Ahdi Hadrich

Virginie Dulong

Christophe Rihouey

Béatrice Labat

Luc Picton

Didier Le Cerf
email: didier.lecerf@univ-rouen.fr

Biomimetic hydrogel by enzymatic crosslinking of pullulan grafted with ferulic acid

Keywords: hydrogels, polysaccharide, enzyme, antioxidant property

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

Introduction

Hydrogels represent a peculiar class of materials. consisting in 3D structures usually based on cross-linked polymer networks. They are characterized by high capacity to absorb aqueous solution and by specific mechanical properties highly dependent on the degree of crosslinking or content interactions [START_REF] Wong | Effect of crosslinking agent concentration on the properties of unmedicated hydrogels[END_REF]. Hydrogels have known a very substantial expansion due to their unique properties and value which aroused manufacturers' interest in different fields specially biomedical [START_REF] Hoffman | Hydrogels for biomedical applications[END_REF], pharmaceutical [START_REF] Kashyap | Hydrogels for pharmaceutical and biomedical applications[END_REF] and cosmetic [START_REF] Patravale | Novel cosmetic delivery systems: an application update[END_REF]. Beyond their current uses, hydrogels are more and more envisaged in many technological and innovative applications such as scaffolds for tissue engineering [START_REF] Moreira Teixeira | Enzyme-catalyzed crosslinkable hydrogels: Emerging strategies for tissue engineering[END_REF], entrapping of drugs or enzymes [START_REF] Gasmi | Hydrolysis of pullulan by entrapped pullulanase in Ca/alginate beads[END_REF], functional contact lenses [START_REF] Hu | Functional hydrogel contact lens for drug delivery in the application of oculopathy therapy[END_REF], 3D bio-printing [START_REF] Tasoglu | Bioprinting for stem cell research[END_REF], anti-aging masks [START_REF] An | Primary irritation index and safety zone of cosmetics: retrospective analysis of skin patch tests in 7440 Korean women during 12 years[END_REF] or sunscreen cream or mascara [START_REF] Yahia | History and applications of hydrogels[END_REF]. For sustainable development considerations and in particular the limitation of the fossil source of polymers, hydrogels based on natural polymers or their derivatives constitute an interesting alternative. In this way, polysaccharides are primary candidates and research around their chemical crosslinking pathways is dynamic and active.

However, the conventional reagents used in such chemical crosslinks are generally dangerous or toxic such as epichlorohydrin [START_REF] O'connor | The crosslinking of polysaccharides with polyamines and dextranpolyallylamine antibacterial hydrogels[END_REF] and glutaraldehyde [START_REF] Choi | Crosslinking of PVA/alginate carriers by glutaraldehyde with improved mechanical strength and enhanced inhibition of deammonification sludge[END_REF]. Other more safe crosslinkers like sodium trimetaphosphate (STMP) can also be used but the crosslinking pathway requires working at high pH which limits the applications of such systems [START_REF] Lack | Hydrogels based on pullulan crosslinked with sodium trimetaphosphate (STMP): rheological study[END_REF]. Therefore, it is common to use a new generation of hydrogels based on natural derivatives with biomimetic ways of crosslinking as in the vegetal reign and more precisely in structure of some cereal as wheat [START_REF] Naczk | Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis[END_REF]. It was widely described that ferulic acid (FA) is the most abundant phenolic compound in such structures linked by ester bond to cell wall polysaccharides chiefly arabinoxylans ones [START_REF] Bento-Silva | Relevance, structure and analysis of ferulic acid in maize cell walls[END_REF]. Number of studies has shown very interesting properties correlated to the role of FA as antioxidant, antiallergic, antimicrobial, anti-inflammatory, antiviral, antithrombotic, antidiabetic, anticancer, antihepatotoxic (Kumar & Pruthi, 2014). In addition to these properties, and as the main idea of our concept, FA presents the capacity to dimerize by oxidation in the presence of enzyme such as laccase [START_REF] Adelakun | Laccase-catalyzed dimerization of ferulic acid amplifies antioxidant activity[END_REF], widely distributed in plants, fungi, bacteria and insects [START_REF] Rivera-Hoyos | Fungal laccases[END_REF] and forming with its homologues, the protein superfamily of multicopper oxidases [START_REF] Sirim | The Laccase Engineering Database: a classification and analysis system for laccases and related multicopper oxidases[END_REF]. Thus, it was demonstrated that grain and cell wall contain dimers of FA, which have leading to the crosslinking of arabinoxylans chains alone or together with lignin. Such natural enzymatic crosslinking may be useful for plant in order to strengthen their structure [START_REF] Naczk | Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis[END_REF] or to elaborate hydrogels around the seeds to fix water needed for growing [START_REF] Vismeh | Profiling of diferulates (plant cell wall cross-linkers) using ultrahigh-performance liquid chromatography-tandem mass spectrometry[END_REF]. Using such arabinoxylans naturally grafted FA with its characteristics for new application fields appears difficult, unsuitable or not versatile for many reasons: delicate extraction with low yield, few global amounts for envisaging large field of applications, very few amount of grafted FA, impossibility to modulate the FA content (i.e. to control both swelling and mechanical properties) and finally impossibility to decline such process on other type of polysaccharides which may be more suitable for specific applications.

