Feriel Boussema

Andrew J Gross

Fatma Hmida

Brahim Ayed

Hatem Majdoub

Serge Cosnier

Abderrazak Maaref

Michael Holzinger
email: michael.holzinger@univ-grenoble-alpes.fr

Dawson-type polyoxometalate nanoclusters confined in a carbon nanotube matrix as efficient redox mediators for enzymatic glucose biofuel cell anodes and glucose biosensors

Keywords: Electron transfer mediator, Dawson type polyoxometalates, glucose biofuel cells, glucose dehydrogenase, multi walled carbon nanotubes

come     Dawson-type polyoxometalate nanoclusters confined in a carbon nanotube matrix as efficient redox mediators for enzymatic glucose biofuel cell anodes and glucose biosensors

Introduction.

The possibility of exchanging electrons between immobilized redox enzymes and electrodes clearly improves the efficiency of biosensors and is essential for biofuel cell applications (Saboe et al. 2017).

Achieving efficient electron transfer with glucose oxidizing enzymes with flavin adenine dinucleotide (FAD) cofactors is a particular challenge since this redox unit is covered by a thick protein shell that makes direct electron transfer difficult, if not impossible. One of the key issues is that electron transfer is exponentially dependent on the distance between the redox active center and the electrode material (Marcus and Sutin 1985). One of the most common enzymes for glucose oxidation is glucose oxidase (GOx). Its FAD unit is protected by a glycosylated protein shell (total mass ~160 kDa) which is responsible for its outstanding stabilities and activity over several pH values (Wilson and Turner 1992). However, the fact that GOx produces hydrogen peroxide via oxygen reduction, after glucose oxidation, is on the one hand a positive aspect for electrochemical biosensing (Ferri et al. 2011), but on the other hand, a drawback for biofuel cell anodes. Therefore, FAD-dependent glucose dehydrogenases (FAD-GDHs) are becoming prominent alternatives for the bioelectrocatalytic oxidation of glucose since they exhibit similar activity to GOx, whilst also benefiting from being insensitive to oxygen which avoids detrimental hydrogen peroxide production (Zafar et al. 2012).

For the wiring of FAD-dependent enzymes, the mediated electron transfer (MET) strategy, which uses a redox active compound of appropriate potential, is essential for many types of glucose biosensors and biofuel cells (Zhao et al. 2015). MET-based glucose biosensors can operate at convenient redox potentials without affecting the enzyme's activity (Saleem et al. 2015). Ferrocene and ferricyanide derivatives are commonly used mediators in glucose biosensor setups (Wang 2002) but they are inappropriate as anodic mediators in glucose biofuel cells since the glucose oxidation overpotential is much too high to achieve reasonable fuel cell potentials. For the bioconversion of energy, the development of redox mediators with potentials close to the enzyme's cofactor (here, FAD), and which exhibit fast electron transfer kinetics, still remains of high interest. Historically, the Heller group first described MET with GOx using an osmium-based redox polymer [START_REF] Gregg | [END_REF]). Since the redox potential of osmium complexes can be adjusted by altering the ligand structure, more appropriate osmium-based redox hydrogels have been synthesized for glucose biofuel cell applications (Heller 1992;Mano et al. 2002;Pinyou et al. 2016). More recently, quinones have emerged as prominent alternatives to metal organic complexes due to efficient electron transfer kinetics and the possibility to improve operational lifetimes (Gross et al. 2017;Milton et al. 2015;Reuillard et al. 2015).

Another class of compounds with tunable redox potentials are polyoxometalates (POMs). POMs belong to a large family of inorganic nanoclusters which comprise of a metal-oxygen framework and have attracted interest in many fields such as catalysis (Wang et al. 2015) and electrocatalysis [START_REF] Freire | Polyoxometalate-based Modified Electrodes for Electrocatalysis: From Molecule Sensing to Renewable Energy-related Applications[END_REF]Hamidi et al. 2008). POMs are attractive as they can be rationally designed to give a wide range of complex materials with tailored catalytic, electronic, and structural properties (Long et al. 2007;Miras et al. 2012;Zhou et al. 2007). POMs can also provide rich electrochemical processes with reversible multi-step and multi-electron transfers (Ueda 2015). The various electrocatalytic properties of POMs may be envisioned for energy conversion (Ji et al. 2015) or molecular sensing [START_REF] Freire | Polyoxometalate-based Modified Electrodes for Electrocatalysis: From Molecule Sensing to Renewable Energy-related Applications[END_REF]. For biomedical applications, POM composites and alloys have shown promising performances for the electrochemical detection of biomolecules such as ascorbic acid (vitamin c), dopamine (Zhou et al. 2013) (Zhang et al. 2014) and NADH, the reduced form of the coenzyme nicotinamide adenine dinucleotide (Ammam and Easton 2013). The first use of POMs with enzymes for glucose sensing was described by Turdean et al. (Turdean et al. 2002) 2H2O, and 2-hydroxy-6-methylpyridine were purchased from Sigma-Aldrich. Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH, 1150 U mg -1 solid) was obtained from Sekisui Diagnostics (UK). Multi-walled carbon nanotubes (MWCNTs, 9.5 nm diameter, 1.5 μm length, purity >95%), obtained from Nanocyl, were used as received without any purification step. Distilled water was obtained by water purification to a resistivity of 15 MΩ cm using a Millipore Ultrapure system. Glucose solutions were left to mutarotate overnight to β-D-glucose prior to use.

Electrochemical measurements were performed in an electrochemical cell with a conventional three-electrode configuration using an Autolab pgstat100 potentiostat with Nova 2.0 software. A GCE working electrode was used together with a platinum wire counter electrode, and a saturated calomel reference electrode (SCE). Before each experiment, the surface of the glassy carbon electrode (GCE, 3 mm diameter) was polished with 2µm diameter diamond paste (MECAPREX Press PM) then rinsed with distilled water to remove any residual paste. Finally, the electrodes were sonicated for 5 min in ethanol and 5 min in acetone, followed by a thorough washing step with distilled water and ethanol. IR spectra were recorded using a Nicolet 470FT-IR spectrophotometer with pressed KBr pellets. SEM images were recorded using a FEI QUANTA-FEG 250 microscope with an Everhardt Thornley SED (secondary electron detector), an accelerating voltage of 3 kV, and a working distance of 8.0 mm.

Synthesis of (C4H10N)6 (P2Mo18O62) . 4H2O

The POM (C 4 H 10 N) 6 (P 2 Mo 18 O 62 ) . 4H 2 O, was synthesized as described in our recent work (Hmida et al. 2015). Briefly Na2 MoO4 . 2H2O (1 g, 4 mmol) was dissolved in water (25 mL) with stirring. H3PO4 (0.5 mL, 0.2 mmol) and pyrrolidine (0.2 mmol) were then added to the mixture (a light turbidity occurred which was allowed to clear with time before the next drop was added) and the pH was adjusted to about 2.5-2.6 by adding 6 mmolL -1 HCl. The mixture was refluxed at 353K for 3 h and then cooled to room temperature. Slow evaporation of the solvent at room temperature led to green prismatic crystals.

(C4H10N)6 (P2Mo18O62) . 4H2O was characterized by single crystal X-ray analysis and IR spectroscopy (see supplementary material).

Synthesis of (C6H8NO)4 (P2W18O62) . 6H2O

(C6H8NO)4 [H2P2W18O62] 6H2O, was synthesized by dissolving Na2WO4 . 2H2O (0.33 g, 1 mmol) in 10 mL of water. The solution was heated at 80 °C for 45 min and the pH was adjusted to 4.5 with aqueous HCl (5 mmolL -1 ) after 0.3 ml of H3PO4 was added. Then, a solution of 2-hydroxy-6-methylpyridine (0.5 g, 6 mmol) in water (10 mL) was added dropwise (correspondingly, when the solution cleared, the next drop was added). The final pH was adjusted to 4.5 by addition of diluted HCl (5 mmolL -1 ). The mixture was transformed to a Teflon-lined stainless steel autoclave (18 mL) and kept at 140 °C for five days. After the autoclave had cooled down to room temperature, yellow block crystals were filtered off and washed with distilled water. This compound was characterized by single crystal X-ray analysis, UV-Vis, and IR spectroscopy (see supplementary material).

Preparation of the bioelectrodes

The MWCNT-based electrodes were prepared in several steps. First, MWCNTs were dispersed in NMP by 30 min sonication of 5 mg of nanotubes in 1 mL of NMP until a homogenous black suspension was obtained. Then, the solution was drop-casted on glassy carbon electrodes to obtain a MWCNT film on the electrode. The MWCNT electrodes were functionalized with PMA via incubation in a 1-pyrenemethylamine hydrochloride (1 mg mL -1 in DMF) solution for 60 min). The solvents were removed under vacuum and the electrodes were then washed with McIlvaine buffer. The functionalized electrodes were then incubated in a 10 -4 molL -1 solution of P 2 W 18 or P 2 Mo 18 in H 2 SO 4 (1 molL -1 ). After intensive rinsing to remove the acid, the electrodes were finally incubated in a 5 mg mL

Results and discussion

Electrochemical behavior of P2W18-and P2Mo18-modified electrodes

The electrochemical behavior of the polyoxometalates, P2W18 and P2Mo18, was first characterized in acidic solution (10 -4 molL -1 , 0.1 molL -1 H2SO4) using cyclic voltammetry (CV) with bare GCE as the working electrode. In Figure 1A, P2W18 shows four reversible redox systems with a half-wave potential, E½ = 0.04 V, -0.12 V, -0.39 V, and -0.64 V vs SCE, assigned to two one-electron and two two-electron reductions (Keita and Nadjo 1987;Wang et al. 2011) according to the equations:

P2W18 VI O62 6-+ e -⇋ P2W17 VI W V O62 7- (I) (1) P2W17 VI W V O62 7-+ e -⇋ P2W16 VI W V 2O62 8- (II) (2) P2W16 VI W2 V O62 8-+2e -+2H + ⇋ H2P2W14 VI W4 V O62 8- (III) (3)
H2P2W14 VI W4 V O62 8-+ 2e -+2H + ⇋ H4P2W12 VI W6 V O62 8- (VI) (4)
P2Mo18 shows three reversible redox systems at E½ = 0.31 V, 0.18 V, and 0.01 V vs SCE, which correspond to three two-electron reductions, as previously described (Hmida et al. 2015;Zhou et al. 2007) (Figure 1B):

P2Mo18 VI O62 6-+ 2e -+ 2H + ⇋ H2P2Mo16 VI Mo2 V O62 6- (I) (5) H2P2Mo16 VI Mo2 V O62 6-+2e -+2H + ⇋H4P2Mo VI 14Mo4 V O62 6-(II) (6) H4P2Mo14 VI Mo4 V O62 6-+2e -+2H + ⇋H6P2Mo12 VI Mo6 V O62 6-(III) (7)
With the aim to study the polyoxometalates for mediated electron transfer with FAD-GDH, we studied their electrochemical behavior in McIlvaine buffer at pH 7.0 after confinement in a MWCNT matrix. In the potential range of 0.2 V and -0.8 V vs SCE, P2W18 shows four reversible redox systems at E½ = -0.04

V, -0.20 V, -0.57 V, and -0.67 V (Figure 1C) according to equations ( 1 Since the electrochemical reductions of P2Mo18 are two-electron two-proton processes, the increased pH from pH 1.0 to pH 7.0 led to a clear negative shift of the reversible redox waves, as shown in Figure 1D compared to Figure 1C (Ammam and Easton 2011). The two systems corresponding to equations ( 5) and ( 6), situated at E½ = -0.04 V and -0.22 V, shifted perfectly into the potential range of interest. The third two-electron redox couple (equation ( 7)) could not be resolved for the P2Mo18-modified electrode even after extending the potential window, consistent with instability and irreversibility for this process (not shown). It should be noted that the PMA-functionalized MWCNT deposits provide an efficient platform for the immobilization of the two polyoxometalate compounds. By potential cycling at different scan rates, a linear dependency of the peak current versus the scan rate was observed for both types of POMmodified electrodes, confirming that the electroactive POMs are surface bound (supplementary material: The surface coverage (Γ) of electroactive P2W18 or P2Mo18 clusters at the surface of GCE was determined by integration of the charge under the polyoxometalate reduction peaks after subtracting the capacitive current of the MWCNTs, according to Laviron's equation:

Ipf = n 2 F 2 AΓυ/4RT (8)
where n is the number of electrons involved in the redox process, F is the Faraday's constant, A is the geometric surface area of the electrode (0.071 cm²), R is the gas constant, T is the temperature, and ν is the scan rate. The average surface coverage of P2W18 was 1.6 × 10 -9 mol cm -2 while that of P2Mo18 was 1.2 × 10 -8 mol cm -2 based on voltammograms recorded at 5 mV s -1 .

