Luanda Lins
email: luandaqmc@gmail.com

Florence Wianny

Colette Dehay

Jacques Jestin

Watson Loh

Adhesive Sponge Based on Supramolecular Dimers Interactions as Scaffolds for Neural Stem Cells

Keywords: Supramolecular, 2-ureido-4-pyrimidone, Sponge-like scaffolds, Neural Tissue Engineering

Improving cell-material interactions of non-adhesive scaffolds is crucial for the success of biomaterials in tissue engineering. Due to their high surface area and open pore structure, sponges are widely reported as absorbent materials for biomedical engineering. The biocompatibility and biodegradability of polysaccharides sponges, coupled with the chemical functionalities of supramolecular dimers, make them promising combinations for the development of adhesive scaffolds. Here, a supramolecular tactic based on (UPy)-modified polysaccharide associated with threedimensional structure of sponges was developed to reach enhanced cellular adhesion.

For this purpose, three approaches were examined individually in order to accomplish this goal. In the first approach, the backbone polysaccharides with non-cell adhesive properties were modified via a modular tactic using UPy-dimers. Hereupon, the Page 1 of 50 ACS Paragon Plus Environment Biomacromolecules physical-chemical characterizations of the supramolecular sponges were performed,

showing that the presence of supramolecular dimers improved their mechanical properties and induced different architectures. In addition, small-angle neutron scattering (SANS) measurements and rheology experiments revealed that the UPydimers into agarose backbone are able to reorganize in thinning aggregates. It is also demonstrated that the resulted UPy-agarose (AGA-UPy) motifs in surfaces can promote cell adhesion. Finally, the last approach showed the great potential for use of this novel material in bioadhesive scaffolds indicating that neural stem cells show a spreading bias in soft materials and that cell adhesion was enhanced for all UPy-modified sponges compared to the reference, i.e. unmodified sponges. Thus, by functionalizing sponge surfaces with UPy-dimers, an adhesive supramolecular scaffold is developed which opens the opportunity its use improving neural tissues regeneration.

Introduction

Neural tissue engineering (NTE) is an emerging area at the interfaces of chemistry, engineering materials, neuroscience, and biology, where research tools in cell biology are combined with engineering biomaterials principles to facilitate the cell adhesion on scaffold for regeneration or repair of neural tissues. The combination of neural stem cells, biomaterial scaffolds, and bioadhesive molecules may contribute to cultivate cells in vitro before being implanted into damaged tissue. The scaffold implanted can stimulate the self-healing capability of the tissue for neural system repair in different ways. Besides, monkey Neural Stem Cells (NSCs) on a suitable scaffold may be implanted on the brain with a lessened risk of immune rejection, representing invaluable implements for preclinical studies involving neural tissue engineering [START_REF] Lins | Development of Bioresorbable Hydrophilic-Hydrophobic Electrospun Scaffolds for Neural Tissue Engineering[END_REF][START_REF] Lins | Effect of polyvinylidene fluoride electrospun fiber orientation on neural stem cell differentiation[END_REF] .

There seems to be a general consensus that a three-dimensional scaffold with a suitable microstructure is essential to promote cell adhesion, proliferation, and driving tissue regeneration as well as to support oxygen exchange and nutrient diffusion. Additionally, scaffolds need to exhibit rough surfaces and high porosity to promote chemical bonding with the tissue associated with the proper mechanical features to support the cells and to meet the required achievement of the tissues at the site of implantation. Thanks to their large surface area, high absorption capacity, and high porosity, sponges or aerogels are good candidates as scaffolds for these cells growth [START_REF] Erdem | Fabrication and characterization of soft macroporous Jeffamine cryogels as potential materials for tissue applications[END_REF][START_REF] Hixon | A comprehensive review of cryogels and their roles in tissue engineering applications[END_REF] .

Superabsorbent and porous scaffolds, such as sponges or aerogels, can be yielded at frozen temperatures from polymeric hydrogels, of which the hydrophilic structure provides them adept of holding huge amounts of water in their threedimensional structures. So far, polysaccharide hydrogels have been explored as biomaterials for three-dimensional cell encapsulation and transplantation. This is due to fact that these hydrogels can resist to compressive forces on the matrix while permitting a fast diffusion of nutrients between the blood and tissue cells. Agarose is a typical example of hydrogel that forms a non-cytotoxic support. It may be utilized to reproduce the natural cells environment, as extracellular matrix (ECM). However, agarose and other hydrophilic polysaccharides form a stiff inert hydrogel that does not carry any biological information, thus the cells cannot adhere to the surface of these scaffolds [START_REF] Forget | Polysaccharide hydrogels with tunable stiffness and provasculogenic properties via α-helix to β-sheet switch in secondary structure[END_REF] .

Therefore, great challenges still remain to transform hydrophilic polymer-based hydrogels to equivalent bioadhesive sponges.

One strategy to enhance the integration of cells and tissues into scaffolds, involves the use of adhesion molecules by functionalization of the scaffold surfaces.

Biomaterials may be adapted with small recognition motifs that mimic the ECM to endorse cell binding. Therein, incorporating adhesion molecules into polymers backbone is an excellent methodology for growing tissue integration of neural biomaterials [START_REF] Rao | Adhesion molecule-modified biomaterials for neural tissue engineering[END_REF] . It has been already reported that grafting short motifs to hydrogels improves neural cell adhesion, while conserving a matrix with similar properties to those found in vivo [START_REF] Lévesque | Synthesis of cell-adhesive dextran hydrogels and macroporous scaffolds[END_REF][START_REF] Huang | Functional improvement and neurogenesis after collagen-GAG matrix implantation into surgical brain trauma[END_REF][START_REF] Bellamkonda | Laminin oligopeptide derivatized agarose gels allow three-dimensional neurite extension in vitro[END_REF] . In this present work, supramolecular dimer 2-ureido-4(1H)pyrimidone (UPy) was used as a source of active functional groups into agarosederivative chains, resulting in an improvement in the scaffold physical properties and greater ability in neural cell adhesion on surfaces. UPy-dimer is a self-complementary quadruple hydrogen bonding motif based on donor-acceptor array, obtaining a molecule with four hydrogen-bonding sites and providing homo-complementary sequences [START_REF] Hirschberg | Helical Supramolecular Aggregates Based on Ureidopyrimidinone Quadruple Hydrogen Bonding[END_REF] .

Recent articles have shown that supramolecular materials based on UPy-dimers quadrupole-hydrogen bonds are signally appropriate for yielding such bioactive scaffolds owing to their low-temperature processability, adhesive capacity, biocompatible behavior, and favorable degradation [START_REF] Dankers | A modular and supramolecular approach to bioactive scaffolds for tissue engineering[END_REF][START_REF] Dankers | Chemical and biological properties of supramolecular polymer systems based on oligocaprolactones[END_REF][START_REF] Guan | Modular Domain Structure: A Biomimetic Strategy for Advanced Polymeric Materials[END_REF][START_REF] Ren | Facile Preparation of Linear Polyurethane from UPy-Capped Poly(dl-Lactic Acid) Macrodiol[END_REF][START_REF] Liu | Quadruple hydrogen bonds and thermo-triggered hydrophobic interactions generate dynamic hydrogels to modulate transplanted cell retention[END_REF] . Particularly, the functionalization of scaffolds surfaces with UPy-dimers can promote rapid communication with integrins through physical adsorption by intermediate proteins and other biological molecules favoring cells signal transduction [START_REF] Liu | Quadruple hydrogen bonds and thermo-triggered hydrophobic interactions generate dynamic hydrogels to modulate transplanted cell retention[END_REF] . Thereby, the dynamic nature of the supramolecular hydrogen bonds agrees with a modular tactic to gaining control over cellular performance and activity both in vitro and in vivo.

The interactions via supramolecular hydrogen bonds have attracted attention in science biomaterials due their enormous impact on our daily life. The pairing of nucleic acid strands in DNA is possibly the most sophisticated example of supramolecular interactions in nature, as it is based in cooperative multiple hydrogen bonding between bases and hydrophobic interactions. Likewise UPy, the combination of auto-associative systems, as -NH 2 , -O, -H, and -N form dynamic dimers. Faced with this phenomenon, UPy-dimers can also be used as a new alternative 'crosslinker' for polysaccharides chains to adjust the microstructure of the sponge. The occurrence of dimers junction zones from the hydrogen bonds and hydrophobic interactions between polymer chains renders the material softer and less brittle. The simplest methodology to creating a surface-based supramolecular structure in sponges is to use UPy-dimers in agarose backbone capable of creating an extended hydrogen bond network without the need of adding any additional agents. The hypothesis is that the mildly different chemical structures of the agarose linked to UPy will result in a substantial difference in the sponge microarchitecture providing the same rapid gelation; this difference will then alter the behavior of neural stem cells seeded in the scaffolds. We report here a structurally new type of sponge-like scaffold, a supramolecular sponge, derived from homo-complimentary hydrogen bonding. This is a modular method based on the supramolecular dimers to form sponges-like scaffolds with tailorable properties to help adhesion and cell growth in scaffolds.

Experimental section

Materials

Agarose for molecular biology (low EEO), 2-Amino-4-hydroxy-6methylpyrimidine, 1,6-Diisocyanatohexane 98 %, Pentane, Pyridine, N,N-Dimethylformamide -anhydrous, 99.8 % were purchased SIGMA-Aldrich.

Synthesis

2-(6-isocyanatohexylaminocarbonylamino)-6-methyl-4(1H)-pyrimidinone (UPy-NCO):

The synthesis of 2-(6-isocyanatohexylaminocarbonylamino)-6-methyl-4 (1H)pyrimidinone (UPy-NCO) was adapted from Meijer et al [START_REF] Folmer | Supramolecular Polymer Materials: Chain Extension of Telechelic Polymers Using a Reactive Hydrogen-Bonding Synthon[END_REF] . A mixture of 2-amino-4-hydroxy-6-methylpyrimidine (5 g, 0.03995 mol) in excess of 1,6-hexanediisocyanate (50 g, 0.2972 mols) and pyridine (3 g, 3.06 mL) was agitated during 16 hours at 100 °C under argon atmosphere. A white solid was obtained after pyridine removal and excess diisocyanate by filtration in a sintered-glass filter and then a succession of washes with pentane (500 mL). The product obtained was then dried at 50 ° C to remove any trace of water (in a Schlenk tube under argon). A yield of 95.5 % was determined.

General procedure for the synthesis of Agarose-UPy

The general synthesis of agarose backbone functionalization with UPy (AGA-Br, AGA-N 3 , AGA-NH 2 , and AGA-UPy) is detailed in the in the Supporting Information.

Preparation of sponge-like scaffolds with alternating degrees of supramolecular cross-linking

For the preparation of supramolecular crosslinked gels, 0.25 g of different ratios between neat agarose and synthetized AGA-UPy were dispersed in 5.0 mL deionized water and stirred at 90 °C until a fully dispersion was obtained. To obtain a lower and higher degree of supramolecular cross-linking, the 90:10 and 50:50 ratios of agarose and synthetized AGA-UPy (AGA:AGA-UPy) were chosen for this study. For better analysis of the results, neat agarose hydrogel was also prepared and characterized. The gelation of the samples was confirmed by the standard inversion test. To prepare sponges, the mixtures with AGA-UPy and neat agarose were poured into PP molds at -20 °C during 24 h, and subsequently freeze-dried to remove ice by sublimation.

