high concentration of superoxide is provided in one go at the beginning of the kinetics measurement. In these direct methods for the kinetics evaluation (PR or SF), superoxide can be given in excess to the catalyst, ensuring several turnovers are possible. In the MCF assay, which is an indirect assay, the superoxide is provided at a continuous slow rate close to that encountered in biological systems. Indirect and direct methods are thus complementary.

a log(k cat ) determined per mole NPs.

b Method using the production of superoxide with xanthine oxidase and a fluorescent or EPR-based detection, with respectively hydroethidine or 5,5-dimethyl-1-pyridine N-oxide.

c MCF-type assay, using superoxide generated upon the UV irradiation of riboflavin and methionine and NBT as a detector.

Scheme 1

Structures of the SOD mimics mentioned here. Cp stands for compound, Pep for peptides, BNP for boehmite nanoparticles, SNP for silica nanoparticles, S for sarcosyl, MCM-41 for mesoporous MCM-41 silica, SBA-15 for mesoporous SBA-15. Core structures of five SOD mimic families-porphyrins, poly-aza-macrocycles (PAM), ligands amine centered, salan-type ligands, salen-type ligands, bimetallic-are given with variable side chains (R x ). For some of the molecules, the name/code from the original papers is also reminded. . Note that similar anticancer activity has also been described for Mn-porphyrins and has already been extensively reviewed [START_REF] Batinic-Haberle | H2O2-Driven anticancer activity of Mn porphyrins and the underlying molecular pathways[END_REF]63].

Studies in cells: pathways beyond direct antioxidant effects and physicochemical characterization in cells An indirect mode of action enhancing the antioxidant cellular defenses

Recently, an increasing interest has grown about potential 'side reactions' performed more specifically by Mn-porphyrins (MnPs) with reactive oxygen species (ROS) or cellular compounds (ONOO À , H 2 O 2 , H 2 S, CO 3 -, ascorbate, and GSH) [START_REF] Batinic-Haberle | An educational overview of the chemistry, biochemistry and therapeutic aspects of Mn porphyrinsfrom superoxide dismutation to H2O2-driven pathways[END_REF] and their mechanism of action in cells [7,18,63]. As early as 2004, an increase in extracellular SOD and MnSOD was reported [64] after incubating cells with MnPs, suggesting that these compounds may act not solely as a direct antioxidant by reducing ROS concentration but also indirectly: they may induce antioxidant cell feedback through a prooxidant effect, enhancing the protective cell arsenal. Batinic et al. have reported such a pro-oxidant effect of MnPs in 2012 [65,66]. They have extensively shown then that besides their reactions against ROS and reactive nitrogen species (RNS), MnPs display an effect on thiols regulation and cellular sulfur metabolism [7,18,63,67], which activates the Nrf2 [68] pathway. This is interesting as the Keap1-Nrf2 pathway [69,70] constitutes the major defense system against electrophilic and oxidative stress: Keap1 acts as a redox sensor through the oxidation or modification of its cysteine residues upon oxidative/electrophilic stress, which results in the Nrf2-induced up-regulation of several antioxidant enzymes (see Figure 1). It shows that compounds endowed with an overall antioxidant cellular activity may actually be active by the induction of cellular antioxidant feedback via the Keap1-Nrf2 pathway through a pro-oxidant effect. Interestingly, this activation would constitute a late but long-lasting effect, as opposed to an immediate catalytic antioxidant direct effect, but possibly lasting shorter if the antioxidant catalyst is metabolized.

Examples of SOD-mimics inducing this Nrf2 pathway, or not

MnTnBuOE-2-PyP 5þ (Cp3) is an example of a SOD mimics inducing such an activity. This Mn-porphyrin has been shown to upregulate the expression of enzymes under the control of Nrf2, including NQO1, catalase and MnSOD, through Nrf2 activation in hematopoietic stem cells in transgenic mice [67]. The activation of Nrf2 is suggested to occur via Keap1's Sglutathionylation. Similarly, a polyazamacrocyclic MnSOD mimic, M40403 (Cp20), was shown to increase the expression level of Nrf2 in the skin of diabetic rats [71]. But this scheme is not general. In contrast, the salan-type SOD mimics Cp29-31 reduce the overexpression of mitochondrial MnSOD associated with lipopolysaccharide-induced oxidative stress in intestinal epithelial cells (IECs), indicating that these SOD mimics can complement mitochondrial MnSOD. This result pinpoints a direct antioxidant effect as opposed to an activation of the Nrf2 pathway. No effect on cytosolic CuSOD or catalase was recorded, showing the importance of mitochondria and the specific management of superoxide [41e44].

Detection in cells

The biological efficiency of SOD mimics is linked to their intrinsic SOD activity and also to other parameters such as cellular uptake, subcellular distribution, and speciation in biological environments that are intricate and abound with Lewis bases [6].