Morphological studies of P2Mo18/PMA/MWCNTs and P2W18/PMA/MWCNTs deposits

To gain more insight into the observed electrochemical behavior of the P2Mo18/PMA/MWCNTs and P2W18/PMA/MWCNTs electrodes, and, in particular, the difference of surface coverage by almost a factor of 10, we performed morphological studies using scanning electron microscopy (SEM). Figure 2 shows representative SEM images of PMA/MWCNTs deposits before and after modification with the POMs.

The SEM image of PMA-modified MWCNTs (Figure 2A) shows a typical porous morphology of entangled CNTs without traces of PMA residues. After deposition of P2Mo18, the deposit appears very undulated with small aggregates homogeneously incorporated in the MWCNT matrix, as seen in Figure 2B. It seems that the protonation of PMA in acidic media provokes a swelling of the MWCNT deposit which enables good penetration of P2Mo18 in the structure. After drying, small POM particles are formed with a homogenous distribution which indicates a good initial surface coverage of P2Mo18 on the PMA/MWCNTs substrate. For the P2W18/PMA/MWCNTs samples, a similar undulation is observed (Figure 2C) but fewer particles are observed and they are larger. Here, P2W18 seems to interact much less with the PMA/MWCNTs matrix, even when the same swelling effect of the MWCNT structure is observed. It can be assumed that, under these preparation conditions, the formed 2-hydroxy-6-methylpyridinium ligand provokes electrostatic repulsion with the protonated PMA-MWCNT layer, thus leading to the formation of larger aggregates and less effective inclusion of P2W18. This argument also explains the ten-fold lower surface coverage of electroactive P2W18 revealed by the electrochemical studies.

Mediated electron transfer between FAD-GDH and MWCNTs with P2W18 and P2Mo18

The anodic onset potentials of redox system (I) for both types of POM/PMA/MWCNTs composites are in the suitable range for electron mediation with FAD-dependent oxidizing enzymes. The mediated bioelectrocatalysis of P2W18-modified electrodes shows an open circuit potential (OCP) at -0.08 V vs SCE and a catalytic current density of 0.34 mA cm -² at 0.2 V. In contrast, the voltammograms recorded at P2Mo18-modified electrodes showed much higher current densities of 4.7 mA cm -2 at 0.2 V, and an attractively lower OCP at -0.1 V vs SCE. The higher performance for P2Mo18 in terms of both the onset potential and catalytic glucose oxidation current are most likely due to the improved stability and incorporation of P2Mo18 in the MWCNT matrix which resulted in higher amounts of immobilized POM.

Figure 4A displays the bioelectrocatalytic behavior of FAD-GDH/P2W18/PMA/MWCNTs and FAD-GDH/P2Mo18/PMA /MWCNTs electrodes at increasing glucose concentrations. Increasing current densities were observed for both electrodes with increasing concentrations. At low glucose concentrations the P2W18-modified electrode is clearly less performing than the electrode modified with P2Mo18. No further current increase was measured at glucose concentrations higher than 10 mmolL -1 for the P2W18 electrodes, which reached a maximum current density of 0.4 mAcm -2 . The comparatively low catalytic current at glucose saturation can again be attributed to the reduced amount of this POM on the MWCNT electrode.

The maximum current density observed was reached at 4.5 mAcm -2 with P2Mo18-modified electrodes at glucose concentrations higher than 50 mmolL -1 . Figure 4B shows the calibration curves, obtained from chronoamperometric measurements, which enabled the determination of the sensitivity, linear range and detection limit of the electrodes for glucose detection. The FAD-GDH/P2Mo18/PMA /MWCNT electrodes exhibited the best glucose biosensor performance with the highest sensitivity of 0.198 ± 0.015 mAcm -2

Lmmol -1 and a larger linear range between 1 mmolL -1 and 20 mmolL -1 ). The biosensor based on FAD-GDH/P 2 W 18 /PMA /MWCNT electrodes exhibited significantly lower sensitivity of 0.034 ± 0.008 mAcm - 2 Lmmol -1 . The difference in catalytic glucose oxidation performance between the two POM composites corresponds approximately to the difference of the surface coverage. It can therefore be assumed that P2W18 might have similar enzyme wiring capacities as P2Mo18. However, several attempts were made to adjust the surface coverage of P2W18, but unfortunately, this was without conclusive results.

Stability tests

The operational stability is one of the key parameters of bioanodes. The FAD-GDH/P2Mo18/PMA/MWCNTs and the FAD-GDH/P2W18/PMA/MWCNTs electrodes were investigated in a 5 mmolL -1 glucose solution for the duration of each test. Figure 5 shows the results of each stability test performed during one month. The bioelectrocatalytic currents were recorded almost every day at 0.2

V vs SCE after a 30 second discharge. Measurements were performed after 30 seconds to allow the current to stabilize and to eliminate the capacitive current of the MWCNT electrodes. Following a 45% loss of the initial current density after the first two weeks, the FAD-GDH/P2Mo18/PMA/MWCNTs electrode performance decreased more gradually. A catalytic current density of 0.68 mA cm -2 was still observed after a total of 32 days (ca. 30% of the initial current). For comparison, we recently reported a quinone-based FAD-GDH-modified MWCNT bioanode which showed high catalytic current densities for glucose oxidation but poor stability with only 48% of the initial current remaining after 5 days or ca. 20% after 9 days (Gross et al. 2017). Similar storage stability for an FADGDH electrode has been observed using an

Os polymer mediator with 56% of the initial current observed after 6 days (Zafar et al. 2012). The dramatic improvement in stability for the POM electrodes, compared to the quinone and osmium-based examples from the literature, may be attributed to better physical entrapment of the mediator and enzyme in the electrode matrix. The positively-charged pyrrolidinium and pyridine cations of the POMs may also contribute to improved stabilization of the enzyme via electrostatic interactions. The steady loss of catalytic performance for the P2Mo18 electrode can be related to various reasons. For example, the pyrene-contain-ing PMA molecule, the POM, or the FAD-GDH enzyme can desorb, and/or the enzyme can lose its catalytic activity due to denaturation and inhibition. Also, the steadily decreasing pH, resulting from the enzymatic production and accumulation of gluconic acid, may shift the redox system (I) of P2Mo18 to potentials where efficient electron mediation. Increased acidity may also be responsible for deactivation of FAD-GDH. For the FAD-GDH/P2W18/PMA/MWCNTs electrode, due to the initial low catalytic current, the performance decrease appears more gradual up until no bioelectrocatalytic current could be measured after 25 days. The same arguments as those given for the P2Mo18 electrode be taken to explain this performance loss.

Conclusions

(C4H10N)6[P2Mo18O62] . 4H2O was evaluated as an electron shuttle for the electrical wiring and regeneration of FAD-dependent GDH, and revealed excellent bioelectrocatalytic performance in terms of catalytic current density, low onset potential and 1 month stability. This POM was compared under identical conditions with a second type of POM, (C6H8NO)4[H2P2W18O62] . 6 H2O, for construction of bioanodes, for biofuel cell applications, or bioelectrodes for glucose biosensors. At this stage of investigation, the tungsten-based POM seems to have similar wiring capacities for FAD-GDH wiring but appeared less performing compared to the molybdenum-based POM due to immobilization issues, leading to reduced quantities by a factor 10. To our knowledge, this is the first example of using a polyoxometalate as a redox mediator for electron transfer between an oxidoreductase enzyme and an electrode material. The excellent bioelectrocatalytic current densities on the order of several mA's and the long term stability of these glucosemediating electrodes make these organic-inorganic hybrid materials very promising candidates for glucose biofuel cell and biosensor applications. The possibility of fine tuning the redox potentials, and the enhanced long term stabilities, combine the beneficial properties of commonly used inorganic (e.g. Os complexes (refs)) and organic (e.g. quinones) (Gross et al. 2017;Milton et al. 2015) mediators. The structural and electronic versatility of POMs, including their redox properties, size and charges, promise to enable improved electrical wiring of a wide range of enzyme-electrode interfaces.

Supplementary material: All methods, chemicals, materials, and the synthesis and characterization of the POM compounds, can be found in the supplementary material document.

  Scheme 1: Compounds and conditions for the synthesis of the Dawson type POMs ((C 4 H 10 N) 6 [P2Mo18O62] . 4H2O and (C6H8NO)4 [H2P2W18O62] . 6H2O) and their integration on a CNT bioelectrode for mediated electron transfer with FAD-GDH and bioelectrocatalytic glucose oxidation.

  ) to (4). The first two electron reductions are almost independent of pH(Sadakane and Steckhan 1998) and are slightly shifted to negative values compared to the voltammograms recorded in acidic solution. Both redox systems are situated in the potential range of interest for mediated electron transfer with FAD-GDH. The redox system III/III', related to equation (3), is the most affected by the pH change and exhibited a 180 mV shift to lower potentials. In spite of the pH dependent redox process shown in equation (4), the E½ value corresponding to the IV/IV' peaks is less affected by the pH change(Ammam 2013).

Figure

  Figure S6 and S7). Without PMA modification of the MWCNTs, no signals related to the POMs were

Figure 1 .

 1 Figure 1. CV scans of (A) P2W18 and (B) P2Mo18 in acidic aqueous solution (10 -4 mol.L -1 , 0.1 molL -1 H2SO4) at bare GCE. Scan rate: 100 mV s -1 . CV scans of (C) P2W18/PMA/MWCNTs and (D) P2Mo18/PMA/MWCNTs in McIlvaine buffer at pH 7.0. Scan rate: 10 mV s -1 .

Figure 2 :

 2 Figure 2: Representative SEM images of A) PMA/MWCNTs, B) P2Mo18/PMA/MWCNTs and C) P2W18/PMA/MWCNTs.

  FAD-GDH was used to evaluate the wiring capacities of POMs after its immobilization on the electrodes by simple incubation in enzyme-containing McIlvaine buffer solution overnight. To evaluate the bioelectrocatalytic glucose oxidation activity of the hybrid nanocluster-enzyme modified electrodes, the catalytic responses of the FAD-GDH/P2W18/PMA/MWCNTs and FAD-GDH/P2Mo18/PMA/MWCNTs electrodes were compared. The cyclic voltammograms of different modified electrodes were studied in McIlvaine buffer at pH 7 in the presence and absence of 100 mmol.L -1 glucose at a scan rate of 5 mV s -1 .

