Saidu Sani

Mélissa Messe

Quentin Fuchs

Marina Pierrevelcin

Patrice Laquerriere

Natacha Entz-Werle

Damien Reita

Nelly Etienne-Selloum

Véronique Bruban

Laurence Choulier

Natacha Entz- Werle

Sophie Martin

Monique Dontenwill
email: monique.dontenwill@unistra.fr

Biological Relevance of RGD-Integrin Subtype-Specific Ligands in Cancer

teaching and research institutions in France or abroad, or from public or private research centers.

resolved. Selective ligands may help to reconsider integrins as therapeutic targets in specific clinical settings.

INTRODUCTION

Integrins are heterodimeric transmembrane proteins recognized first as adhesion molecules on specific extracellular matrix (ECM) components. They were further assigned to a true receptor family as their ability to initiate intracellular signaling pathways was largely emphasized. In the hallmarks of cancer as defined by Hanahan and Weinberg in 2000 [1] , integrins were acknowledged as actors of self-sufficiency growth signaling, as regulators of anti-apoptotic signaling, angiogenesis and adaptation to new environments during invasion and metastasis of tumor cells. New hallmarks were added ten years later [2] ,in particular, the contribution of the tumor microenvironment (TME) to tumorigenesis. The TME does not only contain ECM but also recruits immune and stromal cells able to facilitate tumor growth. Changes in the repertoire and expression level of integrins in these non tumoral cells were described to support aggressive tumoral phenotypes [3] . Integrins were recognized early as therapeutic targets in cancers [4][5][6] . The most studied integrins in oncology belong to the RGDintegrin subfamily with αvβ3/β5 and α5β1 integrins in the spotlight for the two last decades [7] . The RGD (Arg-Gly-Asp) peptidic motif is found in ECM proteins (fibronectin, vitronectin, osteopontin, thrombospondin among others) and represents a selective binding site for integrins to ECM. Since activation of integrins by adhesion to their ECM ligands was shown to be essential to non-tumoral adherent cell survival [8,9] , similar effect was expected for tumoral cells. Disrupting the interactions between integrins and ECM through RGD-like compounds appeared therefore a valuable strategy to trigger tumor cell apoptosis. Research in the design and characterization of integrin antagonists as cancer drug candidates has expanded rapidly [10] . Cilengitide, a cyclic RGD peptide, was the first αvβ3/β5 integrin antagonist that reached the clinic [11] . Despite numerous preclinical encouraging data, cilengitide failed to improve glioblastoma patient's survival in phase II/III randomized clinical trials [12,13] . No better outcomes were obtained with Volociximab or MINT1526A, both α5β1 integrin inhibitory antibodies, in several clinical settings [14,15] . These disappointing results impeded considerably research on RGD-integrins as therapeutic targets in oncology. However, recent reviews on integrins and cancer emphasize the need to reconsider the topic [6,7,[16][17][18][19][20] . In this review we will focus mainly on RGD-integrins αvβ3 and α5β1 as most ligands have been designed for them. We will support the idea that integrins should still be considered as targets for anticancer therapies. In addition, ligands specific for definite subtypes of integrins will help in understanding how and where integrins must be targeted and will shed more light on the complexity of the topic.

RGD-integrins in cancer

2.1.

Integrins in focal adhesion and cell migration

Common rules of integrins have been extensively reviewed elsewhere (for a recent review see [21] ). In human, 18 α and 8 β subunits can combine to form heterodimeric transmembrane proteins with specific ligand recognition properties. They share an architecture comprising an extracellular domain, a transmembrane region, and a generally short intracellular part. If specific adhesion to ligands lies in the composition of the αβ extracellular head, integrins signal inside the cells by structural changes and recruitment of cytoplasmic proteins in complexes named adhesomes [22] . Such complexes contain about 2000 proteins among which a highly dynamic "consensus adhesome" of 60 proteins may control integrin dependent cell adhesion and signaling [23] . Mechanical forces can influence the assembly/disassembly of these macro-molecular structures adding a further level of complexity [24] . Recent work pointed that β3 integrins were more strongly bound to their substrate in adhesion sites submitted to high tension than β1 integrins [25] . The dynamic nanoscale organization of integrins and their regulators within focal adhesion points was shown to differ between β3and β1-integrins in the control of signaling during cellular functions [26,27] . Although both integrins recognize fibronectin, striking differences between α5β1 and αvβ3 integrin signaling pathways have been described in normal/tumoral cells. Most data addressed the question of focal adhesion point maturation and cell migration. αvβ3 integrins were associated with persistent migration through activation of Rac-mDia1, whereas α5β1 integrins were linked to RhoA-Rock-MyoDII pathway and random migration [28] . The role of both fibronectin-binding integrin classes can begin with an initial competition followed by a cooperative crosstalk [29] .

Alternatively, blocking αvβ3 integrin may activate α5β1 integrin recycling back to membrane and increase cell migration [30] . Distinct mechanisms and/or efficient crosstalk between both integrins have emerged over the years [31] . New insights on focal adhesion maturation have been made possible using ligands specifically designed to target either αvβ3 or α5β1 integrins. It was shown that focal adhesion maturation on α5β1 integrin-selective substrates is dependent on αvβ3 integrin recruitment [32][33][34] . Most of the above described knowledges were obtained in non-tumoral cells and translation to cancer cells may be even more decisive for their functional behavior as the expression of integrins and the ratio between αvβ3 and α5β1 integrin levels are specifically altered in tumoral tissues as is the case for the expression of their common ECM ligand fibronectin [35] .

Integrins in hallmarks of cancer

Thirty years ago integrin αvβ3 emerged as a marker of tumoral neo-angiogenesis which was inhibited by specific antagonists such as cilengitide in preclinical models [36][37][38] . These results were challenged as enhanced pathological angiogenesis and increased primary tumor growth were observed in mice lacking β3 or β3/β5 integrins [39] . Specific overexpression of Integrin β3 in tumor cells suppressed tumor growth in a human model of gliomagenesis [40] . Additionally, it was shown that nanomolar concentrations of RGD-mimetic αvβ3/αvβ5 inhibitors can enhance the growth of tumors in vivo by promoting VEGF-mediated angiogenesis [41] . These data may explain in part why the therapies with these inhibitors have failed in humans [12,13] . Other integrins participate in angiogenesis [42] and the proangiogenic function of α5β1 integrin has been clearly demonstrated [43][44][45] . It was shown that reduced expression of the α5 subunit is associated with reduced blood vessel formation and tumor growth [46] and that α5β1 integrin was overexpressed in tumoral vessels [47] . It will be essential to reconsider and to revisit fundamental biology underlying how these integrins coordinate angiogenesis to successfully target them [48] .

Beside their role in angiogenesis, both integrins are overexpressed in many tumoral cells and thought to participate in enhanced survival and resistance to therapies [7,20,[49][50][51][52] . This overexpression is mainly characterized at the mRNA level but less data exist at the protein level in cohorts of patients. The large transcriptomic databanks available nowadays for different tumor types help not only to define the inter-patient heterogeneity but also to correlate specific gene overexpression in tumor subtypes to patient survival. They have been used for high grade brain tumors (glioblastoma, GBM) for example [53] . In these tumors, α5 and β1 integrin mRNAs are overexpressed in GBM versus normal brain but even more in the GBM mesenchymal subgroup [54] ; it was less subgroup dependent for αv and β3 integrin mRNA [55] . α5 and β1 genes are included in the GBM mesenchymal signature so that they represent biomarkers of this aggressive subtype. Interestingly α5 integrin is also overexpressed in subclones of pediatric brain tumors [56] . Extensive evaluation and comparison of integrin gene expression in different tumors and tumor subtypes would be a first step towards efficient antiintegrin therapeutic strategies. Even if determination of integrin proteins in cell lines is a prerequisite to characterize functional implication in vitro, the missing link is however the characterization of integrins at the protein level in clinical studies. Few data on small-to medium-sized patient cohorts are available in the literature. Immunohistochemical analysis of αv or β3 integrin protein expression in such cohorts were recently reported in osteosarcoma [57] , hepatocellular carcinoma [58] , leukemia [59] and the relationships with patient survival established. A retrospective immunohistochemical analysis of αvβ3, αvβ5 or αvβ8 integrins in the glioblastoma patient cohorts of cilengitide clinical trials was done. It revealed that only αvβ3 integrin expression in tumoral cells may predict benefit from cilengitide inhibition in a subset of patients with glioblastoma lacking MGMT promoter methylation [60] . Similarly, high levels of α5 or β1 integrin protein expression corresponded to worse survival in oral carcinoma [START_REF] Deng | α5/ITGA5 Promotes The Proliferation, Migration, Invasion And Progression Of Oral Squamous Carcinoma By Epithelial-Mesenchymal Transition<[END_REF] , triple negative breast cancer [START_REF] Ou-Yang | [END_REF] , pancreatic cancer [63] , osteosarcoma [64] and colorectal cancer [65] . In the last period, data concerning integrin expressions and functions in nontumoral cells in the microenvironment of tumors have been increasingly reported. Roles of integrins in Cancer Associated Fibroblasts (CAF) have been reviewed recently [66] . Engineered mice models allowed deletion of integrin gene in specific population of cells. It was shown that acute deletion of β3 integrin in endothelial cells transiently diminished tumor growth and angiogenesis but long term deletion was ineffective [67] . Specific deletion of this gene in myeloid cells resulted in enhanced tumor growth [68] and increase of M2 macrophages at the tumor site thus promoting pro-tumoral functions in the microenvironment [69] . In another work, it was shown that tumor-associated -macrophages (TAM) accumulation correlated with tumor cell expression of αvβ3. This characteristic was used to design an αvβ3 antibodydependent cellular cytotoxicity (ADCC) against tumoral cells [70] . Two recent works underlined the expression of α5 integrin in the stroma of pancreatic [71] and colorectal tumors [72] . In the former study, inhibition of this integrin in pancreatic stellate cells attenuates tumorigenicity and potentiates efficacy of chemotherapy; in the latter, integrin depletion reduced the ability of CAF to promote cancer cell migration presumably by the down regulation of fibronectin.