Characterizations

The FTIR of 6-methyl-isocytosine and 2-(6-isocyanatohexylaminocarbonylamino)-6-methyl-4(1H)-pyrimidinone were finely ground and then mixed with potassium (KBr) in order to form pellets by vacuum pressure. The spectra were collected at a resolution of 32 scans and 4 cm -1 with Nicolet iS10 thermo scientific spectrometer in transmission mode from 4000 -650 cm -1 . The Small-angle neutron scattering (SANS ) measurements were performed on the small-angle spectrometer PA20 spectrometer of Leon Brillouin Laboratory (LLB, Saclay, France). The three configurations are defined by a constant wavelength λ= 6 Å, three sample-to-detector distances (2, 8, and 19 m). These setups enable to cover a total q-range from 2.10 -3 to 0.3 Å -1 . Hydrogels were placed into standart Hellma cells in a sample changer and measured under ambient conditions. The 2D patterns were reduced to 1D spectra, I(q) versus q, after a radial averaging around the center of the scattered beam. Neutron beam transmission, standard corrections by sample thickness, electronic background, empty beam signal subtraction, detector efficiency, and subtraction of incoherent scattering were applied to get the scattered intensities on an absolute scale (cm -1 ).

To measure the water retention capacity, the sponges were placed in water at 37 °C overnight, then the excessive surface water was removed by gently tapping the sponges on a paper towel and W wet was recorded. Sponges were dried at 60 ºC overnight, and the W dry was measured. The succeeding equation was used to calculate the percent water holding of the sponges: (Equation 2)

𝑊𝑅 (%) = 𝑊 𝑤𝑒𝑡 -𝑊 𝑑𝑟𝑦 𝑊 𝑑𝑟𝑦 𝑥 100
Scanning Electron Microscopy (SEM Philips XL30) was used to detect the surface changes in sponges induced by UPy-dimers addition.

Neural stem cell culture

Neural Stem Cells:

Neural Stem Cells (NSCs) were previously isolated in our laboratory from monkey embryonic stem cells (ESCs) [START_REF] Wianny | Derivation and Cloning of a Novel Rhesus Embryonic Stem Cell Line Stably Expressing Tau-Green Fluorescent Protein[END_REF] . Concisely, monkey ESCs were cultured to confluency during 15 days with daily medium change. Neuroepithelial-like cells spontaneously emerging in culture were designated manually and transferred into gelatin coated dishes (0.1 %; Sigma), and cultured in "NSC medium" (N2 supplement, DMEM/F12, 1 % nonessential amino acids, 2mM L-glutamine, 0.1mM β-mercaptoethanol (Invitrogen), with 20 ng/ml FGF2 (Millipore) and 20 ng/ml EGF (Millipore). NSCs used in this study stably express the TAU-green fluorescent fusion protein (TAU-GFP), which binds the GFP to microtubules. For amplification, NSCs were trypsinized (trypsin 0,025 %, EDTA, 0,1 g/L) (Invitrogen). Trypsin was inhibited by addition of Trypsin Inhibitor (Invitrogen) and cells were resuspended in NSC medium.

Cell loading:

Sponge-like scaffolds were UV treated for 30 minutes, and 2-mm thick sections were placed into 24-well dishes. NSCs were harvested and resuspended at a density of 2 10 6 NSCs/ml in NSC medium. An aliquot of 50 L was added on top of each spongelike sample. After 2 mins incubation, another 50 L aliquot was added (2.10 5 NSCs total). Sponge-like scaffolds were let to swell for 5 mins in the incubator at 37 °C and 900 l of NSC medium was added. The loaded samples were incubated at 37 °C, 5 % CO 2 , 5 % O 2 , for 3 days, replacing medium every other day.

Immunofluorescent stainings

Sponge-like scaffolds were fixed by immersion in 2 % paraformaldehyde in cold phosphate-buffer at 4 °C for 30 min, and embedded in 3 % low gelling agarose (Sigma) in HBSS (Life Technologies) supplemented with glucose 18 %, MgSO 4 and CaCl 2 (Sigma) at 37 °C. 100 μm thick slices were prepared with a vibratome (Leica VT1000S). Slices were permeabilized with Triton X-100 (0.1 % in Tris-buffered saline (TBS).

Aspecific binding was blocked by incubation in normal donkey serum (10% in TBS) (Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 20 min at room temperature (RT). Slices were incubated overnight at 4 °C with anti-GFP (1/800, A10262, Life Technologies) diluted in antibody diluent (Dako, Glostrup, Denmark).

After three rinses in TBS, they were incubated with affinity-purified donkey anti-Chicken conjugated to Alexa488 (Life Technologies) for 1 h at RT. To stain F-actin, slices were incubated with Phalloidin-Alexa Fluor 488 (Life Technologies) diluted in antibody diluent, for 45 mins at RT. Nuclei were counterstained with 4', 6-diamidino-2phenylindole (DAPI) (D103; 1/5000; Life Technologies) for 3 mins. Slices were mounted on slides in Fluoromount G (Clinisciences). Images were acquired by confocal microscopy (Leica, TCS SP at 10X, 20X, and 40X objectives). Z-stack images were acquired at 5 to 10 μm intervals.

Results and Discussion

Synthesis and characterization of the associative unit and precursor comonomer of a supramolecular foams network

Synthesis and characterization of 2-(6-isocyanatohexylaminocarbonylamino)-6-methyl-4(1H)-pyrimidinone (UPy-NCO)

Ureidopyrimidinone dimers (UPy) are formed by strong quadruple hydrogen bonds capable of physically crosslinking a material network and at the same time, these physical links can be reversibly broken and reformed. This principle was chosen by Meijer et al. who have synthesized isocytosine derivatives able of associating in the same way as the pyrimidines and purine sequences, i.e. donor-acceptor homocomplementary interaction (DDAA) [START_REF] Beijer | Strong Dimerization of Ureidopyrimidones via Quadruple Hydrogen Bonding[END_REF] . Isocytosine has an amine function, whose reaction with an isocyanate forms a new hydrogen bond by the creation of a urea function. This gives the site four hydrogen bonds, whose spatial arrangement is favored by the presence of an intramolecular hydrogen bond. The UPy donor-acceptor interaction (DA) between neutral electron-deficient and electron-rich dimers can be fruitfully used to the self-assembly of various interconnected supramolecular materials.

The product which we will be named in this paper as UPy-NCO is derived from the condensation reaction of isocytosine in hexane diisocyanate (as shown in Figure S1) 20, 21 . The excess of diisocyanate (dilution effect) combined with a substitution effect (decreased reactivity of the second isocyanate function) promoted the formation of monofunctional species. On the basis of 1 H NMR analysis (Figure S1), the UPy moieties composition was confirmed showing a good agreement with the proposed structure. The attendance of peaks at δ= 10.18, 11.85, and 13.01 ppm which were used to ascribe the NH proton signals, confirming the 4[1H]-pyrimidinone dimer geometry in solution [START_REF] Beijer | Strong Dimerization of Ureidopyrimidones via Quadruple Hydrogen Bonding[END_REF] .

The product functional groups have characteristic absorbance peaks at perfectly identifiable wavelengths as shown in Figure 1. After synthesis, the NH 2 peak at ~3000 cm -1 disappeared in the spectrum of UPy-NCO, and new peaks emerged at ~3400-3200 cm-1, are assigned to the NH vibrations (amide band I, and II) [START_REF] Folmer | Supramolecular Polymer Materials: Chain Extension of Telechelic Polymers Using a Reactive Hydrogen-Bonding Synthon[END_REF][START_REF] Beijer | Strong Dimerization of Ureidopyrimidones via Quadruple Hydrogen Bonding[END_REF] . Noteworthy, a band at 2283 cm -1 reveals the presence of the isocyanate function [START_REF] Jangizehi | Supramolecular Polymer Based on Poly (Ethylene-co-Vinyl Alcohol)-g-Ureidopyrimidinone: Self-Assembly and Thermo-Reversibility[END_REF] . The analysis of the carbonyl group region in UPy-NCO reveals the presence of two 1700 cm -1 (amide I) absorption bands originating from the formation of the urea function and to the carbonyl isocytosine at1666 cm -1 . Finally, the presence of the two 1600 and 1522 cm -1 bands characteristic of the amide II vibration modes indicates the presence of a N, N' bisubstituted urea function [START_REF] Jangizehi | Supramolecular Polymer Based on Poly (Ethylene-co-Vinyl Alcohol)-g-Ureidopyrimidinone: Self-Assembly and Thermo-Reversibility[END_REF] .

Supramolecular Agarose Functionalization: Agarose-UPy

Agarose polysaccharide was selected as a cryogel base for scaffolds building due to its ability to immobilize water molecules and its inertness. Polysaccharides with functionalized groups, e.g., SH, NH 2 and COOH, have many medical, biological, and biotechnological applications. Amino-polysaccharides can be used as synthetic tools owing to their structural specificity to isolate carbohydrate-binding proteins and biomolecules [START_REF] Kondaveeti | Modification of agarose: 6-Aminoagarose mediated syntheses of fluorogenic pyridine carboxylic acid amides[END_REF] . The high regio-selective introduction of the amino group in agarose and their derivatives is extremely important to coupling reactions. Here, we present a facile synthetic approach using 'click chemistry' to produce functional 6-amino-agarose (AGA-NH 2 ) via 6-bromo-deoxyagarose (AGA-Br). In this synthetic regio-selective approach, unmodified agarose was first brominated with carbon tetrabromide and triphenylphosphine; and then azidated with sodium azide to afford 6-azido-6deoxyagarose (AGA-N 3 ). The synthetic route for 6-amino-6-agarose from (1,3) linked To further confirm the coupling reaction to form UPy-grafted agarose, the solid state [START_REF] Guan | Modular Domain Structure: A Biomimetic Strategy for Advanced Polymeric Materials[END_REF] C NMR spectra of products were examined. As shown in Figure S4, the [START_REF] Guan | Modular Domain Structure: A Biomimetic Strategy for Advanced Polymeric Materials[END_REF] C NMR solid-state spectra of the unmodified agarose as well as its peak assignments was confirmed by the literature [START_REF] Wang | Oriented covalent immobilization of recombinant protein A on the glutaraldehyde activated agarose support[END_REF][START_REF] Meena | Preparation, characterization and benchmarking of agarose from Gracilaria dura of Indian waters[END_REF][START_REF] Zumbrink | Analysis of affinity supports by 13C CP/MAS NMR spectroscopy: Application to carbonyldiimidazole-and novel tresyl chloride-synthesized agarose and silica gels[END_REF] . The differences observed between unmodified agarose and AGA-UPy samples were perceptible in CP-MAS [START_REF] Guan | Modular Domain Structure: A Biomimetic Strategy for Advanced Polymeric Materials[END_REF] C NMR spectra. For UPy signals, we can see peaks in the range of 50-30 ppm corresponding to the hydrocarbons present in the isocyanate structure; but we can mainly focus on the peak corresponding to the reactive isocyanate group present at 121 ppm [START_REF] Hosseinpourpia | Modification of Pea Starch and Dextrin Polymers with Isocyanate Functional Groups[END_REF][START_REF] Despres | 13C NMR investigation of the reaction in water of UF resins with blocked emulsifiable isocyanates[END_REF] . Isocyanates are very reactive compounds which react almost spontaneously with amino groups to form urea. In the AGA-UPy spectrum, the absence of the isocyanate peak is clear, indicating the subsequent coupling reaction between AGA-NH 2 and UPy-NCO, yielding the AGA-UPy product. Certainly, this is caused by the urea linkage between agarose and UPymoieties, as shown in scheme 1.

