Dambarudhar Parida

Camille Bakkali-Hassani

Éric Lebraud

Christophe Schatz

Stéphane Grelier

Daniel Taton

Joan Vignolle
email: joan.vignolle@enscbp.fr

Eric Lebraud

Tuning the activity and selectivity of polymerised ionic liquid-stabilised ruthenium nanoparticles through anion exchange reactions

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

Introduction

The development of highly active, selective and recyclable catalysts is crucial in the context of energy savings and sustainable chemistry. [1][2][3][4][5][6][7] Metallic nanoparticles (MNPs) are promising candidates because of their unique properties that stem from their nanometric size and the presence of highenergy surface atoms. [8][9][10][11][12][13] As MNPs prove thermodynamically unstable respective to bulk metal, stabilisers such as polymers, ligands, surfactants or ionic liquids (ILs) are required to ensure colloidal stability. [14][15][16][17][18] In addition, stabilisers may also strongly influence the catalytic properties of MNPs, owing to their multiple interaction with the MNPs surface. [19][20][21][22][23][24][25] In this regard, polymeric stabilisers generally lead to highly stable dispersion of MNPs thanks to their steric protection. [26][27][28][29] Meanwhile, steric congestion may limit the access of substrates to the surface, thus decreasing catalytic performances. In contrast, with molecular ILs as stabilisers, very active MNPs are formed. [30][31][32][33] Nevertheless, such dispersions remain poorly stable under forcing conditions, 33 which can be overcome by adding some polymer. 34 Polymerised ionic liquids (PILs) are polymeric versions of ionic liquids (ILs), where cations (or anions) are tethered to the polymer backbone and counter-ions are mobile. [35][36][37][38][39] This class of poly(electrolytes), which combines the properties of polymers with those of ILs, behaves as powerful electro-steric stabilisers for MNPs. [40][41][42][43][44] Hence, PILs, notably those based on the imidazolium cation, generally lead to PILs-stabilised MNPs catalytic systems, denoted as M@PIL NPs, with enhanced activity and improved stability relative to molecular ILstabilised MNPs. 43 Applications of M@PIL NPs-type catalysts (M = Rh, Pd, Pt, Ni, Au and Ag) have proven efficient in a variety of chemical transformations, including Suzuki,

We wish to report herein that simple PILs based on poly(1butyl-3-vinyl imidazolium), associated with different anions, such as Cl, Br, I, bis(trifluoromethyl)sulfonimide (NTf 2 ) act as very efficient stabiliser for RuNPs both in water and organic media. More importantly, their catalytic activity can be tuned by simple counter-anions exchange reactions (Figure 2). Hence, this effect provides an innovative and powerful means to reversibly turn the catalytic activity of RuNPs on and off. Switching the chemo-selectivity of Ru@PIL NPs in the hydrogenation of styrene from ethylbenzene to ethylcyclohexane can also be accomplished upon I -/NTf 2 -anion exchange (Figure 2). On the basis of XPS and DLS analysis, this anion effect could result from the specific interactions between the counter-anions and/or the imidazolium moieties of the PIL stabiliser and the Ru surface, as well as from the relative solvation and hydrophobicity of the PIL corona.

Fig. 2 Tuning the activity and selectivity of polymerised ionic liquid-stabilised Ru nanoparticles by anion exchange. NTf2 -bis(trifluoromethyl)sulfonimide.

Results and discussion

The ability of poly(1-Butyl-3-vinyl-imidazolium chloride), denoted as PIL(Cl), to stabilise RuNPs was first investigated. PIL(Cl) was prepared by free radical polymerisation of the corresponding N-vinyl imidazolium chloride ionic liquid monomer at 70°C in iso-propanol. The corresponding Ru@PIL(Cl) NPs were synthesized by a polyol process (Fig. 3), 71 that is by reduction of RuCl 3 in ethylene glycol at 170 °C, in presence of different amount of PIL(Cl) (for details, see SI). Using a PIL(Cl)/Ru molar ratio of 50, 10 and 2, mono-disperse NPs of 1.5 nm (1Cl), 2.6 nm (2Cl) and 2.7 nm (3Cl) were formed respectively, according to TEM analysis (Fig. 4). XRD analysis revealed that 1Cl, 2Cl and 3Cl were polycrystalline, the size of the crystalline domains, as determined from the Debye-Scherrer relation, being smaller than the size measured by TEM (1.1 nm, 1.3 nm and 1.4 nm for 1Cl, 2Cl and 3Cl respectively; see Table S4). As an assessment of their stability in water, ζ-potential was then determined. All Ru@PIL(Cl) NPs exhibited positive values as expected from their poly-cationic nature (Fig. 4d). The ζ-potential also increased with the PIL/Ru ratio in the order 3Cl (35 mV) < 2Cl (49 mV) < 1Cl (56 mV), indicating the higher stability of 1Cl. The Ru(0) and Ru(IV) contents could be evaluated to 88% (BE = 461.4 eV) and 12% (BE = 463.4 eV) by XPS analysis of 2Cl upon deconvolution of the Ru 3p3/2 signals (Fig. S18, Table S7). To investigate the influence of the PIL counter-anions on the RuNPs catalytic properties, different Ru@PIL NPs with Br, I and NTf 2 anions were also prepared. 2Br and 2I were generated in a similar way as 2Cl, using PIL(Br) and PIL(I) as stabilisers respectively, with a PIL(X)/Ru ratio equal to 10. In contrast, 2NTf 2 was accessed by anion metathesis between 2Br and LiNTf 2 because of the insolubility of PIL(NTf 2 ) in ethylene glycol (Fig. 2). Importantly, no trace of residual bromide was detected in 2NTf 2 by XPS analysis (Fig. S14 & S15), evidencing that the anion exchange was quantitative. Alternatively, 2Cl could be used instead of 2Br for the anion metathesis, the anion exchange being also complete in this case (Fig. S15b). The size of the RuNPs was not affected by the anion exchange reaction but was found to decrease in the order: 2Cl (2.6 nm) > 2Br/2NTf 2 (2.3 nm) > 2I (1.6 nm) according to TEM analysis, highlighting the influence of the PIL counter-anions over the NPs size (Fig. S5 andS6). Although the origin of this effect is not clearly understood, it may result from the nature and intensity of the PIL/Ru and cation/anion interactions (vide infra). Hydrogenation were performed with 0.166 mol.% of Ru catalyst and 6 wt% of phenol in 5 mL of water, at 110 °C, under 25 bars of H2. The conversion was determined by 1 H NMR. The selectivity was confirmed by GC-MS analysis. *Aggregation of NPs and/or bulk metal formation was observed. a IL: 1-butyl-3ethyl-imidazolium bromide. b See SI for their preparation and characterization.

Hydrogenation of phenol with Ru@PIL(X) NPs in water (X = Cl, Br)

The catalytic activity of 1-3Cl was first investigated for the hydrogenation of phenol in water as model reaction (Table 1). 72 Reactions were performed at 110 °C for 25 min, using 25 bars of H 2 and 0.166 mol.% of Ru loading. Under those conditions, 2Cl appeared as the most efficient catalyst, providing full substrate conversion, as well as complete selectivity in favour of cyclohexanol (entry 2). In comparison, 1Cl and 3Cl displayed a moderate conversion (62 %, entry 1) and a poor stability (entry 3), respectively, highlighting that an intermediate ratio PIL/Ru ratio of 10 was the optimal balance. The same ratio was thus kept throughout the study. The formal replacing of Cl¯ by Br¯ had virtually no influence on the catalytic properties of such Ru@PIL(X) systems, as indicated by the full substrate conversion and full selectivity toward cyclohexanol obtained with 2Br, under similar conditions (entry 4). For comparison purpose, RuNPs either electrostatically stabilised by an homologous monomeric ionic liquid, namely, 1-Butyl-3-ethylimidazolium bromide, or sterically stabilised with neutral PVP were prepared by the same polyol process (see part 2b in SI), yielding to waterdispersible RuNPs of 1.8 and 2.7 nm respectively. In both cases, aggregation of the NPs and bulk metal formation were observed (Table 1, entry 5 and 6) under the standard conditions (110°C, 25 min, 25 bars of H 2 ). In contrast, 2Br and 2Cl remained perfectly stable. The recyclability of Ru@PIL NPs was also briefly examined. Quantitative phenol conversion could thus be achieved over 7 cycles with 2Cl, without any sign of NP aggregation according to TEM analysis (Fig. S24 & 25). Although hydrogenation of aromatics generally involves heterogeneous species as catalysts, a CS 2 poisoning experiment was performed to confirm the heterogeneous nature of the catalyst. 73 Thus, upon addition of CS 2 to the catalytic solution containing 2Cl, no further increase of the conversion with time was noted (Fig. S26), in agreement with a surface-mediated catalysis.

Switching of the catalytic activity by anion exchange

As Ru@PIL(X)NPs (2Cl, 2Br, 2NTf 2 and 2I) display different solubility depending on the type of counter-anions introduced, methanol was selected as common solvent to compare their catalytic activity under semi-heterogeneous conditions. Hydrogenation reactions were performed at 110 °C for 1 h, using 25 bars of H 2 and phenol as substrate. Under those conditions, 2I proved completely inactive, while 2Br, 2Cl and 2NTf 2 afforded low substrate conversion of 2, 6 and 11% respectively (Table 2). Ru@PVP 100* 100 incomplete in this case. Thus, the catalytic activity of Ru@PIL(X) NPs could be switched on and off thanks to the reversible exchange of the PIL counter-anions. Although the influence of some anions over the catalytic properties of MNPs has been reported, 69 this anion exchange-mediated reversible switching of the catalytic activity is unprecedented to the best of our knowledge. This reversible behaviour originates from the specific properties of the PIL stabilisers, which combine a macromolecular structure and multiple, relatively weak anioncation interactions. As already reported for other MNPs, this anion exchange reaction also allows the polarity of the catalysts to be readily modified (Table S2), 56,65 leading to quantitative hydrogenation of toluene with 2NTf 2 as catalyst in THF as solvent (Table S10).

Switching of the catalytic selectivity by anion exchange

The inability of 2I to hydrogenate aromatics (Table 2), prompted us to investigate the selective hydrogenation of C=C bonds using styrene as model substrate (Table 3). Thus, hydrogenation of styrene was performed at 40 °C under 15 bars of H 2 , in presence of a catalytic amount of 2I (0.33 mol% of Ru). Under these conditions, we were pleased to observe full substrate conversion and complete selectivity for ethylbenzene (EB) (Table 3, entry 2).

Table 3. Hydrogenation of styrene (ST) to ethylbenzene (EB) and ethylcyclohexane (EC) using Ru@PIL(X) NPs (X = Br, I, NTf2) as catalysts.

Hydrogenation in methanol (5 ml methanol and 6 wt% of styrene) at 40 °C for 6 hours under 15 bar of H2 and 0.33 mol.% of Ru loading. GC-MS was used to determine conversion and chemo-selectivity of different catalysts. a 2NTf2 was prepared by anion exchange between 2I and AgNTf2. b Commercial catalyst.