Overall cellular uptake of metal complexes can be quantified using techniques such as inductive-coupled plasma mass spectrometry ICP-MS [42,44] whatever the metal ion (Mn, Fe, Cu, Zn, Ni), or specific techniques such as EPR, available for instance in the case of S = 5/2 Mn II [41e43,72], EPR and ICP-MS estimates being shown to be consistent [42]. In the case of MnPs, UVevis spectroscopy of cell lysates [44,63,73] also gave estimates similar to ICP-MS [44]. Quantification of MnP in mice tissues, after injection or oral gavage, was performed using LC-MS/MS [START_REF] Leu | CNS bioavailability and radiation protection of normal hippocampal neurogenesis by a lipophilic Mn porphyrin-based superoxide dismutase mimic, MnTnBuOE-2-PyP5+[END_REF][START_REF] Weitner | Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics, MnTE-2-PyP5+ and MnTnHex-2-PyP5+[END_REF], and the trend of their penetration in the brain matched with the lipophilicity, the more lipophilic compounds (MnTnBuOE-2-PyP 5þ (Cp3), MnTnHex-2-PyP 5þ (Cp2)) being more concentrated in brain tissues such as cortex and hippocampus, whereas MnTE-2-PyP 5þ (Cp1) was not detected in the brain. The Mn oxidation state in MnPs was investigated using resonant Raman spectroscopy in the 1250-1550 cm À1 range in whole endothelial cells: MnTnBuOE-2-PyP 5þ (Cp3) and MnTnHex-2-PyP 5þ (Cp2) were predominantly in their reduced Mn(II) form and present at the mitochondria, as seen by cellular fractionation [START_REF] Carballal | Manganese porphyrin redox state in endothelial cells: resonance Raman studies and implications for antioxidant protection towards peroxynitrite[END_REF]. The weaker stability of the PMn(II) redox state could make it prone to exchanges with Zn(II) [START_REF] Spasojevi C | Mn porphyrin-based superoxide dismutase (SOD) mimic, MnIIITE-2-PyP5+, targets mouse heart mitochondria[END_REF][START_REF] Spasojevi C | Pharmacokinetics of the potent redox-modulating manganese porphyrin, MnTE-2-PyP5+, in plasma and major organs of B6C3F1 mice[END_REF], which does not seem to occur in cells, probably due to the low bioavailability of Zn(II) being sequestered at the active site of enzymes and mainly in the nucleus. Last but not least, X-ray absorption spectroscopy (XAS) can be used to quantify the relative content of metal complexes directly in cells prepared for X-ray fluorescence microscopy (XFM) (see below) [41, [START_REF] Weekley | Cellular fates of manganese(II) pentaazamacrocyclic superoxide dismutase (SOD) mimetics: fluorescently labeled MnSOD mimetics, X-ray absorption spectroscopy, and X-ray fluorescence microscopy studies[END_REF][START_REF] Aitken | Intracellular targeting and pharmacological activity of the superoxide dismutase mimics MnTE-2-PyP 5+ and MnTnHex-2-PyP 5+ regulated by their porphyrin ring substituents[END_REF] or in whole-cell pellets (freeze-dried) [START_REF] Weekley | Cellular fates of manganese(II) pentaazamacrocyclic superoxide dismutase (SOD) mimetics: fluorescently labeled MnSOD mimetics, X-ray absorption spectroscopy, and X-ray fluorescence microscopy studies[END_REF]. Note that applications of XAS and XFM in bioinorganic research have been reviewed recently [START_REF] Lovett | Application of X-ray absorption and Xray fluorescence techniques to the study of metallodrug action[END_REF].

Distribution in cells is also a key to the understanding of bioactivity. A first paper showed different cellular distributions for two MnPs. MnTE-2-PyP 5þ (Cp1) was Schematic representation of the two possible pathways from the literature for the SOD mimics cellular activity. Pathway A corresponds to the direct effect of SOD mimics, with their ability to catalytically dismutate superoxide. Pathway B is associated with a pro-oxidant effect of SOD mimics: as they produce H 2 O 2 , they can activate the Nrf2-signaling pathway. Nrf2 is continuously produced in cells, and Keap1, by binding Nrf2 in a particular conformation, promotes its degradation by the proteasome. Oxidative or chemical stress is sensed by modification of Keap1 cysteines. Their covalent modification/oxidation by electrophiles or oxidants (S cov or S ox ) leads to an inhibition of Nrf2 degradation by the proteasome. Nrf2 then accumulates in the cytosol and translocate to the nucleus, where it activates genes of a series of proteins, including antioxidant proteins such as catalase or SODs. The balance between these two pathways depends on the speed of dismutation (k cat ) in comparison with the cellular capacity to detoxify H 2 O 2 and also on the H 2 O 2 content of the cell. Pathway A may occur as soon as the SOD mimics enter the cells or the mitochondria but is also prone to decrease in time due to the SOD mimic degradation. In contrast, Pathway B requires time to activate protein synthesis (from 30 min to several hours), but this has a long-lasting effect as it works by boosting the cellular antioxidant feedback.