D-β-galactose (1,4) linked-α-
X-Ray photoelectron spectroscopy (XPS) is a highly sensitive tool which can provide data about the atomic composition and speciation of each atom on grafted The high-resolution XPS spectra of N1s (Figure 3) confirms amino-agarose functionalization and coupling reaction between NH 2 and isocyanate groups of UPy-NCO moieties. Evidently, since unmodified agarose is composed solely of carbon and oxygen, no peak in N (1s) spectra are detectable. Instead, the spectra of modified agarose by NH 2 and afterwards by UPy hihlights three peaks at 399.2 (-NH 2 ), 400.3 (-NH), and 401.7 eV (NH 3 + ), confirming the modification of agarose [START_REF] Ravi | EDTA-functionalized KCC-1 and KIT-6 mesoporous silicas for Nd3+ ion recovery from aqueous solutions[END_REF] . The synthesized AGA-NH 2 are strongly dependent on the protonation state of the amine, and the XPS spectrum highlighted two different types of nitrogen corresponding to the presence of deprotonated primary amine (-NH 2 , 399.5 eV) as well as the protonated ammonium (-NH 3 + , 401.5 eV). These same results have been described in the literature for systems such as chitosan or glucosamine. For example, Wu et al. have highlighted by XPS measurements the formation of the deprotonated and protonated forms of the glucosamine at 399.5 eV and 401.5 eV, respectively [START_REF] Wu | Electrical Writing onto a Dynamically Responsive Polysaccharide Medium: Patterning Structure and Function into a Reconfigurable Medium[END_REF][START_REF] Liu | Chitosan-Coated Electrodes for Bimodal Sensing: Selective Post-Electrode Film Reaction for Spectroelectrochemical Analysis[END_REF] . The estimated amine groups substitution using XPS can be calculated by Equation 1 (experimental section), and showed that NH2 functionalization after synthesis is estimated at 30%. After the coupling reaction, the peak attributed to the NH 2 is no longer observed and instead, two peaks appear. The peak at 400.1 eV is attributed to secondary amide resulting from the coupling reaction (urea formation) between the isocyanate function of the UPy-NCO moieties and the primary amine at the agarose chain. Finally, N (s1) peak at 401.0 eV corresponding to the primary amine of the UPy-moieties [START_REF] Gauche | Colloidal Behavior of Cellulose Nanocrystals Grafted with Poly(2-alkyl-2-oxazoline)s[END_REF] . In conclusion, the modification of the agarose with amine groups was achieved and supramolecular AGA-UPy was successfully synthesized.

Thermal Properties of AGA-UPy

For a better AGA-UPy covalent linkage analysis, TGA was used to highlight the decomposition mechanisms of UPy-NCO and modified-agarose, i.e., AGA-NH 2 , AGA-UPy, and AGA-NH 2 /UPy physical mixture. Thus, the TGA thermograms was presented in Figure 4 and the decomposition temperatures of all the systems are reported in Table S2. Three distinct degradation steps were observed for ureidopyrimidinone compound in Figure 4-A. The first degradation occurs between 170 °C and 268 °C, representing 13 wt % and corresponds to the decomposition of the hexamethylene diisocyanate. Then, different degradation mechanisms take place between 210 °C and 400 °C corresponding to degradation of 59 wt % of UPy. In fact, it is well-known that the increase of temperature may result in the formation of isocyanurate functions from the isocyanate functions leading to the vaporization of these compounds [START_REF] Lapprand | Reactivity of isocyanates with urethanes: Conditions for allophanate formation[END_REF][START_REF] Nambu | Synthesis of novel aromatic isocyanurates by the fluoridecatalyzed selective trimerization of isocyanates[END_REF][START_REF] Duong | N-Heterocyclic Carbenes as Highly Efficient Catalysts for the Cyclotrimerization of Isocyanates[END_REF] . The thermal behavior of UPy-NCO, AGA-NH 2 /UPy-NCO physical mixture, and agarose functionalized with ureidopyrimidinone (AGA-UPy) were also investigated by DSC. As can be seen in Figure 4-B, a single endothermic peak around 83 °C with an enthalpy of 2.3 J/g was observed for the synthesized monofunctional ureidopyrimidine (UPy-NCO). This phenomenon highlights a transition from mesophase to the isotropic state. UPy-dimers are capable of establishing H-bond in an orientational order forming a line joining the centers of nearest-neighbor molecules resulting in a nematic mesophase [START_REF] Andrienko | Introduction to liquid crystals[END_REF] . In fact, many authors have widely reported the appearance of this endothermic peak by DSC for mono-and bifunctional UPy-NCO [START_REF] Hirschberg | Helical Supramolecular Aggregates Based on Ureidopyrimidinone Quadruple Hydrogen Bonding[END_REF][START_REF] Folmer | Supramolecular Polymer Materials: Chain Extension of Telechelic Polymers Using a Reactive Hydrogen-Bonding Synthon[END_REF][START_REF] Delgado | Combining block copolymers and hydrogen bonding for poly(lactide) toughening[END_REF] . For example, Hirschberg et al.

have shown that supramolecular aggregates based on UPy quadruple H-bonding built homeotropic monodomains in the liquid-crystalline state. As a consequence of this interaction, they observed a phase transition between 90 °C and 242 °C due to the highly viscous isotropic melt of UPy-dimers [START_REF] Hirschberg | Helical Supramolecular Aggregates Based on Ureidopyrimidinone Quadruple Hydrogen Bonding[END_REF] .

Regarding AGA-NH 2 , AGA-UPy and AGA-NH 2 /UPy physical mixture, endothermic peaks at 112 °C, 92 °C, and 57 °C were obtained, respectively. In addition, differences in the peaks area were observed as a reflex of their different ability to retain water as well as the strength of polymer-water interaction. Polysaccharides are wellknown to display excellent ability to be easily hydrated due to their strong interactions with water molecules [START_REF] Watase | Differential scanning calorimetry, rheology, x-ray, and NMR of very concentrated agarose gels[END_REF][START_REF] Watase | Rheology, DSC and Volume or Weight Change Induced by Immersion in Solvents for Agarose and Kappa-Carrageenan Gels[END_REF] . Despite the water evaporation during the rise in temperature, the enthalpies and temperature corresponding to the endothermic peaks should undergo changes as a consequence of the functionalization of agarose [START_REF] Kittur | Characterization of chitin, chitosan and their carboxymethyl derivatives by differential scanning calorimetry[END_REF] . Thus, large and similar enthalpies are observed for AGA-NH 2 (340 J/g) and AGA-NH 2 /UPy-NCO (222 J/g) provide that the amount of mass introduced at the beginning is taken into account. In both cases, these results are consistent with the literature where the presence of amine (-NH 2 ) and quaternary ammonium (-NH 3 + ) in the polysaccharide structure induced an increase in the amount of bound water [START_REF] Demarger-Andre | Chitosan carboxylic acid salts in solution and in the solid state[END_REF] .

Otherwise, for AGA-UPy a significant decrease in enthalpy (94 J/g) and a shift to lower temperatures (57 °C) may also be observed indicating a disappearance of amine functional groups. This event can be explained by the formation of urea functions issue from the reaction between the isocyanate functions of UPy-NCO and the amine functions of agarose. In addition, the decrease in the exothermic peak temperature (57 °C instead of 112 °C) confirms the agarose hydrophobization characterized by the disappearance of the presence of bound water. In conclusion, the study of different functionalized agaroses by their thermal behavior shows an increase in the hydrophobicity of the agarose due to its functionalization by the monofunctional ureidopyridimidinone (UPy-NCO) compound.

Effect of Crosslinking Dimerization

Multi-functional materials displaying supramolecular interactions are wellknown as dynamic materials and are inspired by biological systems due to their easy adaptability. Increasingly, different classes of interactions have been investigated to design materials based on crosslinked physical interactions such as hydrogen bonds [START_REF] Braccini | Chain-Chain Interactions for Methyl Polygalacturonate: Models for High Methyl-Esterified Pectin Junction Zones[END_REF] , ionic [START_REF] Chung | Preparation of alginate/galactosylated chitosan scaffold for hepatocyte attachment[END_REF] , host-guest recognition [START_REF] Charlot | Specific Interactions in Model Charged Polysaccharide Systems[END_REF] . Recently, H-bonding as well as weak hydrophobic interactions were shown to improve the stability of physically cross-linked hydrogels formed by telechelic PEG-hydroxylase enzymes [START_REF] Nadgorny | 3D-printing of dynamic self-healing cryogels with tuneable properties[END_REF][START_REF] Cheng | Supramolecular Polymer Hydrogels for Drug-Induced Tissue Regeneration[END_REF] . On the basis of these perceptions, we synthesized AGA-UPy which was mixed with unmodified agarose at different ratios to reduce the brittleness of this gel from non-covalent agarose backbone interactions.

Agarose was selected owing to its excellent water solubility, excluding the requirement for organic solvents. More importantly, its fast rate of gelation ensures the different modeling of these non-cytotoxic 3D hydrogels and efficient encapsulation of active principles, growth factors, cells and so on. On the other hand, the human and animal cells can not adhere to the polysaccharide due to the formation of a rigid, hydrophilic, and inert hydrogel. The introduction of hydrophobic-and H-bonding interactions into polysaccharide hydrogels is expected to reproduce the natural environment of cells, which provide gels the capability to flow [START_REF] Mihajlovic | Tough Supramolecular Hydrogel Based on Strong Hydrophobic Interactions in a Multiblock Segmented Copolymer[END_REF] . In light of designing materials that are simple to prepare and process for cell scaffolds, we were inspired by H-bonds of UPy to design a hydrogel and subsequently supramolecular sponges-like scaffold capable of absorbing and fixing biological fluids such as cells, growth factors and culture medium. The hydrogels were prepared from a hot aqueous solution and for all compositions the gelatinization was verified by visual observation arisen in a few seconds at 37 o C. For all samples, under oscillating shear for all samples (Figure 5-A), the storage modulus (G') of the hydrogels was found to be far higher than the loss modulus (G''), indicating solid-like behavior. Frequency sweep experiments revealed that hydrogel with 50 % of AGA-UPy (50:50) exhibits frequency dependent behavior with nonlinear increase of G' and G'', which is a characteristic of dynamic interaction in gels [START_REF] Nadgorny | 3D-printing of dynamic self-healing cryogels with tuneable properties[END_REF] .

Likewise others classical supramolecular counterparts, a non-linear behavior is observed for the sample with 50 % AGA-UPy in Figure 5-B. A characteristic of supramolecular networks when oscillation frequencies increase is that their dynamics is driven by different timescales; just as relaxation of supramolecular interactions, followed by organization/association of supramolecular interactions, and then breakage of supramolecular associations or inter-chain crosslink [START_REF] Seiffert | Physical chemistry of supramolecular polymer networks[END_REF] . The networks with higher amounts of UPy (50:50) show firstly a chain relaxation, a plateau at frequencies between 2 and 20 rad/s, then bonds-dissociation upon increasing frequency. This association effect occurs due to the organization of the chains owing to force application, producing a transient state. Afterwards, a breakage of supramolecular bonds occurs and the gel moves to a viscous-fluid state. Similarly, Li et al. presented that macroscopic viscosity and shear behavior of a low molecular weight dye in polymer melts are oriented by association and dissociation of H-bonding motif in UPy functional group [START_REF] Li | Thermally activated diffusion in reversibly associating polymers[END_REF] . Those authors prepared reversibly associating copolymers containing UPymotif and reported that stress relaxation is therefore dominated by the rate of UPy association-dissociation.