On the basis of these considerations, the chemical grafting of FA with well-defined and controlled amounts on any polysaccharides represents a great challenge. It could open the way to very large possibilities of modulate hydrogels crosslinked with enzymatic eco-friendly pathway. In this context, our approach is to create natural and versatile hydrogels after chemical grafting of FA whatever the polysaccharide, following by a controlled cross-linking between polysaccharide grafted chains performed by dimerization of FA under laccase action.

Polysaccharides can be neutral (arabinoxylan, pullulan, cellulose…), anionic (alginate, hyaluronic acid…) and more rarely cationic for artificial polysaccharides (chitosan, diethylaminoethyldextran…). To cover practically all polysaccharides, we decided to work on two model polysaccharides, a neutral one as pullulan (PUL) and an anionic one as carboxymethylpullulan (CMP). This choice is motivated since the chemical pathways for grafting the ferulic acid are different if the polysaccharide evidence carboxylic acid functions or only hydroxyl ones. Our first approach has been oriented toward anionic polysaccharides and achieved outcomes using CMP were already reported [START_REF] Dulong | Enzymatic cross-linking of carboxymethylpullulan grafted with ferulic acid[END_REF] showing very nice results. In this previous study, CMP-FA were obtained by activation of the carboxylate functions of CMP with the well-known EDC/NHS activators.

Here, the purpose is to covalently graft FA with controlled amounts on pullulan, a watersoluble neutral polysaccharide issued from fermentation of black yeast like Aureobasidium pullulans. It consists of linear and flexible macromolecular chains composed of maltotriose units linked by α-1,6 [START_REF] Kato | Solution properties and chain flexibility of pullulan in aqueous solution[END_REF]. Pullulan is being explored for various applications especially in biomedical due to its biocompatibility (Singh, (CDI, considered as an eco-friendly reagent [START_REF] Métro | 1,1′-Carbonyldiimidazole and mechanochemistry: a shining green combination[END_REF] in two steps by creating an ester bond between FA and PUL [START_REF] Wen | Corn starch ferulates with antioxidant properties prepared by N,N′-carbonyldiimidazole-mediated grafting procedure[END_REF]. The efficiency of the reaction was qualitatively and quantitatively verified by characterizing PUL-FA products using several techniques (FTIR and 1 H NMR). The behavior in aqueous solution was studied with size exclusion chromatography (SEC) coupling on-line with several detectors.

Then laccase from Trametes versicolor was used to crosslink PUL chains to obtain hydrogels.

Kinetics and mechanical properties were analyzed with rheology. Swelling, antioxidant and cytotoxicity properties were also investigated.

Experimental

Materials

Pullulan (PUL) was provided by Hayashibara Biochemical Laboratory (Japan) and ferulic acid was purchased from Acros Organics (France). N,N'-carbonyldiimidazole (CDI) and Laccase [EC 1.10.3.2] from Trametes versicolor were supplied by Sigma-Aldrich (France).

All other materials are listed in SI file.