Imaging SOD mimics in cells. 2a. Elemental distribution of P, K, Mn, Re, and Zn in an HT29-MD2 cell incubated with Cp30. The phosphorus (P) and zinc (Zn, K-lines) maps were used to identify the nucleus area. The overlay (top right) corresponds to the Mn (magenta) and Re (L-lines) (green) maps. The regions corresponding to an overlap of both elements are displayed in white. Intestinal epithelial cells HT29-MD2 were incubated for 2 h with Cp30 (100 mM, 0.02% DMSO) before cryofixation and freeze-drying. Images were recorded on the 2-ID-D beamline of the APS synchrotron (excitation at 12.0 keV chosen to excite both Re and Mn; integration time, 2 s per pixel; pixel size, 200 nm). Scale bar, 3 mm. integration time, 2.4 s/pixel; pixel size, 300 nm). Scale bar, 3 mm. The overlay shows that part of the Re signal overlaps with the MitotrackerTM, suggesting that in these areas, Cp30 is localized in the mitochondria. This correlates with EPR titration that shows an accumulation of Mn in these organelles as compared to control cells, but to a lesser extent than Mn1 (Cp29). 2c. Distributions of the pyrene tag and manganese, which are similar in SH-SY5Y cells treated with 200 mM Cp18 for 24 h. (a) Fixed cells imaged by both fluorescence microscopy (pyrene tag, left) and low-resolution XFM (manganese, right). found evenly distributed, while MnTnHex-2-PyP 5þ (Cp2), known to be accumulated five times more than MnTE-2-PyP 5þ in the mitochondria in vivo [START_REF] Miriyala | Manganese superoxide dismutase, MnSOD and its mimics[END_REF][START_REF] Spasojevi C | Mn porphyrin-based superoxide dismutase (SOD) mimic, MnIIITE-2-PyP5+, targets mouse heart mitochondria[END_REF], displayed a perinuclear distribution within A549 cells [START_REF] Aitken | Intracellular targeting and pharmacological activity of the superoxide dismutase mimics MnTE-2-PyP 5+ and MnTnHex-2-PyP 5+ regulated by their porphyrin ring substituents[END_REF]. Mathieu et al. have imaged SOD mimics Cp29-33 in freeze-dried HT29-MD2 cells. Whereas Cp29 was homogeneously distributed in cells [41], Cp30 showed a punctuated distribution localized around the nucleus (Figure 2a) [42]. Using quantification (ICP-MS) in a mitochondria-enriched fraction and by colocalization studies with a fluorescent mitochondriatracker imaged prior to XFM (Figure 2b), Cp30 was found co-localized with this organelle [42]. In a comprehensive paper, Weekley et al. followed a range of Mn-PAMs (Cp17-19), functionalized with a fluorescent probe or not, using XAS of whole cells frozen pellets (see above) and XFM (Figure 2c). Some of the Mn-PAMs were shown to decompose into Mn(II)phosphate [START_REF] Weekley | Cellular fates of manganese(II) pentaazamacrocyclic superoxide dismutase (SOD) mimetics: fluorescently labeled MnSOD mimetics, X-ray absorption spectroscopy, and X-ray fluorescence microscopy studies[END_REF]. In both studies [41,[START_REF] Weekley | Cellular fates of manganese(II) pentaazamacrocyclic superoxide dismutase (SOD) mimetics: fluorescently labeled MnSOD mimetics, X-ray absorption spectroscopy, and X-ray fluorescence microscopy studies[END_REF], integration over the cell to determine a range of intracellular concentrations was used, from which a relative scaling for the penetration of various compounds, including MnCl 2 , was proposed. Mathieu et al. suggested an indirect way to question the speciation of a Mncomplex (Cp30) using XFM imaging, tagging the ligand with heavy metal (by conjugation to a Re-trisCO probe [START_REF] Hostachy | Re(I) carbonyl complexes: multimodal platforms for inorganic chemical biology[END_REF]). The Re(ligand) and Mn X-ray fluorescence maps, recorded above the edges of both Re and Mn, were overlapping, but only partly: a few areas showed only Re, revealing some dissociation in cells [42]. In this study, the distribution was also studied both by cell fractionation and XFM, XFM presenting the advantage of fewer steps for sample preparation, which can minimize risks of artefactual contamination or relocalization. Weekley et al. have also studied the distribution of the Mn-PAMs functionalized by a fluorescent probe using both XFM [START_REF] Weekley | Cellular fates of manganese(II) pentaazamacrocyclic superoxide dismutase (SOD) mimetics: fluorescently labeled MnSOD mimetics, X-ray absorption spectroscopy, and X-ray fluorescence microscopy studies[END_REF] and classical fluorescence (Figure 2c), showing that the fluorescent probes could be cleaved in cells. They conclude with the importance of studying the fate of the complexes in cells.