Figure

  Figure 3A and 3B show an illustration of the bioelectrocatalytic oxidation of glucose to gluconolactone

Figure 3 :

 3 Figure 3: (A, B) Schematic presentation of the bioelectrocatalytic oxidation of glucose by FAD-GDH via mediated electron transfer with (A) P2W18-and (B) P2Mo18-modified MWCNT electrodes. (C, D) CV scans at FAD-GDH/P2W18/PMA/MWCNTs and FAD-GDH/P2Mo18/PMA/MWCNTs electrodes recorded (a) without and (b) in the presence of 100 mmol.L -1 glucose. Voltammograms were recorded in McIlvaine buffer at pH 7.0 at a scan rate of 5 mV.s -1 .

Figure 4 :

 4 Figure 4: (A) Current density-time responses at increasing glucose concentrations for (a) FAD-GDH/P2W18/PMA/MWCNTs and (b) FAD-GDH/P2Mo18/PMA/MWCNTs electrodes (B) Catalytic current density evolution as a function of glucose concentration for (a) FAD-GDH/P2W18/PMA /MWCNTs and (b) FAD-GDH/P2Mo18/PMA/MWCNTs electrodes. Chronoamperometric experiments were performed at a fixed potential of 0.2 V vs. SCE in McIlvaine buffer at pH 7.0. Catalytic currents in (B) were measured after 30 seconds discharge.

Figure 5 .

 5 Figure 5. Stability of (a, red) FAD-GDH/P 2 Mo 18 /PMA/MWCNTs and (b, black) FAD-GDH/P2W18/PMA/MWCNTs electrodes. Catalytic current densities were measured by chronoamperometry at 0.2 V vs SCE after 30 s discharge in McIlvaine buffer at pH 7.0 in the presence of 5 mmolL -1 glucose.

  . A composite graphite electrode was prepared via electropolymerization of 1,8-

	2016) described the use of a alpha-metatungstate POM cluster, H2W12O40 6-, as an electrocatalyst for the
	reduction of hydrogen peroxide, produced by GOx. A good correlation between the glucose concentration
	and cathodic current density at -0.8 V vs SCE was observed. Very importantly, in in these examples, the
	redox activity of the POM clusters was used simply as an electrocatalysis and not exploited for electron
	transfer between the electrode and the enzyme (i.e. for bioelectrocatalysis).
	We want to introduce here, for the first time, two organic-inorganic POM materials
	((C4H10N)6[P2Mo18O62] . 4H2O (P2Mo18) and (C6H8NO)4[H2P2W18O62] . 6H2O) (P2W18) for efficient elec-
	tron transfer between FAD-GDH and a pyrenemethylamine (PMA) functionalized multiwalled carbon
	nanotube (PMA/MWCNT) electrode. The POM cluster polyanions, with charge compensating organic
	cations, were investigated as an efficient assembly with MWCNT electrodes for improved stability at
	neutral pH and as potential electron relays for MET-based glucose biosensors and biofuel cell anodes.
	2. Experimental
	2.1. Reagents and apparatus
	1-Pyrenemethylamine hydrochloride (PMA,95%), D-glucose (≥99.5%), citric acid, monosodium phos-
	phate monohydrate (NaH2PO4, ≥98%), disodium hydrogen phosphate heptahydrate (Na2HPO4, 98-
	102%), N,N-dimethylformamide (DMF, 99.9%), N-Methyl-2-pyrrolidone (NMP), Na2MoO4, Na2WO4 .
	diaminonaphthalene	in	the	presence	of	a	trilacunary	Keggin	type	POM,
	Na6H2O(Fe4[H4Fe4(PMo9O34)2(H2O)2] . H2O. The electrode was modified with a biocatalytic layer com-
	prising glutaraldehyde crosslinked GOx / BSA (bovine serum albumin). The POM electrode demonstrated
	efficient electrocatalytic reduction of enzymatically produced hydrogen peroxide at -0.04 V (vs. Ag/AgCl.
	KClsat) and showed very satisfying long term stability. More recently, Sahraoui et al. (Sahraoui et al.

Acknowledgment

We gratefully acknowledge funding from the Agence Nationale de la Recherche with the project CAROUCELL (ANR-13-BIOME-0003-02). The authors wish also to acknowledge support from LabEx ARCANE (ANR-11-LABX-0003-01), the PHC Utique program n° 14G1206 (CMCU), n° 30583QD (Campus France), the Institute Carnot PolyNat in Grenoble, the ANR project Mocca Cell (ANR-15-JTIC-0002-01), all for financial support, and the ICMG Chemistry Nanobio Platform, Grenoble, for providing facilities (PCN-ICMG).


Justine Guerin

Claire Soligot

Jennifer Burgain

Marion Huguet

Gregory Francius

Sofiane El-Kirat-Chatel

Faustine Gomand

Sarah Lebeer

Yves Le Roux

Frédéric Borges

Sarah Lebeer De

Frederic Borges

Joël Scher

Claire Gaiani
email: claire.gaiani@univ-lorraine.fr

Adhesive interactions between milk

Keywords: Lactobacillus rhamnosus GG, Milk fat globule membrane, pili, Atomic force microscopy, Cellular adhesion

Introduction

Pili are long and flexible proteinaceous filaments expressed at the surface of many Gram positive and Gram negative bacteria [START_REF] Kline | Bacterial Adhesins in Host-Microbe Interactions[END_REF]. Recently, pili were imaged at the surface of the model lactic acid bacterium (LAB) L. rhamnosus GG (LGG) [START_REF] Tripathi | Deciphering the Nanometer-Scale Organization and Assembly of Lactobacillus rhamnosus GG Pili Using Atomic Force Microscopy[END_REF][START_REF] Reunanen | Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG[END_REF]. LGG has two pili gene clusters in its genome, known as the spaFED and spaCBA, encoding two distinct kinds of pili: SpaFED pili and SpaCBA pili. Only the spaCBA gene cluster is actually expressed in standard laboratory growth conditions [START_REF] Reunanen | Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG[END_REF][START_REF] Douillard | Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics[END_REF]. LGG contains between 10 to 50 SpaCBA pili per cell, each with an approximate length of 1 µm [START_REF] Reunanen | Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG[END_REF][START_REF] Kankainen | Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human-mucus binding protein[END_REF][START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF]. The SpaCBA pili is composed of three pilin subunits [START_REF] Reunanen | Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG[END_REF]. SpaA is the major subunit and constitutes the backbone of the pilus. SpaB is mostly found at the base of the pilus, at the anchorage point with the cell wall, and few of them are also found in the pilus backbone. The SpaC subunits are present along the pilus and at the pilus tip [START_REF] Reunanen | Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG[END_REF]. SpaCBA pilus glycosylation was recently demonstrated and it was suggested that the SpaC pilins are the ones carrying glycosylation [START_REF] Tytgat | Probiotic Gut Microbiota Isolate Interacts with Dendritic Cells via Glycosylated Heterotrimeric Pili[END_REF]. SpaCBA pili are key players in promoting LGG adhesion to a variety of human substrates such as human mucus [START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF], collagen [START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF] or intestinal epithelial cells [START_REF] Sullan | Single-cell force spectroscopy of pilimediated adhesion[END_REF]. Adhesive and mechanical properties of pili when interacting with extracellular components of host epithelial layers (mucin or collagen) have been investigated using a non-invasive single cell force spectroscopy method [START_REF] Sullan | Single-cell force spectroscopy of pilimediated adhesion[END_REF].

SpaCBA pili adhesive properties are often attributed to the SpaC subunits. Two adhesive mechanisms were recently proposed. The first mechanism involves a molecular zipper featuring low forces with multiple SpaC subunits disposed along the pilus. The second model proposes a nanospring mechanism implying high forces at the tip of the pilus [START_REF] Tripathi | Adhesion and Nanomechanics of Pili from the Probiotic Lactobacillus rhamnosus GG[END_REF]. Recently, the key role of SpaCBA pili in LGG adhesion to Caco-2 intestinal epithelial cells was confirmed within a study involving LGG surface mutants [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF]. The exopolysaccharides (EPS) layer of the LGG surface interfered with the SpaCBA pili during the occurrence of the adhesion phenomenon to epithelial intestinal cells. The use of a LGG mutant without exopolysaccharides but with increasingly exposed pili improved the adhesion of LGG to epithelial intestinal cells compared to the LGG WT strain [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF].

Milk and a wide range of fermented milk products are often used in the industry as LAB delivery systems, which are likely to feature health benefits for the host [START_REF] Dalli | Industrial Production of Active Probiotics for Food Enrichment[END_REF]. Bacterial adhesion to epithelial intestinal cells is influenced by the presence of dairy components. Several studies demonstrated the ability of milk components to modulate pathogenic and probiotic bacteria adhesion to epithelial intestinal cells [START_REF] Volštátová | Effect Of Hydrolyzed Milk On The Adhesion Of Lactobacilli To Intestinal Cells[END_REF][START_REF] Volstatova | Milk digesta and milk protein fractions influence the adherence of Lactobacillus gasseri R and Lactobacillus casei FMP to human cultured cells[END_REF]. The addition of acid-hydrolyzed milk decreases the adhesion of Lactobacillus gasseri R and Lactobacillus plantarum S2 to a co-culture of Caco-2 and HT29-MTX cells [START_REF] Volštátová | Effect Of Hydrolyzed Milk On The Adhesion Of Lactobacilli To Intestinal Cells[END_REF]. The adhesion of L. gasseri R is also negatively affected by the addition of UHT-treated milk or rennet caseins. On the contrary, the addition of bovine serum albumin or whey proteins increases the adhesion of both L. gasseri R and L. casei FMP strains [START_REF] Volstatova | Milk digesta and milk protein fractions influence the adherence of Lactobacillus gasseri R and Lactobacillus casei FMP to human cultured cells[END_REF]. These studies show that milk components have the ability to modulate the adhesion of probiotic strains to epithelial intestinal cells. The same behavior was observed for pathogenic bacteria. Defatted milk fat globule membrane (MFGM) prevented E. coli O157 : H7 infection by inhibiting bacteria attachment to Caco-2 cells [START_REF] Ross | Defatted bovine milk fat globule membrane inhibits association of enterohaemorrhagic Escherichia coli O157:H7 with human HT-29 cells[END_REF]. In another study, bovine mucin extracted from the MFGM inhibited the adhesion of enteric pathogens to Caco-2 cells [START_REF] Parker | Bovine Muc1 inhibits binding of enteric bacteria to Caco-2 cells[END_REF][START_REF] Sando | Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria[END_REF]. Dairy products may thus influence bacterial attachment to epithelial intestinal cells but to our best knowledge, no mechanism has yet been described to explain this phenomenon. Interactions between bacterial surface molecule and dairy matrices may negatively impact the attachment of bacteria to intestinal cells.

Adhesive interactions between SpaCBA pili and food matrices, including milk components, are yet poorly studied. The MFGM has gained considerable attention due to its nutritional and technological properties [START_REF] Dewettinck | Nutritional and technological aspects of milk fat globule membrane material[END_REF]. The MFGM is a thin membrane surrounding the milk fat globule.