In the case of 2Br, styrene was fully converted into a mixture of 64% of EB and 36% of ethylcyclohexane (EC), highlighting a lack of chemo-selectivity in this case. In sharp contrast, when 2NTf 2 was used under identical conditions, both the vinyl group and the aromatic ring were quantitatively reduced (entry 3). Interestingly, 2NTf 2 could be generated from 2I using AgNTf 2 (SI Sec. 2c), which enabled the chemo-selective hydrogenation of styrene to be switched from EB to EC, upon I¯/NTf 2 ¯ anion exchange, while maintaining quantitative conversion (Fig. S16). For comparison, hydrogenation of styrene was also performed with Ru/C (5%) under the same conditions, leading to a mixture of EB (16%) and EC (84%) (Entry 4), and thus evidencing the superior selectivity of Ru@PIL(X) NPs. Overall, those results not only demonstrated the interest of Ru@PIL(X) NPs catalytic systems relative to commercial catalysts, but also further highlighted the influence of PIL counter-anions over the catalytic properties.

Investigation of the PIL(X)-RuNPs interactions in the solid state

To gain more insight into the influence of the nature of PILs on NP properties, XPS analysis was performed on all Ru@PIL(X) NPs, i.e. 2Cl, 2Br, 2I and 2NTf 2 (prepared from 2Br by anion exchange), as well as on corresponding PIL(X) stabilisers (X = Cl, Br, I, NTf 2 ; Fig. 5). Although this analysis is performed in the solid state, it provides qualitative information on the nature and relative strength of PIL-Ru interactions. Except iodide (I¯), all anions were found to interact with the Ru surface as evidenced by the shift toward higher binding energy (BE) of the signals associated to Cl(2p), Br(3p), N(1s)NTf 2 compared to their respective BE in the free polymer (Fig. S21).

More specifically, the strongest interaction with the Ru surface was observed for both Br and NTf 2 anions (∆BE = +0.4). In the case of I¯, no detectable shift in the BE I(3d) was observed between PIL(I) and the corresponding RuNPs 2I, suggesting the absence of interaction between I¯ and the Ru surface.

The environment around the imidazolium cation was next scrutinized by analysing the BE of the N(1s)Im. In the free PIL(X), anions interact solely with imidazolium cations, likely by H-bonding with the C 2 -H and C 4,5 -H. 74 The BE N(1s)Im was found to increase with an increase of the polarizability of the anion in the order: Cl (401.2 eV)< Br (401.4 eV)< NTf 2 (401.6 eV) < I (401.8 eV) (Fig. 5a-d and Table S8). Thus, the most polarizable NTf 2 ¯ and I¯ anions only weakly interact with the imidazolium cation, leading to a cationic structure, where the electrons are tightly bound to the N atom. In contrast, the low BE N(1s)Im observed for Cl reflects its strong interaction with the imidazolium moiety via H-bonding, which virtually led to a partial transfer of the positive charge from the nitrogen atom to the proton. In Ru@PIL(X) NPs (X= Cl, Br and NTf 2 ), the signal corresponding to N(1s)Im was shifted to higher BE relative to that of their respective PIL(X), indicating a weakening of the anion-cation interactions in presence of Ru. This effect was more pronounced for 2NTf 2 (∆ BE = +0.6) than for 2Cl (∆ BE = +0.4) and 2Br (∆ BE = +0.2) (Fig. 5a, b and d and fig. 6a,b). In sharp contrast, a 0.2 eV shift toward lower BE was noted for the N(1s)Im of 2I, relative to that of PIL(I) (Fig. 5c), suggesting an interaction between imidazolium cations and the Ru surface (Fig. 6c). 75,76 From a catalysis point of view, such interactions would prevent aromatic substrates from adsorbing onto the flat surface of faces and to undergo hydrogenation of the aromatic moiety (fig. S23), as observed experimentally with 2I and toluene and styrene as substrates. However, binding of styrene via the alkene moiety to edges or corners of RuNPs would not be hindered, 8,77,78 allowing for complete EB selectivity with 2I as catalyst. Note also that the smaller size of 2I relative to 2Cl and 2Br observed by TEM (Fig. S5) may also originate from this peculiar imidazolium-Ru interaction, which would inhibit NP growth during their synthesis.

Influence of the PIL counter-anions (X) over the properties of Ru@PIL(X) NPs in solution

Dynamic light scattering (DLS) was performed on 2Br, 2NTf 2 and 2I in MeOH in order to investigate the influence of the counter-anions over the solution properties of NPs. The intensity-weighted size distributions were found to be bimodal for 2Br and 2NTf 2 , suggesting the presence of some aggregation that likely resulted from inter-particular hydrogen bonding between imidazolium cations and respective counteranions (fig. 7a). 74 However, the number-weighted size distributions of 2Br and 2NTf 2 indicated that the proportion of these aggregates was very small, a single population at 8 and 12 nm, corresponding to isolated NPs, being observed for 2Br and 2NTf 2 respectively (fig. 7b). In contrast, only aggregates of about 180 nm were observed for 2I on both types of size distribution (fig. 6a,b), which could result from the peculiar binding mode of PIL(I) to Ru that involves interactions between the poly(cationic) chains and the Ru surface, as demonstrated by XPS analysis (fig. 6c).

All these data clearly evidenced that the nature of PILs counter-anions has a dramatic impact on the nature and strength of the PIL-Ru and anion-cation interactions, both in the solid state and in solution. While the selectivity observed with 2I could originate from the peculiar imidazolium-Ru interaction, the higher activity of 2NTf 2 , relative to 2Br, in the hydrogenation of toluene and styrene could be ascribed to a more favourable micro-environment due to the better solvation of the PIL corona on one hand (Fig. 7b), and, on the other hand, to the relative hydrophobicity due to the presence of n-butyl substituents on imidazolium units and NTf 2 -counteranions (Fig. 6b).

Conclusions

Easily accessible imidazolium-based polymerised ionic liquids (PILs) behave as powerful electro-steric stabilisers for RuNPs, affording highly active systems for the hydrogenation of aromatic substrates in different solvents such as water, MeOH or THF. In comparison to more conventional stabilisers, PILs provide a tailorable micro-environment around the metallic surface, which enable for reversibly controlling the catalytic properties of the corresponding RuNPs by simple exchange of the PIL counter-anions. Hence, this strategy allows, not only the catalytic activity to be turned on and off, but also the chemo-selectivity of RuNPs to be switched depending on the nature of the counter-anions. According to XPS and DLS analysis, influence of the PIL over the activity and selectivity of RuNPs stems from the relative hydrophobicity and solvation of the PIL corona and from the nature (via the anion or the cation) and strength of PIL-Ru interactions, respectively. Overall, this work demonstrates that, conceptually, different activity and selectivity can be reached from a single catalyst through anion exchange reactions. The variety of cations and anions available offers a bright horizon to tailor the metal surface properties of MNPs, via simple anionic metathesis. Future work is underway to extend this concept to other

Experimental and methods

Preparation of poly(ionic liquid)s (PILs) and Ru@PIL(X) NPs

Polymerised ionic liquids PIL(X) (X = Cl, Br, I) were prepared by free radical polymerisation of the corresponding 1-butyl-3vinyl-imidazolium ionic liquid (ILs) monomers at 70°C for 24h, using AIBN as initiator (IL/AIBN = 200). 1 H NMR analysis after polymerisation confirmed 100% conversion of IL(Cl) and IL(Br). Conversion of 75% was obtained in the case of IL(I) after 48 hours of polymerisation. Poly(1-Butyl-3-vinylimidazolium chloride) (PIL(Cl)) and Poly(1-Butyl-3-vinylimidazolium bromide) (PIL(Br)) were precipitated repeatedly in diethyl ether and dried under vacuum (35 °C) till a constant weight was obtained (quantitative yields). PIL(I) was precipitated once in diethyl ether and then taken for dialysis against methanol to remove the remaining IL(I) monomer. After 48 hours of dialysis, PIL(I) was collected by precipitation in diethyl ether and dried under vacuum (35 °C) till a constant weight was obtained with a final yield of 60%. All PIL(X) (X = Cl, Br, I) were analysed by 1 H NMR to confirm the absence of any residual monomer (Fig. S1). Macromolecular characteristics of the PILs were determined by size exclusion chromatography (SEC) (table S1). Prior to SEC analysis, all PILs were converted to PIL(NTf 2 ) by anion exchange to make them soluble in the mobile phase (THF) (SI, sec. 2a). PIL(X)-stabilised RuNPs (Ru@PIL(X) NPs) (X = Cl, Br & I) were prepared using RuCl 3 ×H 2 O and RuBr 3 ×H 2 O as metal precursors and PIL(Cl), PIL(Br) or PIL(I) as stabilisers via polyol process. Different molar ratio between PIL(X) (repeating IL(X) units) to metal salt (i.e. 50:1, 10:1, 2:1) were used to synthesise Ru@PIL NPs. Typically, 400 mg of PIL(X = Cl, Br & I) and required quantity of metal salt were dissolved in 40 ml ethylene glycol in a 200 ml Schlenk flask. After stirring for 4 hours at 750 rpm, the resulting dark yellow solution was degassed with argon and immersed in an oil bath pre-heated at 170 °C. The dark yellow solution slowly turned black, indicating the formation of Ru(0)NPs. Heating was continued for 1.5 hours to ensure complete conversion of ruthenium salt to Ru(0). Complete reduction of Ru(+2) salts and formation of NPs was confirmed by UV-vis spectroscopy analysis (see SI). Disappearance of the broad absorption band in the spectrum of the precursor after reduction indicates complete reduction of Ru(+2) into Ru(0). A representative example is given in Fig. S1 with the reduction of RuCl 3 in presence of PIL(Cl). PILs stabilised NPs were collected by centrifuging 5 ml of NP solution with 30 ml mixture of diethyl ether and acetone (50:50). After this initial centrifugation, RuNPs were dissolved in acetone (15 mL) and diethyl ether was added (20 mL); the resulting suspension was then subjected to centrifugation. This step was repeated once. The resulting sticky black product was next dissolved in 5 mL of methanol and centrifuged with 25 mL of diethyl ether. The resulting shiny black product was vacuum dried at 35 °C (0.8 mmHg) till a constant weight was obtained (24 -48h). This step ensures the removing of the different solvents, including ethylene glycol (b.p. = 197 °C). All centrifugations were carried out at 8000 rpm for 15 min at 15 °C. The resulting shiny black products 1Cl (PIL(Cl)/Ru = 50), 2Cl (PIL(Cl)/Ru = 10), 3Cl (PIL(Cl)/Ru = 2), 2Br (PIL(Br)/Ru = 10) and 2I (PIL(I)/Ru = 10) were vacuum dried at 35 °C till a constant weight was obtained.

To obtain hydrophobic Ru@PIL(NTf 2 ) NPs, i.e. 2NTf 2 , a Br¯ / NTf 2 ¯ anion exchange was performed from 2Br. In a typical anion exchange procedure, 10 ml aqueous solution of LiNTf 2 (4 molar equivalent) was added dropwise to 30 ml aqueous solution (1.5 mg/ml) of 2Br under vigorous stirring, leading to the slow precipitation of 2NTf 2 . To ensure complete exchange, the mixture was stirred for 16 hours under ambient temperature. All operations were carried out under argon atmosphere. Resulting precipitates were washed several times with water and then dried under vacuum at 35 °C until a constant weight was obtained. The solubility of the different RuNPs was examined in a variety of solvents (Table S2). Alternatively, 2NTf 2 could be prepared from 2Cl, following the same procedure. The reverse process, involving NTf 2 ¯ /Br¯ anion exchange, could also be performed on 2NTf 2 . In a typical procedure, excess of LiBr (4 molar equivalent) pre-dissolved in acetone was added dropwise to 2NTf 2 solution in acetone (1.5 mg/ml) and stirred overnight under inert atmosphere. Upon anion exchange, 2Br' precipitated out of the solution. Removal of excess LiBr was accomplished by washing the precipitate with acetone. 2I could also undergo a complete I¯/ NTf 2 ¯anion exchange by using AgNTf 2 as the source of NTf 2 ¯. Here, methanol was used as the solvent during anion exchange and AgI was obtained as the solid precipitate. AgI was removed by centrifugation to obtain a solution of 2NTf 2 , which was then evaporated under vacuum to obtain 2NTf 2 (SI, sec. 2c).