Grafting of FA on PUL

As mentioned previously, a two-step reaction was conducted to get PUL-FA. The first step is to activate FA by CDI. For example, in a two-necked round-bottomed flask equipped with a magnetic stirrer and connected to the N2 line, equivalent moles of FA and CDI (1.5 mmol) were dissolved in 20 mL of anhydrous DMSO. The reaction was kept under stirring (375 rpm) at 60°C for 16 hours. In the second step, 1.6 g of dried pullulan was dissolved in anhydrous DMSO (60 mL) and added slowly to the previous solution to form PUL-FA through an esterification reaction. Then, the mixture was let at 60°C with continuous stirring for 6 hours.

At the end of reaction, the PUL-FA derivative was precipitated in five volumes of acetone and after filtration it was stirred in the same volume of acetone for 24 hours to remove unreacted FA, CDI and imidazole. After that, a second filtration was performed and the obtained product was dried under vacuum at 50°C for 3 days. To eliminate residual traces of solvent, the yellowish powder obtained was dissolved in milli-Q water and dialyzed against milli-Q water until low conductivity of dialysis water (equivalent to milli-Q water one). Finally, PUL-FA derivatives were lyophilized and stored at 4°C.

Fourier transform infrared spectroscopy

FTIR spectra were carried out using a Nicolet IS50 FT-IR spectrometer (Thermo Scientific, USA) over a wavenumber range of 600-4000 cm -1 by accumulating 128 scans at a resolution of 4 cm -1 .

Nuclear magnetic resonance spectroscopy

1 H NMR spectra were recorded on a Bruker Avance AC-P 300 MHz spectrometer (USA) using D2O/NaOD (85/15, v/v) as solvent. The substitution degree of FA onto pullulan (DSFA) was determined based on the integral ratio of aromatic and methine protons of FA to α-1,4 anomeric protons of pullulan's anhydroglucose unit (AGU) via Eq. 1:

= , Eq. 1
The amount of FA can be also expressed as FA equivalent (FAeq) in mg per g of PUL-FA by Eq. 2.

! "# = $ ×&' $ ( )*
Eq. 2 with MFA, the molar mass of FA.

Macromolecular characteristics

The absolute average number and weight molar masses (respectively Mn and Mw) and molar masses distribution of PUL-FA derivatives were determined using on-line coupling a size exclusion chromatograph (SEC), a multi-angle light scattering system (MALS), a viscosity detector (VD), an ultraviolet spectrophotometer (UV) and a differential refractive index detector (DRI). The details were provided in Supplementary Information.

Laccase from Trametes versicolor activity

Laccase activity was determined using the oxidation method of syringaldazine [START_REF] Figueroa-Espinoza | Oxidative cross-linking of pentosans by a fungal laccase and horseradish peroxidase: mechanism of linkage between feruloylated arabinoxylans[END_REF]. The protocol is described in the SI file.

Preparation of PUL-FA hydrogels

A series of PUL-FA hydrogels was prepared by biomimetic enzymatic crosslinking with following procedures. Different amounts of PUL-FA samples were dissolved, for 24h at room temperature, in 2 mL of 0.1 M citrate-phosphate buffer (pH= 5.5) leading to three different concentrations (40, 70 and 100 g/L). Then, laccase solution at 0.5, 1 and 2 nkat was added.

For rheological analysis, the solution is mixed manually (during a precise time added to the analysis time) and immediately poured into the geometry of the rheometer to investigate the gelling kinetics in situ.

For other analysis, the crosslinking reaction was kept at room temperature during 24h. The obtained hydrogels were thoroughly washed with milli-Q water, frozen and freeze-dried for one week.

Rheological measurements

Rheological experiments were carried out using a Discovery Hybrid Rheometer HR2 (TA Instrument, U.K.) with standard-size double concentric cylinder geometry in dynamic mode.

Crosslinking reactions were performed by recording both elastic modulus (G′) and viscous modulus (G″), time dependent, at 1 Hz with 2% strain (in the linear viscoelastic domain).

Temperature (25°C) was kept constant using a Peltier and experiments were reproduced twice with good accuracy. Obtained data were analyzed with TRIOS V 4.1.1.33003® software.