MFGM is composed of phospholipids, sphingolipids, and membrane-specific proteins, mainly glycoproteins. A recent review describes the potential role of the glycoprotein mucin 1 (MUC1) in the adhesion phenomenon occurring between bacteria and MFGM [START_REF] Guerin | Milk fat globule membrane glycoproteins: Valuable ingredients for lactic acid bacteria encapsulation?[END_REF][START_REF] Douëllou | Invited review: Anti-adhesive properties of bovine oligosaccharides and bovine milk fat globule membrane-associated glycoconjugates against bacterial food enteropathogens[END_REF]. In fact, many similarities are observed between the bovine MFGM mucin and human intestinal mucin [START_REF] Patton | The epithelial mucin, MUC1, of milk, mammary gland and other tissues[END_REF][START_REF] Bansil | Mucin structure, aggregation, physiological functions and biomedical applications[END_REF].

Bacterial adhesive properties in relation to intestinal mucin have already been described [START_REF] Nishiyama | Adhesion Properties of Lactic Acid Bacteria on Intestinal Mucin[END_REF], whereas surprisingly, the adhesive properties in relation to MFGM have been poorly studied.

Microscopic methods were used to decipher the adhesive phenomena occurring between MFGM and bacteria [START_REF] Tunick | Rheology and microstructure of low-fat Mozzarella cheese[END_REF][START_REF] Laloy | Influence of the fat content of Cheddar cheese on retention and localization of starters[END_REF][START_REF] Lopez | Lipolysis during ripening of Emmental cheese considering organization of fat and preferential localization of bacteria[END_REF][START_REF] Hickey | Growth and location of bacterial colonies within dairy foods using microscopy techniques: a review[END_REF]. Similarly, the role of MUC1 in adhesion with pathogenic bacteria has already been described [START_REF] Parker | Bovine Muc1 inhibits binding of enteric bacteria to Caco-2 cells[END_REF][START_REF] Sando | Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria[END_REF] but the interactive behaviors of MFGM or MUC1 were never studied at the molecular scale.

In this study, a biophysical approach was first implemented to understand how LGG adheres to MFGM using atomic force microscopy (AFM). Then, a biological study was performed to understand how the adhesion of LGG to Caco-2 TC7 intestinal epithelial cells can be modulated by MFGM. For this purpose, LGG surface mutants were employed to decipher how SpaCBA pili influence LGG adhesion to intestinal epithelial cells in the presence or absence of milk components (i.e. MFGM).

Materials and Methods

Materials

The wild type strain L. rhamnosus GG (ATCC 53103) (LGG WT) and two of its surface mutants were used in this study. The two mutants were the pili-depleted spaCBA mutant CMPG5357 (called

LGG spaCBA in this study) [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF] and the EPS-deficient welE mutant CMPG5351 (called LGG welE) [START_REF] Lebeer | Exopolysaccharides of Lactobacillus rhamnosus GG form a protective shield against innate immune factors in the intestine[END_REF][START_REF] Lebeer | Identification of a gene cluster for the biosynthesis of a long galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase[END_REF].

The isolation of MFGM was adapted from [START_REF] Le | Effect of washing conditions on the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from milk[END_REF] with some modifications [START_REF] Le | Effect of washing conditions on the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from milk[END_REF]. For each experimental run, 40 L of raw bovine milk was collected from a stirred cooling tank from a local farm (farm of La Bouzule, Laneuvelotte, France). Milk was first heated at 50 °C to favorize milk creaming. Then, fat globules were separated from skimmed milk using a cream separator (Elecrem1, Elecrem, Fresnes, France). After one night at 4 °C, the cream was churned using a mixer to obtain two fractions: butter and buttermilk. The buttermilk containing the MFGM was added to the final MFGM solution. The butter was melted at 50 °C after adding distilled water (half of the equivalent volume w / w). The total mixture was centrifuged at 3300 g for 10 min. The supernatant containing butter oil was eliminated and the butter serum containing the MFGM fraction was rinsed with 20 g of distilled water and was centrifuged at 3300 g for 10 min. The suspension of MFGM containing the butter serum was collected and stored in a freezer for one night. The MFGM powder was obtained after freeze-drying the butter serum.

The purified gastric mucin was purchased from Sigma-Aldrich (Mucin from porcine stomach, M1778, France).

The Caco-2 TC7 TC7 cells were obtained from Pr. Yves Leroux (team MRCA, laboratoire URAFPA, Nancy, France).

Atomic force microscopy

Bacteria culture.

LGG culture was performed as described by [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF] [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF]. Preculture of LGG (WT and its surface mutants) was prepared by inoculating 9 ml of fresh MRS broth with 100 µl of a bacterial stock solution and placed overnight at 37 °C without stirring. This preculture was used to inoculate 9 ml of fresh MRS broth and the growth was performed at 37 °C to reach an optical density of 1.2 at 660 nm. Bacteria were centrifuged at 3000 g for 10 min and resuspended in 1 ml of phosphate-buffered saline (PBS) buffer (pH 6.8).

Preparation of bacterial surfaces. The bacterial suspension was deposed on a mica coated with a gold layer functionalized with a NH3 terminated-PEG linker (PT.BORO.PEG.NH3, Novascan, Ames, Iowa, USA) for 15 h at 4 °C (pH 6.8). The mica was rinsed with PBS before use. AFM topographic images confirmed the presence and good coverage of LGG on the mica surface (data not shown).

Preparation of proteins probes. AFM probes with borosilicate glass particles (2 µm) used in this study were purchased from Novascan (PT.BORO.PEG.NH3, Ames, Iowa, USA). These probes were coated with gold and modified with a NH3 terminated-PEG linker (PT.BORO.PEG.NH3, Novascan, Ames, Iowa, USA). The spring constant of these probes was 0.01 N/m. MFGM or purified mucin were prepared in distilled water at a concentration of 1 % (w/w) and adhered on the probe by immersion at 4°C for 15 h. The time for adsorption was much longer than the time necessary for the proteins to adsorb on the tip. Preliminary experiments confirmed that the tip was covered with proteins (data not shown). Tips were rinsed with milli-Q-grade water before use.

Atomic force microscopy measurement. The bacteria were adhered on a functionalized mica and the AFM probe was brought in contact with the bacterial surface so as to form a molecule-molecule complex governed by intermolecular bonds. When the AFM probe retracts from the substrate surface, the force required for dissociation i.e. the unbinding force can be defined as the rupture force occurring when the probe dissociates from the sample (i.e. when the interaction bonds break) [START_REF] Lee | Atomic force microscopy: Determination of unbinding force, off rate and energy barrier for protein-ligand interaction[END_REF].

Force measurements were performed at room temperature in PBS buffer (pH 6.8) using an Asylum MFP-3D atomic force microscope (Santa Barbara, CA, USA) controlled by the operation software IGOR Pro 6.04 (Wavemetrics, Lake Osewego, OR, USA) as described by [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF] [START_REF] Guerin | Pili of Lactobacillus rhamnosus GG mediate interaction with β-lactoglobulin[END_REF].

The cantilever spring constant with borosilicate particle was first determined using the thermal calibration method which provided a value of 0.01 N/m [START_REF] Lévy | Measuring the spring constant of atomic force microscope cantilevers: thermal fluctuations and other methods[END_REF]. For each experiment, the force map was recorded on a 10 × 10 µm² surface corresponding to 32 × 32 points, i.e. 1024 force curves. AFM force-distance curves were obtained a retraction speed of 400 mm/s.

Curves processing.

For each AFM force measurements a force map containing 1024 force curves was recorded. Retraction curves were analyzed to determine the presence of specific adhesive occurrences between bacteria and milk proteins. Specific adhesion revealed the presence of stretching biomolecule signatures on the retraction curve. For each set of experimental conditions, 100 adhesive force curves were selected and the corresponding retractions were analyzed to determine the percentage of adhesive events with specific biomolecules stretching signatures. In this study, the percentage of adhesive events represented are the percentage of specific adhesive phenomena presenting retraction curves which proved to feature specific biomolecules stretching signatures.

Bacterial adhesion to Caco2 TC7 cells

Epithelial cell lines and culture conditions. Epithelial cells Caco-2 TC7 were grown in Dulbecco's modified Eagle's Minimal Essential Medium with 4.5 g/L glucose (DMEM Glutamax, Fisher Scientific) supplemented with 20 % (v/v) of heat-inactivated fetal calf serum (FCS; Gibco®) and 1 % (v/v) of penicillin-streptomycin (5 000 U/ml penicillin and 5,000 μg/ml streptomycin, Gibco®) and 1 % (v/v) non-essential amino acid ×100 (Gibco®). Cells were conserved in DMEM medium supplemented with FCS and 10 % glycerol in liquid nitrogen.

The Caco-2 TC7 culture was adapted from [START_REF] Kebouchi | Implication of sortase-dependent proteins of Streptococcus thermophilus in adhesion to human intestinal epithelial cell lines and bile salt tolerance[END_REF] [START_REF] Kebouchi | Implication of sortase-dependent proteins of Streptococcus thermophilus in adhesion to human intestinal epithelial cell lines and bile salt tolerance[END_REF]. Briefly, cells were thawed and seeded in plastic flasks (75 cm², Dutscher). After 4 days, cells were trypsinized with 0.05 % of trypsin-EDTA (×1, Gibco®). After 5 min of contact with trypsin, DMEM medium with FCS was added to the culture to inhibit the trypsin action. Cells were recovered after by centrifugation at 1000 rpm for 5 min at 37 °C. The cells pellet was suspended in DMEM (20 % FCS, 1 % antibiotic and 1 % amino acid) and seeded in six well culture plates (Dutscher) at a final concentration of 6 × 10 4 cell per cm². Cells were maintained at 37 °C in a humidified atmosphere with 10 % CO2 and the culture medium was changed every day. The differentiated cell monolayers were obtained after 22 culture days. Two days before the adhesion tests, DMEM without antibiotics was used to avoid bacteria death during the occurrence of the adhesion phenomenon.

LGG cultures conditions. Bacterial stocks were stored at -80 °C in MRS broth with 20 % (v / v) glycerol. Pre-culture of LGG (WT and its surface mutants) were prepared by inoculating 50 ml of MRS broth with 1 ml of bacterial stock and placed at 37 °C for 8 h without stirring. These precultures were used to inoculate 200 ml of fresh MRS broth; bacterial growth was led for 12 h at 37 °C. The cultures were centrifuged at 3000 g for 10 min at room temperature. The resulting pellets were washed with HBSS (Hanks' Balanced Salt Solution, Fisher Scientific) to remove the excess of medium and centrifuged at 3000 g for 10 min.

To study the role of surface components in the adhesion phenomenon occurring between LGG and Caco-2 TC7, the LGG pellet (WT and its surface mutants) were directly resuspended in DMEM medium supplemented with only 1 % of amino acid (without FCS and antibiotic) at a bacterial concentration around 10 9 CFU/ml.

To study the effect of MFGM addition on LGG adhesion, the bacterial pellets were resuspended in a solution of MFGM (5 mg/ml) in DMEM medium supplemented with only 1 % of amino acid, for 1 h at 37 °C without stirring. The bacteria mixtures were then centrifuged at 500 g for 10 min to obtain bacteria cells pellets. The supernatant containing unbound MFGM was removed and the pellets containing bacteria cells were resuspended in DMEM media (1% amino acid) at a concentration around 10 9 CFU/ml.

LGG survival in DMEM. Bacterial viability in cells culture media (DMEM + 1 % amino acids) was determined by comparing the number of LGG initially present in the DMEM with 1 % amino acids solution and the number of LGG in the medium after incubating for 2 h at 37 °C (Figure S1).

In both cases, solutions containing the bacteria were mixed for 1 min using a vortex homogenizer.

Samples were serially diluted in tryptone salt broth and plated on MRS agar. After 48 h incubation at 37 °C, cell counts were performed and expressed using CFU/ml (Colony Forming Units/ml).