Conclusion

All these studies highlight the importance of studying SOD mimics in biological environments. Cellular and in vivo studies are key to the understanding of their bioactivity, which is a prelude to their therapeutic applications [7, [START_REF] Batinic-Haberle | SOD therapeutics: latest insights into their structure-activity relationships and impact on the cellular redox-based signaling pathways[END_REF][START_REF] Bonetta | Potential therapeutic applications of MnSODs and SOD-mimetics[END_REF][START_REF] Olson | Effects of manganese porphyrins on cellular sulfur metabolism[END_REF]63]. Note that Cp29 was shown to alleviate inflammation in a colitis mice model [41] or reduce the side effects of a treatment by oxaliplatin [START_REF] Guillaumot | Oxaliplatin-induced neuropathy: the preventive effect of a new super-oxide dismutase modulator[END_REF]. Interestingly, Mn-porphyrins (MnTE-2-PyP 5þ (Cp1) and MnTnBuOE-2-PyP 5þ (Cp3)) [7] and Mn-PAMs (Cp20-21) [7,[START_REF] Anderson | Phase IIb, randomized, double-blind trial of GC4419 versus placebo to reduce severe oral mucositis due to concurrent radiotherapy and cisplatin for head and neck cancer[END_REF] have advanced to several clinical trials, for anticancer (as sensitizer or radioprotector) or anti-inflammatory applications. Some recent applications of SOD mimics in the context of COVID were investigated to reduce inflammation and cytokine storm [START_REF] Karlsson | May mangafodipir or other SOD mimetics contribute to better care in COVID-19 patients?[END_REF], undergoing clinical trials (NCT04555096). Overall, the story of SOD mimics and recent developments show that both tool box of the chemists and the knowledge of the cellular and biological pathways are important in the development of compounds mimicking SODs.

Papers of particular interest, published within the period of review, have been highlighted as: * of special interest * * of outstanding interest 1. Note that superoxide can also be written O 2 to stress its radical nature, but this is not mandatory. Similarly, one could write O 2 to insist on the bi-radical nature of dioxygen, but this is rarely used. . Martínez-Camarena Á, Llinares JM, Domenech-Carbó A, Alarcón J, García-España E: A step forward in the development of superoxide dismutase mimetic nanozymes: the effect of the charge of the surface on antioxidant activity. RSC Adv 2019, 9:41549-41560. An 8-fold enhancement in SOD activity was demonstrated with several binuclear Cu 2+ azacyclophanes complexes were grafted onto boehmite nanoparticles. This effect was attributed to their positive z-potential since no similar outcome was observed with silica nanoparticles having a negative z-potential. The reproduction of the positively charged funnel of native SODs to attract superoxide seems to be a promising strategy to improve the SODm abilities of existing mimics. The acidity in an aqueous medium of highly charged Mn(P) and Fe(P) (+9 and + 8) with similar redox potentials was found to be different and thought to be important for the protonation of coordinated substrates in the catalytic cycle. In addition to the overall charge to attract negatively charged substrates, the substitutional lability of axial water ligands should play a decisive role in ligand binding for an inner-sphere pathway.

discovery of an efficient peptidyl copper complex Pep1 that exhibited good stability, suitable redox potential, and excellent intrinsic activity. Cell assays showed that Pep1 had beneficial effects against oxidative stress. Interestingly, this complex was found to be inactive as a catalase mimic, while another which was selected with a catalase assay (in another paper [38]) was inactive for SOD activity, validating the specificity of the methodology with regards to activity. This study on an SOD mimic (Mn1) with intestinal epithelial cells (IEC) included its bioactivity and investigation of the intracellular fate (quantification by EPR in acidified cell lysates, localization by micro-Xfluorescence (XFM) in freeze-dried cells, speciation in cell lysates using MS/MS). In IEC activated by LPS, Mn1 showed antiinflammatory activity (as measured by IL8 and COX2 levels). An increase in active MnSOD expression upon LPS was recorded, which was not observed by co-incubation of LPS with Mn1, indicating that Mn1 acted as a direct antioxidant and is able to compensate for MnSOD. Mn1 was also able to reduce inflammation in vivo in chemoinduced acute colitis in mice. Spectroscopic and magnetic analyses of these materials confirmed the metal centers of the complexes have kept the coordination sphere after insertion into the MCM-41 silica matrix. These complexes@MCM-41 showed a SOD activity increased by one order of magnitude in comparison to the nonencapsulated complexes (see ref. [START_REF] Daier | Synthesis, characterization and activity of imidazolate-bridged and Schiff-base dinuclear complexes as models of Cu,Zn-SOD. A comparative study[END_REF]). This enhancement SOD was attributed to a matrix effect, the close-fitting of the complexes into the nanochannels of MCM-41 silica favoring the Cu active site and HImZn(or Cu) group to stay in close proximity during catalysis. 61. Yang P, Tao J, Chen F, Chen Y, He J, Shen K, Zhao P, Li Y: Multienzyme-mimic ultrafine alloyed nanoparticles in metal organic frameworks for enhanced chemodynamic therapy. Small 2021, 17:2005865.