Figure 5-C displays the strain dependence of the elastic and loss modulus for hydrogels, measured at an oscillation frequency f = 15 Hz. At the lowest strains the response is solid-like, with a G' value that is greater than the G'', while at the highest frequencies the loss modulus dominates the response for samples with AGA-UPy (50:50 and 90:10) indicating viscous-like behavior. A striking feature of supramolecular networks is that as the strain increases at fixed ω, the loss modulus initially upsurges to a peak before finally decreasing at the largest strains indicating that dissipative processes occur [START_REF] Seiffert | Physical chemistry of supramolecular polymer networks[END_REF] . This behavior may be elucidated as a consequence of a slow structural H-bonding relaxation process that is predictable in dimers-interaction at very low strain deformation. Instead, unmodified agarose hydrogel displays no liquid-like characteristics showing a brittle behavior, breaking a rheological yield strain at 1 %.

However, the hydrogels with 10 % (90:10) and 50 % (50:50) retain an overall solid-like behavior, with G' superior than G" and only feebly dependency on γ (%). Hereupon a few percent of applied strain amplitude, G' decrease significantly and G'' grows until G' = G'' at a 'fluidization' strain [START_REF] Knowlton | A microscopic view of the yielding transition in concentrated emulsions[END_REF] . Beyond the critical strain, both moduli drop with strain, but the G'' dominates indicating a fluid-like behavior. The critical strain crossover for samples with 10 % UPy (90:10) is γ= 6 %, while for hydrogels with 50 % of AGA-UPy (50:50) the fluidization crossover occurs at γ=14 % due to the greater dimer interaction.

Hydrogels with H-bond-crosslinked supramolecular networks (UPy) showed the ability to switch between solid-like and liquid-like states, belonging to the family of shear-thinning hydrogels [START_REF] Glassman | Reinforcement of Shear Thinning Protein Hydrogels by Responsive Block Copolymer Self-Assembly[END_REF] . The shear-thinning is attributed to networks with rapidly associating-dissociating disentanglements or fast decrosslinking kinetics [START_REF] Seiffert | Physical chemistry of supramolecular polymer networks[END_REF][START_REF] Loontjens | Novel supramolecular polymer networks based on melamine-and imide-containing oligomers[END_REF] . When the linkage chain relaxation arises quicker than the reconnection of supramolecular interactions, shear-thinning appears due to the transformation of intramolecular to intermolecular bonds, as shown in Figure 5-D. Similar results have been achieved by Lehn et al. for hydrogen-bonded supramolecular networks associated with an intense decrease of the viscosity of semidilute solutions [START_REF] Buhler | Dynamical properties of semidilute solutions of hydrogen-bonded supramolecular polymers[END_REF] . In the opposite case, hydrogels with only unmodified agarose showed shear-thickening feature, where intertwined helices chains become more stressed when submitted to mechanical stress. On the macroscopic scale, the effect of shear-enhancement in shear-thickening hydrogels is a fracture resulting in the brittle and stiff materials. Accordingly, the used supramolecular dimers with crosslinked domains was seen to increase the gel strain capacity to high deformation, leading to gels with superior energy dissipation and enhanced resistance to shear deformation.

To further confirm the supramolecular dimers interactions, SANS measurements were performed and the following functional form is fitted to the data (Figure 6). We quantitatively evaluated the SANS patterns by Beaucage model, where the unified equation with two terms is used in order to describe a complex morphology in a wide range of I(q) [START_REF] Beaucage | Small-Angle Scattering from Polymeric Mass Fractals of Arbitrary Mass-Fractal Dimension[END_REF] . The correlation lengths (ξ), radius of gyration (R g ) and fractal dimension (D f ) in hydrogels were determined by equation 3. The first term of equation is an Ornstein-Zernike function that describes the polymer concentration fluctuation [START_REF] Bin Imran | Extremely stretchable thermosensitive hydrogels by introducing slide-ring polyrotaxane cross-linkers and ionic groups into the polymer network[END_REF] .

The second term is described by Guinier's law, which describes excess scattering from a spatial inhomogeneity [START_REF] Shibayama | Small-angle neutron scattering on polymer gels: phase behavior, inhomogeneities and deformation mechanisms[END_REF] .

(Equation 3)

𝐼(𝑞) = 1 1 + 𝑞 2 𝜉 2 + exp ( -𝑞 2 𝑅 𝑔 2 3 )
Because agarose chains are able to form mechanically robust hydrogels, AGA-UPy hydrogels scattering contributions show a microphase separation behavior from the UPy-dimers, driven by hydrogen-bonds and hydrophobic interactions. The introduction of physical crosslinkers intrinsically leads to regions which are less accessible to water.

According to Mihajlovic and co-workers, the physical crosslinker junctions for the UPy-dimers produce spherical inhomogeneities as nano or microdomains [START_REF] Mihajlovic | Tough Supramolecular Hydrogel Based on Strong Hydrophobic Interactions in a Multiblock Segmented Copolymer[END_REF] . These dimers act as crosslinking points that are bridged and surrounded by agarose chains forming supramolecular aggregates, as shown schematically in Figure 6. Mainly in hydrogels with 50 % AGA-UPy (50:50), due to a great dimers fraction used, the correlation peak is much more pronounced because the phase separation is more distinct. Thereby, we managed to study morphology of the aggregates as well as their correlation distances. The influence of dimers on the formation of supramolecular aggregates can be illustrated by the ξ values. For unmodified agarose, the ξ value is 12.6 nm, while the incorporation of UPy-dimers doubled it. The increased of correlation length in 10 % AGA-UPy (90:10) arises from the interaction between dimers and can be associated to the medium spacing between the hydrophobic domains or dimer aggregates, which reduces in 50 % AGA-UPy (50:50) and becomes more defined upon an increase in dimer volume fraction (e.g. ξ = 29.6 nm for AGA:AGA-UPy 90:10 versus ξ = 27.2 nm for AGA:AGA-UPy 50:50). The formation of these supramolecular aggregates has also the consequence of increasing the radius of gyration, we can observe values of Rg = 44.6 nm, 114.4 nm, and 127.00 nm for unmodified-AGA, AGA:AGA-UPy 90:10, and AGA:AGA-UPy 50:50, respectively. This Rg variation agrees with the results obtained in rheology and the scheme shown in Figure 5-D.

The fractal dimension (D f ) in hydrogels may be detected for either surface or mass fractals. Experimentally, information illustrating the aggregate sizes and structures of the hydrogels by SANS may be acquired by quantifying the density gradient of the hydrogel [START_REF] Singh | Internal structures of agargelatin co-hydrogels by light scattering, small-angle neutron scattering and rheology[END_REF] . The Guinier equation provides the fractal dimension of the network structure, D f values ranging between 2.0 and 2.6 inferring the formation of dense network domains [START_REF] Newbloom | Mesoscale Morphology and Charge Transport in Colloidal Networks of Poly(3-hexylthiophene)[END_REF] . This behavior is credited to the interface between the aggregates.

As expected, D f decreases with increasing H-bonding interaction as the dimers volume fraction in the gel is greater. To sum up, SANS and rheology measurements revealed the aggregation into connected structures from dimers interactions.

Sponges-like scaffold formation and their architecture

It is common understanding that a three-dimensional setting with a suitable microstructure is crucial to promote pre-vascularization for oxygen exchange driving tissue regeneration, as well as to protect cells from mechanical stress. Besides, the ability to absorb and retain biological fluids in sponge-like scaffolds leads to material properties which are comparable to some physic-chemical properties of soft tissue. Hydrogels obtained by the sol-gel process are probable candidates to be turned into sponges by freeze drying in order to promote a vascularized cell support. These hydrogels should be able to avert the pore collapse phenomenon retaining the porous texture of the wet material [START_REF] Zhao | Biopolymer Aerogels and Foams: Chemistry, Properties, and Applications[END_REF] . However, the development of many hydrophilic polymerbased hydrogels, and subsequently sponges, for cell growth and tissue regeneration, sometimes is not achievable because of their poor cell attachment properties.

Taking this into account, we report a structurally new type of sponges, supramolecular sponges, derived from hydrogels formed by homo-complimentary hydrogen bonding. The UPy-dimers were used as an alternative supramolecular crosslinker to modify the microstructure and surface of the sponge. Hydrogels with AGA-UPy provide the same rate of rapid gelation of agarose hydrogels as scaffold, which allowed us to analyze the influence of microstructure on cell attachment within the three types of sponges: unmodified agarose, and sponges with 10 wt% (90:10) and 50 wt% (50:50) of synthesized AGA-UPy. The hypothesis was that these different amounts of supramolecular crosslinking would result in a great difference in sponge microarchitecture (Figure 7), hence this difference will alter the attachment of neural stem cells cultured in the scaffolds.

As mentioned in the previous section, the unmodified agarose and synthesized AGA-UPy conjugate were mixed; then hydrogels were rapidly generated within few seconds by simply cooling the solutions. All the cubical hydrogel samples were prepared inside plastic capillaries (with an internal diameter of 1 cm). The unique structure of sponge scaffold-like has been generated by removal of the liquid component of gel by freeze-drying. The fast-freezing kinetic leads to a greater nucleation rate of ice crystals and consequently to vast number of small crystals. In the conventional freezing method the freezing rate is slower favoring crystal growth, thereby a larger pore size is reached [START_REF] Martoïa | Cellulose nanofibril foams: Links between ice-templating conditions, microstructures and mechanical properties[END_REF] . In order to obtain a great porosity and firmer polysaccharide walls, we chose to produce sponges of agarose crosslinked with supramolecular UPy to compare with unmodified agarose by the slow freezing (24 h) and conventional freezing-dry methods (24 h). This method suggests the creation of ice crystals in the microscopic spatial sphere (Figure 7) and the formation of completely random polymer walls. Followed by freezing-drying process, the water elimination yields micro-sized pores, resulting in sponges-like materials. Macroscopically, all sponges show a three-dimensional (3D) marshmallow-like shape. However, we can observe that a more dense structure in aerogel with 10 % AGA-UPy is formed. The microstructure of highly porous supramolecular AGA-UPy sponges was investigated via transmission electron microscope tomography and SEM images (Figures S6). A series of images was then acquired, segmented, and reconstructed in order to create a three-dimensional (3D) model of the aerogel (Figure 8). X-ray microtomography confirmed the 3D structure, monolithic and porous structure. The stacks of two-dimensional XMT images from the top of sponges are shown in Figure 8. These images visibly show colorful walls corresponding to the AGA and AGA-UPy solid structure and black areas consistent to the air voids. Sponges-like materials displayed the anisotropic structures of the pores that may easily be seen when the sample is illustrated along different axes (XY-axis and XZ-axis).

The sponges show an sophisticated open cellular architecture of interconnected pores. In all samples, especially the sample with unmodified AGA, the polymer chains have been collapsed to form a sheet-like structure during the sublimation process.

Physical significance to determine the porosity in sponges is unclear because real pores may have different geometry. Conventional freezing process causes a random growth of ice crystals and, as a consequence, an irregularity in the pore structure. However, we found that a smaller porosity was formed in unmodified agarose sponges (87 % ± 0.44).