All the non-exhaustive recent findings described in the first part of this review suggest that we need to better understand the roles of RGD-integrins in tumors. Encouraging preclinical discoveries with integrin inhibitors did not translate to clinical successes due to the complexity of integrin biology. We will have to consider not only integrins in tumoral cells and vessels but also in the tumor microenvironment including the immune system (Figure 1). For this point, preclinical modeling of the clinical reality remains a challenge. The overexpression of some integrins in treatment-resistant cells or at metastasic sites must be thoroughly characterized.

We will have to refine the positive or negative crosstalk between different integrins and their relationships in specific tumor area or in specific subset of patients. We need to compare different integrin subtypes more systematically to address their respective contribution to tumorigenicity and optimize the use of specific inhibitors. The availability of specific and selective integrin ligands is of great importance to go further on.

RGD-integrin ligands

Three main classes of integrin ligands have been proposed: antibodies, RGD-derived peptides and RGD-mimetic small molecules (Figures 2 and3). More recently, aptamers (short nucleic acid sequences) have been included in this list [73,74] . While antibodies may be highly integrin subtype-specific by nature, RGD-derived ligands may recognize all RGD-binding integrins. Efforts towards achieving high selectivity of RGD-integrin ligands were actively made during the last two decades with, for example, the pioneer works of H. Kessler's group. Although recognizing the same tripeptide sequence in their natural ligands, similarities and differences between RGD-integrins have been highlighted based on their crystal structures. The structures of the extracellular fragment of integrins αvβ3, αIIbβ3 and α5β1 were respectively resolved in 2001 [75,76] , 2004 [77] and 2012 [78] helping to understand how ligands fit in the integrin binding sites.

3.1. Specificity, selectivity, and activity of integrin ligands.

Until today, integrin ligands were mainly tested for their capacity to bind integrins and to disrupt their interactions with their preferred natural ligands, using as a readout the role of integrin as adhesion proteins. For the evaluation of large libraries of compounds, tests must be rapid and reliable for lead characterization and optimization. They are based either on cell adhesion assays or on purified soluble integrins adhesion assays on specific ECM substrates. Results (expressed as IC50 values) obtained by both approaches are strongly variable depending on several parameters. Rational design of such tests appears crucial to compare ligands for one integrin or one ligand for different integrins. Recent works give insights in this topic [79,80] . Concerning activity, the notion of integrin antagonism versus agonism is fairly taken into account in the first steps of ligand selection. This point becomes critical in the field of oncology where disruption of cell adherence does not automatically relate to cell death. Moreover, RGD-like compounds may behave as true agonists mimicking the ECM natural ligand-dependent activation of integrins and subsequent pro-tumoral signaling pathways [41] . Very interestingly, recent developments on integrin pure antagonists have been published supporting a new area in integrin ligand research [81][82][83][84] . Based on electron microscopy, X-ray chrystallography and receptor priming studies, it was shown that these new classes of integrin antagonists do not induce the integrin conformational changes associated with activation. Specific antibodies against RGD-integrins contributed to study integrin activation-state regulation, integrin biology and integrin-based therapeutics. In contrast with peptides or peptide-mimetics acting essentially at the RGD binding domain, they cover a large set of epitopes localized in different structural parts of the α or β integrin subunits [85] . They can be classified into three main groups: stimulatory or activation-specific, inhibitory and nonfunctional antibodies as largely described by the group of M. Humphries [86][87][88] . It must be emphasized here that integrin conformations are known to be variable in relation to their functional status which addressed mainly their capacity to adhere to ECM ligands. The bent, the extended closed and the extended open global conformations are likely to be shared by the majority of integrins. Using electron microscopy and biophysical thermodynamic analysis on α5β1 integrin and with the help of specific antibodies, it was shown that only the extendedopen conformation mediates adhesion to fibronectin and that intrinsic affinity depends on specific integrin conformational states [89,[START_REF] Su | Proc. Natl. Acad. Sci[END_REF] . Antibodies against α5β1 and αvβ3 integrins are thus widely used in preclinical studies to depict fundamental cues, label or inhibit these integrins. Among inhibitory antibodies, some reached early phases clinical trials (volociximab for α5β1 integrin and Intratumumab/CNTO95 or abituzumab for αvβx integrin) [START_REF] Almokadem | [END_REF][START_REF] Hussain | on behalf of the[END_REF] . But despite being well tolerated, they failed to progress to phase II/III trials.

Antibodies

RGD-derived peptides

Cilengitide, a cyclic pentapeptide, developed twenty years ago by Horst Kessler and his group, became the prototype of RGD-derived peptides with enhanced selectivity towards αvβ3 integrin. The story of cilengitide is extensively described in [93] explaining the different steps of development from a linear, flexible and non-selective RGD peptide to a cyclic, rigid and selective one. In the search for peptides with increased selectivity either towards αvβ3 or α5β1 integrins, multiple strategies have been explored using isoDGR motifs [94,95] , cyclic azapeptides [96] or di-N-methylation of cilengitide [97] . Cilengitide remains the reference compound in the field and integrin binding characteristics of new integrin ligands are often compared to it. Other groups worked around the cyclic pentapeptide structure. For example, cyclic RGD pseudopentapeptide incorporating bicyclic lactams were described [98] and further optimized leading to potent antagonists of αvβ3/β5 integrins [98][99][100][101] . Series of cyclooctapeptides including RGD have also been described. Based on One-bead One-Compound combinatorial library technology, the lead compound LXW7 was discovered [102] and optimized as LXW64 and LXZ2 [103,104] . These compounds proved able to selectively label tumoral cells expressing αvβ3 in vitro and in vivo. Recently, a novel specific integrin αvβ3 targeting linear pentapeptide, RWrNK, was described [105] . Its development used a structure-based pharmacophore method integrated with molecular docking. Bifunctional diketopiperazines were introduced into cyclic peptidomimetics containing the RGD sequence (DKP-RGD) leading to compounds specific for αvβ3/β5 integrins [106] . Interestingly, these compounds exhibited binding affinities towards α5β1 integrin but always lower than for αvβ3 integrin [107] . Thus using several approaches, numerous peptidic ligands specific/ selective for αvβ3/β5 integrins have been made available unlike those for α5β1 integrin. The isoDGR peptide library [95] was screened to confer to the small lead pentapeptide selectivity towards the fibronectin-binding integrins α5β1 and αvβ6 [94] . Through sequential N-methylation, the biselective c(phg-isoDGRk) was converted to c(phg-isoDGR-(NMe)k) which appeared as a selective α5β1 integrin ligand 10.1002/cbic.202000626

Accepted Manuscript

ChemBioChem

This article is protected by copyright. All rights reserved. [108] . It should be noted that other peptides highly selective for α5β1 integrin have also been proposed without an RGD sequence. The first example is the Cys-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys (CRRETAWAC) peptide originally discovered in 1993 from the screening of a phage-display library in which heptapeptides were flanked by cysteine residues, thus making the inserts potentially cyclic [109] . Further investigations showed that residues of the α5 subunit involved in recognition of RRETAWA are predicted to lie close to those involved in RGD binding but do not completely overlap [110] . The second example is ATN161, a capped five amino-acid peptide derived from the synergy region PHSRN of fibronectin (which contributes to high affinity recognition of fibronectin by α5β1 integrin). It was shown to be very selective for α5β1 integrin over αvβ3/β5/β6/β8 integrins in an ELISA test using soluble purified integrins [79] . Compared to other integrin ligands, ATN-161 has the particularity of not competing with the fibronectin binding and thus is unable to detach the cells from the matrix. It was included in two clinical trials (for advanced renal cancer and recurrent glioblastoma) but no results were posted to date.