Mathieu E, Bernard

Patriarca

*

. Zhang L, Zhang Y, Wang Z, Cao F, Sang Y, Dong K, Pu F, Ren J, Qu X: Constructing metal-organic framework nanodots as bio-inspired artificial superoxide dismutase for alleviating endotoxemia. Mater Horiz 2019, 6:1682-1687. A Cu-Pd@MIL101 MOF pegylated was shown to display a 'multienzyme'-like activity, namely, peroxidase from pH 4 to 6, SOD, and capacity to deplete GSH. Its cellular uptake was determined along with the HO formation under a hypoxic tumor environment. The SOD-like nanozymes produced a higher flow in H 2 O 2 . In the tumor environment, where the level of H 2 O 2 is close to the toxic threshold, this can lead to toxicity, whereas no toxicity is seen in noncancerous cells, which have a lower level of H 2 O 2 . The nanozymes showed toxicity for breast cancer cell, with increase in lipid peroxidation, but had no effect on the proliferation of normal cells (HUVEC and LO 2 ). These nanozymes derived from MOF are thus promising for tumor chemotherapy. 129-138. Hematopoietic stem/progenitor cells (HSPCs) are known to be sensitive to dioxygen, with a prolonged lifetime under hypoxia. A redoxactive SOD-mimic, MnTnBuOE-2-PyP 5+ (Cp3), which has been shown in an earlier work to accumulate in the mitochondria, was found to increase the number of bone marrow HSPCs in freshly isolated mouse bone marrow cells or mice and to improve the function of longterm engraftment and multilineage differentiation of the HSCs; these can be associated with ROS regulation. MnP also induced a reduction in oxidative phosphorylation. Using an oxidative stress transcription factor activation profiling array and quantification of the protein under Nrf2-control, it was shown that Cp3 (20 mM, 1h, 5% O 2 , bone marrow nucleated cell) significantly upregulated the Nrf2/ARE system (ARE = antioxidant response element). These effects suggested that Cp3 acted as a mild pro-oxidant, elevating H 2 O 2 in bone marrow cells.

Figure 1

 1 Figure 1

  This figure shows the un-even and punctuated distribution in cells of Cp30. It was colocalized with a Mito Tracker previously mapped in classical fluorescence (not shown). The merging of Re and Mn maps shows areas where Re does not colocalize with Mn, revealing that partial decoordination occurred. 2b. Confocal fluorescence images of HT29-MD2 cells incubated with a mitochondrial marker (MitotrackerTM, fluorescence) and X-fluorescence maps of Cp30 (Re, X-fluorescence). Intestinal epithelial cells HT29-MD2 were incubated for 1h30 with Cp30 (100 mM, 0.02% DMSO) before the addition of MitotrackerTM deep red (200 nM, 30 min), washing with 50 mM EDTA, cryofixation, and freeze-drying. Confocal fluorescence images of MitotrackerTM deep red (ex 633 nm, em 645-750 nm) were recorded with an open pinhole. X-fluorescence (Re) images were recorded on the Nanoscopium beamline of SOLEIL synchrotron (excitation at 14.1 keV;

  (b) Confocal fluorescence microscopy image of the pyrene tag (blue) in living cells (bright field) treated with Cp18. (c) High-resolution elemental maps of manganese in chemically fixed (PFA) cells. Figure 2a and b from Ref. [42] and Figure 2c from Ref. [79].

  2. Sheng Y, Abreu IA, Cabelli DE, Maroney MJ, Miller A-F, Teixeira M, Valentine JS: Superoxide dismutases and superoxide reductases. Chem Rev 2014, 114:3854-3918. 3. Wang Y, Branicky R, No€ e A, Hekimi S: Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling. J Cell Biol 2018, 217:1915-1928. 4. Gardner PR, Rainer I, Epstein LB, White CW: Superoxide radical and iron modulate aconitase activity in mammalian cells. J Biol Chem 1995, 270:13399-13405. 5. Gleason JE, Galaleldeen A, Peterson RL, Taylor AB, Holloway SP, Waninger-Saroni J, Cormack BP, Cabelli DE, Hart PJ, Culotta VC: Candida albicans SOD5 represents the prototype of an unprecedented class of Cu-only superoxide dismutases required for pathogen defense. Proc Natl Acad Sci USA 2014, 111:5866-5871. 6. Policar C: Mimicking SODs, why and how: bio-inspired manganese complexes as SOD mimics. In Redox active therapeutics. Edited by Batini c Haberle I, Rebouças JS, Spasojevi c IHaberle I, Tome ME: Thiol regulation by Mn porphyrins, commonly known as SOD mimics. Redox Biol 2019, 25:101139. A very comprehensive review on the cellular effects of Mn-porphyrins, covering their diverse non-redox dependent reactivity. In particular, their potential in the modulation of the cellular redox regulatory signaling networks via proteins S-glutathionylation is delineated. 8. Durot S, Policar C, Cisnetti F, Lambert F, Renault J-P, Pelosi G, Blain G, Korri-Youssoufi H, Mahy J-P: Series of Mn complexes based on N-centered ligands and superoxidereactivity in * *