MTT toxicity assay.

The effects of MFGM on Caco-2 TC7 cell viability was determined through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) toxicity assays. MTT assays involve the reduction of the water soluble MTT, a yellow tetrazolium salt, into a blue formazan dye precipitate, that can be extracted using an organic solvent. The reduction is realized by the mitochondria of metabolically active cells. MTT assays with 22-day Caco-2 TC7 monolayer culture were assessed as described by [START_REF] Bu | Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant[END_REF] [START_REF] Bu | Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant[END_REF]. Briefly, cells were treated with 4 ml of different MFGM solutions (0.5 and 5 mg/ml in DMEM without FCS and antibiotic) for 2 h at 37 °C. The MFGM solution was then removed and the differentiated Caco-2 TC7 cell monolayers were gently rinsed with 1 ml of HBSS. Cells were then incubated with 3 ml of DMEM (20% FCS, 1 % amino acid and 1 % antibiotic) and 300 µl of MTT solution (5 mg/ml in PBS) for 3.5 h at 37 °C; the medium was then removed and 2 ml of DMSO per well were added. Solutions containing cells were diluted ten times and transferred on 96-square angled bottom well plates. The plates were covered from light and incubated with orbital shaking (150 rpm) for 10 min. Absorbance was then measured using a microplate reader at a wavelength of 570 nm.

Adhesion tests. Before adhesion, the differentiated Caco-2 TC7 cell monolayers were gently rinsed with 3 ml of HBSS; 4 ml of bacterial suspension (bacteria alone or bacteria incubated with MFGM) were then added to the cells at a concentration of 10 9 CFU/ml to reach a bacteria cells to epithelial cells ratio of 1000:1 [START_REF] Kebouchi | Implication of sortase-dependent proteins of Streptococcus thermophilus in adhesion to human intestinal epithelial cell lines and bile salt tolerance[END_REF]. The number of bacteria present in the initial solutions were determined by bacterial enumeration. Bacteria were incubated on epithelial cells during 2 h at 37 °C and the supernatant of each well was removed. The cell monolayers were gently rinsed with HBSS (4 × 1 ml) to obtain non-adherent bacterial suspensions. Caco-2 TC7 cells were then scraped with 3 × 1 ml of Triton® (×100, Sigma) at 0.1 % (v / v). The solution containing Caco-2 TC7 cells with adherent bacteria was passed three times through a needle and then incubated for 30 min at room temperature. Bacterial enumeration was used to evaluate both the number of non-adherent and adherent bacteria in the supernatant. In both cases, the solution containing the bacteria was mixed during 1 min using a vortex homogenizer. Samples were serially diluted in tryptone salt broth and plated on MRS agar. After a 48 h-incubation period at 37 °C, cell counts were determined and expressed using CFU/ml. The number of adherent bacteria per cell was determined using the following equation:

Statistical tests.

Experimental data obtained in this study were all analyzed using a one-way analysis of variance (ANOVA) followed by a Tukey's test. Comparisons were performed between LGG WT and the mutant strains. Results were expressed as means ± SEM (Standard Error of the Mean). Differences were considered statistically significant when P value < 0.05. In the figures, means ± SEM followed by a different superscript letter indicate a significant difference (P<0.05).

Results and Discussion

Key role of pili in the adhesive interactions occurring between LGG and MFGM.

In the present study, adhesive events between LGG and MFGM were measured using AFM to understand how LGG adheres to MFGM. AFM probes were functionalized with MFGM and bacteria strains were immobilized on mica. The three strains used in this work are LGG wildtype (WT), LGG spaCBA mutant [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF] (pili-depleted), and LGG welE mutant [START_REF] Lebeer | Exopolysaccharides of Lactobacillus rhamnosus GG form a protective shield against innate immune factors in the intestine[END_REF][START_REF] Lebeer | Identification of a gene cluster for the biosynthesis of a long galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase[END_REF] (EPS-depleted).

Their percentage of adhesive events presenting specific biomolecule stretching signatures with the MFGM probes were 75.5 ± 1.5, 7.0 ± 3.5 and 99.0 ± 1.0 %, respectively (Figure 1). The adhesive events of the pili-depleted spaCBA mutant were thus drastically decreased in comparison with those occurring for the wild type strain. On the contrary, when SpaCBA pili were overexposed (for the EPS-deficient welE mutant), the adhesive events significantly increased compared to the wild type strain. It is known that pili located on the bacterial surface of the LGG welE mutant are not embedded anymore within the EPS layer [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF], therefore the pili in this mutant are probably fully available to establish contact points with MFGM, explaining the increased number of adhesive events [START_REF] Burgain | Impacts of pH-mediated EPS structure on probiotic bacterial pili-whey proteins interactions[END_REF]. These results highlight the key role of SpaCBA pili in the adhesion phenomenon occurring between LGG and MFGM.

To confirm the role of the SpaCBA pilus in the adhesion phenomenon occurring between LGG and MFGM, force profiles of AFM retraction curves were then analyzed for each strain (Figure 2).

Retraction curves recorded during adhesion between MFGM and

LGG WT presented several rupture peaks with a mean number of ruptures of 1.9 ± 0.0, a mean maximal rupture force of 0.5 ± 0.0 nN, and a maximal rupture distance of 5.9 ± 0.1 µm (Figure 2A). These signatures are characteristic of stretched biomolecules, which would be present on the bacterial surface [START_REF] Dufrêne | Sticky microbes: forces in microbial cell adhesion[END_REF]. To identify the biomolecule stretched during the adhesion with MFGM, LGG surface mutants were used. The specific signatures recorded for LGG spaCBA mutant on retraction curves were rare (7 %) and totally different from those observed with LGG WT (Figure 2B), thus confirming a key role of the SpaCBA pili in the adhesion phenomenon occurring between LGG and MFGM. In the absence of pili, the mean number of ruptures was 1.0 ± 0.0, the mean maximal rupture force was 0.1 ± 0.0 nN, and the mean maximal rupture distance was 0.3 ± 0.1 µm. Signatures recorded during biomolecules stretching between the LGG spaCBA mutant and MFGM appear similar to saccharide molecules signatures; they could therefore correspond to the EPS [START_REF] Francius | Stretching polysaccharides on live cells using single molecule force spectroscopy[END_REF] stretching behavior , which are more accessible in the absence of pili. In contrast, the specific signatures recorded on the retraction curves for the LGG welE mutant contained multiple rupture peaks. The number of rupture peaks and the force parameters increased compared to those recorded for LGG WT. These signatures have a mean number of ruptures of 5.8 ± 0.7, a mean maximal rupture force of 1.2 ± 0.1 nN, and a mean maximal rupture distance of 8.4 ± 1.1 µm. Since the SpaCBA pili are overexposed in the absence of EPS for the LGG welE mutant [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF], the increase number of adhesion peaks and the increased force may be explained by the better accessibility of the MFGM adhesion sites to the pili. All these results thus point towards the SpaCBA pilus being the key macromolecule engaged in the adhesion of LGG to MFGM.

The MFGM is composed of many glycoproteins; one of them is a kind of mucin called MUC1.

Many similarities in the structure and composition of mucin MUC1 and purified mucin [START_REF] Patton | The epithelial mucin, MUC1, of milk, mammary gland and other tissues[END_REF][START_REF] Bansil | Mucin structure, aggregation, physiological functions and biomedical applications[END_REF] have been found. The signatures observed during the retraction of the MFGM-functionalized probe from the LGG welE mutant's surface are similar to those observed during the gastrointestinal mucin stretching in another study [START_REF] Gunning | Mining the "glycocode"--exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy[END_REF]; this indicates that MUC1 (from MFGM) may be implied in the adhesion phenomenon with LGG. Mucins are highly glycosylated proteins, with multiple lectinadhesive sites. [START_REF] Gunning | Mining the "glycocode"--exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy[END_REF] explained the multiple adhesion peaks by repeated attachment-detachment cycles of the lectin-functionalized AFM tip present on the mucin chains [START_REF] Gunning | Mining the "glycocode"--exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy[END_REF]. These events are achieved with the presence of multiple binding sites along the mucin molecule. During adhesion between MFGM and the LGG welE mutant, the relative large number of adhesion peaks observed on the retraction curve may be the result of repeatedly attachment and detachment of mucin MUC1 on the LGG welE mutant cells due to the increased number of adhesive sites on pili for this mutant. [START_REF] Gunning | Mining the "glycocode"--exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy[END_REF] used the term "glycocode" to talk about signatures with multiple adhesion peaks, typical of glycosylated molecule-stretching as for mucin.

To support our hypothesis concerning the role of mucin MUC1 in the adhesion phenomenon involving MFGM and LGG, forces measurements with purified mucin from porcine stomach were performed using AFM. The adhesive events percentages were 58.5 ± 11.5, 13.0 ± 1.0, and 86.0 ± 2.0 % with LGG WT, LGG spaCBA mutant and LGG welE mutant, respectively (Figure 3). Similar as MFGM, adhesive events decreased for the LGG spaCBA mutant compared to LGG WT.

On the contrary, adhesive events significantly increased with the LGG welE mutant compared to

LGG WT. These results suggest a key role for the SpaCBA pilus in LGG adhesion with purified intestinal mucin. Retraction curves recorded during the occurrence of the adhesion phenomenon between the purified mucin and LGG WT (Figure 4A), the LGG spaCBA mutant (Figure 4B), or the LGG welE mutant (Figure 4C) confirmed the role of the SpaCBA pilus in adhesion.

The retraction curves recorded for LGG WT presented the specific signatures of stretching biomolecules with a mean number of ruptures of 2.2 ± 0.1, a mean maximal rupture force of 0.8 ± 0.1 nN, and a mean maximal distance of rupture of 0.7 ± 0.0 µm (Figure 4A). The similar signatures observed on the retraction curves of LGG WT when interacting with both MFGM or purified mucin (Figure 5A) suggest that the mucin MUC1 may be the biomolecule implicated in the adhesion between LGG and MFGM. For the pili-depleted strain, the LGG spaCBA mutant, the specific signatures on retraction curves are rare (13.0 %) and totally different from those observed with LGG WT (Figure 4B); this indicates that SpaCBA pili are essential for adhesion to occur between purified mucin and LGG. Retraction curves observed between the LGG welE mutant and purified mucin contained multiple rupture peaks similar to those observed for MFGM (Figure 4C), yet the maximal rupture forces was higher for purified mucin (1.4 ± 0.0 nN of mean value). This could probably due to differences in purity levels; purified mucin is indeed highly purified, whereas MFGM contains milk mucin but also many other biomolecules such as glycolipids and other glycoproteins. Some part of the retraction curves recorded for MFGM and the purified mucin were similar and correspond to the "glycocode" (Figure 5B), meaning that the adhesion between LGG SpaCBA pili and MFGM may be governed by the mucin MUC1. The maximal distance of rupture was very important during adhesion between the LGG welE mutant and MFGM with a value of 8.4 ± 0.2 µm. The adhesion between the LGG welE mutant and purified mucin lead to a maximal distance of rupture of only 1.9 ± 0.2 µm and ruptures observed on retraction curves which returned several times to the baseline. Because MFGM is composed of many different biomolecules, this could mean that different biomolecules, including MUC1, are successively stretched during the retraction of the MFGM-functionalized AFM probe from the LGG welE mutant surface.