Panels of selective RGD-derived peptides are available. Few of them have been studied as true antagonists of integrin signaling pathways and pro-tumoral effects. However, knowledge on the mode of binding of RGD-containing peptidomimetics has largely increased with the advent of computer-assisted docking studies in the crystal structure of the integrin binding sites. This helped and will further improve the design of new ligands.

Small molecules as RGD-mimetics.

In parallel with the design of RGD-derived peptides, search for RGD-mimetics, led to the discovery of selective more stable and bioavailable small molecules as integrin ligands. The first goal of research was to obtain selectivity for αvβ3 versus αIIbβ3 integrins. This latter RGDintegrin is involved in platelets regulation and undesirable antagonist side effects had to be avoided. The second goal was to evaluate the possibility of distinguishing the αvβ3 integrin from the α5β1 in order to have highly selective ligands even though their RGD binding site are highly homologous. Both goals were achieved by pharmaceutical and academic groups. A review recapitulated the αvβ3 antagonists available in 2000 [111] . The field has progressed and began to focus also on α5β1 integrin. The initial series of αvβ3 integrin ligands were generally also recognized by α5β1 integrin. A series of nonpeptide integrin ligands containing spirocyclic scaffolds was described including SJ749 and SJ755, the first highly selective molecules for α5β1 integrin [112,113] . Their biological effects have been characterized : they behave as potential enhancers of antibiotic efficacy by interfering with α5β1 integrin/fibronectin/M1 proteindependent bacterial entry in epithelial cells [START_REF] Cue | Proc. Natl. Acad. Sci[END_REF] , as blockers of angiogenesis [START_REF] Kim | [END_REF] and as inhibitors of proliferation and migration of glioma cells [116,117] . Docking of SJ749 into a built 3D model of the binding domain of α5β1 integrin has permitted the identification of two potentially important and unique regions of this integrin compared to others [118] . This model was largely used for the rational design of highly active and selective ligands for α5β1 and αvβ3 integrins achieving a selectivity ratio as high as 10000 [119][120][121] . The differentiation between the α5β1 and αvβ3 integrin-dependent glioma migration modes was possible using such ligands and revealed that αvβ3 integrin antagonists increased single cell migration whereas α5β1 integrin antagonists decreased it [122] . Additionally, we showed that, unlike αvβ3 integrin antagonists, those selective for α5β1 integrin pushed glioma cells towards apoptosis when combined with chemotherapy or p53 activators [123][124][125] . Starting from a virtual combinatorial library designed to cover the chemical space specific for RGD-like compounds, Stragies et al synthesized dual α5β1/αvβ3 specific inhibitors [126] . Optimization of one lead gave JSM6427 which exhibits 10000 times more affinity for α5β1 versus αvβ3 and showed inhibitory activity in several models of pathological angiogenesis [127][128][129] . It was further developed for the treatment of macular degeneration but did not overpass the phase 1 clinical trial.

More recently, new β-lactam derivatives were designed to target integrins. Interestingly, the concept of integrin agonists has gradually emerged since compounds with azetidinone as the only cyclic framework increased integrin-dependent cell adhesion rather than decreasing it [130] . Substituent variations around the β-lactam core led to the discovery of selective compounds for different integrins including α5β1 and αvβ3/β5. Similar selectivity ranges were obtained for these integrins by competitive solid-phase binding assays using purified integrins and cell-adhesion based assays [131,132] . Increase in cell adhesion by integrin "agonists" was corroborated by an increase in integrin-dependent ERK signaling pathway. First characterized as racemic compounds, it was demonstrated that only (S)-enantiomers maintain the agonist activity thus revealing an important stereochemical requirement for integrin recognition and activation [133] . Whether integrin agonists could play a role as therapeutic drugs in oncology remains to be demonstrated [134] .

Taking together all the data concerning these RGD-mimetic ligands, structural determinants discriminating different RGD-binding integrins have been characterized based on compound structure-activity relationships and computer-assisted docking on crystallographic integrin models. As is the case for the RGD-derived peptides, few of these original ligands are currently under investigations to check their potential anti-tumoral effects. The main knowledge concerns their ability to inhibit/increase integrin-dependent cell adhesion. Analysis of their roles on integrin signaling pathways has become an urgent need to more clearly decipher the biological cues of specific RGD-integrins in tumoral or surrounding stromal cells.

Current applications of RGD-ligands

Huge efforts to get more and more selective RGD-ligands led to interesting libraries of compounds. Due to the failure of the lead cilengitide to improve patient survival in clinical trials, none of them appears to be developed as anti-cancer therapeutics. Currently they are being exploited as useful tools either as tumor diagnostic or tumor targeting markers. They also serve for functionalization of biomaterials helping to solve several selective integrindependent cell phenotypes.

RGD-ligands for tumor diagnostic

As stated above, the input of RGD-integrins in hallmarks of cancer is largely acknowledged. Their heterogeneous expression in patient tumors closed the way to use their antagonists as therapeutics in unselected cohort of patients. Characterization of the integrin expression panel by a noninvasive way may be of importance to delineate patient subpopulations and adapt therapies. From early 2000 to 2015, huge efforts to characterize Positron Emission Tomography (PET) markers for αvβ3 integrin were made. Radiolabeled cyclic RGD peptides (with 18 F, 64 Cu/ 68 Ga tracers) were thoroughly designed and characterized (for review see [135] and [136] ). Several were investigated in the clinic [137][138][139] . By contrast very few radiotracers specific for α5β1 integrin are available (Table 1) as this integrin moved in the forefront of cancer research more recently, in particular for its unambiguous role in neoangiogenesis. The first α5β1-selective integrin antagonist useful as a PET tracer was described in 2013 by the group of H. Kessler [140] . A selective α5β1 integrin peptidomimetic (described in [119,141] ) was functionalized with the NODAGA chelator, labelled with 68 Ga and named FR366 [142] . FR366 has a high affinity for α5β1 integrin, a specific integrin uptake in vivo and a good tumor-tobackground contrast. A further step was achieved by trimerization of an azide-functionalized pseudopeptide [143] using a 1-pot click chemistry procedure with a TRAP chelator scaffold [144] . The compound 68 Ga-Aquibeprin obtained by this way has affinity and selectivity for α5β1 integrins and gave high contrast PET imaging in vivo [145,146] . A similar approach was used with c(phg-isoDGR-(NMe)k peptide (see above) which was conjugated to pentynoic acid on the lysine side chain and then trimerized by the TRAP chelator. In a proof of concept experiment this compound labeled with 68 Ga behaved as a potential PET agent for noninvasive imaging of α5β1 expressing tumors [108] . Other PET peptidic radiotracers for α5β1 integrin were proposed (based on CRETTAVAC or KSSPHSRN(SG)5RGDSP linear peptides) but they lack an efficient accumulation in α5β1 integrin-positive tumors in vivo [147,148] .

RGD-ligands for tumor targeting

Integrins appear as valuable entry door for anti-cancer drugs in a tumor selective way. Based on the overexpression of αvβ3 and α5β1 integrins in tumor vasculature and cells and their ability to get internalized through endosomes [149] , strategies have been developed to achieve specific transport for therapeutics. RGD-ligands coupled to cytotoxic drugs can serve as direct carriers and are intensively investigated. The drug is coupled to the RGD peptide by a linker which may be cleaved inside the cell to allow the therapeutic effect (for review see [150,151] .

Examples of RGD-ligands and cytotoxic drug complexes can be found in two recent reviews ( [152,153] . Recent advances in the field included dual-functional complexes that incorporate one fluorophore on one side for imaging and a cytotoxic pro-drug on the other side [154] . But currently intense efforts are mainly directed towards nanocarriers functionalized with RGDligands. We will not develop here this topic as recent reviews are available to which readers can refer [155][156][157][158][159][160][161] .

Although huge literature exists concerning the applications available with RGD-ligands for targeting tumors, they appear, to our knowledge, non-dedicated to address specifically the selectivity of RGD-integrins. The RGD-ligands mainly used in these studies have been designed to target αvβ3 integrins. From a biologist point of view, further improvements will be achieved when tailored systems will be proposed to target specifically either αvβ3 or α5β1 integrin expressing tumors.

Summary and outlook

In this review we focused on two RGD-integrins which are important players in oncology, the αvβ3 and α5β1 integrins. It is important to note that other RGD-integrins become under spotlight as for example the αvβ6 integrin [162] . Selective Inhibitors and PET tracers have been designed [163][164][165][166][167] which will be useful to detect and treat epithelial carcinoma for example. Integrins remain therapeutic targets in oncology (as assessed by 2 recent reviews [168,169] ) but we have to reconsider their roles in the area of personalized medicine.