  24. Domergue J, Pécaut J, Proux O, Lebrun C, Gateau C, Le Goff A, Maldivi P, Duboc C, Delangle P: Mononuclear Ni(II) complexes with a S3O coordination sphere based on a tripodal cysteine-rich ligand: pH tuning of the superoxide dismutase activity. Inorg Chem 2019, 58:12775-12785. 25. Kenkel I, Franke A, Dürr M, Zahl A, Dücker-Benfer C, Langer J, Filipovi c MR, Yu M, Puchta R, Fiedler SR, et al.: Switching between inner-and outer-sphere PCET mechanisms of smallmolecule activation: superoxide dismutation and oxygen/ superoxide reduction reactivity deriving from the same manganese complex. J Am Chem Soc 2017, 139:1472-1484. 26 * . Franke A, Scheitler A, Kenkel I, Lippert R, Zahl A, Balbinot D, Jux N, Ivanovi c-Burmazovi c I: Positive charge on porphyrin ligand and nature of metal center define basic physicochemical properties of cationic manganese and iron porphyrins in aqueous solution. Inorg Chem 2019, 58:9618-9630.

  38. Coulibaly K, Thauvin M, Melenbacher A, Testard C, Trigoni E, Vincent A, Stillman MJ, Vriz S, Policar C, Delsuc N: A di-copper peptidyl complex mimics the activity of catalase, a key antioxidant metalloenzyme. Inorg Chem 2021, 60:9309-9319. 39 * * . Ward MB, Scheitler A, Yu M, Senft L, Zillmann AS, Gorden JD, Schwartz DD, Ivanovi c-Burmazovi c I, Goldsmith CR: Superoxide dismutase activity enabled by a redox-active ligand rather than metal. Nat Chem 2018, 10:1207-1212. An unusual Zn(II) SODm complex, with a redox-inactive Zn metal center, was shown to exhibit strong superoxide dismutase activity, which was attributed to the redox-active quinol ligand. The antioxidant activity was characterized using: (a) a McCord and Fridovich derived assay, a competitive assay using xanthine-xanthine oxidase to produce a slow flow of superoxide and lucigenin or cytochrome c for detection, (b) a DPPH assay to characterize the radical quenching property, and (c) a stopped-flow full kinetics approach, showing that the catalytic reaction was first order in superoxide. A mechanistic pathway (UV-vis and MS) involving Zn(II)-ligand radical species was proposed. 40. Cabelli DE: Redox activity goes organic. Nat Chem 2018, 10: 1173-1174. 41 * * . Mathieu E, Bernard A-S, Delsuc N, Quévrain E, Gazzah G, Lai B, Chain F, Langella P, Bachelet M, Masliah J, et al.: A cell-penetrant manganese superoxide dismutase (MnSOD) mimic is able to complement MnSOD and exerts an antiinflammatory effect on cellular and animal models of inflammatory bowel diseases. Inorg Chem 2017, 56:2545-2555.

  68. Nrf2 stands for nuclear factor erythroid 2-related factor 2. [date unknown], 69. Keap1 stands for Kelch-like ECH-associated protein 1. [date unknown], 70. Yamamoto M, Kensler TW, Motohashi H: The KEAP1-NRF2 system: a thiol-based sensor-effector apparatus for maintaining redox homeostasis. Physiol Rev 2018, 98:1169-1203.