Characterizations of nanoparticles

Ru content of all NP samples was determined by thermo gravimetric analysis (TGA) using a TGA Q-500 from TA instruments. TGA was performed in two steps: in the first step, samples were heated till 600 °C under nitrogen to remove PILs. In the next step, air was introduced after 600 °C to facilitate removal of residual char by combustion. The residual weight obtained around 615 °C was considered as the Ru content as all carbon-based matter had been removed.

Particle sizes of RuNPs were determined by recording Transmission electron microscopic (TEM) image by Hitachi 7650 TEM operating at 80 kV in high-resolution mode. Samples for TEM analyses were prepared by putting a drop of NP solution (0.5 mg/ml in methanol) on a carbon coated copper grid and drying it for 90 minutes before analysis. Particle size and distribution were determined by measuring 200 particles at random locations by Image J software.

XRD patterns were recorded on a PANalitycal X'pert MPD-PRO Bragg-Brentano θ-θ geometry diffractometer equipped with a secondary monochromator and an X'celerator detector over an angular range of 2θ = 20-80°. The Cu-Kα radiation was generated at 45 KV and 40 mA (λ = 0.15418 nm).

Particle size and ζ-potentials of NPs were determined by dynamic light scattering at 25 °C at a concentration of 0.5 mg/ml using Malvern Zetasizer ZS equipment. All the particle size analysis was carried out using methanol as the solvent. ζpotentials were measured using folded capillary cells (DTS 1070). Based on the solubility, both water and methanol were used as solvent.

X-ray photoelectron spectroscopy (XPS) analysis was carried out in a K-Alpha X-ray Photoelectron Spectrometer from Thermo Fisher Scientific equipped with monochromatized AlKα source (hν=1486.6 eV). The full spectra (0-1350eV) and high-resolution spectra were recorded with constant pass energy of 200 eV and 40 eV respectively. Ar+ sputtering was used for depth profiles and high-resolution spectra were processed with AVANTAGE software. All scans were corrected considering C(1s) as the reference (285.0 eV).

Hydrogenation procedure

Hydrogenation of unsaturated hydrocarbons was carried out in high pressure Parr reactor with internal volume of 45 ml and equipped with two valves, a manometer and a magnetic stirrer. In a typical hydrogenation, 5 ml of solvent were taken in a Schlenk to which 6 wt% of substrate and required quantity of NP catalyst were added. In the first method, solution of substrate and catalyst were charged into the reactor and the reactor was flushed three times with H 2 prior to sealing it with desired pressure. Then the reactor was immersed in the oil bath at the set temperature and reaction start time was noted when stirring speed reached 1000 rpm. Reaction was stopped by cooling the reactor by liquid nitrogen and releasing the H 2 pressure. In the second method of hydrogenation (preconditioning route), solution of substrate and catalyst was preheated to reaction temperature prior to flushing and sealing it with desired H 2 pressure. 1 H NMR analysis was used to determine conversion of substrate. In case of phenol hydrogenation, diethyl ether was used to extract the product and after evaporating diethyl ether at room temperature (15 min), product was analysed by 1 H NMR. When organic solvents were used as a solvent for hydrogenation, aliquots were diluted with deuterated solvents for 1 H NMR analysis. Conversion and selectivity of some hydrogenation reactions were also determined by a Thermo Scientific GC-MS with Trace Ultra GC and Trace DSQ MS. Instrument was equipped with a RESTEK Rtx-5 Sil MS column (0.25mm × 30m) and helium (0.8 ml/min) was used as the carrier gas.

Catalyst recycling

NP recycling for hydrogenation was carried out with 2Cl using phenol as a substrate. After 25 min. of hydrogenation, product was extracted with diethyl ether. Then, required quantity of phenol was added to the NP solution and reactor was closed and was flushed thrice with H 2 before sealing it at a desired pressure. Reactor was immersed in an oil bath at required temperature and start time was noted when stirring speed reached 1000 rpm. After completion of hydrogenation, the same procedure was repeated for the next cycle.

Materials

1-Vinyl-3-imidazolium, 1-chlorobutane, 1-bromobutane, 1-iodobutane, and Poly(vinylpyrrolidone) (MW 55000 g/mol) (PVP) were purchased from Sigma Aldrich, France. Ruthenium chloride hydrate (99.9%) and ruthenium bromide hydrate (99.9%) were purchased from Strem Chemicals. Lithium bis(trifluoromethanesulfonimide) (LiNTf2) was purchased from TCI France. Ru on Carbon (Ru/C 5%) was purchased from Alfa Aesar France and used as received. HPLC grade solvents were used for synthesis, purification and analysis. All reagents and solvents were used without further purification unless otherwise mentioned.

General procedures (a) Synthesis of Ionic liquids (monomers) and Polyionic liquids

1-butyl-3-vinylimidazolium halides (Cl, Br and I) were synthesized following methods reported in the literature. 1,2 NMR data match those reported in the literature. [1][2][3] Polymerizations of ionic liquids (ILs) were carried out in isopropanol at 70 °C for 24 hours using azobis(2-methylpropionitrile) (AIBN) as thermal initiator. In all cases molar ratio between IL and AIBN was 200 and polymerization was carried out under an inert atmosphere. 1 H NMR analysis after polymerization confirmed 100% conversion of IL(Cl) and IL(Br). Conversion of 75% was obtained in the case of IL(I) after 48 hours of polymerization. Poly(1-butyl-3-vinylimidazolium chloride) (PIL(Cl)) and Poly(1-butyl-3-vinylimidazolium bromide) (PIL(Br)) were precipitated repeatedly in diethyl ether and dried under vacuum (35 °C) till a constant weight was obtained (quantitative yields). PIL(I) was precipitated once in diethyl ether and then taken for dialysis in methanol to remove the remaining IL(I) monomer. After 48 hours of dialysis, PIL(I) was collected by precipitation in diethyl ether and dried under vacuum (35 °C) till a constant weight was obtained with a yield of 60%. Finally, PILS was analyzed by 1 H NMR to confirm the absence of any unreacted monomers, and 1 H NMR spectra of PIL(Cl), PIL(Br), and PIL(I) are given in Figure S1.

Molecular weight and polydispersity index of PILs were determined by the procedure reported by Matyjaszewski et. al. 4 A GPC system equipped with PSS SDV Linear S (5 µm) column was used at eluent flow-rate of 1 ml/minute. THF containing 10 mmol/L of LiNTf2 was used as eluent. A viscometer, a multi-angle light scattering detector, and a RI detector from Wyatt Technology were used to acquire elution traces. ASTRA 6.1 software was used for the processing of elution traces. Macromolecular characteristics of different PILs are given in Table S1. For GPC sample preparation anion exchange was carried out on all synthesized PILs to achieve bis(trifluoromethyl)sulfonimide (NTf2) anion. Typically, 4 molar equivalent LiNTf2 dissolved in water (10 ml) was added to a 20 ml aqueous solution of PIL(X) (X is Cl or Br) (10 mg/ml) under stirring. The mixture was stirred for 24 hours to ensure a complete exchange of halide anions (Cl¯ or Br¯) by NTf2¯. Then the precipitate was washed several times with distilled water to remove LiBr and excess LiNTf2. The product was then dissolved in acetone, precipitated in water, and centrifuged. PIL(NTf2) obtained was dried under vacuum (35 °C) till a constant weight was obtained (quantitative yield). For anion exchange on PIL(I), AgNTf2 was used instead of LiNTf2. In this case, 2 molar equivalent AgNTf2 dissolved in methanol (10 ml) was added to 20 ml methanol solution of PIL(I) (10 mg/ml) under stirring. After 24 hours, the mixture was centrifuged at 8000 rpm for 15 minutes to remove insoluble AgI. Then, the solution was concentrated by evaporation of methanol and the polymer was recovered by precipitation in water. To ensure complete removal of AgNTf2, PIL was precipitated twice in water. Finally, it was dried under vacuum (35 °C) till a constant weight was obtained.

(b) Preparation of Ru nanoparticles

RuCl3×H2O and RuBr3×H2O were used as metal precursors and PIL(Cl), PIL(Br) or PIL(I) as stabilizers for the synthesis of PIL(X)-stabilized Ru nanoparticles (NPs) (X = Cl, Br, I). The different molar ratio between PIL(X) (IL(X) units) to metal salt (i.e. 50:1, 10:1, 2:1) were used to synthesize PIL(X)@Ru NPs, namely 1-3Cl, 2Br and 2I via polyol process. 5,6 Typically, 400 mg of PIL(X) and the required quantity of metal salt were dissolved in 40 ml ethylene glycol in a 200 ml schlenk flask. After stirring for 4 hours at 750 rpm, the resulting dark yellow solution was degassed with argon and immersed in an oil bath preheated at 170 °C. The dark yellow solution slowly turned black, indicating the formation of Ru(0)NPs. Heating was continued for 1.5 hours to ensure complete conversion of ruthenium salt to Ru(0) and formation of NPs which was confirmed by UV-vis spectroscopy analysis. The disappearance of the broad absorption band in the spectrum of the precursor after reduction indicates complete reduction of Ru(+2) into Ru(0). A representative example is given in Figure S3 with the reduction of RuCl3 in presence of PIL(Cl). PILs stabilized NPs were collected by centrifuging 5 ml of NP solution with 30 ml of a mixture of diethyl ether and acetone (50:50). After this initial centrifugation, RuNPs were dissolved in acetone (15 ml) and diethyl ether was added (20 ml); the resulting suspension was then subjected to centrifugation. This step was repeated one more time. The resulting sticky black product was next dissolved in 5 ml of methanol and centrifuged with 25 ml of diethyl ether. All centrifugations were carried out at 8000 rpm for 15 min at 15 °C. The resulting shiny black product was vacuum (0.8 mmHg) dried at 35 °C till a constant weight was obtained (24 -48h). This step ensures the removing of the different solvents, including ethylene glycol (b.p. = 197°C). The same procedure was followed for the preparation of PVP-stabilized RuNPs and 1-Butyl-3-methylimidazolium bromide-stabilized RuNPs.

(c) Anion exchange on PILs stabilized Ru NPs Anion exchange via LiNTf2:

To change the solubility and study the effect of anion on the catalytic activity of Ru NPs, anion exchange was carried out on 2Br. In a typical anion exchange reaction, 10 ml aqueous solution of LiNTf2 (4 molar equivalent) was added dropwise to 30 ml aqueous solution (1.5 mg/ml) of 2Br under vigorous stirring, leading to the slow precipitation of 2NTf2. To ensure complete anion exchange, the mixture was stirred for 16 hours under ambient temperature (see also XPS data in Figures S14 andS15). All operations were carried out under an argon atmosphere. The resulting precipitates were washed several times with water and then dried under vacuum at 35 °C till a constant weight was obtained. After anion exchange, NPs became hydrophobic and soluble in organic solvents, as given in the solubility chart (Table S1). The same procedure was used for the preparation of 2NTf2 and 2PF6 from 2Br. For the preparation of 2PF6, LiPF6 was used as the salt.