Our goal in this review was to give the biologist point of view regarding the chemical approaches to design selective compounds differentiating RGD-integrins in preclinical and clinical settings. According to the data (even the recent ones), the αvβ3 integrin still remains the gold standard tumoral target. Efforts are however made towards other integrins. This is an important point in order to have a better knowledge of the complex integrin world. To go further, we have to set up a virtuous circle by increasing the knowledge on one particular integrin using selective ligands and inversely to take into account this knowledge to design new ligands with improved efficacy. The biological tests are crucial as are the preclinical models. It is time to progress towards models that better reflect the clinical reality to test integrin antagonists. For example, replacing 2D by 3D sphere cultures and hence by organoids/tumoroids to test integrin antagonists is nowadays accessible steps. In addition, we have to develop high/medium throughput assays aiming to characterize the biological effects of RGD-ligands on not only integrin/cell adhesion but also on oncogenic signaling pathways.

We have also to keep in mind that integrin antagonists/agonists may inhibit/activate general mechanisms which are under the control of several players. It will be of great interest to evaluate more thoroughly the benefit of their association with other targeted therapies.

Transdisciplinary networks involving biologists, clinicians and chemists will greatly help to go through preclinical investigations towards clinical benefits with integrin ligands. Cilengitide was the first RGD-containing cyclic peptide reaching the clinic for glioblastoma treatment [93]. LXW7 is a cyclooctapeptide described in [102]. C(phg-isoDGR-NMe)k) is described in [108]. SJ755 was one of the first small molecule specific for α5β1 integrin [112]. Compound 1 is described in [141] and compound 17 is an α5β1 integrin agonist [131].

Legends to figures

  radiotracers selective for α5β1 integrin. Different xenografted tumoral cell lines have been used with differential expression of α5β1 and αvβ3 integrins. RKO cells : α5β1 +, αvβ3 -; M21 cells: α5β1 +/-and αvβ3 +; DU145 cells: α5β1 +, αvβ3 -; B16-F10: α5β1+, αvβ3 +/-; SW48 cells: α5β1 -, αvβ3 +/-. 10.1002/cbic.202000626 Accepted Manuscript ChemBioChem This article is protected by copyright. All rights reserved.

Figure 1 :

 1 Figure 1: Integrins in hallmarks of cancer. Altered expressions of integrins are detected in tumor cells but also in non tumoral cells in the tumor microenvironment. Integrin-activated signaling pathways have pro-tumoral functions.

Figure 2 :

 2 Figure 2: RGD-integrin ligands. Integrins αvβ3 and α5β1 both recognize Fibronectin (FN). Ligands able to interfere with FN-integrin complexes have been developed mainly as integrin antagonists of cell adherence (RGD-based peptides, RGD-mimetics). Antibodies recognize epitopes in-or outside the RGD binding sites and are useful to mark integrins in inactivated or activated states. Non-RGD peptides have been developed based on the FN synergy site (PHSRN) recognized by α5β1 integrin.

Figure 3 :

 3 Figure3: Chemical structures of some RGD-integrin ligands. Cilengitide was the first RGD-containing cyclic peptide reaching the clinic for glioblastoma treatment[93]. LXW7 is a cyclooctapeptide described in[102]. C(phg-isoDGR-NMe)k) is described in[108]. SJ755 was one of the first small molecule specific for α5β1 integrin[112]. Compound 1 is described in[141] and compound 17 is an α5β1 integrin agonist[131].

  protected by copyright. All rights reserved.

  Figure 1

Figure 2 10.

 2 Figure 2

  Figure 3

This article is protected by copyright. All rights reserved.

Acknowledgements

We are thankful for financial support from Institut National du Cancer (grant INCA_11527), Ligue contre le Cancer (CCIR Est), Association pour la Recherche contre le Cancer, CNRS (grant 80 PRIME), the University of Strasbourg (France), the Alex Akwueme Federal University Ndufu-Alike Ikwo (Nigeria), the Association Lyons Club de Niederbronn Les Bains.

Conflict of interest

The authors have no conflict of interest to declare. Keywords : RGD-integrins, ligand design, inhibitors, peptides, cancer

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Caroline Norsica
email: caroline.norsic@univ-orleans.fr

Jean-Michel Tatibouëta
email: jean.michel.tatibouet@univ-poitiers.fr

Catherine Batiot-Dupeyrata
email: catherine.batiot.dupeyrat@univ-poitiers.fr

Elodie Fourréa
email: elodie.fourre@univ-poitiers.fr

Methanol oxidation in dry and humid air by dielectric barrier discharge plasma combined with MnO2-CuO based catalysts

Keywords: Non thermal plasma, humidity, methanol, oxidation, metal oxide, TPR, ozone Highlights

Methanol oxidation in dry and humid air by dielectric barrier discharge plasma combined with MnO2-CuO based catalysts

Caroline Norsic, Jean-Michel Tatibouet, Catherine Batiot-Dupeyrat,

•

Humidity in plasma alone enhanced CO2 selectivity Within the possible decontamination ways to clean gas exhaust, non thermal atmospheric plasma (NTAP) is a renowned process, that does no longer need to prove its worth and has been applied to the treatment of numerous polluted gases with a large diversity of reactor configurations [START_REF] Vandenbroucke | Non-thermal plasmas for non-catalytic and catalytic VOC abatement[END_REF][START_REF] Xiao | Non-Thermal Plasmas for VOCs Abatement[END_REF][START_REF] Schiavon | Non-thermal Plasma as an Innovative Option for the Abatement of Volatile Organic Compounds: a Review[END_REF]. In fact, NTAP shows a high efficiency when treating effluents with low concentration pollutants (ppm level). Within the diversity of species generated in an air plasma, atomic oxygen, nitrogen and, in presence of humidity, OH radicals are produced.

These highly reactive species react with each other and with other molecules present in the gas phase to generate ozone and trigger radical oxidation reactions if organic molecules such as volatile organic compounds (VOC) are present. These oxidation processes allows the elimination of pollutants present in air. However, NTAP suffers from several drawbacks such as weak energy efficiency, mineralization and generation of ozone and nitrous oxides (NOx).

One way, which has been developed extensively, to overcome these disadvantages is by placing a catalyst inside or post discharge. Depending on the VOC chemical structure, nonthermal plasma reactor layout and catalyst chemical nature, several conclusions were drawn in respect of VOC removal efficiency (RE), ozone concentration, carbon mass balance (CMB), by-products selectivites, specific energy or discharge behavior [START_REF] Van Durme | Abatement and degradation pathways of toluene in indoor air by positive corona discharge[END_REF][START_REF] Van Durme | Efficient toluene abatement in indoor air by a plasma catalytic hybrid system[END_REF][START_REF] Vandenbroucke | Combination of non-thermal plasma and Pd/LaMnO3 for dilute trichloroethylene abatement[END_REF][START_REF] Karatum | A comparative study of dilute VOCs treatment in a nonthermal plasma reactor[END_REF][START_REF] Sivachandiran | Isopropanol saturated TiO2 surface regeneration by non-thermal plasma: Influence of air relative humidity[END_REF][START_REF] Sugasawa | Additive Effect of Water on the Decomposition of VOCs in Nonthermal Plasma[END_REF][START_REF] Van Durme | Post-plasma catalytic technology for the removal of toluene from indoor air: Effect of humidity[END_REF][START_REF] Futamura | Additive Effect on Energy Efficiency and Byproduct Distribution in VOC Decomposition with Nonthermal Plasma[END_REF][START_REF] Lock | Methanol and Dimethyl Sulfide Removal by Pulsed Corona Part I: Experiment[END_REF][START_REF] Yu-Fang | Humidity Effect on Toluene Decomposition in a Wire-plate Dielectric Barrier Discharge Reactor[END_REF][START_REF] Falkenstein | Microdischarge behaviour in the silent discharge of nitrogen-oxygen and water-air mixtures[END_REF]. Treatment by non thermal plasma alone succeeds in removing low concentration pollutants at low energy consumption compared to conventional catalytic processes. However, the process is sensitive to humidity and since it is non selective, unwanted by-products are generated (ozone, NOx, amongst others depending on the pollutant). When a catalyst is placed in the plasma discharge, and depending of its chemical nature, a synergistic effect is observed leading to high removal efficiencies. In post plasma configuration, it improves the oxidation reactions by ozone activation leading to higher carbon oxides (COx) selectivities and low residual ozone concentration.

Additionally, it is difficult to dissociate humidity levels from gas emission when controlling the levels of contaminated air flows, under real operating conditions, arising either from industrial or household environments. When treatment of polluted gas is considered, either via classical (thermal, catalytic oxidation, adsorption…) or slightly more unconventional ways (such as non thermal plasma, photocatalysis...), water should be taken into account, as it will affect the reaction selectivities and efficiencies.