SpaCBA pilus play a key role in LGG adhesion to epithelial intestinal cells. A recent study

based on the LGG surface mutants suggests that SpaCBA pili play a key role in LGG adhesion to Caco-2 epithelial cells [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF]. To validate our culture cells model and confirm the role of bacterial surface components in LGG adhesion to epithelial intestinal cells, the adhesion phenomena occurring between LGG (LGG WT and two of its surface mutants) and Caco-2 TC7 cells was first checked (Figure 6).

All results are obtained by counting the number of adhering bacteria per intestinal cell. Our data revealed that the adhesion of LGG to Caco-2 TC7 cells was drastically reduced for the LGG spaCBA mutant compared to the WT strain (9.1 ± 1.1 bacteria per cell, versus 19.7 ± 3.8, P < 0.05).

On the contrary, the adhesion capacity of the LGG welE mutant to Caco-2 TC7 cells was significantly increased when compared to the WT strain capacity (42.6 ± 4.7 bacteria per cell, versus 19.7 ± 3.8, P < 0.05). [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF] demonstrated and explained the improved adhesive capacities of the EPS-deficient welE mutant by the increased exposure of the SpaC subunits from the pili SpaCBA [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF]. Our results validate the culture cell adhesion experiment and confirm the key role of pilus SpaCBA in LGG adhesion to Caco-2 TC7 epithelial intestinal cells which has been described in the literature [START_REF] Lebeer | Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells[END_REF].

Impact of MFGM on LGG adhesion to Caco 2 TC7 epithelial cells

When comparing proteins-pilus SpaCBA interactions vs. cell-pilus SpaCBA interactions, the stability of proteins during the gastrointestinal digestion is assumed. It was indeed recently demonstrated that some MFGM proteins are resistant to human enzymatic gastrointestinal digestion [START_REF] Le | Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion[END_REF]. Some glycoproteins such as the mucin MUC1, the cluster of differentiation 36, and a significant part of the periodic acid Schiff 6/7 presented a high resistance to proteases thanks to their glycosylation level. Therefore, MFGM has been chosen to study the impact of food matrix composition on bacterial adhesion.

The impact of MFGM addition on LGG adhesion capacity to Caco-2 TC7 epithelial intestinal cells was investigated by comparing the adhesive capacities of LGG in the presence or the absence of MFGM (Figure 7), after first validating the nontoxic effect of MFGM on Caco-2 TC7 epithelial intestinal cells viability via a cell integrity MTT assay (Figure S2). The key role of the pilus SpaCBA on LGG adhesion to Caco-2 TC7 cells was confirmed by reproducible and repeatable experiments. The number of adherent bacteria per cell was 19.4 ± 2.9, 8.4 ± 0.5 and 29.2 ± 3.1 for

LGG WT, the LGG spaCBA mutant and the LGG welE mutant, respectively. The presence of MFGM drastically decreased the adhesion capacity of LGG WT and the LGG welE mutant with a number of adherent bacteria per cell of 8.5 ± 0.2 and 9.0 ± 1.2, respectively. On the contrary, the adhesion capacity of the LGG spaCBA mutant was not impacted by the presence of MFGM (8.1 ± 1.6 adherent bacteria per cell).

The presence of MFGM only modified the adhesive capacities of LGG WT and the LGG welE mutant. These two strains express SpaCBA pili on their surface. By interacting with pili, MFGM may mask SpaCBA pili adhesion sites to epithelial intestinal cells and thus inhibit the adhesion of

LGG WT and the LGG welE mutant to Caco-2 TC7 epithelial intestinal cells (Figure 7).

Additionally, as expected, MFGM does not influence the adhesion to Caco-2 TC7 epithelial intestinal cells for this mutant, in agreement with the fact that no adhesive events were observed between MFGM and the LGG spaCBA mutant as shown above. The number of adherent bacteria per cell for the wild type strain and the welE mutant in the presence of MFGM was similar to the basal level of adherent bacteria per cell expressed for strains without pili. MFGM thus appears to block SpaCBA pili adhesion sites to cells but other surface molecules present on LGG WT and the welE mutant surfaces still allow adhesive phenomena to occur between WT and welE strains and the cells. These molecules are probably the same as those expressed on the spaCBA strain surface. Therefore a competition is likely to occur between the dairy matrix and epithelial intestinal cells for the adhesion to LGG SpaCBA pili.

Few studies have compared the efficacy of probiotics in different food matrices and little is known about how the dairy matrix impacts probiotic functionality [START_REF] Sanders | Food formats for effective delivery of probiotics[END_REF][START_REF] Marco | Environmental factors influencing the efficacy of probiotic bacteria[END_REF]. Dairy products count amongst the most popular matrices used for probiotic delivery because they provide a physiochemical barrier against gastric acid [START_REF] Wang | Comparative analysis of the gene expression profile of probiotic Lactobacillus casei Zhang with and without fermented milk as a vehicle during transit in a simulated gastrointestinal tract[END_REF]. Milk matrices help bacteria to survive when passing through the stomach thanks to the buffering capacity of milk proteins. Milk proteins are able to create a pH gradient around bacteria permitting a gradual exposure of bacteria to the external low pH leading to an increase in bacterial survival [START_REF] Würth | Protective effect of milk protein based microencapsulation on bacterial survival in simulated gastric juice versus the murine gastrointestinal system[END_REF][START_REF] Heidebach | Microencapsulation of probiotic cells by means of rennetgelation of milk proteins[END_REF]. The delivery matrix can also modify the functional traits at the site of delivery in the gut but very few studies look at this aspect. For example, the functionality of L. casei BL23 is improved in milk. When L. casei BL23 is added in milk, its effect on reducing the development of colitis is improved [START_REF] Lee | Attenuation of Colitis by Lactobacillus casei BL23 Is Dependent on the Dairy Delivery Matrix[END_REF]. The adhesion of LGG to Caco-2 TC7 epithelial intestinal cells is also reinforced after incubation in yogurt vs. ice cream and the binding properties of bacteria are correlated to the product storage time [START_REF] Deepika | Effect of food models and low-temperature storage on the adhesion of Lactobacillus rhamnosus GG to Caco-2 cells[END_REF]. Our study brought to light new knowledge on how food matrices impact probiotic efficacy. The consumption of LGG with MFGM may significantly decrease the adhesion of LGG to Caco-2 TC7 cells, possibly lowering the resulting/associated health impacts.

Conclusion

The ability of MFGM to inhibit the attachment of LGG to host cells was demonstrated. By using two complementary approaches (biophysical and biological), a mechanism was proposed to explain how MFGM can impact bacterial adhesion to intestinal epithelial cells (Figure 8). The biophysical approach using AFM revealed that LGG is able to interact with MFGM through SpaCBA pili. The biological approach using cellular adhesion highlighted that LGG SpaCBA pili also play a key role in the intestinal epithelial cells adhesion phenomenon. When bacteria integrated within a native dairy matrix (as it is the case for some MFGM glycoproteins) reach the intestine, pili adhesive sites are thus probably blocked by milk proteins such as MFGM and therefore bacterial adhesion to intestinal cells can be inhibited or limited. As many lactic acid bacteria are incorporated into dairy products, much research should be performed in order to check if bacterial functionality is preserved in the presence of dairy components. This knowledge on adhesive mechanisms may also be important when formulating new nutraceutical products when the goal is to inhibit the adhesion of pathogenic bacteria to the intestine.

  Number of adherent bacteria/cell = Number of total adherent bacteria (𝐶𝐹𝑈 𝑚𝑙 ⁄ ) × Triton volume (ml) Number of cells in the wellTwo independent experiment series were performed in triplicate.

Figure 1 Figure 2 Figure 3

 123 Figure 1

5 Figure 6

 56 Figure 4 587

Figure 7

 7 Figure 7

  Figure 8

Figure 1 :

 1 Figure 1: Adhesive events quantification between MFGM and LGG WT and two surface mutants

Figure 2 :

 2 Figure 2: Adhesion between MFGM AFM probes and LGG WT, LGG spaCBA, and LGG welE

Figure 3 :

 3 Figure 3: Adhesive events quantification between purified mucin and LGG WT, LGG spaCBA

Figure 4 :

 4 Figure 4: Adhesion between purified mucin and LGG WT and LGG spaCBA and LGG welE

Figure 5 :Figure 6 :Figure 7 :Figure 8 :

 5678 Figure 5 : Representative retraction curves observed during adhesion between LGG WT (A) or

Supporting Information. The LGG survival in DMEM + 1 % amino acid was determined in Figure S1. The nontoxic effect of MFGM on Caco-2 TC7 epithelial intestinal cells viability was determined via a cell integrity MTT assay (Figure S2).


Robin Ramos

Jérémy M Zimbron

Serge Thorimbert

Lise-Marie Chamoreau

Annie Munier

Candice Botuha

Anthi Karaiskou

Michèle Salmain

Joëlle Sobczak-Thépot

Insights into the antiproliferative mechanism of (C^N)-chelated half-sandwich iridium complexes

Transition metal-based anticancer compounds, as alternative to platinum derivatives, are raising scientific interest as they may present distinct although poorly understood mechanisms of action. We used a structure-activity relationship-based methodology to investigate the chemical and biological features of a series of ten (C^N)-chelated half-sandwich iridium III complexes of the general formula [IrCp*(phox)Cl], where (phox) is a 2-phenyloxazoline ligand forming a 5-membered metallacycle. This series of compounds undergoes a fast exchange of their chlorido ligand once solubilised in DMSO. They were cytotoxic to HeLa cells with IC50 values in the micromolar range and induced a rapid activation of caspase-3, an apoptosis marker. In vitro, the oxidative power of all the complexes towards NADH was highlighted but only the complexes bearing substituents on the oxazoline ring were able to produce H2O2 at the micromolar range. However, we demonstrated using a powerful HyPer protein redox sensor-based flow cytometry assay that most complexes rapidly raised intracellular levels of H2O2. Hence, this study shows that oxidative stress can partly explain the cytotoxicity of these complexes on the HeLa cell line and gives a first entry to their mechanism of action

Introduction

Platinum-based compounds in association with other cytotoxic agents are still among the most frequently prescribed anticancer drugs, despite their severe side effects and resistance developed by many tumours. Alternative metalbased compounds, among which various ruthenium II and ruthenium III complexes, [START_REF] Zeng | The development of anticancer ruthenium( II ) complexes: from single molecule compounds to nanomaterials[END_REF] are raising scientific interest as they may present original multi-targeting mechanisms of action (MoA) that stand out from the classical DNA-damage inducers and antimitotics.

Iridium complexes provide other potentially useful alternatives to platinum-based anticancer drugs. In the iridium(III) family, the octahedral, bis-cyclometallated complexes of the general formula [Ir III (C^N) [START_REF] Caporale | Cyclometalated iridium(III) complexes for life science[END_REF] (N^N)] + , X -are well known for their luminescence properties owing to metal-to-ligand charge transfer (MLCT) transitions making them popular imaging probes as well as photodynamic therapy agents. [START_REF] Caporale | Cyclometalated iridium(III) complexes for life science[END_REF][START_REF] Zhang | Nucleus-Targeted Organoiridium-Albumin Conjugate for Photodynamic Cancer Therapy[END_REF] The second series, namely the so-called half-sandwich complexes, presents a different coordination pattern with a pseudo-tetrahedral geometry. The iridium centre is coordinated by a pentasubstituted cyclopentadienyl ligand (typically pentamethyl cyclopentadienyl, Cp*), a chelating bidentate ligand and often a chlorido ligand occupying the last coordination position. The lability of this last ligand makes the complexes reactive towards various substrates, hence their initial application as hydrogenation and C-H activation catalysts.