Anion exchange via AgNTf2: 2I being not soluble in water, a mixture of methanol (MeOH) and water (70-30) was used to obtain a stable solution of 2I. The addition of a LiNTf2 solution (MeOH + H2O) to the solution of 2I (following a similar procedure to that described for 2Cl and 2Br) led to quick precipitation of the product. In this case, XPS analysis revealed that only 20% of iodide was replaced by NTf2. Therefore, the anion exchange procedure was modified to achieve complete exchange. In a typical procedure, 30 mg of 2I was dissolved in 15 ml of methanol. 200 mg AgNTf2 pre-dissolved in 15 ml of methanol was slowly added to the solution and stirred overnight. All the operation was carried out under an argon atmosphere. The mixture was centrifuged at 8000 rpm for 15 minutes to separate AgI (light yellow precipitate). The clear black solution obtained was reduced to ~5 ml by evaporation. Excess of AgNTf2 was removed by dialysis of the solution in distilled water. After 24 hours, the resulting black precipitate was collected and washed several times with distilled water. It is worth mentioning here that, during dialysis, Ru(0) may get converted to its oxide and can adversely affect the catalytic activity of NPs. To reduce the amount of Ru oxide in the final sample, dialyzed NPs were subjected to an additional reduction step. In this step, a 5 ml solution of dialyzed NP (in methanol) was stirred for 16 hrs at 50 °C under 5 bars of hydrogen pressure. Then it was dried under vacuum at 35 °C till a constant weight was obtained.

2BrꞋ was obtained by anion exchange between 2NTf2 and an excess of LiBr. In this case, 2NTf2 was dissolved in acetone (1.5 mg/ml) and LiBr (4 molar equivalent) pre-dissolved in acetone was added dropwise under stirring. The mixture was stirred for 16 hours to ensure a complete exchange of NTf2¯ with Br¯. Upon anion exchange, 2BrꞋ precipitated out of the solution, after being washed with acetone and centrifuged twice to remove excess LiBr, 2BrꞋ was dried under vacuum at 35 °C till a constant weight was obtained. *2NTf2 is partially soluble in ethyl acetate. † 2PF6 is not soluble at room temperature but forms a sticky substance at high temperature (around 100 ⁰C)

Anions

Ru content determination by TGA

Ru content of all NP samples was determined by thermogravimetric analysis (TGA) using a TGA Q-500 from TA instruments. A typical TGA curve of PIL(Cl) (a), 2Cl (b), and 2Br (c) are shown in Figure S4. Initially, samples were heated in a nitrogen environment till 600 °C and subsequently in the air till 750 °C. Residual weight obtained after the introduction of air was considered as the Ru content.

!$.+)b!G"5!=.3-,!)c<87-3)9+'!./!&<C3!!d##X!L-'!<')>!L8+@!,)'()*+!+.!=.9.=),8*!<98+"! ! ! ! ! ! ! ! ! ! ! ! ! ! ) ) ! ! ! ! ! M1!N8$3H

ζ-potentials of Ru NPs

ζ-potentials of all NPs were measured at 25 °C by Malvern Zetasizer ZS. ζ-potentials were measured in water for 1-3Cl, 2Br, and PVP@RuNP (Table S5) and in MeOH for 1-3Cl, 2Br, 2I, 2NTf2 and PVP@RuNPs (Table S6).

XPS analysis

X-ray photoelectron spectroscopy (XPS) analysis was carried out on PIL(X) and PIL(X)@RuNPs to study the nature of the interaction between PILs and RuNPs. K-Alpha X-ray Photoelectron Spectrometer from Thermo Fisher Scientific equipped with monochromatized AlKα source (hν=1486.6 eV) was used for surface analysis. The full spectra (0-1350eV) and highresolution spectra were recorded with a constant pass energy of 200 eV and 40 eV respectively. Ar+ sputtering was used for depth profiles. High-resolution spectra were processed and fitted with AVANTAGE software from Thermo Fisher Scientific. All the scans were corrected considering C(1s) as the reference (285.0 eV). 12,13 The signals corresponding to the binding energy (BE) of all halogen atoms (Cl, Br, I) appear as (non-symmetrical) doublets because of spin-orbit couplings. The value of the BE of the anion was given according to the most intense component of the doublet that appears at the lowest BE (Table S8). All halogens feature both in the "free" PIL(X) (X= Cl, Br, I) and in the corresponding PIL(X)@RuNPs (2Cl, 2Br, 2I) a minor signal that appears at relatively higher BE compared to the most intense signals. Although it is difficult to attribute a specific environment to each signal, we may hypothesize that the difference in BE between the major and minor signals could be related to the number or nature of interactions between the different anions and the C2-H, C4-H, and C5-H by H-bonding. 18 The major signal appearing at the lowest BE would then correspond to a situation where the anion is involved in a fewer H-bond (1 or 2) compared to the minor signal at higher BE, which would be involved in multiple H-bonds (2 or more). For simplicity, only discussions about the BE of the major signals and their shifts between the polymer and the corresponding NP have been included in the main text. In the case of the NTf2 anion, binding to the Ru surface could occur either via the N atom or via the O atoms 18 but XPS analysis does not allow to rule out one of those interactions.

In the case of the iodide anion, the absence of a shift in the BE of I(3p) between PIL(I) and the respective NP (2I) suggests that I¯ do not interact significantly with the metal surface. In contrast, the shift of the N(1s) of the imidazolium moieties N(1s)Im toward lower BE observed in the XPS spectrum of 2I (compared to that observed in PIL(I)) suggests an interaction between the imidazolium cation and the Ru surface. We may hypothesize that the imidazolium cation lies parallel to the surface by analogy with the stabilization of gold NPs in imidazolium-based ILs (see Figure S23). 20 However, the interaction of the imidazolium cation with the Ru surface via the C4-H/C5-H or via the C2-H might also be postulated (Figure S23). 19 As a result of this cation-surface interaction, the surface becomes seriously hindered, which certainly prevents aromatic moieties to adsorb by "p-stacking" onto the flat surface. Nevertheless, this interaction does not prevent the binding of alkenes to the edges or corners of 2I, which allows unsaturated substrates, such as styrene, to undergo selective hydrogenation of the C=C bond. 3)!@4>,.F)9-+8.9!*4*3)'!<9>),!+@)!@4>,.F)9-+8.9!*.9>8+8.9'!02@)9.3!@4>,.F)9-+8.9!-+!55G!PC! /.,!NO!=89K!<'89F!NO!;-,!./!##!-9>!G"5SS!=. &"d(5)=$%) ! M)bIU)"5"01.'.)7L$5)(<"L$%"&'54)*'(&,10)(&,(%)85$)<"F77-9Q)R+AIG)"5"01.'.)H".)"0.$)F"%%'(*)$7&)&$)F$5='%-) F$5<(%.'$5.)8@'47%()G\C9Q) ! ! ! !"#$%&'(*N"!23.+!'@.L89F!(@)9.3!*.97),'8.9!89!+@)!-;')9*)!0>.++)>!389)1!-9>!(,)')9*)!0'.38>!389)1!./!CJ#"!^/+),!+@)!->>8+8.9!./!CJ#!0-+!5G! =891K!*.97),'8.9!,)=-89)>!<9*@-9F)>!L@8*@!8'!+@)!*@-,-*+),8'+8*!./!@)+),.F)9).<'!@4>,.F)9-+8.9"!#4>,.F)9-+8.9!L-'!*-,,8)>!.<+!-+!G"5SS! =.3j!&<!3.->89FK!NO!;-,!##K!:G!PC"!!

(b) Recycling of PIL stabilized Ru NP (2Cl) during hydrogenation of phenol in water

Fig. 3

 3 Fig. 3 Preparation of PIL(X)s-stabilised RuNPs (1-3X) by polyol process and anion exchange reactions (X = Cl, Br, I, NTf2).

Fig. 4

 4 Fig. 4 TEM image and size histogram of (a) 1Cl, (b) 2Cl, (c) 3Cl and (d) Zeta potential of 1-3Cl in water at 25 °C.

Fig. 5

 5 Fig. 5 Overlay of high resolution XPS scans in N(1s) region showing (a) PIL(Cl) and 2Cl, (b) PIL(Br) and 2Br, (c) PIL(I) and 2I (d) PIL(NTf2) and 2NTf2.

  Author version of article published in NanoscaleNanoscale, 2022, 14, DOI: 10.1039/D1NR07628K | 5

Fig. 6

 6 Fig.6 Schematic representation of the interaction between PIL(X) and the Ru surface with (a) PIL(X=Cl or Br), (b) PIL(NTf2) and (c) PIL(I).

Fig. 7 .

 7 Fig.7. DLS of 2Br, 2NTf2 and 2I shown in (a) intensity-weighted size distributions and (b) number-weighted size distributions.

Figure S1. 1 H

 1 Figure S1.1 H NMR spectra of (a) Poly(1-vinyl-3-butylimidazolium chloride) (PIL(Cl)) and (b) PIL(Br) and (c) PIL(I). Traces of isopropanol (from the synthesis) appearing at 1.04 ppm are visible.

Figure S2 .

 S2 Figure S2. DLS size of PIL(Cl), PIL(NTf2) and PIL(I) shown in (a) intensity-average and (b) number average. DLS experiment was carried out at 25 °C using methanol as the common solvent for PILs.

Figure S4 .

 S4 Figure S4. TGA curve of (a) PIL(Cl) (b) 2Cl and (c) 2Br showing weight loss with temperature. Residual weight after 600 °C was considered as the Ru content of the samples.

  !)*AA$37.*#(!INEOJ!'3..%$(&!#A!E-!?B&! ! ! !"#$%&'(P"!2.L>),!d&I!(-++),9!./!&<!$2'!'49+@)'8?)>!<'89F!2AB0C31"!E@)!()-[!*.,,)'(.9>89F!+.!&<!05G51!(3-9)!*-9!;)!'))9!-+!H:P!0Ne1" ,&"-! f8+@!-9!89*,)-')!89!(,)*<,'.,!0&<C3!1!3.->89F!/,.=!)B.!+.!>B.K!+@)!'@-,()989F!./!+@)!()-[!/.,!+@)!5G5!(3-9)!8'!89!-F,))=)9+!L8+@!-9!89*,)-')! 89!$2!'8?)"! ! ! !"#$%&'(Q"!2.L>),!d&I!(-++),9!./!*C%!-9>!*="!A9!+@)!*-')!./!*='05"N!9=!;4!Ea%K!Y8F<,)!J:1K!+@)!'8?)'!./!+@)!*,4'+-3389)!>.=-89'!-,)!+..! '=-33!+.!.;'),7)!-!'8F9-3" "" !!! ! !

Figure S10 .

 S10 Figure S10. XPS survey scan of (a) PIL(Cl), (b) PIL(Br) and (c) PIL(I).

Figure S11 .

 S11 Figure S11. XPS survey scan of PIL(Cl) stabilized Ru NP (2Cl).

Figure S12 .Figure S13 .

 S12S13 Figure S12. XPS survey scan of PIL(Br) stabilized Ru NP (2Br).

Figure S14 .Figure S15 .Figure S16 .Figure S17 .

 S14S15S16S17 Figure S14. XPS survey scan of 2NTf2 NPs obtained after anion exchange between 2Br and LiNTf2. The absence of any characteristic peak for Br is a sign of the complete exchange of Br¯ by NTf2¯.

Figure S18 .Figure S19 .

 S18S19 Figure S18. High resolution Ru(3p) scan of (a) 2Cl, (b) 2Br and (c) 2I. Deconvolution of Ru(3p) shows different oxidation states of Ru present in PILs stabilized NPs.From the figures, it is evident that NPs are mainly composed of Ru(0) (at 461.4 eV) and a small proportion of Ru(4+) towards higher BE (463.4 eV).[14][15][16]

Figure S20 .