In a plasma alone configuration, when no catalyst was present in the discharge or post discharge, opposite effects were observed depending on the relative humidity (RH) percentages present in the gas stream [START_REF] Van Durme | Abatement and degradation pathways of toluene in indoor air by positive corona discharge[END_REF][START_REF] Karatum | A comparative study of dilute VOCs treatment in a nonthermal plasma reactor[END_REF][START_REF] Lock | Methanol and Dimethyl Sulfide Removal by Pulsed Corona Part I: Experiment[END_REF][START_REF] Yu-Fang | Humidity Effect on Toluene Decomposition in a Wire-plate Dielectric Barrier Discharge Reactor[END_REF]. In a low humid environment, the formation of highly oxidative hydroxyl radicals ( • OH) increases due to the dissociation of water molecules by impact with electrons or oxygen radicals, O(1D). Ono and Oda [START_REF] Ono | Measurement of hydroxyl radicals in pulsed corona discharge[END_REF] measured the • OH concentration as a function of humidity and observed a maximum [ • OH] at RH < 1 % in a pulsed corona discharge. In the case of toluene elimination [START_REF] Van Durme | Abatement and degradation pathways of toluene in indoor air by positive corona discharge[END_REF], since • OH radicals have the highest reaction rate coefficient (k = 5.7 x 10 -12 molecule cm -3 .s -1 ) within the group of plasma oxidizing species (for atomic oxygen k = 7.6 x 10 -14 molecule cm -3 .s -1 and ozone k = 3.9 x 10 -22 molecule cm -3 .s -1 ), an increase of the VOC removal efficiency was observed. Depending on the type of plasma reactor and pollutant treated, a positive effect on the removal efficiency (RE) was detected with fairly different RH contents. Lock et al. [START_REF] Lock | Methanol and Dimethyl Sulfide Removal by Pulsed Corona Part I: Experiment[END_REF] observed an increase of the RE with RH up to 2 % in a corona reactor for methanol and dimethyl sulfide elimination. Regarding the treatment of toluene effluent, the limit was drawn at 0.2 % (mol %) in a dielectric barrier discharge (DBD) wire plate reactor [START_REF] Yu-Fang | Humidity Effect on Toluene Decomposition in a Wire-plate Dielectric Barrier Discharge Reactor[END_REF], 26 % in a direct current (DC) positive corona discharge [START_REF] Van Durme | Abatement and degradation pathways of toluene in indoor air by positive corona discharge[END_REF]. Surprisingly, Karatum et al. [START_REF] Karatum | A comparative study of dilute VOCs treatment in a nonthermal plasma reactor[END_REF] showed that a RH of 30 % did not alter the RE of ethylbenzene in a volumic cylindrical DBD.

However, removal efficiencies and ozone concentrations are decreasing when the relative humidity exceeds the values cited above, independently of the reactor configuration or VOC.

Several arguments were given to explain this phenomena. Water content has a strong impact on the plasma discharge electronic density. Excess humidity limits the current at constant applied voltage due to the modification of dielectric's surface resistance and reduces the transferred charges between the electrodes and thus reducing of the volume of plasma [START_REF] Falkenstein | Microdischarge behaviour in the silent discharge of nitrogen-oxygen and water-air mixtures[END_REF].

Additionally, a reduction of highly energetic electrons concentration was observed due to their inelastic collisions with water molecules [START_REF] Ono | Measurement of hydroxyl radicals in pulsed corona discharge[END_REF]. These arguments were confirmed by other authors such as Van Durme et al. [START_REF] Van Durme | Abatement and degradation pathways of toluene in indoor air by positive corona discharge[END_REF]. They reported that toluene abatement, in a plasma alone configuration, was enhanced in presence of relatively low RH contents (25 -27 %). However, at higher humidity levels (50 %), this effect was impeded by the reduction of the plasma electric field and mobility of charge carriers in presence of water, thus altering the discharge chemical composition [START_REF] Van Durme | Abatement and degradation pathways of toluene in indoor air by positive corona discharge[END_REF][START_REF] Fouad | Effect of humidity on positive corona discharge in a three electrode system[END_REF]. If the advantage of increasing • OH radical content with low humidity levels improved the removal efficiency, this effect was counteracted at higher RH due to the decrease of the number of microdischarges, especially for VOCs that can be directly decomposed by electronic impact. Furthermore, the ozone formation in the plasma discharge was reduced under the influence of humidity. O ( 1 D) radicals (from O2 dissociation) reacts with H2O to produce • OH radicals. The H • radicals issued from water dissociation can also account for the increased HO2 formation. The resulting H • , • OH and • HO2 are responsible for the decrease of ozone concentration. These radicals react with ozone to form O2 [START_REF] Van Durme | Abatement and degradation pathways of toluene in indoor air by positive corona discharge[END_REF][START_REF] Lovascio | Experimental Study and Kinetic Modeling for Ethanol Treatment by Air Dielectric Barrier Discharges[END_REF][START_REF] Zhu | Decomposition of benzene by non-thermal plasma processing: Photocatalyst and ozone effect[END_REF]. By-products, COx selectivities and carbon balance were also modified by the RH. If water showed a negative effect on the RE, it improved by-products oxidation, carbon balance and COx selectivities [START_REF] Lock | Methanol and Dimethyl Sulfide Removal by Pulsed Corona Part I: Experiment[END_REF][START_REF] Yu-Fang | Humidity Effect on Toluene Decomposition in a Wire-plate Dielectric Barrier Discharge Reactor[END_REF][START_REF] Dinh | Plasma-catalysis of low TCE concentration in air using LaMnO3+δ as catalyst[END_REF].

When a catalyst is combined to the plasma under humid conditions, in plasma or post plasma, a decrease of the VOC removal efficiency was observed, independently of reactor type or VOC chemical structure [START_REF] Van Durme | Efficient toluene abatement in indoor air by a plasma catalytic hybrid system[END_REF][START_REF] Vandenbroucke | Combination of non-thermal plasma and Pd/LaMnO3 for dilute trichloroethylene abatement[END_REF][START_REF] Sivachandiran | Isopropanol saturated TiO2 surface regeneration by non-thermal plasma: Influence of air relative humidity[END_REF][START_REF] Sugasawa | Additive Effect of Water on the Decomposition of VOCs in Nonthermal Plasma[END_REF][START_REF] Van Durme | Post-plasma catalytic technology for the removal of toluene from indoor air: Effect of humidity[END_REF][START_REF] Futamura | Additive Effect on Energy Efficiency and Byproduct Distribution in VOC Decomposition with Nonthermal Plasma[END_REF][START_REF] Zhu | Decomposition of benzene by non-thermal plasma processing: Photocatalyst and ozone effect[END_REF]. The beneficial effect of the catalyst was hindered by surface coverage with water molecules thus limiting access to active sites [START_REF] Vandenbroucke | Combination of non-thermal plasma and Pd/LaMnO3 for dilute trichloroethylene abatement[END_REF][START_REF] Sivachandiran | Isopropanol saturated TiO2 surface regeneration by non-thermal plasma: Influence of air relative humidity[END_REF][START_REF] Zhu | Decomposition of benzene by non-thermal plasma processing: Photocatalyst and ozone effect[END_REF][START_REF] Dinh | Plasma-catalysis of low TCE concentration in air using LaMnO3+δ as catalyst[END_REF][START_REF] Einaga | Structural changes in alumina-supported manganese oxides during ozone decomposition[END_REF]. This work is in the continuity of a study started on methanol elimination by non thermal plasma combined to catalysis at atmospheric pressure, in dry air conditions [START_REF] Norsic | Non thermal plasma assisted catalysis of methanol oxidation on Mn, Ce and Cu oxides supported on γ-Al2O3[END_REF]. The effect of humidity on the methanol conversion, by products selectivities and ozone concentration is investigated. A high level of relative humidity is chosen, 35 % as it is representative of indoor air levels. The plasma reactor was then associated to MnO2 and CuO/MnO2 based catalysts placed in post discharge and the effect of the catalyst metal oxide ratio, preparation method and methanol initial concentration were evaluated.

Experimental

Plasma reactor configuration

The NTAP reactor consisted in a surface dielectric barrier discharge (DBD) reactor. Two electrodes (one copper comb and one copper plate stuck on the glass plate) were separated from each other by a 2 mm thick glass plate acting as a dielectric barrier. Details of the reactor and experimental setup can be found in [START_REF] Norsic | Non thermal plasma assisted catalysis of methanol oxidation on Mn, Ce and Cu oxides supported on γ-Al2O3[END_REF]. 4g of catalyst were placed in the post discharge (PP) area in a 1.6 cm inner diameter glass tube. The space occupied by the catalyst was 4.1 cm which corresponded to a residence time in the catalytic reactor of 100 ms. The NTAP feeding gas was provided by an evaporating system (Serv'Instrumentation) with controlled pollutant concentrations. The gas flow rate was kept constant (5 L.min -1 , residence time in plasma alone: 0.36 s), whereas the methanol concentration could be varied from 25 to 200 ppmv. The presence of a manometer on the set-up allowed the control of the pressure drop.

Thanks to the structure of the catalyst support, consisting of alumina balls of 2 mm diameter, the system remained at atmospheric pressure. The water vapor was controlled by a humidity transmitter (Vaisala HTM330) with a constant content of 35 % RH which roughly corresponded to 8050 ppmv at 20°C.