Observing the area around the pores, we can noticed that sponges prepared from unmodified agarose are formed of walls with different thicknesses ranging from 24 µm to 88 µm. Sponges with 10 % AGA-UPy supramolecular crosslinking possess thinner walls, thicknesses with a non-homogeneous size distribution, and a slight increase in porosity, 90 % of porosity (± 0.96). The pores for these sponges (90:10) are also anisotropic similar to unmodified sponges, but smaller. The structures are a highly interconnected network and the pores are heterogeneous in shape and size (watch the video on SI, 90_10). When the amount of AGA-UPy increases, the wall thickness of sponges became homogeneous and the sponges lie in mesoporous range. The presence of strong hydrogen bonding of UPy-dimers may cause greater interaction with water and a better distribution of the chains. Thereby, when the agarose sponges were reinforced with 50 % of supramolecular AGA-UPy a remarkably change in wall structure can be noticed. Most of wall thicknesses for this sponge (50:50) are in the range of 24 µm to 40 µm and the porosity increases to 92 % (±0.47). Indeed, the sponges obtained from agarose with 50 % AGA-UPy (50:50) did not show external volumetric shrinkage (watch the video on SI, 50_50). In unmodified agarose sponges, the wall is thickness and random because of the stiffer structure of the agarose hydrogels. Therefore, reinforcement by AGA-UPy resulted in a better distributed and flexible structure. Regardless the proportion of AGA-UPy used in the sponge production, the density and linear shrinkage for all sponges did not change significantly during the freeze-drying process. As mentioned above, unmodified agarose sponges are also porous and show low-density structure, but the major drawback is that these sponges are incompressible and extreme brittle. The mechanical strength of sponges-like scaffolds is a very important parameter for cell storage and transportation. Supramolecular crosslinking by UPy molecules can sideline these drawbacks improving the malleability, compressibility, and flexibility without compromising their original properties. The purpose of adding supramolecular dimers to the material is to enhance the intra and intermolecular interaction of the polysaccharide backbones. Therein, mechanical properties of sponges were evaluated by analysing their microstructure network. The sponges with AGA-UPy can bear compression strain of 45 % without permanent deformation or mechanical destruction (Figure S5). By contrast, strain of unmodified agarose sponge increased as stress increased; this caused greater internal damage and thus slower recovery. The stresses of the sponge were about 50 kPa at 45 % strain for samples with AGA-UPy and 5 kPa at 35 % strain for unmodified agarose in the first cycle (Figure S5). Such higher strain of crosslink supramolecular demonstrates the elastic nature of the scaffold and thereby supports its use to culture of neural stem cells (NSCs). The soft and elastic nature of the synthesized AGA-UPy sponges provides an appropriate physical property for neural tissue engineering.

Water uptake is another crucial factor for nutrient and metabolite transport in cell scaffolds. It is also crucial to determine the ability of the sponges to retain biological fluids and cells adhesion. To examine whether this has not been compromised due to the addition of UPy dimers, the water uptake properties of the supramolecular sponges were compared to those of unmodified sponges. In terms of physical properties, there was no significant change in sponges swelling using different UPy concentrations. Unmodified agarose sponges were found to maintain water up to thirty times their own weight. Moreover, there was no major difference in water retention for sponges after the addition of AGA-UPy (26 times). The higher UPy concentration (50:50) yielded lower water retention, which demonstrates their hydrophobicity (11 times). Despite their lower water retention capacity, sponges with 50 % AGA-UPy show a good balance between more hydrophobic interfaces (desirable for cell adhesion) and adsorption capacity of biological fluids, such as proteins, culture medium, cells and growth factors.

Supramolecular dimers effects on Neural Stem Cell Adhesion

Neural stem cells (NSCs) play a therapeutic role in neurodegenerative diseases and may contribute to functional recovery. Upon transplantation, NSCs substitute lost cells and throw molecules that stimulate the survival of the remaining local cells. Nonetheless, neural-based therapies still face several obstacles as the majority of cells die post-allogeneic transplantation [START_REF] Lins | Effect of polyvinylidene fluoride electrospun fiber orientation on neural stem cell differentiation[END_REF][START_REF] Delcroix | Adult cell therapy for brain neuronal damages and the role of tissue engineering[END_REF] . To circumvent this problem, earlier studies have been developed to yield good 3D substrates for neural tissue engineering, such as biodegradable, bioresorbable, and piezoelectric scaffolds [START_REF] Lins | Development of Bioresorbable Hydrophilic-Hydrophobic Electrospun Scaffolds for Neural Tissue Engineering[END_REF][START_REF] Lins | Effect of polyvinylidene fluoride electrospun fiber orientation on neural stem cell differentiation[END_REF] . Herein, 3D substrates with supramolecular UPy-dimers were assessed as support for NSCs. As described above, we have developed an efficient sponge-like scaffold capable of rapidly absorbing cells, growth factors, and culture medium into the supramolecular-crosslinked biomaterial.

These sponges after the immediate incorporation of cells and culture medium turn into an elegant shear-thinning hydrogels.

Upgrading cell adhesive potential of non-adhesive scaffold is an important goal in neural tissue engineering. It is well-known that neuronal cells interact with the substrate through adhesion molecules, wherein integrin receptors form a major class of proteins that mediates cell adhesion into extracellular matrix (ECM) [START_REF] Arregui | Characterization of neural cell adhesion sites: point contacts are the sites of interaction between integrins and the cytoskeleton in PC12 cells[END_REF] . Integrins receptors are transmembrane α-β heterodimers from members of the large α and β gene families intricate in the regulation of survival, proliferation, migration, and differentiation [START_REF] Arregui | Characterization of neural cell adhesion sites: point contacts are the sites of interaction between integrins and the cytoskeleton in PC12 cells[END_REF][START_REF] Hynes | Integrins: Versatility, modulation, and signaling in cell adhesion[END_REF][START_REF] Hall | Integrins are markers of human neural stem cells[END_REF] . In mammals, integrins are composed of 8 α and 8 β subunits that heterodimerize to form 24 unique receptors [START_REF] Clark | Integrins and signal transduction pathways: the road taken[END_REF][START_REF] Kuo | Chapter Three -Focal Adhesions Function as a Mechanosensor[END_REF] . In neural cells, these integrins receptor engagement and clustering is controlled by ligands from the ECM (or scaffold, in the case) leading to the formation of focal adhesions. Therefore, molecules of interest in scaffold can help create a better connection between integrin receptors and surfaces, which are able to promote actin filaments in cells [START_REF] Rao | Adhesion molecule-modified biomaterials for neural tissue engineering[END_REF][START_REF] Akiyama | Integrins in cell adhesion and signaling[END_REF] . Biomaterials can be modified with molecules-recognition motifs to enhance neural tissue integration and promote ECM production. Towards this end, the inclusion of supramolecular UPy-dimers into the sponges has the dual objective of improving the mechanical properties, while simultaneously incorporating adhesion segments. UPy-dimers represent here one class of adhesion molecules that can promote a better neural cells attachment. These adhesion properties in scaffold mimic the microenvironment of natural neural tissue resulting in better cell behavior.

In order to evaluate the cell affinity and attachment in supramolecular-dimers scaffolds, NSCs were seeded into the sponge-like scaffolds and cultured for 3 days. In previous work, we have already demonstrated that primate NSCs exhibit high proliferation rate and can be expanded continuously in long term culture [START_REF] Lins | Development of Bioresorbable Hydrophilic-Hydrophobic Electrospun Scaffolds for Neural Tissue Engineering[END_REF][START_REF] Wianny | Derivation and Cloning of a Novel Rhesus Embryonic Stem Cell Line Stably Expressing Tau-Green Fluorescent Protein[END_REF] . These primate NSCs express SOX2, a classical marker of NSCs, as well as the proliferative marker, Ki67. In this study, the immunofluorescent analyses (Figure S7) show that the vast majority of NSCs cultured in Neat AGA and AGA modified with UPy expresses SOX2, suggesting that sponges-like scaffolds sustain the proliferation and maintenance of primate NSCs. We also took advantage of our NSCs expressing TAU-tagged GFP, which labels microtubule components of the cytoskeleton, including long cell processes and other subcellular structures, thereby enabling fine visualization of cell morphology.

Complementary staining of Filamentous-actin (F-actin) allowed finely detailed visualization of interactions between cells, as well as between cells and scaffold.

Confocal microscopy images after cell seeding, allowed us to evaluate cells distribution and attachment into scaffolds (Figure 9, blue channel=cells-dapi and green channel=GFP). From fluorescence images in Figure 9-A and 9-B, it is clear that NSCs seeded into unmodified agarose sponge exhibit a poor contact with the scaffold surface.

Cell images on these sponges show that cell-cell adhesion is stronger than scaffold adhesion, forming clusters. The initial cell adhesion on surfaces is crucial for survival and growth, as the attachment determines cytoskeletal organization, cell shape, motility, proliferation and differentiation [START_REF] Jembrek | Atomic force microscopy as an advanced tool in neuroscience[END_REF] . As a result of this poor attachment we can observe in Due to the greater amount and better distribution of AGA-UPy into sponges, we observed a spread-out distribution of NSCs along the scaffold surface and throughout scaffold (Figure S8-B). Whereas, in an overview of the throughout unmodified agarose scaffold a large void space was observed with a sporadic cells distribution (Figure S8-A). In order to better show the integration of the cells in three dimensions, z-stack images were taken at 3 µm intervals through the scaffolds (Figure S9), which show that cells infiltrate within AGA:UPy scaffolds establishing contacts at different levels in the deepness of the scaffolds (indicated by the red arrows). In contrast, very few contacts were observed between the cells and Neat agarose. As above-mentioned, cells request an essential adhesion area on a substrate to survive, and the nature of this contact area may create the formation of associates with the outside environment [START_REF] Rao | Adhesion molecule-modified biomaterials for neural tissue engineering[END_REF][START_REF] Chen | Geometric Control of Cell Life and Death[END_REF] . The development and proliferation of neural cells in suitable scaffold usually follows the following steps: (1) preliminary cell attachment, (2) cell spreading, (3) organization of the actin cytoskeleton, and (4) formation of specific focal contacts [START_REF] Rao | Adhesion molecule-modified biomaterials for neural tissue engineering[END_REF] . Actin organization and focal adhesions mobilization are driven by a grouping of extrinsic and intrinsic signals, counting extracellular matrix proteins, growth factors, and extracellular molecular protagonists, such as neural cell adhesion molecules [START_REF] Loubet | Neuritogenesis: the prion protein controls β1 integrin signaling activity[END_REF] . UPy dimers can mediate α-β heterodimerization for the neural cells attachment via a non-receptor-mediated cell link mechanism, i.e., from its quadruple hydrogen-bonded system, hydrophobic interactions or by its functional amine groups [START_REF] Zustiak | Protein-hydrogel interactions in tissue engineering: mechanisms and applications[END_REF] .