 S20 Figure S20. High-resolution scan of Ru(3p) showing different oxidation states of Ru present in 2BrꞋ.

Figure S21 . 2 Figure S22 .

 S212S22 Figure S21. Overlay of high resolution XPS scan of (a) Cl(2p) region of PIL(Cl) and 2Cl, (b) Br(3d) region of PIL(Br) and 2Br, (c) I(3d) region of PIL(I) and 2I and of (d) N(1s)Im and N(1s)NTf2 in PIL(NTf2) and 2NTf2.

(

  %&'(*>"!J*@)=-+8*!,)(,)')9+-+8.9'!./!(.''8;3)!89+),-*+8.9'!;)+L))9!+@)!8=8>-?.38<=!*-+8.9!-9>!+@)!&<$2!'<,/-*)!89!+@)!*-')!./!*="! ! ! ! ! +,-.&' (P"! E@)! ;89>89F! )9),F4! ./! >8//),)9+! )3)=)9+'! 89! 2AB0d1!0d!o!C3K!D,K!AK!$E/#1! -9>! 89! +@)! *.,,)'(.9>89F!2AB!'+-;838?)>!&<!$2'"!!

Figure S24 .

 S24 Figure S24. Activity and selectivity of 2Cl during recycling. Phenol was used as a substrate at 0.166 mol% Ru loading, 25 bars of H2, and 110 °C. Hydrogenation time was kept 25 minutes for every cycle. Grey bars represent phenol conversion and the white bar indicates cyclohexanol selectivity. At every cycle, complete hydrogenation of phenol to cyclohexanol indicates remarkable stability of PIL stabilized Ru nanoparticles.

  %&'(*M"!Ea%!8=-F)!./!*B.!-/+),!R+@!@4>,.F)9-+8.9!*4*3)"!Y,.=!+@)!(-,+8*3)!'8?)!-9>!>8'+,8;<+8.9K!8+!*-9!;)!*.9/8,=)>!+@-+!2AB'!'+-;838?)>! &<$2'!>.!9.+!-FF3.=),-+)!)7)9!-/+),!=<3+8(

  3"j!./!&<1"! ! ! !0#,C"'"1%*',9-+*-&+&=,8%,C$!, J$)F$5='%-)&,(),(&(%$4(5($7.)5"&7%()$=)U7)b/)F"&"01.&.:)+G")L$'.$5'54)&(.&)H".)F"%%'(*)$7&)$5)\+0)*7%'54),1*%$4(5"&'$5) $=)L,(5$0)'5)H"&(%Q "!#$""$ M1*%$4(5"&'$5)H".).&"%&(*)".)L(%)&,()L%$F(*7%()*(.F%'#(*)'5).(F&'$5)\*Q)J,()%("F&'$5)&(-L(%"&7%()H".) -"'5&"'5(*) "&) BD) c+Q)`=&(%) &,() *(.'%(*) &'-(:) &,() %("F&$%) H".) *(L%(..7%'S(*) &$) F$00(F&) &,() ."-L0() =$%) *(&(%-'5'54) L,(5$0) F$5<(%.'$5Q)`&)&,'.)&'-(:)+G")*'..$0<(*)'5)JM@)H".)'5Z(F&(*)8DQ^)-$0"%)(67'<"0(5&.)%(0"&'<()&$)U79)'5&$)&,()%("F&$%)=$00$H(*) #1)%(AL%(..7%'S"&'$5)H'&,)M"Q)J,(5)&,()%("F&$%)H".)'--(%.(*)'5)&,()L%(,("&(*)$'0)#"&,)8BD)c+9)&$).&"%&)&,(),1*%$4(5"&'$5Q)`=&(%) P),$7%.:)&,()%("F&$%)H".)F$$0(*)*$H5)"5*)*(L%(..7%'S(*)&$).&$L)&,()%("F&'$5Q)J,()L%$*7F&)H".)(?&%"F&(*)H'&,)*'(&,10)(&,(%)"5*)

Table 1 .

 1 Hydrogenation of phenol in water catalysed by RuNPs.

Table 2 .

 2 Influence

	Entry	NP	Phenol Conversion (%)	Toluene Conversion (%)
	1	2Cl	6	10
	2	2Br	2	2
	3	2I	0	0
	4 a	2NTf2	11	70
	5 b	2BrꞋ	-	5
	Hydrogenation of phenol and toluene (6 wt% in 5 mL of methanol) were
	performed at 110 °C for 60 min using 0.33 mol.% of Ru catalyst and 25 bars of H2.
	In all cases, conversion was determined by GC-MS.

of PIL counter-anions over the catalytic activity of Ru@PIL(X) NPs (X = Cl, Br, I, NTf2) for the hydrogenation of phenol and toluene.

a 2NTf2 was prepared by anion exchange of 2Br with LiNTf2. b 2Br' was prepared by anion exchange of 2NTf2 with LiBr.

	Entry	NP	Conversion (%)	Selectivity (b %)
	1	1Cl	62	88
	2	2Cl	100	100
	3	3Cl	100*	100
	4	2Br	100	100
	5 a	Ru@IL	100*	100
	6 b
				Although higher conversion was observed upon Br/NTf 2 anion
				exchange (2 and 11% for 2Br and 2NTf 2 respectively, see
				entries 2 and 4), phenol conversion remained very low
				regardless of the catalyst. Toluene was next tested to evaluate
				the influence of the PIL counter-anions in the case of
				hydrophobic substrates. Here again, under the same
				experimental conditions, 2I proved completely inactive, while
				2Cl and 2Br afforded 10 and 2% of methylcyclohexane
				respectively (Table 2). To our delight, 2NTf 2 , resulting from a
				Br/NTf 2 anion exchange, provided 70% of conversion. Thus,
				the simple replacement of the hydrophilic bromide by the
				hydrophobic and weakly coordinating NTf 2 anion allowed the
				catalytic activity to be dramatically increased in the case of
				toluene. Remarkably, this process is reversible as the addition
				of LiBr to 2NTf 2 turned the catalyst back to its inactive form 2Br' (conv. ~ 5%) (Fig. S27). However, in contrast to the Br -
				/NTf 2	-anion exchange, 8% of NTf 2 -remained in 2Br' according
				to XPS analysis (Figure S17), highlighting that the exchange was

Table S1 .

 S1 Molecular weight (Mw and Mn) and polydispersity index (PDI) of synthesized PILs. UV-Vis spectra of precursor solution (RuCl3+PIL(Cl)) showing the characteristic absorption band (dotted line) of RuCl3 around 365 nm and diffused absorption band was observed after reduction (solid line). Diffused absorption indicates the complete reduction of RuCl3 and formation of very small Ru nanoparticles.7,8

	Figure S3.
	Entries	Sample	Mw (g/mol)		Mn (g/mol)	PDI
	1	PIL(Cl)	59760			33200		1.8
	2	PIL(Br)	72576			34560		2.1
	3	PIL(I)	59280			45600		1.3
	4	PVP	55000 (Sigma)	-		-
								a
		Absorbance (a.u)
		200	300	400	500	600	700	800
			Wavelength (nm)

Table S3 .

 S3 Ru content of NP samples determined by TGA.

	Entries	Sample	Ru content (%)
	1	1Cl	9.8
	2	2Cl	18.1
	3	3Cl	29
	4	2Br	17.1
	5	2I	11.2
	6 *	IL(Cl)@RuNP	13.9
	7 #	PVP@RuNP	10.1
	8	2NTf2	9.3
	9 †	2NTf2	5.7
	10	2BrꞋ	17.6

*Ru NPs obtained by using 1-Butyl-3-ethylimidazolium bromide (IL).

# PVP stabilized Ru NPs. † Obtained from 2I via anion exchange with AgNTf2.

Table S5 .

 S5 ζ-potential of PILs and PVP stabilized Ru NPs in water.

	Sample	ζ-potentials in water (mV)
	1Cl	55.5 ± 2.4
	2Cl	49.4 ± 0.5
	3Cl	34.9 ± 1.6
	2Br	58.3 ± 1.1
	PVP@RuNP	8.2 ± 0.4
	Note: ζ-potential was measured at 25 °C.

Table S6 .

 S6 ζ-potentials of PILs and PVP stabilized Ru NPs in methanol.

	Sample	ζ-potential (mV)
	1Cl	38.0 ± 1.4
	2Cl	38.0 ± 0.8
	3Cl	37.2 ± 1.3
	2Br	40.0 ± 1.1
	2NTf2	41.8 ± 1.1
	2I	40.1 ± 1.1
	PVP@RuNP	2.6 ± 0.3
	Note: ζ-potential was measured at 25 °C.

Table S7 .

 S7 Different oxidation states of Ru and their proportion in NPs determined by deconvolution of Ru(3p) scan.

			Position of peaks and Ru(0) composition
	Entry	Sample	Ru(0) [Area %]	Ru(4+) [Area %]	Ru(X+) [Area %]
	1	2Cl	461.3 [88.4]	463.4 [11.6]	-
	2	2Br	461.4 [91.5]	463.4 [8.5]	-
	3	2NTf2	461.4 [85.1]	463.4 [10.3]	465.9 [4.6]
	4	2BrꞋ	461.4 [82.7]	463.4 [15.7]	465.9 [1.6]
	5	2I	461.4 [90.3]	463.4 [9.

Table S9 .

 S9 Hydrogenation of Phenol in water by PILs, ionic liquid and PVP stabilized Ru NPs.Hydrogenation were performed at 110 °C for 25 min, using 25 bars of H2, 0.166 mol.% of Ru and water as solvent. Conversion was determined by determined by 1 H NMR.

	Entries	Catalyst	Conversion (%) Selectivity (b %) Remark
	1	2Br	100	100	-
	2	2Cl	100	100	The catalyst was recycled
					(fig. S24)
	3	IL(Cl)@RuNPs 100	100	Formation of bulk metal
	4	PVP@Ru	100	100	Aggregation of NPs

| Nanoscale, 2022, 14, DOI: 10.1039/D1NR07628K Author version of article published in Nanoscale

| Nanoscale, 2022, 14, DOI: 10.1039/D1NR07628K Author version of article published in Nanoscale

Acknowledgements

The authors are grateful to CNRS and to the Agence Nationale de la Recherche (ANR), ANR JCJC (CATAPILS Project ANR-14-CE05-0027-01) for financial support. We are extremely thankful to Christine Labrugere-Sarroste of PLACAMAT UMS-3626 for XPS analysis and helpful discussions. Dr Olivier Sandre is warmly acknowledged for his help on fitting XRD data. We extend special thanks to Bordeaux Imaging Center for TEM analysis.

Author version of article published in Nanoscale

Nanoscale, 2022, 14, DOI: 10.1039/D1NR07628K | 7

Conflicts of interest

There are no conflicts to declare.

Supplementary Information

Tuning the activity and selectivity of polymerised ionic liquidstabilised ruthenium nanoparticles through anion exchange reactions

Dambarudhar Parida,

(d) Toluene hydrogenation using 2NTf2

Table S10. Influence of solvent on the catalytic activity of 2NTf2 during hydrogenation of Toluene.