Catalyst preparation

The section detailing the catalyst preparation can be found in [START_REF] Norsic | Non thermal plasma assisted catalysis of methanol oxidation on Mn, Ce and Cu oxides supported on γ-Al2O3[END_REF]. Briefly, a solution of Mn(NO3)2.4H2O was mixed with 2 mm alumina balls and stirred for 4 hours in a rotary evaporator under reduced pressure. The catalyst was dried at 110 °C and calcined at 300 °C.

The volume of precursor salt solution and weight of alumina were adjusted for a metal loading of 5 wt. %.

The mixed oxides of CuO/MnO2 were prepared by successive impregnation of alumina balls by Mn or Cu nitrate solutions, separated by drying at 110 °C and calcinations at 300 °C as previously described for single metal oxide catalyst. Two procedures have been used

according to the order of processing (copper first or manganese first). Metal content and catalysts characterizations are gathered in Table 1. The metal is cited in the order of impregnation. For example, Si-5Cu-5Mn corresponds to a catalyst prepared by successive impregnation of copper followed by manganese. 1 Explained in the following section

Analytical methods

The energy density of the discharge was set at 20 J.L -1 and its calculation is issued from the Manley method [START_REF] Manley | The Electric Characteristics of the Ozonator Discharge[END_REF]. This method has been used in many reported studies. It links the voltage between the electrodes, U, to the charge Q (measured via a capacitor placed in series) of the system. The plot of these series of data results in the so-called Lissajous curve whose integration leads to the energy per cycle (Ec) transferred through the gas. The discharge power (P) is obtained by multiplying the frequency (f) to the energy Ec. Dividing the power (P) by the gas flow rate (Q) results in the energy density, Ed (equation 1), which is a recurrent measure to compare plasma processes [START_REF] Norsic | Non thermal plasma assisted catalysis of methanol oxidation on Mn, Ce and Cu oxides supported on γ-Al2O3[END_REF][START_REF] Biganzoli | Experimental investigation of Lissajous figure shapes in planar and surface dielectric barrier discharges[END_REF].

𝐸 𝑑 (𝐽. 𝐿 -1 ) = 𝑃 (𝑊𝑎𝑡𝑡) 𝑄 (𝐿. 𝑠 -1 )
The specific surface area of the samples was measured by the Brunauer-Emmett-Teller (BET) method after calcination of the catalyst at 400 °C. The metal loading was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The catalysts were also characterized by hydrogen temperature programmed reduction (H2-TPR) [START_REF] Norsic | Non thermal plasma assisted catalysis of methanol oxidation on Mn, Ce and Cu oxides supported on γ-Al2O3[END_REF].

Methanol conversion, X (%) and by-products selectivities, S (%) were calculated as follow:

𝑋(%) = [𝑋] 0 -[𝑋]
[𝑋] 0 with [X]0 = input methanol concentration and [X] = ouput methanol concentration

𝑆 𝑥 (%) = [𝑅].(𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐶𝑎𝑟𝑏𝑜𝑛 𝑜𝑓 𝑅) [𝑋] 0 -[𝑋]
× 100

with [R] output concentration of product R
All the experiments were repeated 3 times and standard deviation of ±2 % was estimated. An ozone analyzer (ES, Dualbeam 205) allowed the online measurement of the ozone upstream and downstream the catalyst bed.

X-Ray diffraction pattern were obtained from a Siemens D5005 diffractometer using the KCu1 = 1.5406 Å. The 2 data within the 0 ° and 90 ° with a step size of 0.08 ° and an integration time of 90s per step. Unfortunately, the structural characterization by XRD was not usable. XRD diffraction lines of the catalysts show only alumina lines suggesting that either the amount of oxides is too low to obtain a visible diffraction pattern or the oxides are mainly amorphous.

Results and discussion

Influence of the impregnation method

In order to select the best way to prepare the catalysts, the RE for methanol elimination of the different catalysts, in presence of humidity, were compared. The results are shown in Figures 2 and3. The successive Cu/Mn impregnations procedure with, in first, copper impregnation seems to lead to better performances than the reverse (Figure 2). The SI-5Cu-5Mn catalyst appears to be slightly more efficient in carbon balance recovery, methanol conversion, ozone residual concentration and CO2 selectivity than the corresponding catalyst SI-5Mn-5Cu

prepared by manganese firstly impregnated, which leads also to formaldehyde. According to the total H2 uptake experimental values, it is possible to estimate the respective percentages of Mn IV and Mn III for each sample by assuming the total reduction of copper (Cu II O + H2  Cu° + H2O) and the reduction of manganese species to Mn II (Mn IV O2 + H2  Mn II O + H2O and Mn III O1.5 + 1/2H2  Mn II O + 1/2H2O). The results are gathered in Table 2. to Mn III for the peak at the lowest temperature and to the reduction of Mn III to Mn II for the highest one. The reduction profile of the SI-5Cu-5Mn sample which presents a single peak at low temperature could suggest that a strong interaction exists between copper and manganese increasing the reducibility of Mn III [START_REF] Ye | Influence of the preparation method on the activity of copper-manganese oxides for toluene total oxidation[END_REF].

By varying the Cu/Mn atomic ratio, it appears that the percentage of Mn IV increases with the copper content for the samples prepared by successive impregnation with copper first.

The TPR profiles of the samples prepared by copper first impregnation are shown on Figure 4 along with profiles of manganese and copper oxides alone. For the Cu/Mn atomic ratio higher than 1, the reduction behavior is similar to single oxides, with the presence of only one reduction peak at low temperature. However, for the lowest ratio (Cu/Mn = 0.37) a single peak is also observed but at higher temperature (380-400°C) and could indicate the formation of a defined compound more stable than single oxides of copper and manganese. This compound could be a copper manganese spinel (CuMn2O4) for the lowest Cu/Mn atomic ratio which is destabilized when the copper content increases [START_REF] Spassova | Coprecipitated CuO-MnOx Catalysts for Low-Temperature CO-NO and CO-NO-O2 Reactions[END_REF]. The MnO2 catalyst (Mn/Al) profile (Figure 4), as described in our previous publication, shows a main reduction peak at 300°C and a small shoulder between 350-480°C, resulting from the reduction of MnO2 to Mn2O3/Mn3O4 then MnO [START_REF] Jarrige | Plasma-enhanced catalysis of propane and isopropyl alcohol at ambient temperature on a MnO2-based catalyst[END_REF] or from the reduction of Mn5O8 to Mn3O4 then MnO as observed by Ye and al. [START_REF] Ye | Influence of the preparation method on the activity of copper-manganese oxides for toluene total oxidation[END_REF].

It was demonstrated that copper addition tends to maintain MnO2 oxide phase, while higher manganese oxide content leads to Mn2O3 phase, hindering the copper oxide effect [START_REF] Saqer | Catalytic oxidation of toluene over binary mixtures of copper, manganese and cerium oxides supported on γ-Al2O3[END_REF].

It was suggested, in the case of mixed oxides, that the activity depends on the redox couple Mn 3+ + Cu 2+  Mn 4+ + Cu + [START_REF] Qian | Structure-activity relationship of CuO/MnO2 catalysts in CO oxidation[END_REF] which could be present on manganese copper spinel structure (Mn 3+ oxidation state). We can expect that the highest interactions between copper and manganese cations lead to the highest catalytic activity, as observed for the SI-5Cu-5Mn and SI-25Cu-75Mn catalysts. To notice, these samples contain mainly manganese as Mn III (Table 2). The MnO2 catalyst (Mn/Al) profile (Figure 4), as described in our previous publication, shows a main reduction peak at 300°C and a small shoulder between 350-480°C, resulting from the reduction of MnO2 to Mn2O3/Mn3O4 then MnO [START_REF] Jarrige | Plasma-enhanced catalysis of propane and isopropyl alcohol at ambient temperature on a MnO2-based catalyst[END_REF] or from the reduction of Mn5O8 to Mn3O4 then MnO as observed by Ye and al. [START_REF] Ye | Influence of the preparation method on the activity of copper-manganese oxides for toluene total oxidation[END_REF].

The percentage of loaded metal oxide was also evaluated in terms of methanol RE (Figure 5).

It appeared that the ozone degradation is dependent on the percentage of loaded manganese resulting in only 17 ppm left after one passage over SI-25Cu-75Mn, while it remains at 32ppm over SI-75Cu-25Mn. Methanol conversion increases as a function of ozone degradation, indicating a reaction in which the plasma oxidizing species (O • , • OH, • HO2) are given first place for methanol conversion. As specified above, Cu/Mn redox couple and MnO2 phase availability are responsible of ozone degradation into oxidizing species. Only little effect is observed on the carbon balance between the different metal oxide ratios which remains close to 90% except SI-25Cu-75Mn that drops at 80 %. The drop in carbon balance could be related to formic acid formation. According to the fair methanol RE, carbon balance and selectivity to by-products (especially to CO2), the catalyst prepared via successive impregnation with CuO first, SI-5Cu-5Mn, was chosen as reference catalyst and further investigated.