Swelling properties

For the kinetics of swelling, pre-weighted dried hydrogel (m0) was immersed in 20 mL of milli-Q water at room temperature. For each kinetics time point, the swollen hydrogel was taken out of the solution and weighted (mt) after the elimination of water's excess by Kimtech delicate task wipes until swelling equilibrium was reached. The water content Qt (g/g) was calculated with Eq. 3:

+ , = -,.-/ -/ Eq. 3
The equilibrium water content Qe is obtained when Qt stayed constant. Each experiment was carried out in triplicate and results agreed within ± 0.04 g/g standard error.

Antioxidant activity and cytotoxicity

In order to evaluate the properties of FA, PUL and PUL-FAs, antioxidant and cytotoxicity assays were realized. Furthermore, FA present inside hydrogels has the form of dimers (DiFA). For this reason, we have enzymatically developed these dimers (see SI file) to also estimate their activities.

Antioxidant properties: DPPH free radical scavenging assay

FA, DiFA, PUL and PUL-FAs were measured in terms of radical scavenging ability using the stable radical DPPH• [START_REF] Parejo | Comparison between the radical scavenging activity and antioxidant activity of six distilled and nondistilled mediterranean herbs and aromatic plants[END_REF]. The procedure is detailed in the SI file.

Cytotoxicity assays

The cytotoxicity effect of FA, DiFA, and PUL-FAs were established on mouse fibroblasts cells (L929-ATCC ® CCL-1) according to the protocol described in the SI file.

Results and discussion

Synthesis of PUL-FA

To get PUL-FA, several reaction protocols were tested according to experiments inspired from literature with an appropriate adjustment on our reagents. Carbodiimide mediated coupling reaction using 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide hydrochloride and N-hydroxysuccinimide was used in different solvents and co-solvents [START_REF] Woranuch | Ferulic acid-coupled chitosan: Thermal stability and utilization as an antioxidant for biodegradable active packaging film[END_REF].The Mitsunobu reaction using the coupling reagents triphenylphosphine and diethylazodicarboxylate was also tested in DMSO and DMF [START_REF] But | The Mitsunobu reaction: origin, mechanism, improvements, and applications[END_REF]. Furthermore enzymatic esterification applying lipase from Rhizomucor miehei was also performed [START_REF] Schär | High yielding and direct enzymatic lipophilization of ferulic acid using lipase from Rhizomucor miehei[END_REF]. Results achieved from all those reactions were unfortunately insignificant and the grafting reaction was invalid or non-reproducible. This could be explained by the solubility difference between hydrophilic PUL and hydrophobic FA and could be due also to the low reactivity of some coupling reagent in used solvents. However, the solution was found using an imidazole-mediated coupling reaction in anhydrous DMSO applying CDI, as illustrated in Fig. 1. Obtained PUL-FAs (with good yield) are yellowish that get darker as DS values increase. Their water solubility is highly dependent on DS; the more their DS increase, the more their water solubility decrease and vice versa. To obtain water-soluble PUL-FA, DS of 0.1 should not be exceeded. The productive parameters used to synthesize PUL-FA with different DS are summarized in Table 1. b Degree of substitution of FA determined by NMR (Eq. 1) (standard deviation 5%).

c FA content expressed in mg of FA/g of PUL-FA derivatives (Eq. 2).

Fourier transform infrared spectroscopy

FTIR spectral analyses were carried out to confirm the grafting of FA onto PUL (see the SI file, Fig. SI-3).

Nuclear magnetic resonance spectroscopy

1 H NMR was used to figure out the DSFA onto pullulan. The spectra and the peak attribution of PUL, and PUL-FA-10% are given in SI (Fig. SI-4). Obtained values (Table 1) were lower than the theoretical ones due to low reactivity and steric hindrance on PUL