Entries

Ru NP Solvent Conversion of toluene (%)

Activity switching of Ru NPs


Sisi Li

Momoko Yoshioka

Junjun Li

Li Liu

Shixin Ye

Ken-Ichiro Kamei

Yong Chen

Nanocasting of fibrous morphology on a substrate for long-term propagation of human induced pluripotent stem cells

Keywords: Nanocasting, Nanofiber, Substrate, Extracellular matrix, Stem cells

Human-induced pluripotent stem cells (hiPSCs) can be self-renewed for many generations on nanofibrous substrates. Herein, a casting method is developed to replicate the nanofibrous morphology into a thin layer of polymethylsiloxane (PDMS). The template is obtained by electrospinning and chemical crosslinking of gelatin nanofibers on a glass slide. The replicas of the template are surface-functionalized by gelatin and used for propagation of hiPSCs over tenth generations. The performance of the propagated hiPSCs is checked by immunofluorescence imaging, flowcytometry, and RT-PCR, confirming the utility of the method. The results are also compared with those obtained using electrospun nanofiber substrates. Inherently, the PDMS replicas is of low stiffness and can be reproduced easily. Compared to other patterning techniques, casting is more flexible and cost-effective, suggesting that this method might find applications in cell-based assays that rely on stringent consideration of both substrate stiffness and surface morphology.

Introduction

Human-induced pluripotent stem cells (hiPSCs) are now widely used for disease modeling, drug testing, and regenerative medicine [START_REF] Takahashi | Induction of pluripotent stem cells from adult human fibroblasts by defined factors[END_REF][START_REF] Park | Disease-specific induced pluripotent stem cells[END_REF][START_REF] Sharma | Multi-lineage human iPSC-derived platforms for disease modeling and drug discovery[END_REF][START_REF] Wu | Harnessing the potential of induced pluripotent stem cells for regenerative medicine[END_REF][START_REF] Ahmed | Stem cells based in vitro models: trends and prospects in biomaterials cytotoxicity studies[END_REF], due to their ability to self-renew and differentiate into multiple cell lineages. Indeed, a large number of protocols were proposed for lineage-specific differentiations [START_REF] Ye | Human embryonic and induced pluripotent stem cells: lineage-specific differentiation protocols[END_REF][START_REF] Gunhanlar | A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells[END_REF] and organoid generation [START_REF] Corrò | A brief history of organoids[END_REF]. Particular attention has to be paid, however, to the culture conditions in order to achieve high quality hiPSC products. In the case of hiPSC renewing, for example, feeder cells were initially used [START_REF] Takahashi | Induction of pluripotent stem cells from adult human fibroblasts by defined factors[END_REF] but feeder-free and serum-free culture was found more suitable by using substrates coated with extracellular matrix (ECM) proteins [START_REF] Chan | Emerging Methods for Enhancing Pluripotent Stem Cell Expansion[END_REF].

Matrigel, an ECM mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, is often used as coating agent which is convenient but contain unknown animal factors [START_REF] Hughes | Matrigel: a complex protein mixture required for optimal growth of cell culture[END_REF]. Other coating agents such as recombinant laminin [START_REF] Rodin | Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511[END_REF][START_REF] Miyazaki | Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells[END_REF], recombinant vitronectin [START_REF] Braam | Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self-Renewal via αVβ5 Integrin[END_REF], and synthetic peptides [START_REF] Villa-Diaz | Synthetic polymer coatings for long-term growth of human embryonic stem cells[END_REF][START_REF] Melkoumian | Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells[END_REF] can also be used for multiple passages. In both cases, the cost is an issue and it is desirable to find alternative ways by considering, for example, substrates with appropriate surface morphologies.

Human-induced pluripotent stem cells (hiPSCs) are now widely used for disease modeling, drug testing, and regenerative medicine [START_REF] Takahashi | Induction of pluripotent stem cells from adult human fibroblasts by defined factors[END_REF][START_REF] Park | Disease-specific induced pluripotent stem cells[END_REF][START_REF] Sharma | Multi-lineage human iPSC-derived platforms for disease modeling and drug discovery[END_REF][START_REF] Wu | Harnessing the potential of induced pluripotent stem cells for regenerative medicine[END_REF][START_REF] Ahmed | Stem cells based in vitro models: trends and prospects in biomaterials cytotoxicity studies[END_REF], due to their ability to self-renew and differentiate into multiple cell lineages. Indeed, a large number of protocols were proposed for lineage-specific differentiation [START_REF] Ye | Human embryonic and induced pluripotent stem cells: lineage-specific differentiation protocols[END_REF][START_REF] Gunhanlar | A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells[END_REF] and organoid generation [START_REF] Corrò | A brief history of organoids[END_REF]. Particular attention has to be paid, however, to the culture conditions in order to achieve high-quality hiPSC products. In the case of hiPSC renewing, for example, feeder cells were initially used [START_REF] Takahashi | Induction of pluripotent stem cells from adult human fibroblasts by defined factors[END_REF] but feeder-free and serum-free culture was found more suitable by using substrates coated with extracellular matrix (ECM) proteins [START_REF] Chan | Emerging Methods for Enhancing Pluripotent Stem Cell Expansion[END_REF].

Matrigel, an ECM mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, is often used as a coating agent which is convenient but contains unknown animal factors [START_REF] Hughes | Matrigel: a complex protein mixture required for optimal growth of cell culture[END_REF]. Other coating agents such as recombinant laminin [START_REF] Rodin | Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511[END_REF][START_REF] Miyazaki | Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells[END_REF], recombinant vitronectin [START_REF] Braam | Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self-Renewal via αVβ5 Integrin[END_REF], and synthetic peptides [START_REF] Villa-Diaz | Synthetic polymer coatings for long-term growth of human embryonic stem cells[END_REF][START_REF] Melkoumian | Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells[END_REF] can also be used for multiple passages. In both cases, the cost is an issue and it is desirable to find alternative ways by considering, for example, substrates with appropriate surface morphologies.

Previously, we have shown that long-term expansion of hiPSCs could be achieved by using gelatin nanofiber-covered substrates produced by electrospinning and chemical crosslinking [START_REF] Liu | Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells[END_REF].

Propagation of hiPSCs over more than 20 passages has been demonstrated without introducing any abnormal chromosome. Moreover, the passage of the cells could be done without enzymatic disassociation nor mechanic cutting, thus allowing a better hiPSC expansion. This is because the fact that gelatin is rich in arginine-glycine-aspartate (RGD) peptide sequences and that the nanofibrous morphology is in favor of cell adhesion and signaling pathway activation [START_REF] Clyman | Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen[END_REF].

Moreover, gelatin is widely used in cosmetic, food, and medical industries and can be easily processed. However, previous studies have shown that hiPSCs cannot propagate on flat gelatin layers, due to insufficient cell attachment and rapid loss of pluripotency after a few passages [START_REF] Liu | Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells[END_REF][START_REF] Joddar | Chemically fixed autologous feeder cell-derived niche for human induced pluripotent stem cell culture[END_REF]. This would suggest that hiPSCs are very sensitive to the surface morphology of the gelatin substrate and that the nanofibrous pattern would allow more efficient cell anchoring on the substrate and pluripotency maintenance of the cells [START_REF] Liu | Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells[END_REF]. Generally speaking, the properties of the substrate, including its stiffness, permeability, morphology, and chemical composition, influence to a large extent the behavior of the cells such as adhesion, proliferation, survival, and differentiation [START_REF] Saha | Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions[END_REF][START_REF] Bertucci | Biomaterial engineering for controlling pluripotent stem cell fate[END_REF][START_REF] Cortella | Conditioning of hiPSC-derived cardiomyocytes using surface topography obtained with high throughput technology[END_REF][START_REF] Huethorst | Customizable, engineered substrates for rapid screening of cellular cues[END_REF][START_REF] Li | Engineering-derived approaches for iPSC preparation, expansion, differentiation and applications[END_REF][START_REF] Vignesh | Fabrication of micropatterned alginate-gelatin and k-carrageenan hydrogels of defined shapes using simple wax mould method as a platform for stem cell/induced Pluripotent Stem Cells (iPSC) culture[END_REF][START_REF] Wang | Substrate elasticity dependent colony formation and cardiac differentiation of human induced pluripotent stem cells[END_REF][START_REF] Wang | Fabrication of elastomer pillar arrays with elasticity gradient for cell migration, elongation and patterning[END_REF][START_REF] Li | Culture substrates made of elastomeric micro-tripod arrays for long-term expansion of human pluripotent stem cells[END_REF]. Intuitively, the nanofibrous morphology resembles some parts of in vivo ECMs, which provides more appropriate ligand accessibility and spaces underneath of the cells for diffusional transport of cytokines and large proteins [START_REF] Liu | Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells[END_REF][START_REF] Lim | Electrospun scaffolds for stem cell engineering[END_REF][START_REF] Lu | A 3D microfibrous scaffold for long-term human pluripotent stem cell self-renewal under chemically defined conditions[END_REF][START_REF] Kaitsuka | A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and human pluripotent stem cells to insulin-producing cells[END_REF][START_REF] Wertheim | A microfluidic chip containing 3D nanofibrous scaffolds for culturing human pluripotent stem cells[END_REF].

Electrospinning is a versatile and low-cost technique that works with different types of materials or material compositions [START_REF] Ramakrishna | Science and engineering of electrospun nanofibers for advances in clean energy, water filtration, and regenerative medicine[END_REF][START_REF] Lim | Electrospun scaffolds for stem cell engineering[END_REF][START_REF] Lu | A 3D microfibrous scaffold for long-term human pluripotent stem cell self-renewal under chemically defined conditions[END_REF][START_REF] Kaitsuka | A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and human pluripotent stem cells to insulin-producing cells[END_REF][START_REF] Wertheim | A microfluidic chip containing 3D nanofibrous scaffolds for culturing human pluripotent stem cells[END_REF][START_REF] Kumar | Self-renewal of human embryonic stem cells on defined synthetic electrospun nanofibers[END_REF][START_REF] Teo | Electrospun fibre bundle made of aligned nanofibres over two fixed points[END_REF][START_REF] Yu | Clonal Isolation of Human Pluripotent Stem Cells on Nanofibrous Substrates Reveals an Advanced Subclone for Cardiomyocyte Differentiation[END_REF][START_REF] Kamei | Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments[END_REF][START_REF] Liu | Nano-on-micro fibrous extracellular matrices for scalable expansion of human ES/iPS cells[END_REF]. However, this technique lacks repeatability and reproducibility when applied to collagen nanofiber deposition. Critical control of the environment temperature and humidity is even requested for electrospinning of high-quality gelatin nanofibers [START_REF] Liu | Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells[END_REF]. Herein, we develop a simple and low-cost technique, nanocasting, to reproduce a nanofibrous surface morphology from a substrate (template) defined by electrospinning. Since a template can be used many times in nanocasting, the replicas obtained with the same template may have the same fibrous morphology (positive or negative tones). This would allow us to overcome the lack of reproducibility of electrospun nanofibers. Nanocasting is now widely used to cast polydimethylsiloxane (PDMS) with a template. Previously, we have created artificial lotus leave by casting PDMS with a natural lotus, showing a high fidelity of replication [START_REF] Sun | Artificial lotus leaf by nanocasting[END_REF]. Nevertheless, most of the templates were produced by electron beam lithography and reactive ion etching techniques [START_REF] Chen | Nanofabrication: Conventional and nonconventional methods[END_REF][START_REF] Vieu | Electron beam lithography: resolution limits and applications[END_REF] and natural templates were often used to study the superhydrophobicity of plates or inserts [START_REF] Sun | Artificial lotus leaf by nanocasting[END_REF][START_REF] Yadav | Bio-mimicked hierarchical 2D and 3D structures from natural templates: applications in cell biology[END_REF]. In this work, the template is simply produced by electrospinning and chemical crosslinking techniques, and both negative tone (PDMS replica) and positive tone (PDMS replica² ) replicas were produced in order to compare their performance in long-term hiPSCs propagation, using the nanofiber-covered substrate as control. We will show that the PDMS replica is able to support hiPSC propagation over the tenth generation whereas the PDMS replica² leads to spontaneous differentiation of the hiPSCs after a few passages.