Influence of humidity

Effect of humidity in plasma alone configuration

The addition of 35 % relative humidity in the gas flow led to a decrease of the methanol conversion, from 60 % to 43% and ozone concentration (160 ppm to 75 ppm) but did not affect the carbon balance of the reaction which remained close to 100% (Figure 6). 3). Moreover, the further reactions between hydrogen peroxide and O or • OH radicals are thermodynamically favored and lead to the formation of Moreover, the VOC conversion and ozone concentration were reduced at high humidity levels (level depending on plasma discharge type) due to the modification of the discharge electric field, leading to a decrease of reactive plasma volume and chemical species [START_REF] Falkenstein | Microdischarge behaviour in the silent discharge of nitrogen-oxygen and water-air mixtures[END_REF]. Regarding by-products selectivity, the affinity of formaldehyde to water would favor its direct transformation into CO2 at the expense of CO, according to Sugasawa [START_REF] Sugasawa | Additive Effect of Water on the Decomposition of VOCs in Nonthermal Plasma[END_REF]. The total oxidation of methyl formate and methylal under humid flow contributes to the improved CO2 selectivity.

Effect of humidity in presence of a post plasma (PP) catalyst

As already observed in our previous publication [START_REF] Norsic | Non thermal plasma assisted catalysis of methanol oxidation on Mn, Ce and Cu oxides supported on γ-Al2O3[END_REF], and from the results cited above, a 5 % MnO2/5 % CuO/Al2O3 catalyst presented some of the best activities, such as methanol conversion or ozone degradation, that swayed our catalyst choice to SI-5Cu-5Mn in the study of the humidity impact on methanol elimination efficiency. In order to evaluate the efficiency in presence of a catalyst in post-plasma location, 4 g of SI-5Cu-5Mn catalyst were placed immediately after the plasma reactor in a specific catalytic reactor.

In dry conditions, when 4 g of catalyst were placed in post plasma, the methanol conversion and carbon balance were close to 100 % with only formation of carbon oxides (CO = 43 %; CO2 = 55 %) for the plasma and catalyst reactors connected in series. In presence of humidity (RH = 35 % corresponding to about 8000 ppmv) a large decrease in conversion was observed.

The methanol total conversion decreased from 100 % to 65 % and the carbon balance from 99 % to 89 %, but the CO2 selectivity increased to 83 %, with only 6 % of CO. The most significant effect concerned ozone exiting the plasma reactor which decreased from 160 ppmv in dry conditions to 74 ppmv under 35% of RH.

On the catalyst, the ozone consumption reached 148 ppmv in dry conditions (ozone conversion=92.5%) and only 54 ppmv in presence of humidity (ozone conversion = 73 %).

The residual ozone concentration is higher in presence of humidity (20 ppmv) than in dry conditions (12 ppmv) despite a lower initial concentration entering the catalyst reactor (75 ppm in humid vs 160 ppm in dry conditions). The results are gathered in Tables 4 and5. On the same tables, the calculation of conversions and selectivities are done according to the consumption of methanol for the plasma reactor and to the sum of the consumptions of all the products for the catalyst reactor, taking into account the carbon number for methyl formate and methylal.

For example, in dry conditions (Table 4) the consumption of the products appears to be 20 ppm for methanol, 7 ppm for formaldehyde, 0.5 ppm for methyl formate (corrected to 1 ppm), 0.3 ppm for methylal (corrected to 0.9 ppm) and 0.1 ppm for formic acid, leading to a total consumption of 29 ppm. The value of formic acid formation (formic acid is not detected but its formation is expected according to the possible reaction of oxidation of methanol) is estimated from the carbon balance defect. The CO2 (50%) and CO (47%) selectivities are then calculated as the ratio between their increased concentration through the catalyst reactor (14.5 ppm for CO2 and 13.5 ppm for CO) and the sum of the products consumption (29 ppm).

According to our calculations it appears that the methanol conversion on the catalyst is higher in dry (or pseudo-dry conditions since 51 ppm of water are produced in the plasma reactor due to the methanol oxidation), than in humid conditions in presence of more than 8000 ppm of water, but the selectivity to CO2 is lower (50%) in dry conditions compared to 82% in humid conditions without CO formation. In dry conditions, the selectivity to CO is 47%.

This surprising effect could be correlated with the possible mechanism of ozone and water reaction on the manganese oxide surface. It is well admitted that ozone reacts with manganese surface cations to form Mn-O -or Mn-OO -surface species [START_REF] Li | Mechanism of Ozone Decomposition on a Manganese Oxide Catalyst. 1. In Situ Raman Spectroscopy and Ab Initio Molecular Orbital Calculations[END_REF]. In presence of water the reaction of ozone on the catalyst surface was partly inhibited due to the water adsorption on the manganese active sites [START_REF] Tatibouët | Catalytic oxidation of phenol by hydrogen peroxide over a pillared clay containing iron. Active species and pH effect[END_REF][START_REF] Jia | Facile synthesis of Fe-modified manganese oxide with high content of oxygen vacancies for efficient airborne ozone destruction[END_REF]. A mechanism was proposed by Zhu et al. [START_REF] Zhu | Surface oxygen vacancy induced -MnO2 nanofiber for highly efficient ozone elimination[END_REF] for the reaction of ozone decomposition on manganese oxide in presence of gaseous water. It consists of the slow reaction of ozone on adsorbed water leading to the formation, at the catalyst surface, of hydroxyl species on which further ozone molecules can react to form hydroperoxide species and peroxide species. Nevertheless, the possible formation of hydrogen peroxide cannot be excluded from direct reaction between Mn-O species and water from the gas phase leading to the formation of surface hydroxyl species on which ozone can react [START_REF] Zhu | Surface oxygen vacancy induced -MnO2 nanofiber for highly efficient ozone elimination[END_REF] (Scheme 1) blocking partially the ozone elimination cycle, but allowing the formation of the highly oxidative hydroperoxide species.

Scheme 1: Pathway of ozone elimination in presence of water [START_REF] Zhu | Surface oxygen vacancy induced -MnO2 nanofiber for highly efficient ozone elimination[END_REF] According to this hypothesis, the formic acid formation estimated from the carbon balance defect could result from formaldehyde oxidation by H2O2 or The results are gathered in Figures 7,8 and on Tables 6 and7. In plasma alone and at constant input specific energy (20 J.L -1 ) the methanol conversion decreases with the increase of initial concentration, as already observed. Nevertheless the total amount of methanol transformed increases with the methanol initial concentration and the formaldehyde formation increases to the detriment of CO2 formation (Figure 8). On the catalyst, the amount of methanol transformed (Table 7 (2)) increases with the concentration of methanol at the entrance of catalyst reactor and seems to reach a plateau for a concentration higher than 71 ppmv, similarly to ozone consumption (Table 6 [START_REF] Futamura | Additive Effect on Energy Efficiency and Byproduct Distribution in VOC Decomposition with Nonthermal Plasma[END_REF]).

Regarding the CO2 formation (Table 7 (3)), it goes through a maximum and then decreases for methanol initial concentration higher than 71 ppmv. From the data collected at the entrance and at the exit of the catalytic reactor, it is possible to estimate the amount of the products (CO and HCOH) transformed on the catalyst. Additionally, it is possible to estimate the concentration of CO2 formed on the catalyst coming from methanol and formaldehyde total oxidation. The results obtained by subtracting the CO transformed on the catalyst (the only oxidation product of CO being CO2) to the CO2 formed on the catalyst (column (3)column ( 4)) are plotted on column (5) of the Table 7. It shows that the amount of CO2 from the total oxidation of methanol and formaldehyde (VOC products) depends of the ratio between VOC products and ozone (Table 7 column ( 9)). The selectivity to CO2 from total oxidation of methanol and formaldehyde decreases from 90% to only 2% when the ratio between VOC and ozone decreases from 5.1 to 0.4. Simultaneously, the carbon balance defect (Table 7 (8)), which indicates the formation of undetected product, expected to be formic acid, [START_REF] Dhandapani | Gas phase ozone decomposition catalysts[END_REF][START_REF] Tatibouët | Methanol oxidation as a catalytic surface probe[END_REF], increases from 1.1 to 33.9 ppmv. This behavior is consistent with the usual reaction mechanism of partial methanol oxidation involving successive reaction [START_REF] Dhandapani | Gas phase ozone decomposition catalysts[END_REF][START_REF] Tatibouët | Methanol oxidation as a catalytic surface probe[END_REF]: Scheme 1: Partial oxidation of methanol.

In dry conditions, the higher ozone concentration on the catalyst could favor the formaldehyde and formic acid oxidation to carbon oxides, whereas in presence of water,

CH

CO

either the lower ozone concentration or the presence of hydrogen peroxide resulting of the reaction of activated oxygen species from ozone with water could favor the formation of formic acid instead of the total oxidation of formaldehyde.