Table 1

 1 Names, abbreviations, redox potentials, and catalytic activities of SOD mimics mentioned here.

	SOD mimics	Abbreviation	E 1/2	logk cat (O 2 .-): method detector	References
				(mV vs. NHE)	(pH) = logk cat (buffer)
	Porphyrins
	Mn III TE-2-PyP 5+	Cp1	+228	MCF cyt c (7.8) = 7.76 (P)	[20,86]
	Mn III TnHex-2-PyP 5+	Cp2	+314	MCF cyt c (7.8) = 7.48 (P)	[20]
	Mn III TnBuOE-2-PyP 5+	Cp3	+277	MCF cyt c (7.8) = 7.83 (P)	[12]
	Mn III TBAP 3-	Cp4	-194	MCF cyt c (7.8) = 3.16 (P)	[20,86]
	Fe III (P 2+ ) (H 2 O) (OH) 4+	Cp5	+298	-	[26]
	Mn III (P 2+ ) (H 2 O) 2 5+	Cp6	+270	-	[26]
	Fe III (P 6+ ) (H 2 O) (OH) 8+	Cp7	+249	-	[26]
	Mn III (P 6+ ) (H 2 O) 2 9+	Cp8	+200	-	[26]
	PAM
	[Cu II L1(OH 2 )] 2+	Cp9	-	MCF NBT (7.4) = 7.14 (T)	[28]
	[Cu II L2(OH 2 )] 2+	Cp10	-	MCF NBT (7.4) = 6.86 (T)	[28]
	Cu II	2 -L3	Cp11	+57	MCF NBT (7.4) = 6.73 (H)	[23]
	Cu II	2 -BNP-L3	Cp12	-	MCF NBT (7.4) = 7.63 (H)	[23]
	Cu II	2 -SNP-L3	Cp13	-	MCF NBT (7.4) = 6.09 (H)	[23]
	Cu II	2 -L4	Cp14	-33	MCF NBT (7.4) = 7.07 (H)	[23]
	Cu II	2 -BNP-L4	Cp15	-78	MCF NBT (7.4) = 7.56 (H)	[23]
	Cu II	2 -SNP-L4	Cp16	-	MCF NBT (7.4) = 6.98 (H)	[23]
	Mn II pyane	Cp17	-	SF(7.4) = 7.44 (H)/6.92 (P)	[79]
	Mn II pyane-pyrene	Cp18	-	SF(7.4) = 6.98 (H)/6.46 (P)	[79]
	Mn II pyane-rhodB	Cp19	-	SF(7.4) = 7.08 (H)/6.54 (P)	[79]
	Mn II M40403 (Imisopasem)	Cp20	+525	PR(7.4) = 6.55 (P)	[30]
	Mn II GC4419 (Avasopasem)	Cp21	-	-	-
	Mn II 3,6-PC2A	Cp22	+925	MCF NBT (7.8) = 5.01 (P)	[22]
	Mn II 3,9-PC2A	Cp23	+794	MCF NBT (7.8) = 4.29 (P)	[22]
	Amine Centered
	[Mn II (TMIMA) 2 ] 2+	Cp24	+704	MCF cyt c (7.8) = 6.56 (P)	[8]
	[Mn II (BMPG)] +	Cp25	+549	MCF cyt c (7.8) = 6.68 (P)	[8]
	[Mn II (IPG)] +	Cp26	+759	MCF cyt c (7.8) = 6.28 (P)	[8]
					PR(7.8) = 6.58 (P)
	[Mn II (BIG)] +	Cp27	+789	MCF cyt c (7.8) = 6.18 (P)	[8]
					PR(7.8) = 6.15 (P)	[87]
	[Mn III (PI) 2 ] +	Cp28	+514	MCF cyt c (7.8) = 6.82 (P)	[8]
	Salans
	Mn II 1 +	Cp29	+440	MCF cyt c (7.8) = 6.85 (P)	[42]
					SF(7.4) = 6.70(H)	[88]
	Mn II 1-Re +	Cp30	+487	MCF cyt c (7.8) = 6.81 (P)	[42]
					SF(7.8) = 6.81 (M)
	Mn II 1-R 9 10+	Cp31	+419	SF(7.4) = 6.70 (H)	[43]
	Mn II 1-RW 9 7+	Cp32	-	SF(7.4) = 7.24 (H)	[43]
	Mn II 1-MPP 3+	Cp33	+404	SF(7.4) = 7.34 (H)	[43]
	[Zn II (Hqp1)] +	Cp34	+305	SF(7.4) = 6.53 (H)/7.28 (P)	[39]
	[Mn II (ptp1) (H 2 O)] +	Cp35	-	SF(7.4) = 6.56 (H)/6.04 (P)	[46]
	[Mn II (Hqp1) (H 2 O)] +	Cp36	-	SF(7.4) = 7.99 (H)/6.90 (P)	[46]
	[Mn II (H 2 qp2) (H 2 O)]	Cp37	-	SF(7.4) = 7.08 (H)/7.00 (P)	[46]
	[Mn II (H 3 qp2) (H 2 O)]+
	Salens
	-		Cp38	+91	MCF NBT (7.8) = 6.19 (P)	[27]
	-		Cp39	+159	MCF NBT (7.8) = 6.18 (P)	[27]
	-		Cp40	+274	MCF NBT (7.8) = 5.97 (P)	[27]
	-		Cp41	+315	MCF NBT (7.8) = 6.00 (P)	[27]
	-		Cp42	+454	MCF NBT (7.8) = 5.81 (P)	[27]
	-		Cp43	+720	MCF NBT (7.8) = 6.07 (P)	[27]
	Bimetallic Cu-Zn and Cu-Cu
	[Cu II Zn II (dien) 2 (m-Im)](ClO 4 ) 3	Cp44	-	MCF NBT (7.8) = 6.81 (P)	[89]
	[Cu II Zn II (dien) 2 (m-Im)](ClO 4 ) 3 @MCM-41	Cp45	-	MCF NBT (7.8) = 7.79 (P)	[47]
	[Cu II Zn II (dien) 2 (m-Im)](ClO 4 ) 3 @SBA-15	Cp46	-	MCF NBT (7.8) = 7.40 (P)	[48]
	[Cu II 2 (dien) 2 (m-Im)](ClO 4 ) 3	Cp47	-	MCF NBT (7.8) = 6.89 (P)	[89]
	[Cu II 2 (dien) 2 (m-Im)](ClO 4 ) 3 @MCM-41	Cp48	-	MCF NBT (7.8) = 7.71 (P)	[47]
	[Cu II 2 (dien) 2 (m-Im)](ClO 4 ) 3 @SBA-15	Cp49	-	MCF NBT (7.8) = 7.37 (P)	[48]
	Peptides
					(continued on next page)

with a significant enhancement of the antisuperoxide activity

[49]

, as opposed to the negatively charged silica that produced no enhancement (see Table

1

). This effect was attributed to the positively charged scaffold, which should help in driving O 2 d to the active site, mimicking the positively charged funnel of native enzymes

[23]

.