The UPy moieties provide reactive amine groups allowing site-specific peptide immobilization using cysteine terminated peptides or interaction with amino-terminal domain contained in the nucleotide-binding region [START_REF] Lee | The effect of non-specific interactions on cellular adhesion using model surfaces[END_REF][START_REF] Faucheux | Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies[END_REF][START_REF] Roach | Surface strategies for control of neuronal cell adhesion: A review[END_REF] . These chemoselective moieties can interact with specific integrin peptides in order to preserve receptor recognition, which is reliable with published results on the nonspecific interaction between amine functionalized materials and cell [START_REF] Lévesque | Synthesis of cell-adhesive dextran hydrogels and macroporous scaffolds[END_REF][START_REF] Lee | The effect of non-specific interactions on cellular adhesion using model surfaces[END_REF][START_REF] Faucheux | Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies[END_REF] and more specifically neurons [START_REF] Rao | Adhesion molecule-modified biomaterials for neural tissue engineering[END_REF][START_REF] Roach | Surface strategies for control of neuronal cell adhesion: A review[END_REF] . This outward signal transfer can produce an induced redistribution of integrin in the cell membrane, resultant in a 'cell-scaffold interaction' comparable to the ligand-receptormediated interaction and ECM molecules, such as vitronectin, laminin, collagen or fibronectin [START_REF] Rao | Adhesion molecule-modified biomaterials for neural tissue engineering[END_REF] . Beyond that, the incorporation of specific bioactive molecules can also improve adhesion indirectly, by endorsing the adsorption of ECM proteins through hydrophobic interactions [START_REF] Roach | Surface strategies for control of neuronal cell adhesion: A review[END_REF][START_REF] Bacáková | Cell adhesion on artificial materials for tissue engineering[END_REF] . This adsorption can occur after the initial binding, induced by adhesive molecules, when cells secrete ECM and the mechanotransduction processes is released. It is important to highlight that cells incapable to secrete ECM are unable to sustain binding by this mechanism and undergo apoptosis [START_REF] Rao | Adhesion molecule-modified biomaterials for neural tissue engineering[END_REF][START_REF] Bacáková | Cell adhesion on artificial materials for tissue engineering[END_REF] . Moleculemodified cell adhesion has already been employed in polysaccharides for neurite outgrowth, demonstrating the importance of adhesion molecule selection when creating scaffolds. For example, covalently modified agarose with laminin-derived peptides was revealed to be larger to the original agarose hydrogels in endorsing neural adhesion and axonal growth [START_REF] Bellamkonda | Laminin oligopeptide derivatized agarose gels allow three-dimensional neurite extension in vitro[END_REF] .

Following cell attachment to unmodified AGA, AGA-UPy 50:50, and AGA-UPy 90:10, filamentous-actin was stained in order to visualize the interactions between cell-cell and cell-surface. Cell images of NSCs seeded on unmodified agarose scaffold show that cell-cell adhesion is stronger than scaffold adhesion, with cells forming densely packed cell clusters and a poor contact with the surface (Figure 10-A, -B, and-C). For all samples with AGA-UPy, the NSCs actin appears to associate on UPy-dimers surfaces, as shown in Topology and mechanical compliance of the substrates are two other factors that may have contributed to the initial cells attachment. Surface properties of a scaffold have determinant effects on the NSCs behavior. As shown in Figure S6 (see support information), the surfaces of sponges-like scaffold with 10 % and 50 % of AGA-UPy show significant roughness and surface spreads with waviness and small droplets, while unmodified agarose sponge surfaces are very smooth. The surface roughness can improve cell adhesion due to increased contact area, which is related to the interfacial adhesive force [START_REF] Gentile | Cells preferentially grow on rough substrates[END_REF] . According to Khan et al., increase contacts between cell body and scaffold surface improves subsequently the neural proliferation [START_REF] Khan | Influence of nanoscale surface roughness on neural cell attachment on silicon[END_REF] . Furthermore, Decuzzi and Ferrari have shown that the roughness is contrariwise to the adherent cell stiffness, i.e., cells with less stiff membranes require more surface roughness to initiate their adhesion [START_REF] Decuzzi | The adhesive strength of non-spherical particles mediated by specific interactions[END_REF] . This hypothesis is in agreement with the results obtained here because the brain is a soft tissue, 10 times softer than liver and 50 times softer than muscle [START_REF] Georges | Matrices with Compliance Comparable to that of Brain Tissue Select Neuronal over Glial Growth in Mixed Cortical Cultures[END_REF] .

We demonstrated in the previous section that 50 % AGA-UPy (AGA-UPy 50:50) sponges are softer, showing the greatest ability to create an adhesive matrix for NSCs.

In this context, incorporation of UPy-dimers into the agarose backbone thus becomes a good method for enhancing tissue integration of neural biomaterials, with a great balance between mechanical and adhesive properties.

Conclusions

The approaches discussed in this work improves upon the current sponge-like scaffold technology by providing a material with rapid cellular uptake triggered by the presence of cellular medium, growth factors, proteins, or nutrients. The supramolecular sponges have been shown to be very promising for biomedical applications because they delineate the mandatory use of cross-linking agents that often present toxicity.

Moreover, these supramolecular sponges provide excellent physicochemical properties that render them useful scaffolds for minimally invasive neural implantation. These properties include: a micro-structure that is highly porous with inter-connected pores, shape preservation during gelation, the ability to recall water up to 100 times its weight, mechanical properties resembling those of human soft tissue, and finally, suitable cytocompatibility. Furthermore, UPy-modification was used to improve adhesion of neural stem cells encapsulated within the hydrogel and to guide neural adhesion at the interface with tissue, i.e., on the material surface. Thus, supramolecular sponges may be used as an innovative method for 3D scaffolds application and may also be utilized for many tissue engineering applications that include differentiation of cells in controlled microenvironments.

Acknowledgment

The authors thank FAPESP for financial support #2017/11060-5 in the form of a postdoctoral grant to LCL and the thematic project #2016/25406-5. We also thank the Brazilian Nanotechnology National Laboratory, LNNano, for access to microtomography analyses. The biological application of this work was supported by LABEX CORTEX (ANR-11-LABX-0042), LABEX DEVWECAN (ANR-10-LABX-0061) of the University of Lyon within the program "Investissement d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). We are also grateful for Adèle Fendler's precious help.

1 H

 1 spectra was performed on a Bruker Avance III 400 MHz, 500 MHz or AvanceNEO 600 MHz spectrometer. Samples were dissolved in deuterated DMSO. The coupling constants are indicated in Hz and chemical shifts (δ) are expressed in ppm, relative to internal tetramethylsilane for 1 H nuclei. The solid-state 13 C NMR spectra was recorded at 11.7 T magnetic field using an AVANCE III HD spectrometer at Larmor frequencie ν(13C) = 125.783 MHz using a 4 mm cross-polarization magic angle spinning probe (CP-MAS). MAS spinning speeds for all samples were 10 kHz. The reaction between UPy-NCO and Agarose was evaluated using a K-Alpha+ XPS spectrometer (ThermoFisher Scientific, UK) with monochromatic Al Kα X-ray radiation with pass energy of 200 eV for survey size (400 µm spot) and 50 eV for highenergy resolution core level spectra. Spectra were recorded with an angle of incidence of 30° at two locations and elemental compositions were determined from lowresolution survey measurements with 200 eV for survey and 50 eV for high-energy resolution core level spectra. The Scofield sensitivity factors, analyzer transmission function, and effective attenuation lengths (EALs) for photoelectrons were applied for quantification. EALs were calculated using the standard TPP-2M formalism, which were controlled by means of the well-known photoelectron peaks of metallic Cu, Ag, TGA) of UPy-NCO, AGA-NH 2 , AGA-UPy and the AGA-NH 2 / UPy-NCO physical mixture were recorded on a Q500 thermogravimetric analyzer (TA instruments). The samples were heated from 25 to 800 °C at a rate of 20 °C min -1 under N 2 flow. Differential Scanning Calorimetry measurements (DSC) of neat UPy-NCO, functionalized agarose i.e. AGA-UPy, AGA-NH 2 and the AGA-NH2/UPy-NCO mixture were performed on a Q20 (TA instruments) from 10 to 140 °C to prevent any thermal degradation of the compounds as demonstrated by TGA. The samples were heated or cooled at a rate of 10 K min -1 under N 2 flow of 50 mL min -1 . Hydrogels rheological analyses were carried out using a Rheometric Scientific ARES instrumentation at 37 °C equipped with 30 mm parallel plates, with a 4 mm gap between them (TA Instruments, New Castle, DE, US). The oscillatory responses (G'= elastic modulus and G''= loss/viscous modulus) were determined at low values of strain (0.02 %) over the frequency range 0.1-100 rad/s. The compression strength of the cylindric sponges were measured in a compression testing machine (thermomechanical analyzer TMA 2940 TA Instruments). The tests were performed at a compression at a constant rate of 0.05 N min -1 (20mm DIA cylinder aluminium), equilibrium at 25 °C. The high resolution X-ray micro-computed tomography (Resolution: 1 pixel = 4.5 um) (uCT; model Skyscan 1272, Bruker microCT, Kontich, Belgium) was used to reconstruct three-dimensionally the internal structure of sponges like-scaffold.

Figure 1 .

 1 Figure 1. Infrared spectra of 2-(6-isocyanatohexylaminocarbonylamino)-6-

Schema 1 .

 1 anhydro galactose is shown in Figure S2. Agarose-amine functionalization and characterization are presented in more details at Support Information. The subsequent coupling reaction occurs between electrophilic isocyanate and common nucleophilic end groups of telechelic polymers as amine groups. The molecule denoted (UPy-NCO) readily reacts with amine functions (urea formation) to form the novel AGA-UPy, as shown in Scheme 1. Synthesis of the Agarose-UPy via SN2 from the formation of urea.FT-IR and solid-state 13C NMR were performed to confirm the chemical structure of unmodified agarose, AGA-NH 2 (FigureS3and tableS1), and AGA-UPy. The FTIR spectra in Figure2shows total disappearance of the 2285 cm -1 characteristic isocyanate function (N=C=O) band associated with the appearance of a new 1590 cm -1 peak characteristic of urea, clearly confirming the success of the functionalization[START_REF] Dankers | Convenient Solid-Phase Synthesis of Ureido-Pyrimidinone Modified Peptides[END_REF] . These results indicate the formation of AGA-UPy without presence of UPy-NCO[START_REF] Ren | Facile Preparation of Linear Polyurethane from UPy-Capped Poly(dl-Lactic Acid) Macrodiol[END_REF] . The new bands emerging in the spectral region of 3050 and 2850 cm -1 are characteristic of the secondary amines (N-H) of urea formation. These absorptions bands suggest that they are predominantly present in the form covalently associated with the agarose. In the spectral region from wavenumbers 1700-1500 cm -1 , in the case of AGA-NH 2 , only a single broad band of absorption can be observed. Instead, the functionalization by UPy leads to the appearance of many bands in this region. The presence of UPy characteristic bands (n = 1700-1666-1590-1523 cm -1 ) reveals the presence of the UPy associative entity.

Figure 2 .