4-6

The antiproliferative properties of half-sandwich complexes bearing a C^N chelating ligand were first reported in 2011.

Subsequent reports allowed to delineate the structural parameters (charge, nature of the chelating ligand, degree of lability of the third ligand) governing their cytotoxicity. [START_REF] Lord | Organometallic Iridium Arene Compounds: The Effects of C -Donor Ligands on Anticancer Activity[END_REF][START_REF] Yang | Zwitterionic and cationic half-sandwich iridium( III ) ruthenium( II ) complexes bearing sulfonate groups: synthesis, characterization and their different biological activities[END_REF][START_REF] Zimbron | Synthesis, Photophysical Properties, and Living Cell Imaging of Theranostic Half-Sandwich Iridium-4,4-Difluoro-4bora-3 a ,4 a -diaza-s -indacene (BODIPY) Dyads[END_REF][START_REF] Liu | Potent Half-Sandwich Iridium(III) Anticancer Complexes Containing C ∧ N-Chelated and Pyridine Ligands[END_REF] For some complexes, a relationship between cytotoxicity and rapid raise of total intracellular reactive oxygen species (ROS) was highlighted [START_REF] Liu | The Potent Oxidant Anticancer Activity of Organoiridium Catalysts[END_REF] with concomitant upregulation of antioxidant responses. [START_REF] Hearn | Pharmacogenomic investigations of organo-iridium anticancer complexes reveal novel mechanism of action[END_REF] More generally, the ability of molecules to modulate the oxidation stress represents an innovative strategy to preferentially target cancer cells owing to their unique metabolic properties.

14,15

Because the relationship between cytotoxicity, in vitro generation of H 2 O 2 and intracellular H 2 O 2 levels had never been experimentally demonstrated, we undertook to investigate it on a library of half-sandwich iridium(III) complexes. For this purpose, ten complexes Ir1-10 comprising variously substituted 2-phenyloxazolines (phox) L1-10 as C^N chelating ligands were synthesized and characterized. The phox scaffold was selected owing to the readiness to introduce a wide range of electron-donating or withdrawing substituents on either of the 2 rings and a second stereogenic centre on the oxazoline ring. A structure-activity relationship study was systematically performed regarding their antiproliferative properties on the HeLa cell model together with their ability to elicit the production of hydrogen peroxide in vitro and in vivo.

Using a robust and selective flow cytometric approach with the endogenously expressed protein redox sensor HyPer, we showed that some of the complexes induce a fast and longlasting perturbation of intracellular H 2 O 2 levels. This behaviour was correlated to their ability to catalyse the production of H 2 O 2 from NADH in aerated physiological environment.

Results and Discussion

Synthesis and characterization

The synthetic route to the ten half-sandwich complexes Ir1-10 with the general structure [Cp*(C^N)IrCl] is reported in Scheme 1. The substitution patterns aimed at examining the influence of steric effects and stereochemistry (R

Fig. 1

Molecular structure of Ir1 determined by X-ray crystallography with thermal ellipsoids drawn at 50% probability. The hydrogen atoms have been omitted for clarity. Selected bond lengths (Å): Ir-Cl 2.4084(9), Ir-N 2.076(3), Ir-C 2.054(3), C-Ir-N 77.89 (13).

The phenyl group coordinates the iridium atom from a C-H bond activation reaction which rate was proven to depend on the electronic nature of the ligand. [START_REF] Boutadla | The Scope of Ambiphilic Acetate-Assisted Cyclometallation with Half-Sandwich Complexes of Iridium, Rhodium and Ruthenium[END_REF] The oxazoline's hydrogens in the 1 H NMR spectra of all the complexes were magnetically inequivalent owing to the chiral environment around the iridium atom.

Owing to the presence of another stereogenic centre on chiral ligands L7-10, complexes Ir7 to Ir10, were obtained as mixtures of diastereomers. Ir7-8 were obtained in a 9:1 diastereomeric ratio [START_REF] Boutadla | The Scope of Ambiphilic Acetate-Assisted Cyclometallation with Half-Sandwich Complexes of Iridium, Rhodium and Ruthenium[END_REF] whereas Ir9-10 were obtained in a 1:1 ratio measured by [START_REF] Zeng | The development of anticancer ruthenium( II ) complexes: from single molecule compounds to nanomaterials[END_REF] H NMR in DMSO-d 6 . However, a coordinating environment like DMSO-d 6 induces epimerization of the (phox)-chelated complexes at the iridium centre and the diastereomeric excess quantification is no longer relevant. The molecular structure of Ir1 solved by X-ray diffraction analysis (Fig. 1, Tables S1-2) showed the expected "piano stool" geometry typical of this family of complexes.

Reactivity towards the DMSO vehicle

DMSO is a widely used administration solvent for metal complexes owing to its ability to solubilise highly lipophilic structures and its convenient miscibility with water. Despite its relatively low toxicity, DMSO has been shown to exert a negative effect on the activation process of cisplatin (CDDP) 19 and a rapid formation of a solvento adduct was reported for ruthenium half-sandwich complexes. S3).

Fig. 2

Comparison of the rates of solvolysis of three representative complexes in DMSO-d6. Quantification was done by integration of the characteristic 1 H NMR peak of the Cp* ligand of the two forms (Fig. S1). Half-lives of Ir1-10 are presented in Table S3.

For comparison, the half-sandwich iridium complex carrying the 2-phenylpyridine ligand showed a much lower rate of

C Procedure A or B Ligand R 1 R 2, R 3 Complex L1 a H H, H Ir1 L2 a H Me, Me Ir2 L3 a F H, H Ir3 L4 a OMe H, H Ir4 L5 b NO 2 H, H Ir5 L6 b CH 2 Cl H, H Ir6 L7 a H H, iPr Ir7 L8 a H iPr, H Ir8 L9 a H H, Ph Ir9 L10 a H Ph, H Ir10 . R 1 R 1 R 1 O N Ir S R 1 O Cl DMSO R 2 R 3 R 2 R 2 R 2 R 3 R 3 R 3

L1-10

Ir1-10

exchange with a half-life of 1125 min. [START_REF] Carrasco | Structurally Strained Half-Sandwich Iridium(III) Complexes As Highly Potent Anticancer Agents[END_REF] This structure-based approach demonstrates that the Ir-Cl bond strength is correlated to the electron-withdrawing character of the substituent on the (phox) ligand. Furthermore, the steric effect induced by the substitutions R 2,3 on the oxazoline ring is another parameter affecting the exchange rate since Ir7-8 also present an extended half-life. At equilibrium, the ligand exchange is total for most complexes of the series except for Ir2 and Ir7-8 for which the establishment of a (1:9) ratio was observed between the neutral and cationic forms (Fig. 2).

To check whether the DMSO adducts could further undergo aquation once diluted in the cell-culture medium, a [Ir1-DMSO] + complex was synthesized and isolated as a nitrate salt (see procedure in Fig. S2). Its 1 H NMR spectrum in CD 3 OD/D 2 O

(2:1) showed that no water/DMSO exchange took place. On the other hand, addition of a saturating amount of NaCl (1.1 M) resulted in the coalescence of the two singlets around 2.89 ppm attributed to two methyl groups, marking a very fast exchange between DMSO and Cl -at the NMR timescale.

The high chloride concentration also favoured the partial displacement of DMSO by Cl -affording a mixture of cationic and neutral complexes in 9:1 ratio at equilibrium. This experiment proved the stability of the DMSO adduct, ruling out the formation of an aqua complex and supported the hypothesis that the cationic DMSO complexes are the true administered molecules in the biological assays.

The ability of Ir2 to undergo aquation was also examined by [START_REF] Zeng | The development of anticancer ruthenium( II ) complexes: from single molecule compounds to nanomaterials[END_REF] H NMR in MeOD-d 4 / D 2 O (3:2). Addition of D 2 O caused peak broadening but the chemical shift of all the protons remained the same. Further addition of NaCl restored the initial peaks width (Fig. S3). Therefore, no hydrolysis of the Ir-Cl bond of Ir2 took place in these conditions.

Cytotoxicity assays

The library of iridium complexes was examined for toxicity.

Prior to cell assays, stock solutions of Ir1-10 in DMSO were incubated at room temperature for 24 h to ensure that the equilibrium between the neutral and the cationic forms was reached for each complex. Their IC 50 on the HeLa cancer cell line were evaluated using the MTT cell viability assay. The obtained values are comprised between 3 and 6 µM (Table 1), in good agreement with values previously on the same cell line for half-sandwich iridium complexes bearing anionic C^O chelating ligands. [START_REF] Du | Potent Half-Sandwich Iridium(III) and Ruthenium(II) Anticancer Complexes Containing a P^O-Chelated Ligand[END_REF] This rather narrow range depicts the minor influence of the substituents carried by the chelating ligand on the antiproliferative activity as well as the stereochemistry of the phox ligand. This result dramatically contrasts with previous reports on structurally related complexes for which the substitution pattern greatly influenced the IC 50 values.

23-25

The cell response to Ir2, Ir4, Ir8 and Ir10 was also investigated by a complementary label-free viability assay based on impedance readout, using the xCELLigence Real Time Cell Analysis (RTCA) system that allows the dynamic monitoring of live cell adhesion and proliferation. All compounds induced a dose-dependent and immediate loss of cell adhesion. As illustrated in Fig. S4 for Ir8, cells treated at concentrations close to the IC 50 were able to re-adhere and then to proliferate although at lower rates, as a consequence of either inhibition of cell division or cell death or both (see videomicroscopy SI).

The resulting time-dependent IC 50 was found to reach a minimum after 48 h of treatment and Ir8 was the most active compound of the series with an IC 50 of 3.6 µM (Table 1).

Apoptosis assay

Morphological changes induced by Ir1-10 were suggestive of regulated cell death. Apoptosis is characterised by hierarchical activation of caspases by proteolytic processing, with the effector caspase-3 being a critical executioner that degrades many cellular proteins. We thus used caspase-3 cleavage as a readout of apoptosis in treated samples. Fig. 3 shows the presence of cleaved caspase-3 in iridium-treated samples starting 12 h after drug administration, a fast kinetics compared to conditions of etoposide-induced DNA damage suggesting a different MoA. Overall these results point to regulated cell death induced by the complexes regardless of their substitution pattern. Immunoblot showing accumulation of cleaved caspase-3 fragments at 19 and 17 kDa for the indicated time (h) after addition of 10 µM compound or vehicle (DMSO). Three representative complexes of the series are presented. Etoposide, a clinically approved topoisomerase II inhibitor that induces single and double strand DNA breaks, was used as positive control and showed the presence of the cleaved form of caspase-3 after 24 h. Equal loadings were assessed by Ponceau S staining (Fig. S5).

In vitro catalytic activity

The reaction of various half-sandwich iridium complexes with NADH is widely described in the literature. [START_REF] Okamoto | Transfer Hydrogenation Catalyzed by Organometallic Complexes Using NADH as a Reductant in a Biochemical Context[END_REF] Some structures are able to form metal hydride complexes with concomitant formation of NAD + following a dissociative mechanism according to DFT calculation [START_REF] Ritacco | DFT Investigation of the Mechanism of Action of Organoiridium(III) Complexes As Anticancer Agents[END_REF] . Hydride can then be transferred to various acceptors such as flavin mononucleotide (FMN), 28 pyruvate to form lactate, [START_REF] Betanzos-Lara | Organometallic Ruthenium and Iridium Transfer-Hydrogenation Catalysts Using Coenzyme NADH as a Cofactor[END_REF] proton to form dihydrogen, 30 aldehydes to form alcohols [START_REF] Ngo | Catalytic Hydrogenation of Cytotoxic Aldehydes Using Nicotinamide Adenine Dinucleotide (NADH) in Cell Growth Media[END_REF][START_REF] Bose | Intracellular Transfer Hydrogenation Mediated by Unprotected Organoiridium Catalysts[END_REF] or imines to yield amines. [START_REF] Soetens | η 5 -Pentamethylcyclopentadienyl)iridium Complex Catalyzed Imine Reductions Utilizing the Biomimetic 1,4-NAD(P)H Cofactor and N -Benzyl-1,4-dihydronicotinamide as the Hydride-Transfer Agent[END_REF] It was also shown that dioxygen could act as hydride acceptor to yield hydrogen peroxide which presence after 48 h incubation of NADH and complexes in aerated solutions was qualitatively detected using a peroxides test stick.