Table 1

 1

	(continued )
	SOD mimics	Abbreviation	E 1/2	logk cat (O 2 .-): method detector	References
			(mV vs. NHE)	(pH) = logk cat (buffer)
	OCP1-Cu II Ac-PDHKHHKH-NH 2	Pep1	+250	MCF XTT (7.4) = 7.38 (H)	[37]
	CATm1-Cu II 2 Ac-PHYKHRLH-OH	Pep2	+345	-(no SOD activity, but	[38]
				a catalase activity)

  Metal-based nanomaterials display a high density in metal centers that can act as catalytically active redox sites for catalysis[50]. The SOD-like activities of these materials were evaluated qualitatively or reported per mole of NPs, as k cat or in the percentage of SOD activity (see Table1).

	However, these values are difficult to
	compare with those determined for molecular catalysts,
	as there is a high number of accessible catalytic sites per
	NPs. Nonetheless, these materials display valuable
	antioxidant activity, approaching those of SOD on a per
	mole NPs basis. After the pioneering work of Self et al. on
	ceria nanoparticles [51], later encapsulated in various
	protein scaffolds [52,53], Mn-based nanoparticles (e.g.
	Mn 3 O 4 ) [54e56] or Fe-based Prussian blue [57e59]
	were recently characterized for their multiple catalytic
	activitiesdSOD, catalase and GSH-peroxidasedpos-
	sibly retained in cells or in vivo [55]. Interestingly, Singh
	et al. have evidenced that the supernatants of Mn 3 O

4 NPs were not active, nicely ruling out the possible involvement of leached ion in the activity

[55]

. The activities were dependent on the shape and size of the particles

[55]

. In these condensed systems, the clustering effect of the substrate, proposed in another context

[45]

, could be at stake. Metaleorganic frameworks (MOF) have also been used as a scaffold to build metalloenzyme mimics [50], and a few MOF have been designed, either with dispersed copper [61] or using porphyrin (TBAP, see Scheme 1 for the porphyrine acronym) and copper(II) as building blocks

[62]

with activity reported to 12.5% that of SOD. These MOF are endowed with multiantioxidant activities, but also Fenton pro-oxidant effect

[61,62]

. Some of these materials have been applied as antioxidants or antiinflammatory agents [54e57,59,62], others as anticancer agents, their anticancer activity being related to toxicity associated with pro-oxidant effect due to the overproduction of H 2 O 2 and/or of

HO [60,61]

  63. Batinic-Haberle I, Tovmasyan A, Spasojevic I: Mn porphyrinbased redox-active drugs: differential effects as cancer therapeutics and protectors of normal tissue against oxidative injury. Antioxidants Redox Signal 2018, 29: 1691 -1724. 64. Tse HM, Milton MJ, Piganelli JD: Mechanistic analysis of the immunomodulatory effects of a catalytic antioxidant on antigen-presenting cells: implication for their use in targeting oxidation-reduction reactions in innate immunity. Free Radic Biol Med 2004, 36:233-247. 65. Jaramillo MC, Briehl MM, Crapo JD, Batinic-Haberle I, Tome ME: Manganese porphyrin, MnTE-2-PyP5+, acts as a pro-oxidant to potentiate glucocorticoid-induced apoptosis in lymphoma cells. Free Radic Biol Med 2012, 52:1272-1284. 66. Jaramillo MC, Briehl MM, Batinic-Haberle I, Tome ME: Manganese (III) meso-tetrakis N-ethylpyridinium-2-yl porphyrin acts as a pro-oxidant to inhibit electron transport chain proteins, modulate bioenergetics, and enhance the response to chemotherapy in lymphoma cells. Free Radic Biol Med 2015, 83:89-100. Carroll DW, You Y, Chaiswing L, Wen R, Batinic-Haberle I, Bondada S, Liang Y, St Clair DK: A novel redox regulator, MnTnBuOE-2-PyP5+, enhances normal hematopoietic stem/progenitor cell function. Redox Biol 2017, 12:

	67	. Zhao Y,
	* *

Acknowledgments

The authors wish to acknowledge ENS-PSL, PSL, Sorbonne University, CNRS, ANR, and Qlife for financial support. JB thanks the program Interface pour le vivant (IPV-Sorbonne Universite ´) for his PhD fellowship. Fundings for research on the topic of SOD mimics by the research group 'Metals in Biology d Inorganic Biological and Cellular Chemistry' (https:// www.chimie.ens.fr/bic/), include the projects MAGIC (ANR-15-CE07-0027), METALLOPEPZYME (ANR-16-CE07-0025-01), CATMAN (ANR 20-CE07-0039-01), MAIN (Qlife initiative, ANR Q-life ANR-17-CONV-0005), DEI20151234413 (Fondation pour la recherche me ´dicale), and BACTMAN and ANACOMDA (Mission pour les initiatives transverses et l'interdisciplinarite ´-CNRS) and by a research fellowship from the Association Franc ¸ois Aupetit (AFA) (associations of French patients suffering from inflammatory bowel diseases).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.