Material and methods

Template fabrication

The template was fabricated by electrospinning and chemical crossing of gelatin nanofibers [START_REF] Liu | Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells[END_REF]. Briefly, 10 wt% gelatin powder from porcine skin (G2625, Sigma-Aldrich, France) was dissolved in a solution of acetic acid, ethyl acetate (Sigma-Aldrich, France), and de-ionized (DI) water with the volume ratio of 21:14:10. The solution was loaded into a syringe which is mounted in a syringe pump (KD Scientific). The needle of the syringe which served as spinneret was connected to the anode of a high potential power supply (TechDempaz, Japan) with a bias voltage of 11 KV and a silicon wafer placed 12 cm far away from the spinneret is grounded on which a cover glass (25 mm diameter; Matsunami Glass Ind., Ltd, Japan) was taped for the nanofiber connection. The flow rate of the gelatin solution was fixed at 0.2 ml/h. After electrospinning, gelatin nanofibers were dried in a 37°C vacuum overnight and cross-linked in a 0.2 M mixture of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC; Dojindo, Japan) and Nhydroxysuccinimide (NHS; Sigma-Aldrich, Japan) in ethanol for 4 h. After crosslinking, samples were rinsed with 99.5% ethanol twice and dried in a vacuum at 37°C overnight. The sample fabrication process was used for the fabrication of the culture substates of the control.

Nanocasting and surface coating of the replicas

Firstly, the template was fixed in a petri-dish with adhesion tapes. A prepolymer solution of PDMS at a ratio of 10:1 was poured on the template at 1 mm thickness. After removing air bubbles in a desiccator, the PDMS layer was cured for 2 h in an 80 °C oven, peeled-off from the template, and bonded on a glass slide after surface treating in air plasma. When this 1st (negative tone) replica was used as a template for the casting of a 2nd replica, the surface of PDMS was treated in a vapor of trimethylchlorosilane (TMCS) to facilitate the template separation Then, it was used to produce the 2nd (positive tone) replica using the same casting parameter.

Before being used for hiPSC propagation, the replica (negative and positive) was spin-coated with 0.1 wt% gelatin dissolved in the mixed solution of acetic acid, ethyl acetate, and DI water at a volume ratio of 21:14:10. Before spin-coating, the replica was treated with plasma for 3 min and the gelatin solution was deposited with as spin-coater at 3000 rpm for 60 s. After drying in a 37°C vacuum overnight, it was crosslinked using the same method as gelatin nanofibers. Afterward, the PDMS replicas were used for hiPSCs culture.

hiPSC expansion

253G1 hiPSCs from Kyoto University (CiRA) have been used in this work. Before cell loading, the substrate was rinsed with 99.5% ethanol 3 times for sterilization. Dissociated hiPSCs harvested from gelatin nanofiber substrate were introduced on a PDMS replica or replica 2 at a density of about 4 × 10 4 cells/cm 2 with a widely-used hiPSC culture medium mTeSR1 (Stem Cell Technologies) [START_REF] Ludwig | Feederindependent culture of human embryonic stem cells[END_REF], supplemented with 10 µM ROCK inhibitor (Y-27632; Wako Chemicals, Japan) [START_REF] Watanabe | A ROCK inhibitor permits survival of dissociated human embryonic stem cells[END_REF] The cells were cultured in the 37 °C incubator with 5% CO2 (IP400 CO2 incubator). The medium was changed every day. After 48 h, the ROCK inhibitor was removed. When cells reached 80% of confluency on day 4, they were treated with a cell dissociated no-enzyme solution (GIBCO) for 5 min at 37 °C. Cells were then dissociated by hand pipetting over 10 times to a single cell level and transferred onto a new PDMS replica or replica 2 . Similarly, gelatin nanofiber substrate was used as a control.

Characterization

Atomic Force Microscope (AFM) imaging: Nanofibrous morphology of template and replica were analyzed using an atomic force microscope (Agilent 5500 AFM) in tapping mode with SPM probes (AppNano).

Contact angle measurement:

The contact angle measurement has been done by using the sessile drop method. A 2 µl water droplet was deposited onto the sample, and the water/substrate interface was captured by a CCD camera. The edge of the droplet was then analyzed using a sessile dropfitting model.

Immunocytochemistry: Cells were firstly fixed in 4% paraformaldehyde at room temperature for 30 min; permeabilized with 0.5% Triton X-100 (Wako Chemicals, Japan) in Dulbecco's Modified phosphate-buffered saline (D-PBS; GIBCO) for 30 min; and incubated with blocking solution containing 5% normal goat serum (NGS; Vector Laboratories, Burlingame, CA, USA), 5% normal donkey serum (NDS; Jackson ImmunoResearch, West Grove, PA, USA), 3% fetal bovine serum (FBS; CCB, Japan) and 0.1% Tween 20 (Wako Chemicals, Japan) in D-PBS at 4°C overnight.

Then, cells were incubated with primary antibodies, i.e., 2 µg/ml anti-OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; C-10, SC-5279) and 20 µg/ml anti-SOX2 (Cell Signaling Technology, Danvers, MA, USA) in blocking buffer at 4 °C overnight. Following primary antibody incubation, cells were incubated with appropriate secondary antibodies -1. Total RNA purification: Total cellular RNA was harvested using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. Total RNA concentrations were measured in a NanoDrop1000 machine (ThermoFisher, Japan).

Reverse transcription PCR (RT-PCR):

Total RNA was reverse transcribed using the RT First Strand Kit (Qiagen) and the primer sets described in [START_REF] Liu | Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells[END_REF]. A reaction mixture (25 µl) containing 1 µl cDNA, 0.2µM PCR primers, and 5 units of Taq DNA polymerase (Takara, Japan) in PCR buffer was subjected to amplification in a DNA thermal cycler (Applied Biosystems 7300 Real-Time PCR System). PCR was performed for 30-35 cycles (94°C, 30 sec; 58°C, 30 sec; 72°C, 60 sec) with an initial incubation at 94°C for 5 min and a final extension at 68°C for 3 min. Amplified products (10 µl) were resolved by 1.2 % agarose gel electrophoresis and visualized by ethidium bromide staining. RT-PCR results of glyceraldehyde-3-phosphate dehydrogenase were used for the control.

Scanning Electron Microscope (SEM) imaging: High-resolution images were taken with an SEM (JCM-5000; JEOL Ltd. Japan) operated at 10 kV. Prior to imaging, a 5-nm-thick gold layer was deposited on samples by sputtering (MSP 30T; Shinku Device, Japan).

Results

The fabrication process of gelatin nanofiber substrate has been optimized previously [START_REF] Liu | Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells[END_REF] and adopted in this work for the fabrication of nanocasting templates. Once fabricated, the surface pattern of the template was replicated into PDMS many times (Fig. 1). As can be seen in AFM images of the figure, the PDMS replica displays a complimentary surface morphology of the template. After gelatin coating, the contact angle of the replica (63.2°) is close to that of the gelatin nanofiber substrate (60.4°), both being hydrophilic and suited for cell seeding and cell attachment.

In Fig. S1, the whole fabrication processes of PDMS replica and replica² before and after gelatin coating are shown. The surface roughness of the AFM images was analyzed by assuming a roughness profile of 𝑛 orders. The arithmetic average of absolute values 𝑅 𝑎 and the root mean squared 𝑅 𝑟𝑚𝑠 are calculated,

𝑅 𝑎 = 1 𝑛 ∑ |𝑦 𝑖 | 𝑛 𝑖=1 , 𝑅 𝑟𝑚𝑠 = √ 1 𝑛 ∑ 𝑦 𝑖 2 𝑛 𝑖=1
where yi is the vertical distance from the mean line. As shown in Fig. S1(c), Ra and Rrms of the PDMS replica were smaller that than of the gelatin nanofiber substrate (template), and that of the PDMS replica 2 were further reduced. In both cases, the roughness of the replicas decreases but the contact angle reduces after gelatin-coating. Fig. S2 scanning electron microscopy images of the gelatin nanofiber before and after crosslinking as well as photographs of 2 l water droplets on two types of replicas before and after gelatin deposition. As can be seen, the first-generation replica fairly reproduced the crosslinked nanofiber features but the second-generation degraded the nanofibers' morphology. This is often the case when PDMS is used as a template. Although the quality of the second-generation replica could be improved by using a template of harder material, this most mostly relied on the PDMS nanocasting for the sake of process simplicity. As can also be seen, the contact angles of the two types of replicas are comparable before and after gelatin deposition, suggesting that they have comparable surface roughness though they are both smaller than that of the master.

After seeding, hiPSCs aggregated to form cell clusters in three hours and then cell colonies in one day, on both gelatin nanofiber substrate and PDMS replica, which is characteristic of the hiPSC expansion (Fig. 2a,b). hiPSCs were generated from somatic cells [START_REF] Patel | Advances in reprogramming somatic cells to induced pluripotent stem cells[END_REF]. When just seeded, they had a round shape (Fig. 2a1,b1). On day 1, ROCK inhibitor (Y-27632) was applied and hiPSCs colonies kept growing up to day 4 in E8 and E8flex media (Fig. 2a2-4, 2b2-4). Clearly, these hiPSC colonies were sharp-edged, flat, and ball-like monolayer structures termed "round colonies" (Fig. 2a4,2b4), which is a typical undifferentiated ESC-like morphology [START_REF] Yu | Low cell-matrix adhesion reveals two subtypes of human pluripotent stem cells[END_REF]. No significant differences in the morphology of the cells and colonies could be observed during this period.

The morphologies of hiPSC colonies were also examined after passage. Fig. 2c and2d display the phase contract images of the hiPSC colonies at passages 4, 6, 8, and 13, showing also comparable growth and formation of the hiPSC colonies. Here, the passages were performed at the same time for both types of substrates at 70-80% confluence, suggesting the same growth rate of the hiPSCs over a long period on both types of substrates. However, on PDMS replica 2 some cells appeared in the areas between colonies as marked by the red circles in Fig. S3, suggesting that cells underwent a spontaneous differentiation after three passages. Fig. 3 shows alkaline phosphatase (AP) staining images of the hiPSCs on both types of the substrate at passage 5, confirming their undifferentiated state. In contrast, both AP staining and phase-contrast images of the hiPSCs on gelatin (without crosslinking) coated glass at passages 1 and 2 already showed a bad state of the cells (Fig. S3).

Immunocytochemistry analyses were performed with markers OCT4 and SOX2, which are essential transcription factors of pluripotent stem cells. As shown in Fig. 4, the two types of proteins were highly expressed in hiPSCs on both types of substrates at passage 9, also confirming maintained pluripotency of the hiPSCs cells. In contrast, on PDMS replica 2 , the expression level of OCT4 became relatively low at passage 6, and some cells did not express OCT4 all as marked by small red cells in Fig S4 .   Furthermore, flow cytometric analyses were performed using surface proteins, SSEA4 and TRA-1-60, of the hiPSCs at passage 9 (Fig. 5). As expected, both proteins were highly expressed in the hiPSCs on both types of culture substrate, and quantitatively over 99.9% of the hiPSCs maintained their pluripotency. However, SSEA4 expression on the PDMS replica² substrate is comparatively lower with more than 0.2% cells without expression of neither SSEA4 nor TRA-1-60, suggesting them all in a completely differentiated state.