12,34

The ability of Ir1-10 to catalyse NADH oxidation was firstly investigated by monitoring the absorbance at 340 nm of a 100 µM solution of NADH in 5 mM phosphate buffer/MeOH (99:1) in the presence of 10 mol% complex during 150 min. Reactions were performed with stock solutions of chlorido complexes in MeOH as we found that the Ir-DMSO adduct was less active (Fig. S7), which is consistent with the poor lability of the DMSO ligand (Fig. S2). As NADH oxidation was previously shown to generate various side-products, [START_REF] Bucci | A Single Organoiridium Complex Generating Highly Active Catalysts for both Water Oxidation and NAD + /NADH Transformations[END_REF] results are expressed in concentration of remaining substrate after 150 min (Fig. 4). All the complexes catalysed the oxidation of NADH. Moreover, the moderately contrasted data show again a modest influence of the ligand substitution pattern on this process. Resulting TONs at 2.5 h were calculated for Ir1-10 (Table S4) and all values are higher than 1, confirming the catalytic nature of this reaction.

Secondly, we examined whether oxidation of NADH was accompanied by the production of H 2 O 2 in the presence of O 2 , the iridium complexes thus mimicking NADH oxidase activity (See catalytic pathway in Fig. S8). For this, the production of H 2 O 2 was monitored for the first time using the HRP-linked Amplex Red® assay over 150 min. Since NADH autooxidation is known to give false positive results in this assay, we added 40 U/mL of SOD to prevent the formation of radical intermediates that spontaneously oxidize the Amplex substrate, following recommendations from the literature. [START_REF] Votyakova | Detection of hydrogen peroxide with Amplex Red: interference by NADH and reduced glutathione auto-oxidation[END_REF] Although all of the complexes were able to accept a hydride from coenzyme NADH, only the oxazoline-substituted complexes (Ir2, Ir7-10) were able to transfer their hydride to dissolved O 2 , producing detectable amounts of hydrogen peroxide (Fig. 4). This trend may be due to differences of hydricity, i.e. hydride donor strength, among the complexes [START_REF] Ngo | Structure-activity relationship study of half-sandwich metal complexes in aqueous transfer hydrogenation catalysis[END_REF] which may be linked to the electron-donating property of the alkyl / aryl substituent on the oxazoline ring of the chelating ligand. Reassuringly, the couples of isomers (Ir7-8 and Ir9-10) displayed a similar potency to catalyse the production of H 2 O 2 . These four complexes along with Ir2 display the highest potency towards this overall process, showing that the hydride transfer to form H 2 O 2 is mainly dependent on steric hindrance around the labile position.

Intracellular H 2 O 2 production

Among cellular reactive oxygen species (ROS), hydrogen peroxide is involved in various metabolic pathways and is suspected of having a signalling role in natural biological processes. [START_REF] Rampon | Hydrogen Peroxide and Redox Regulation of Developments[END_REF] According to the literature, iridium half-sandwich complexes including 2-phenylpyridine as chelating ligand were shown to induce oxidative stress (i.e. total ROS and RNS levels increase) in cancer cells and this was suggested to result from the catalytic production of H 2 O 2 . [START_REF] Liu | The Potent Oxidant Anticancer Activity of Organoiridium Catalysts[END_REF] We thus investigated whether the ability of Ir1-10 to catalyse the production of H 2 O 2 in vitro could be correlated to their cytotoxicity and their capacity to elevate intracellular H 2 O 2 . A stable HeLa cell line that constitutively expresses HyPer 40 was exposed to Ir1-10, then reduced and oxidized forms of HyPer were quantified by flow cytometry as the ratio of fluorescence emission at 520 nm after excitation at 405 and 488 nm respectively (Fig. S6).

The median ratios M of the HyPer-expressing cells were used to calculate a HyPer-index [START_REF] Lyublinskaya | Redox environment in stem and differentiated cells: A quantitative approach[END_REF] H which was quantified in % according to Equation ( 1) :

(

where M DMSO and M H₂O₂ correspond to the median ratio values obtained for the negative and positive controls, respectively. This index directly shows the significance of the observed variations upon a 60-min treatment with 10 µM Ir1-10 (Fig. 5a). Dramatic differences between the complexes were immediately observed with Ir2, Ir4, Ir7 and Ir10 displaying high HyPer indexes while Ir1 and Ir5 were fully unable to induce H 2 O 2 production in HeLa cells. Since Ir2 complex displayed the highest HyPer index, the rate of this increase was also monitored over a time course of 80 min. As a positive control, cells exposed to a saturating amount of hydrogen peroxide (Fig. 5b, Fig. S6) exhibited a fast recovery with the ratiometric median parameter decreasing to a basal value within an hour, as a result of cellular detoxification processes. In contrast upon treatment with Ir2 in the same time lapse (Fig. 5c), H 2 O 2 levels rose progressively suggesting an uncompensated production of H 2 O 2 that is presumably catalytic. Moreover, the rate of H 2 O 2 production was also concentration-dependent in the 5 -20 µM range (Fig. S6).

Conclusions

In summary, we have reported the synthesis and characterization of a set of half-sandwich iridium complexes comprising a phenyloxazoline chelating ligand with a wide range of substituents. We examined their outcome in the vehicle and the stability of the resulting DMSO adducts in the presence of water. These adducts are cationic, more lipophilic and thus susceptible to display an enhanced penetration across cellular membranes. The strong coordinating power of DMSO to iridium suggests that the lifetimes of the cationic iridium complexes may be long in the intracellular context.

Altogether, it appears necessary to properly determine the stability of the drug in the vehicle, a feature scarcely described in the literature, since it can dramatically influence its biological activity. This study also highlighted the minor influence of the structural modifications of the (phox) ligand on the overall toxicity of the complexes, all of them inducing cell death by apoptosis. In vitro, all complexes were able to accept a hydride from coenzyme NADH, but they were not equally able to transfer it to dioxygen to produce H 2 O 2 . We quantified for the first time the production of hydrogen peroxide elicited by half-sandwich iridium complexes in two experimental settings, both in vitro and in vivo. We found that the complexes carrying substituents on the oxazoline ring (Ir2, Ir7-10) were the most efficient catalysts of H 2 O 2 production in vitro. Strikingly, among these molecules, Ir2, Ir7 and Ir10 were also proven to generate high levels of H 2 O 2 in living cells during the first hour of treatment. Moreover, the cells' detoxification machinery was not able to compensate this production over time unlike with a simple hydrogen peroxide addition. While this mode of action provides a rationale for the fast cytotoxicity of the compounds that induced intracellular accumulation of H 2 O 2 , it cannot be excluded that other mechanisms be at stake, leading to the fast activation of caspase-3 and cell death by apoptosis. Indeed, neither Ir1 nor Ir5 were efficient in both analyses despite a comparable antiproliferative activity. Recent studies have shown that a structurally related iridium complex exhibited a mitochondrial tropism and induced distortions of the inner membrane. [START_REF] Conesa | Unambiguous Intracellular Localization and Quantification of a Potent Iridium Anticancer Compound by Correlative 3D Cryo X-Ray Imaging[END_REF] A future challenge will be to decipher whether this accumulation of H 2 O 2 results from mitochondrial disruption or is a direct consequence of intracellular transfer hydrogenation catalysed by the iridium complexes.

Scheme 1

 1 Scheme 1 Synthetic route used to access the (phox) ligands L1-10 and the corresponding half-sandwich iridium chlorido complexes Ir1-10, chlorido ligand exchange in DMSO. a Procedure A. a) CH2Cl2, r.t.; b) SOCl2 (4 eq), CH2Cl2, r.t. b Procedure B. a) CH2Cl2, r.t.; b) SOCl2 (4 eq), CH2Cl2, r.t. ; c) NaH (1 eq), THF, r.t. Procedure C. NaOAc (6 eq), CH2Cl2, r.t.

20 The

 20 vehicle used for further biological tests being DMSO, the stability of the complexes in this solvent was investigated by 1 H NMR spectroscopy. The characteristic chemical shift of the Cp* ligand was used to monitor the conversion of Ir1-10 to [Ir-DMSO-d 6 ] + (Fig. S1). At 297K, most complexes undergo a very fast chlorido to DMSO ligand exchange with half-lives below 12 min (Ir1, Ir2, Ir4, Ir6, Ir9-10) albeit with an electronic effect observed for Ir3 and Ir5 that is linked to the σ-I Hammett constant of fluoro and nitro substituents. Ir5's half-life in DMSO-d 6 was the longest of the series, that is 270 min thus 20fold longer than that of Ir3 (Table

Fig. 3

 3 Fig. 3

Fig. 4

 4 Fig. 4Amounts of remaining NADH and H2O2 produced in model physiological environment. Data are shown as mean ± SD of three experiments performed at 37°C in 5 mM phosphate buffer pH 7.4 containing 1% MeOH in the presence of 10 mol% iridium complex. Hydrogen peroxide was quantified using a multiplex measurement of triplicates and standard for each experiment.

Fig. 5 a.

 5 Fig.5a. HyPer index derived from the median values of HeLa cells exposed to 10 µM Ir1-10 for 60 min (at least 5 000 gated events). Statistical significance was compared to the negative control with a Mann-Whitney non-parametric t-test (p < 0.0001) b. Evolution of the HyPer ratios over time after addition of a saturating amount of hydrogen peroxide. c. Evolution of the HyPer ratios over time after addition of 20 µM Ir2, similar results were obtained with lower concentrations and are reported in Fig.S6. Cells treated for 90 min with DMSO were used as negative control.

Table 1

 1 Antiproliferative activity of Ir1-10 towards the HeLa cell line

a Median inhibiting concentration after 96 h of treatment assessed by the MTT cell viability assay. b Median inhibiting concentration after 48 h of treatment assessed by the xCELLigence RTCA system. c Not determined.

mol% iridium complex. Hydrogen peroxide was quantified using a multiplex measurement of triplicates and standard for each experiment.

Acknowledgements

This research was supported by the IPV doctoral program of Sorbonne Université. We wish to acknowledge basic support from CNRS, INSERM and Sorbonne Université, to UMR_8232 and UMR_S938 and competitive funding from LabEx MiChem part of French state funds managed by the ANR within the Investissements d'avenir programme under reference ANR-11-IDEX-004-02 and from INCA-DGOS-Inserm_12560 SiRIC CURAMUS financially supported by the French National Cancer Institute, the French Ministry of Solidarity and Health and Inserm. We thank Dr. Sophie Vriz for the gift of HeLa-HyPer stable cell line. We are indebted to Blaise Gatin-Fraudet and Kévin Passador for supporting technical assistance and data acquisition and to Romain Morichon for videomicroscopy experiments.

Conflicts of interest

There are no conflicts to declare.