 2 Figure 2. FTIR of amine-agarose (AHA-NH 2 ), UPy-NCO precursor, and Agarose-UPy (AGA-UPy).

  agarose chains. Because XPS is a suitable technique for obtaining information of chemical structure of compounds in the solid state, the chemical environment of unmodified agarose, AGA-NH 2 , and AGA-UPy could be analyzed without any interference from interactions in solution. In order to obtain a deeper insight, the high resolution of C1s and N1s spectra are shown in Figure3before and after agarose modification. The deconvolution signals of unmodified agarose, agarose with NH 2 -grafted groups and sequentially coupled by UPy-moieties show that different covalent bonds can be associated with different carbon signal peaks. The survey spectra obtained of C(1s) from unmodified agarose illustrates binding energies at 285.0 eV corresponding to alkane-type carbon C-H/C-C, 286.4 eV associated to C-O of ether groups, and 288.0 eV which can be attributed to C-O of alcohol groups 31 . AGA-NH 2 spectra also exhibited a C1s binding energy peak at 285.0 eV C-O carbon alkanes. Moreover, a shoulder peak at 284.6 eV appears confirming covalent C-N binding from the substitution of OH by NH 2 -groups on agarose-C6 32 . After grafting NH 2 groups into the polysaccharide backbone (C6), the peak corresponding to C-O and C-N became predominant, while the peak at 288.0 markedly decreased, indicating the modification of hydroxyl-containing functional groups[START_REF] Sánchez-Vaquero | Characterization and cytocompatibility of hybrid aminosilane-agarose hydrogel scaffolds[END_REF] . C1s component for remaining hydroxyl groups tends to be shifted to higher binding energy and a weak intensity relative to C-O and C-N, thus being able to be overlapped by these peaks. In AGA-UPy spectra, the successful UPy-functionalization on AGA-NH 2 was also confirmed by similar binding energies for C-N at 284.6 eV, whereas the peak at 288.4 eV was assigned to carboxylic functions (C=O) originating from urea function borne by AGA-UPy binding.

Figure 3 .

 3 Figure 3. Deconvoluted high-resolution C1s and N1s X-ray photoelectron spectra of

Figure 4 .

 4 Figure 4. (A) Weight loss as a function of temperature (TGA) and (B) DSC thermograms:

Figure 5 .

 5 Figure 5. (A) Frequency dependence of G' (full symbols) and G'' (empty symbols) for

Figure 6 .

 6 Figure 6. Schematic of supramolecular network effect on the polymeric

Figure 7 .

 7 Figure 7. Fabrication of sponges which results in the formation of a

Figure 8 .

 8 Figure 8. Three-dimensional reconstructed images from microtomography of aerogels

Figure

  Figure S8-D and -E the DNA defragmentation, the absence of actin fibers, and

Figure 9 .

 9 Figure 9. Integration of NSCs in sponge-like scaffolds. Confocal images of NSCs following culture in (A, B) unmodified agarose; (C, D) 50:50 AGA-AGA:UPy; (E, F) 90:10 AGA-AGA:UPy. (A,C,E) Merge images of nuclei stained with DAPI (blue), GFP (green), and phase contrast images (grey). (B,D,F) GFP staining. Maximum intensity projections of z-stack images are shown. Yellow arrows indicate AGA-UPy aggregates; red arrows indicate cell-scaffold interactions. Scale bars, 10μm.

Figure 10 -

 10 D to -I. When the dimers association formed aggregates into sponges, it is possible to notice that the cells surround these aggregates showing several points of contact. The initial attachment of NSCs onto sponge surfaces modified with UPy-dimers results in immobilization, avoiding detachment in answer to slight shear forces. When the surface of the biomaterial is suitable for the cell attachment, the cell membrane begins to spread along the available surface area, as shown in Figure10-H and -I. This event is succeeding with the formation of a filamentous actin cytoskeleton. Finally, integrins form clusters in response to force applied by the cytoskeleton, known as focal adhesion sites. These trigger signaling trails can influence cell function, viability, and proliferation[START_REF] Alberts | Cell Junctions, Cell Adhesion, and the Extracellular Matrix[END_REF] . SEM images revealed NSCs morphology spreading on AGA-UPy surfaces (Figure10-K and -L). Meanwhile, cells seeded on unmodified agarose sponges appear to repulse the surface (Figure10-J).

Figure 10 .

 10 Figure 10. Interaction between NSCs and sponge-like scaffolds. (A-I), Confocal images of the sponge-like scaffolds following cell attachment, stained for F-Actin. (A, B, C) unmodified agarose; very few contacts are detected between the cells and scaffold surface (yellow dotted line); (D, E, F) 50:50 AGA-AGA:UPy. (G, H, I) 90:10 AGA-

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Supporting Information

The supporting information is available free of charge on the ACS Publications website: Experimental details: General procedure for the synthesis of Agarose-UPy. Figures: 1H-

Author Information

*Corresponding author: L. Lins, e-mail: luandaqmc@gmail.com


Pierre-Marie Gassin
email: pierre-marie.gassin@enscm.fr

Benedicte Prelot

Brian Gregoire

Gaelle Martin-Gassin

Second Harmonic Scattering can Probe Hydration and Specific Ion Effects in Clay Particles

We report Montmorillonite (MMT) particles organization in aqueous solution using polarization resolved second harmonic scattering (SHS). This technique can probe specifically the organization of water in this system. The effect of the colloidal stability of the particle and the nature of the interlayer cations on the SHS Intensity is highlighted. The interlayer hydration has been probed and changes with the order Mg 2+ > Ca 2+ > Li + , Na + , K + > Cs + . Real time SHS evolutions during cesium exchange have been monitored, and different pathways are discussed in regards with the experimental results.

Introduction

Clay minerals and in particular Montmorillonite (MMT) particles are nanostructured materials composed by stacks of negatively charged layers compensated by solvated cations. These materials are currently involved in many environmental and industrial processes because of their catalytic and retention properties. [START_REF] Okumura | Radiocesium interaction with clay minerals: Theory and simulation advances Post-Fukushima[END_REF][START_REF] Wang | Nanoscale Clay Minerals for Functional Ecomaterials: Fabrication, Applications, and Future Trends[END_REF][START_REF] Benny | Clay Mineral Catalysis of Organic Reactions[END_REF][START_REF]Clays in Sedimentary Environments[END_REF] Understanding the fundamentals of clay-water interfaces is a crucial point for the success of those applications. Numerous computational [START_REF] Ho | Revealing Transition States during the Hydration of Clay Minerals[END_REF][START_REF] Honorio | Hydration Phase Diagram of Clay Particles from Molecular Simulations[END_REF] , experimental works using XRD [START_REF] Ferrage | Investigation of smectite hydration properties by modeling experimental X-ray diffraction patterns: Part I. Montmorillonite hydration properties[END_REF] , quasi-elastic neutron scattering [START_REF] Bordallo | Quasi-Elastic Neutron Scattering Studies on Clay Interlayer-Space Highlighting the Effect of the Cation in Confined Water Dynamics[END_REF] or infrared spectroscopy [START_REF] Ly | Cohesive Vibrational and Structural Depiction of Intercalated Water in Montmorillonite[END_REF] studied the swelling, water adsorption and cation exchange properties on MMT under controlled humidity conditions. Those studies contributed to our current-day understanding of the MMT hydration, thickness and interactions between cations. Roughly, MMT crystalline hydrates with one, two, or three molecular layers of water separating the stack layers depending on the controlled relative humidity and the nature of interlayer cations. In aqueous solution, the behavior of MMT colloidal system is quite more complicated because complex chemo-mechanical properties involving competitive effects, determine the whole organization of the mineral plates into an exfoliated or stacked arrangement. The precise interplay between interfacial water structure and ion adsorption/exchange remains experimentally poorly characterized using in situ and in real time probes that are non-invasive, label-free and specific to interfacial water.

Nonlinear optics and Second Harmonic Scattering (SHS) [START_REF] Roke | Nonlinear Light Scattering and Spectroscopy of Particles and Droplets in Liquids[END_REF][START_REF] Eisenthal | Second Harmonic Spectroscopy of Aqueous Nano-and Microparticle Interfaces[END_REF] have been previously used to studied MMT particles in suspension. [START_REF] Yan | Probing the Interface of Microscopic Clay Particles in Aqueous Solution by Second Harmonic Generation[END_REF] This first study demonstrated the contribution of the oriented water around the charged plate in the whole SHS intensity and its dependence with the ionic strength. The colloidal transition between the delaminated/stacked plate and the change in the interlayer hydration during cation exchange was not explored in this former work and will be investigated in this article. In particular, the results presented here will be discussed in term of coherent contribution in the SHS Intensity coming from water correlation induced by the MMT particles. Indeed, in the last three years, numerous works have significantly improved our understanding of the SHS signals concerning the contribution of organized or correlated water in the whole SHS intensity [START_REF] Tocci | Second-Harmonic Scattering as a Probe of Structural Correlations in Liquids[END_REF][START_REF] .; R K | C c "S c -Ha m c Sca g a a f S uc u al C la L qu[END_REF][START_REF] Gonella | Second Harmonic and Sum-Frequency Generation from Aqueous Interfaces Is Modulated by Interference[END_REF][START_REF] Shelton | Long-range orientation correlation in water[END_REF] , particularly in the domain of electrolyte solution [START_REF] Duboisset | Salt-induced Long-to-Short Range Orientational Transition in Water[END_REF][START_REF] Borgis | What Does Second-Harmonic Scattering Measure in Diluted Electrolytes?[END_REF][START_REF] Chen | Electrolytes induce long-range orientational order and free energy changes in the H-bond network of bulk water[END_REF][START_REF] Shelton | Water-water correlations in electrolyte solutions probed by hyper-Rayleigh scattering[END_REF] . Based on this general framework, the global SHS intensity I, for N molecules, each located at position r i , can be written as [START_REF] Borgis | What Does Second-Harmonic Scattering Measure in Diluted Electrolytes?[END_REF][START_REF] Bersohn | Double-Quantum Light Scattering by Molecules[END_REF] (1)

Where β labo is the hyperpolarizability tensor expressed in the laboratory frame, q is the scattering wavevector q=k s -2k i where k i is the fundamental incident wave vector, k s is the harmonic scattered one, u and v are the polarization directions of the incoming and outgoing beams described by the angles γ, Γ (see figure 1 for the notation), and Ω is the orientation of the molecule, define with three Euler angles, in the laboratory frame (XYZ). The brackets <..> stand for the time average. The first part of equation 1 is referred as the incoherent contribution and the second term is referred as the coherent contribution in the SHS intensity. In the case of correlated scattered molecule, this second term can overcome the first term, as it will be the case in systems presented here. Otherwise, for the case of uncorrelated molecule, this second term is zero.

Experimental Methods

Experimental Optics setup:

The figure 1 depicts the notation together with the experimental setup [START_REF] Gassin | Second-Harmonic Scattering in Layered Double Hydroxide Colloids: A Microscopic View of Adsorption and Intercalation[END_REF] . Briefly, this setup was built on a femtosecond Ti-sapphire oscillator laser source providing pulses with a duration of about 100 fs at a repetition rate of 80 MHz (coherent, model Chameleon ultra II). After passing through a low-pass filter, the fundamental beam, set to a fixed wavelength at 800 nm and an averaged power of 600 mW, was focused by a microscope objective (Ealing x10, numerical aperture 0.25.) onto the sample at 2 mm close to the output cell window (Hellma QS 10 mm).

The SH light was collected at 90° by a 10 cm focal length lens and separated from its fundamental counterpart by a high-pass filter. The SH light was detected with a water-cooled CCD camera (Andor, Newton) placed after a spectrometer (Andor, Shamrock 193). The fundamental input beam was linearly p la z a h pu p la za a gl γ wa l c with a rotating half-wave plate. An analyzer, placed in front of the spectrometer, was used to separate w p la za a : cal u pu a fu h am I(γ,V u , h z al output a f I(γ,H u . I fac l a h a g a c h w h h works [START_REF] Tocci | Second-Harmonic Scattering as a Probe of Structural Correlations in Liquids[END_REF][START_REF] Gonella | Second Harmonic and Sum-Frequency Generation from Aqueous Interfaces Is Modulated by Interference[END_REF][START_REF] Borgis | What Does Second-Harmonic Scattering Measure in Diluted Electrolytes?[END_REF][START_REF] Chen | Electrolytes induce long-range orientational order and free energy changes in the H-bond network of bulk water[END_REF] , the SHS intensity I(0°,V out ) is also referred to I SSS and I(0°, H out ) to I PSS , figure

1A summarizes these notations. The experimental data of figure 1 were recorded during 10 seconds for each point. The experimental data of figure 2 were recorded during 10 seconds, each point are repeated 5 times, and the errors bar represents +/-2 standard deviation. In figure 3, each point is recorded during 10 s in the I SSS configuration and the global time monitoring is about 4500 s. All experiment were conducted at 20°C and under stirring condition (a magnetic stirrer operates in the cell measurement). For figures 1 and 2, the solution were equilibrated 12h prior the measurement.