Conclusion

Under humid conditions (37 % RH), close to ambient air concentrations, and at low energy density (20J.L -1 ), non thermal plasma treatment of methanol affects the conversion rate compared to dry air but improves largely the CO2 selectivity in presence of a CuO-MnO2 based catalyst in post plasma configuration. Additionally, selectivities to secondary products are reduced (CO and HCOH) and no methylal, nor methyl formate are yielded at low concentrations (≤ 50ppm). The presence of humidity in the discharge leads to a reduction of ozone concentration which contribute to the partial methanol oxidation. These results could be explained by a reaction between ozone and adsorbed water leading to the formation of hydroperoxide species and/or adsorbed H2O2 more selective to CO2 formation. The presence of these species and a low O3 concentration would favor the formation of formic acid instead of the total oxidation of formaldehyde to CO2. We could also expect that these reactions block partially the cycle of ozone decomposition, therefore explaining the leftover ozone detected at the exit of the plasma catalyst reactor. The impregnation method and metal oxide ratios showed a little effect on the conversion rate of methanol, a 5 % MnO2 /5 % CuO / Al2O3 catalyst prepared by successive impregnations results in the best conversion rates and CO2 selectivity.

Figure 1 :

 1 Figure 1: Experimental set-up.

Figure 2 :

 2 Figure 2: Effect of impregnation method of CuO/MnOx catalysts in post plasma configuration

Figure 3 :

 3 Figure 3 : TPR profiles of the catalysts with different impregnation methods: SI-5Cu-5Mn,

Figure 4 :

 4 Figure 4: TPR profiles of the catalysts with different oxide ratios: SI-5Cu-5Mn, SI-25Cu-

Figure 5 :

 5 Figure 5: Effect of metal oxide ratio of MnO2/CuO catalysts placed in post plasma

Figure 6 :

 6 Figure 6: Effect of water on methanol conversion, ozone concentration and selectivities to secondary products: HCOH; ML: methylal; MF: methyl formate. Conditions: 20J.L -1 , 50 ppm

2 161. 1 H2O

 1 + O  H2O2 H = -143.5 kJ/mol; G = -108.4 kJ/mol H2O + O  2 • OH H = + 70.6 kJ/mol; G = + 65.4 kJ/mol H2O2  2 • OH H = + 214.1 kJ/mol; G = + 175.9 kJ/mol H2O2 + O  • HO2 + • HO H = -72.0 kJ/mol; G = -75.5 kJ/mol H2O2 + • OH  H2O + • HO2 H = -142.6 kJ/mol; G = -140.9 kJ/mol

(

  Experimental conditions: 20 J.L -1 , catalyst = 4 g, total flow rate = 5 L.min -calculated from the carbon balance defect4.3. Reactivity as a function of methanol concentration in humid reaction conditionsWe have already shown[START_REF] Norsic | Non thermal plasma assisted catalysis of methanol oxidation on Mn, Ce and Cu oxides supported on γ-Al2O3[END_REF] that in dry reaction conditions, a strong influence on reaction behavior occurs when the initial methanol concentrations change. The influence of humidity (RH = 35 %) on the reaction behavior was evaluated as a function of initial methanol concentration, both in plasma alone and with the catalytic reactor. The methanol initial concentration was varied from 25 to 200 ppmv and in post plasma catalytic configuration, 4 g of the reference catalyst SI-5Cu-5Mn was used.

Figure 7 :

 7 Figure 7: Effect of methanol initial concentration on methanol conversion and ozone

Figure 8 :

 8 Figure 8: Effect of methanol initial concentration on carbon balance and selectivities to

Table 1 :

 1 Metal oxide content (theoretical and measured from ICP 1 ), BET1 , support nature and preparation methods of the catalysts.

	Catalyst name *	CuO content (wt. %)	MnO2 content (wt. %)	SBET (m 2 .g -1 )	ICP (oxide wt.%)
	Mn/Al	0	5	179	5.0
	Cu/Al	5	0	197	3.9
	SI-5Cu-5Mn 5	5	186	3.7 CuO 4.0 MnO2
	SI-5Mn-5Cu 5	5	187	4.0 CuO 4.2 MnO2
	CI-5Cu5Mn 5	5	191	3.7 CuO 4.2 MnO2
	SI-25Cu-75Mn	2.5	7.5	186	2.5 CuO 7.4 MnO2
	SI-75Cu-25Mn	7.5	2.5	194	7.6 CuO 2.5 MnO2

*SI: successive impregnation; CI: co-impregnation.

Table 2 :

 2 Hydrogen consumption for the oxide reduction (cm 3 H2/g ± 5 %) STP.It appears that the sample prepared by manganese first impregnation contains only Mn IV , whereas the sample prepared by copper impregnation first contains mainly Mn III . The two peaks profile of SI-5Mn-5Cu sample could then be explained by the reduction of Cu II to Cu° and Mn IV

		SI-25Cu-	SI-	SI-75Cu-	SI-5Mn-5Cu CI-5Cu5Mn Mn/Al
		75Mn	5Cu-	25Mn
			5Mn
	Mn (wt%)	4.7	2.5	1.6	2.7	2.7	3.2
	Cu (wt%)	2.0	3.0	6.1	3.2	3.0	0

Table 3 :

 3 • OH and • HO2 which are highly oxidative species. It means that the • OH and • HO2 generation occurs most probably by hydrogen peroxide secondary reactions rather than by water dissociation or direct reaction of O radical with water. Thermodynamic data.

	Product	H°f (kJ/mol)	S° 300K
			(J/mol.K)
	H2O2	-136.1	239.9
	H2O	-241.8	188.8
	°HO	+39.0	183.7
	°HO2	+2.1	229.1
	O°	+249.

Table 4 :

 4 Dry conditions: Summary of the methanol conversion and products selectivity on plasma and catalyst reactors and products concentration after plasma and catalyst reactors. "S product": selectivity to products. (Experimental conditions: 20 J.L -1 , catalyst = 4 g, total flow rate = 5 L.min -1 ).

	• OH [17]:

Table 5 :

 5 Humid conditions: Summary of the methanol conversion and products selectivity on plasma and catalyst reactors and products concentration after plasma and catalyst reactors.

Table 6 :

 6 Methanol conversion and concentration after plasma alone (P), plasma + catalyst (P

	+ C) and catalyst alone (C) with ozone consumption (experimental and calculated from
	observed products formation).
	[MeOH]°	[MeOH]	MeOH	[MeOH]	MeOH	Conv.	Conv.	Conv.	[O3]°	[O3]	O3 (exp.)	O3 (th.)
	(ppmv)	After	transformed	After	transformed	MeOH	MeOH	MeOH	After	After	consumption	consumption
		plasma	by plasma	catalyst	(P + C)	(P) (%)	(C)	(P+C)	plasma	catalyst	on catalyst	on catalyst
		(ppmv)	(ppmv)	(ppmv)	(ppmv)		(%)	(%)	(ppmv)	(ppmv)	(ppmv)	(ppmv)
	(1)	(2)	(3)	(4)	(5)	(6)	(7)	(8)	(9)	(10)	(11)	(12)
	25	12.3	12.7	3.5	21.5	51	71	86	75	27	48	31
	50	27.5	22.5	17.5	32.5	45	36	65	74	20	54	39
	100	71	29	50	50	29	30	50	72	13	59	68
	150	117	33	91.5	58.5	22	22	39	70	10	60	70
	200	160	40	136	64	20	15	32	68	7	61	67

Table 7 :

 7 Methanol and products transformation on the catalyst SI-5Cu-5Mn as a function of the methanol concentration at the entrance of the catalytic reactor.

	(1)	(2)	(3)	(4)	(5)	(6)	(7)	(8)	(9)
	MeOH at the	MeOH	CO2	CO	CO2 from	HCOH	Selectivity to	Carbon	Ratio
	entrance of	transformed	formation on	transformed	total	transformed	CO2 for	balance	.	[O3]° .
	the catalyst	on the	the catalyst	on the	oxidation of	on the	MeOH and	defect	([MeOH]+[HCOH])
	(ppmv)	catalyst	(ppmv)	catalyst	MeOH and	catalyst	HCOH	(ppmv)
		(ppmv)		(ppmv)	HCOH	(ppmv)	oxidation
					(ppmv)		(%)
	12.3	8.8	10.8	0.7	10.1	2.4	90	1.1	5.1
	27.5	10.0	13.8	2.7	11.1	4.5	77	3.6	2.3
	71	21.0	18.1	3.2	14.9	8.4	51	14.0	0.9
	117	25.5	11.9	3.5	8.4	8.6	25	24.6	0.5
	160	24.0	7.2	6.4	0.8	11.6	2	33.9	0.4

Acknowledgment

The authors are thankful to the ANRT for the financial support.