Finally, RT-PCR analyses were performed for hiPSCs on different types of substrates (Fig. 6). Clearly, the expression of pluripotency (OCT4, NANOG, and SOX2) and differentiation (PAX6, BRACHYURY, and AFP) genes of the hiPSCs on PDMS replica maintain similar levels of that on gelatin nanofiber substrates over 13 passages. However, the hiPSCs on PDMS replica² at passage 9 showed a high expression of BRACHYURY, indicating a significant deviation from the pluripotency state of the cells.

Discussions

Previously, we have reported a nanocasting method to replicate a lotus leaf with both micro and nano-scale features which are difficult to be created artificially [START_REF] Sun | Artificial lotus leaf by nanocasting[END_REF]. Based on this approach, we fabricated templates with gelatin nanofibrous morphology. As can be seen in Fig. S2, the electrospun nanofibers after crosslinking showed multiple nanofiber cross-points and the morphology of the template could be fairly reproduced into PDMS. To make the PDMS replica suited for cell culture [START_REF] Lee | Compatibility of mammalian cells on surfaces of poly (dimethylsiloxane)[END_REF] and keep the consistency with our previous work, gelatin-coating was applied. As result, the gelatin-coated PDMS replica became hydrophilic with a contact angle comparable to that of the gelatin nanofiber substrate.

The mechanical properties and in particular the stiffness of the substrate play an important in the cell fate decision of the stem cells [START_REF] Saha | Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions[END_REF][START_REF] Bertucci | Biomaterial engineering for controlling pluripotent stem cell fate[END_REF][START_REF] Cortella | Conditioning of hiPSC-derived cardiomyocytes using surface topography obtained with high throughput technology[END_REF][START_REF] Huethorst | Customizable, engineered substrates for rapid screening of cellular cues[END_REF][START_REF] Li | Engineering-derived approaches for iPSC preparation, expansion, differentiation and applications[END_REF][START_REF] Vignesh | Fabrication of micropatterned alginate-gelatin and k-carrageenan hydrogels of defined shapes using simple wax mould method as a platform for stem cell/induced Pluripotent Stem Cells (iPSC) culture[END_REF][START_REF] Wang | Substrate elasticity dependent colony formation and cardiac differentiation of human induced pluripotent stem cells[END_REF][START_REF] Wang | Fabrication of elastomer pillar arrays with elasticity gradient for cell migration, elongation and patterning[END_REF][START_REF] Li | Culture substrates made of elastomeric micro-tripod arrays for long-term expansion of human pluripotent stem cells[END_REF][START_REF] Ramakrishna | Science and engineering of electrospun nanofibers for advances in clean energy, water filtration, and regenerative medicine[END_REF][START_REF] Lim | Electrospun scaffolds for stem cell engineering[END_REF][START_REF] Lu | A 3D microfibrous scaffold for long-term human pluripotent stem cell self-renewal under chemically defined conditions[END_REF][START_REF] Kaitsuka | A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and human pluripotent stem cells to insulin-producing cells[END_REF][START_REF] Wertheim | A microfluidic chip containing 3D nanofibrous scaffolds for culturing human pluripotent stem cells[END_REF]. Stem cells probe the substrate and regulate the intracellular contractility and stiffness [START_REF] Clyman | Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen[END_REF][START_REF] Wang | Substrate elasticity dependent colony formation and cardiac differentiation of human induced pluripotent stem cells[END_REF]. The pluripotency of the hiPSCs should be associated with their low contractility, suggesting the use of low stiffness substrate for long-term propagation of the substrate [START_REF] Wang | Substrate elasticity dependent colony formation and cardiac differentiation of human induced pluripotent stem cells[END_REF]. It has been recently shown that soft substrates (1.5 or 15 kPa PDMS) caused modulation of several cellular features into a phenotype closer to pluripotent stem cells due to increased expression of pluripotency-related genes [START_REF] Gerardo | Soft culture substrates favor stem-like cellular phenotype and facilitate reprogramming of human mesenchymal stem/stromal cells (hMSCs) through mechanotransduction[END_REF]. In our case, the Young's modulus of the PDMS is in the order of 1 MPa [START_REF] Kim | Enhancement of the thermo-mechanical properties of PDMS molds for the hot embossing of PMMA microfluidic devices[END_REF]. The PDMS replica may have a smaller effective Young's modulus, due to nanofibrous surface morphology for the same reason as the micropillar deflection. Previously, an effective Young's modulus of PDMS pillars was much smaller than that of a bulk PDMS [START_REF] Wang | Substrate elasticity dependent colony formation and cardiac differentiation of human induced pluripotent stem cells[END_REF][START_REF] Wang | Fabrication of elastomer pillar arrays with elasticity gradient for cell migration, elongation and patterning[END_REF]. Although the nanofibrous morphology is significantly different from that of pillars, the bending effect of a featured structure with a lateral force exercised by cells should be qualitatively the same. Roughly speaking, an effective Young's modulus of ~ 211 kPa could be estimated by considering an array of pillars of 1 µm diameter and 1 µm height.

On the other hand, Young's modulus of wetted gelatin is also in the order of 1 MPa [START_REF] Zhang | Gelatin nanofibrous membrane fabricated by electrospinning of aqueous gelatin solution for guided tissue regeneration[END_REF]50]. By using the same technique described in [50], we measured the deflection of a bilayer structure, a monolayer of crosslinked gelatin on a thin PDMS layer, as a function of applied pressure. As result, an effective material Young's modulus in the order of 1 MPa was determined. According to the theory of cellular solids [START_REF] Gibson | Cellular solids[END_REF], the effective Young's modulus of a porous or structured material is always smaller than Young's modulus of the bulk material. For a suspended monolayer of crosslinked gelatin nanofibers, its in-plan effective Young's modulus can be as small as 1 kPa.

When the monolayer is bonded with a PDMS layer, the stiffness should be different [START_REF] Luo | Fabrication and application of silicon-reinforced PDMS masters[END_REF][START_REF] Shi | Investigation of cell culture in microfluidic devices with different bilayer substrates[END_REF] but in the same order of magnitude. In contrast, a nanofiber-covered glass substrate should have a much larger Young's modulus (>10 GPa) [START_REF] Inaba | Young's modulus and compositional parameters of oxide glasses[END_REF]. Finally, cells, and in particular the aggregated hiPSCs are generally not in conformal contact with a rough surface due to the surface tension of the cell membrane. This means that there is more free space for more efficient diffusion of nutrients and metabolites than that of the cells on a flat substrate. Therefore, the nanofibrous morphology is twice in favor of hiPSC culture and proliferation.

We would like to mention that although other types of biomolecules can be used to coat the surface of the PDMS replica, gelatin has been chosen for close comparison with gelatin nanofiber substrate. Furthermore, gelatin is low-cost and easy to process into thin films, nanofibers, microparticles, and encapsulations [START_REF] Guimarães | The stiffness of living tissues and its implications for tissue engineering[END_REF][START_REF] Wu | Cell adhesion and proliferation enhancement by gelatin nanofiber scaffolds[END_REF][START_REF] Bello | Engineering and functionalization of gelatin biomaterials: From cell culture to medical applications[END_REF][START_REF] Li | Fabrication of gelatin nanopatterns for cell culture studies[END_REF]. Depending on the crosslinking, the biodegradability of gelatin can be regulated. On the other hand, gelatin possesses the RGD sequences of collagen and the nanofibrous morphology of patterned gelatin promotes cell adhesion and cell proliferation.

Matrigel is often used for hiPSC culture and self-renewing but it was produced from mouse cells with many unknown factors. Nevertheless, it is rich in ECM composition. Compared to Matrigel, gelatin is perhaps too pure. In this regard, new ECM components may be added and more systematic studies are expected.

Conclusions

We have developed a method to reproduce the nanofibrous morphology of a substrate by nanocasting with PDMS. This method is generic and low-cost. It can be easily implemented and scaled up for different applications. Our results showed that such a negative tome replica of the template could be used as a substrate for hiPSC propagation over tenth generations but the positive tone replica could not be used for long-term hiPSC propagation. The use of PDMS seems to be advantageous not only for easy casting not also for its relatively low stiffness often required in hiPSC differentiation. This method might find application cell-based assays or tissue engineering which are based on stringent considerations on material stiffness and surface morphology.

  5 µg/ml DyLight 488 donkey anti-mouse IgG (Jackson ImmunoResearch) or 0.375 or 3 µg/ml DyLight 649 donkey antirabbit IgG (Jackson ImmunoResearch) in blocking buffer at room temperature for 1 h. Finally, cell nuclei were stained with 300 nM 4, 6-diamidino-2-phenylindole (DAPI; Dojindo, Japan) at room temperature for 30 min. Phase-contrast and fluorescent images of cells were recorded using inverted microscopy (Olympus IX71) equipped with a high-sensitive CCD camera (Olympus U-LH100HGAPO).

  Flow cytometry: hiPSCs cultured on gelatin nanofiber, PDMS replica and PDMS replica 2 substrate were harvested with TrypLE Express. Cells were washed with PBS twice, and cell numbers were counted. For staining with antibodies, cells were diluted to a final concentration of 1 × 10 7 cells/ml in PBS with 2% FCS. Phycoerythrin (PE) anti-human SSEA4 or TRA-1-60 (Stemgent) labeled antibodies were added and incubated at room temperature for 30 min. As a negative control, specific isotype controls (PE Mouse IgG3 Isotype Control (Stemgent) and Dylight 488 mouse IgM Isotype Control (Stemgent)) were used. After removing antibodies by centrifugation at 1000 rpm for 5 min, cells were washed with PBS containing 2% FCS. Finally, cell suspensions were applied to a flow cytometry cell sorting (FACS) Canto II machine (BD Biosciences).

Figure caption Figure 1 .

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Figure 2 .

 2 Figure2. Phase contract images of the hiPSCs on gelatin nanofiber substrate (a1-a4, c1-c4) and PDMS replica (b1-b4, d1-d4) after incubation for 3 h, 1, 2, and 4 days (a1-a4, b1-b4) and at passage 4, 6, 8, and 13 (c1-c4,d1-d4), respectively. Scale bar in (a1) and (b1) represents 50 m; scale bar in (a2-a4, b2-b4, c1-c4, d1-d4) represents 100 m.

Figure 3

 3 Figure 3 Alkaline phosphatase (AP) staining images of the hiPSCs at passage 5 on gelatin nanofiber substrate (a1-a3) and PDMS replica (b1-b3), respectively. The diameter of the culture dish in (a1) and (b1) is 35mm. Scale bar in (a2) and (b2) represents 200 m, scale bar (a3) and (b3) represents 100m.

Figure 4 .

 4 Figure 4. Immunocytochemical images of OCT4 (green) and SOX2 (red) marked hiPSCs at passage 9 on gelatin nanofiber substrate (a1-a3) and PDMS replica, respectively. Cell nuclei stained with DAPI (blue). Scale bar represents 100 m.

Figure 5 .

 5 Figure 5. Flow cytometric results of SSEA4 and TRA-1-60 expression level of the hiPSCs on different types of substrates including gelatin nanofiber substrate, PDMS replica (negative tone), and PDMS replica² (positive tone) at passage 9.

Figure 6 .

 6 Figure 6. RT-PCR results of the hiPSCs on gelatin nanofiber substrate and PDMS replica at passage 13 on PDMS replica² at passage 8, respectively.

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