Materials

In all experiments, the MMT particles are prepared in 18 MΩ.cm water (MilliQ) at the same concentration of 0. This clay mineral exhibits a low octahedral charge and extremely limited tetrahedral substitution. [START_REF] Stucki | Effects of reduction and reoxidation of structural iron on the surface charge and dissolution of dioctahedral smectites[END_REF] Size fractionation was performed by centrifugation to extract the <1µm size fraction. Prior to the measurement, the particles were sonicated during 10 min and kept under permanent agitation. The particles named Ca-MMT in the manuscript were prepared by ionexchange reaction of Na-MMT at room temperature with 1 mol/L aqueous solutions of CaCl 2 .

The suspensions solution was first allowed to stir for 24h and then, the solid fraction was removed from the saline solution by centrifugation and dispersed again in a fresh saline solution at the same concentration. These steps were repeated three times. The excess of CaCl 2 was removed by rinsing several times the solid using distilled water until no Cl anions were measured in solution as indicated by a negative silver nitrate test. For the figure 2 data, the following ions are added in the solution: LiNO 3 , KNO 3 , CsNO 3 , NaNO 3 , Ca(NO 3 ) 2 , Mg(NO 3 ) 2 , from Sigma Aldrich. For the figure 3, ions addition is carried out using a microvolume injection (18 µL) of a 0.8 M solution of NaNO 3 in a 2 ml suspension of 0.25g/L of Na-MMT (figure 3A) or 0.8 M solution of CsNO 3 in a 2 ml suspension of 0.25g/L of Ca-MMT (figure 3B), in such way, the dilution factor is negligible. The solution is rapidly homogeneous thanks to a magnetic stirrer place in the measuring cell. When MMT particles are introduced in water, the SHS intensity is globally enhanced by a factor around two, which is explained by coherent contribution of water coming from its organization around and inside the mineral sheet. The coherent term of equation 1 is thus not zero and contributes in the same order of magnitude as the incoherent term coming from "bulk"

Results and discussion

water. No contribution in the SHS intensity of the mineral materials is considered here, as it will be justified below with the cesium results. When the ionic strength is modified, the behavior of the polarization plot changes with a small decrease in the I(γ,H u y wh a h I(γ,Vout) remains more or less stable. For the case of stacked Ca-MMT particles, h I(γ,Vout) intensity is higher compared to exfoliated Na-MMT system. To study the transition between exfoliated/stacked particles and the possible specific ion effects, figure 2 shows the SHS intensity evolution for initially exfoliated Na-MMT particles by varying ionic strength with different cations. To interpret those curves, two domains of ionic strength (IS) can be considered, low IS (IS<10 - 3 M) and high IS (IS>10 -3 M). In the first domain, no specific ion effects are observed since all curves seem to follow globally the same evolution: the intensity I SSS is nearly constant and intensity I PSS decreases. These evolutions are explained by the interference between water molecules oriented by the electric field induced by the surface charge onto the platelet. This contribution is referred as Diffuse Double Layer (DDL) contribution [START_REF] Gonella | Second Harmonic and Sum-Frequency Generation from Aqueous Interfaces Is Modulated by Interference[END_REF] . This evolution in the 10 -5 -10 -3 M IS range is in agreement with both theoretical model [START_REF] Gonella | Second Harmonic and Sum-Frequency Generation from Aqueous Interfaces Is Modulated by Interference[END_REF] describing the interference in the DDL and with previous experimental work presented by Eisenthal [START_REF] Yan | Probing the Interface of Microscopic Clay Particles in Aqueous Solution by Second Harmonic Generation[END_REF] . At higher IS (10 -3 -10 -2 M), the DDL contribution tends toward zero and the colloidal stability of the MMT particles changes with a transition to a stacked organization [START_REF] Abend | Sol-gel transitions of sodium montmorillonite dispersions[END_REF][START_REF] Lagaly | Colloid chemistry of clay minerals: the coagulation of montmorillonite dispersions[END_REF] . This transition is observed both on the two polarization states I PSS and I SSS . Depending on the cation added in the system, the experimental data exhibit different behaviors coming from the supposed cation exchange and leading to different water organization in the system. When Li + or K + ions are added, no significant deviations are observed experimentally in the SHS evolution compared to the case of sodium ion. When Ca 2+ or Mg 2+ ions are introduced, the I PSS and I SSS increase significantly. The cation exchange induced a stacking of the layer with more correlated water in the interlayer domain. The data obtained at 10 -2 ionic strength may be compared with the case of Ca-MMT presented in fig 1D . The global polarization plots are consistent even if prepared Ca-MMT intensity is a little bit higher, 2.5 compared to 2.2 with the data coming from the in situ Ca-exchange. In the case of prepared Ca-MMT, the stacking is likely better than the organization obtained by in situ cation exchange. When Cesium ion are added, the SHS intensity decreases and the ratio I/I water falls very close to 1 for I SSS and close to 1.4 for I PSS . This probably means that the coherent contribution in equation 1 becomes very small compared to the initial condition. It is interesting to note that the ratio I PSS is slightly higher but nevertheless close to the value measured by Roke and coworker in electrolyte solution at high ionic strength [START_REF] Chen | Electrolytes induce long-range orientational order and free energy changes in the H-bond network of bulk water[END_REF] . These two ratios may be interpreted as the electrolyte contribution and the contribution of organized water around or inside Cs-MMT particles seems negligible. These results likely show that few correlated water molecules are involved in the interlayer domain and around the particles. This is in agreement with a zero water model in the interlayer and with the "ca y cha g ma ch g ff c " wh ch ha c ly c f h c um . [START_REF] Yamamoto | Wh ch yp f clay m al f x c um ff c ly? h "ca y-charge matching ff c[END_REF] Moreover, the cesium results are also consistent with a negligible contribution of the mineral material compared to water contribution. Indeed if the material itself exhibits an intrinsic significant nonlinear response, this contribution would be measured in that case.

- - - - - - - - - - - - - - - - - - - - - - - - Cs+ - - - - - - - - - - - - - - - - - - - - - - - - Cs+ Cs+ Cs+ Cs+ Ca2+ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Ca2+
To summarize this part, increasing ionic strength in the system induces a decrease in the DDL SHS contribution, which is a non-specific ion effect, and induces also a transition from exfoliated to stacked particles. When particles are stacked, the water organization in the interlayer domains exhibits specific ion effect, with an hydration effect varying in the order Cs + < K + , Na + ≈ Li + < Ca 2+ < Mg 2+ . To get in situ information about the transition state during the cation exchange and the global organization of the system, the SHS real time intensity is recorded during the ion exchange.

Figure 3 shows the real-time evolutions when Cs is injected in the system at zero ionic strength for the two different kinds of MMT particles: exfoliated Na-MMT or stacked Ca-MMT. In both cases, the final ionic strength is 7.10 -3 M, and SHS signal evolutions with time are well reproduced. For the case A, the time dependent plot exhibits a first increase during around 1000 seconds. This duration could be considered as the characteristic time of exfoliated/stacked transition. It contains both the ions diffusion/adsorption signal and the stacking platelet characteristic time. This characteristic time is consistent with the SHS evolution obtained for the case of calcium injection onto exfoliated Na-MMT (see additional in the SI). This first increase is followed by an abrupt decrease, which is interpreted as a dehydration step where water is expelled from the interlayer domains. This time duration is likely in the same order of magnitude observed for the previous diffusion/exchange/stacking characteristic times. The equilibrium is reached in around 3000 seconds. In the case B, the evolution shows a more fluctuating decrease and reached an equilibrium state of the same order of magnitude than in case A. These fluctuations [START_REF] Martin-Gassin | Second harmonic generation monitoring of nitric acid extraction by a monoamide at the waterdodecane interface[END_REF] are the highest during the time interval 1000-3000 seconds and are comprised in the range between 2.5 and 1.5 value. We interpreted these fluctuations as a signature of the cation exchange: the cesium cations close progressively the sheet when calcium cations are expelled. Those results are remarkable in the context of recent works about cesium adsorption in clays [START_REF] Okumura | Radiocesium interaction with clay minerals: Theory and simulation advances Post-Fukushima[END_REF] concerning the discussion between different pathway referred a " c xcha g " " c llap -driven exchange ». In the case of exfoliated Na-MMT (figure 3A), the Cs-exchange seems to follow the sequence « collapse induced dehydration ». In the case of initially stacked

Ca-MMT the nature of the fluctuation in the experimental data seems to sign the coexistence between calcium hydrated / cesium dehydrated stacked domain in the system. The sequence " c llap xcha g " a llu a bottom figure 3, may be in agreement with this fluctuating evolution. Nevertheless, these data cannot clearly determine if one pathway between " c xcha g " " c llap -driven exchange » is really preferred. These real time SHS data show that for the two different initial states studied here, the characteristic time of ion exchange is nearly the same. In other words, h " p g" f h ack arrangement (see bottom figure 3) seems not to be a limiting step in the kinetics.

Conclusions

In conclusion, this work demonstrates that SHS is able to probe MMT particles organization and interlayer hydration. The nature of the compensating cation strongly changes this interlayer hydration. Real time SHS has been used to monitor the cation exchange for two different initial states. For initially exfoliated particles, a specific evolution with two steps involving a collapse before dehydration has been highlighted. For initially stacked particles, fluctuations in the SHS intensity are observed during the cation exchange. Future works involving other type of clays particles, for example clays nanotube, are currently planned in order to study the effect of other geometrical water arrangements in such material. Overall, we hope that these studies will stimulate the use of SHS to probe hydration and molecular organization around and inside porous materials.

ASSOCIATED CONTENT Supporting Information. Additional information about the fit parameters presented in figure 1 and complementary real time SHS evolution. The following files are available free of charge.

AUTHOR INFORMATION

Notes

  25 g/L and two types of MMT particles were used. The particles named in the manuscript as Na-MMT are the SWy-3 montmorillonite from the Source Clay Repository of The Clay Mineral Society with structural formula [(Al 2.99 Fe 0.43 Mg 0.52 )(Si 7.97 Al 0.03 )O 20 (OH) 4 ] M + 0.7 .

Figure 1 .

 1 Figure 1. Sketch of the SHS experiment, SHS intensities are recorded at a scattering angle of 90°

Figure 1

 1 Figure 1 presents the polarization resolved SHS intensity for different systems: pure water,

Figure 2 .

 2 Figure 2. Evolution of the normalized SHS intensity in the PSS and SSS polarization states as a

ACKNOWLEDGMENT

The financial support of this work by the ANR project CAMOMILS (ANR-15-CE21-0002) is greatly acknowledged.

Any additional relevant notes should be placed here.

The authors declare no competing financial interests.