Discussion

The amount of FA contained in PUL-FA shows a maximum value of 108.1 mg of FA/g of PUL-FA (Table 1). Besides, if we compare our values to those obtained on CMP (between 8.53 and 83 mg/g) [START_REF] Dulong | Enzymatic cross-linking of carboxymethylpullulan grafted with ferulic acid[END_REF], we notice that our FA contents are quietly higher which confirm the good efficiency of the carbodiimidazole chemistry in esterification reaction. In addition, our values are much higher than those naturally observed on arabinoxylans (between 1.6 and 2.3 mg/g) extracted from endosperm of wheat kernel [START_REF] Carvajal-Millan | Impact of the structure of arabinoxylan gels on their rheological and protein transport properties[END_REF]. This result proves that, we succeeded to graft more FA moieties than in nature and then the possibilities of crosslinking will be more important leading improved higher mechanical properties. However, PUL-FAs contain much lower amounts of FA moieties than those obtained on starch using the same coupling method [START_REF] Wen | Corn starch ferulates with antioxidant properties prepared by N,N′-carbonyldiimidazole-mediated grafting procedure[END_REF]. But in this case, starch derivatives were no water-soluble.

Higher values of FA content are reported on chitosan based on carbodiimide chemistry [START_REF] Woranuch | Preparation, characterization and antioxidant property of water-soluble ferulic acid grafted chitosan[END_REF]. The water-solubility was ensured by the ionic character of the chitosan, but with limit to a maximum concentration of 1.3 g/L. In comparison with PUL, chromatograms of PUL-FAs (Fig. 2A and SI 1) are noticeably different. On one hand, PUL-FAs evidence a new population at low elution volume between 14 and 15.5 mL (i.e. larger hydrodynamic volumes of polymer) together with higher apparent molar masses. This could be related to the presence of aggregated structures due to intermolecular hydrophobic interactions between grafted FA expected in aqueous solution, traducing the amphiphilic character of PUL-FA. On the other hand, one can notice that the major fraction of PUL-FA appears shifted toward higher elution volumes (i.e. smaller hydrodynamic volumes of polymer) compared to PUL. This clearly evidences the presence of a diminishing of PUL-FA size compared to PUL. That could be due either to a degradation of PUL backbone during the chemical modification or to the evidence of intramolecular associations between FA hydrophobic grafted moieties leading to strongly compacted structures. The hypothesis of a degradation can be push aside since the apparent molar masses distributions of PUL-FA fractions appears always parallel and higher than that of PUL.

Physico-chemical behavior of PUL-FA in dilute regime

Moreover, this observation highlights the hypothesis of intramolecular associations since at a same elution volume (i.e. at a same hydrodynamic volume of polymer), PUL-FA always presents higher apparent molar mass than PUL. This is clearly the confirmation of more compact structure for PUL-FA due to FA intramolecular hydrophobic associations.

Furthermore, the absorbance at 280 nm (according to FA phenolic structure) during the elution (Fig. 2B) indicates that the elution profile is comparable to the DRI response showing the homogenous distribution of FA moieties on all the molar masses distribution. This is also observed for the first fraction of PUL-FA at low elution volume corresponding to the aggregates evidencing a considerable amount of associated grafted FA in such structures.

This behavior, classic for amphiphilic polymers, has already been reported with pullulan grafted with guaiacol derivatives [START_REF] Kouassi | Carboxymethylpullulan grafted with aminoguaiacol: synthesis, characterization, and assessment of antibacterial and antioxidant properties[END_REF].

All of the above qualitative conclusions are fully confirmed looking at data in Table 2. No diminishing in average apparent molar masses (Mn and Mw) of PUL-FA is noticeable, evidencing the absence of chemical degradation of pullulan backbone. On the contrary, and compared to PUL, apparent molar masses of PUL-FA become larger as DSFA increases. This This associative behavior was less present with CMP-FA samples due to their anionic nature [START_REF] Dulong | Enzymatic cross-linking of carboxymethylpullulan grafted with ferulic acid[END_REF]. b DPn apparent is the apparent degree of polymerization DPn apparent = Mn / M0 c Integration zone : 15.5 to 18mL

Rheological measurements

The formation of PUL-FA hydrogels was rheologically investigated during the biomimetic crosslinking reaction of PUL-FA by laccase in citrate-phosphate buffer (0.1M, pH=5.5) . Influence of DSFA for PUL-FAs at 70 g/L with laccase at 0.5 nkat. C) Influence of PUL-FA concentration for PUL-FA-6% using laccase at 1 nkat.

All rheological analyses evidence similar profile (Fig. 3). At the beginning, both elastic (G')

and viscous (G") moduli are very low with G' lower than G" corresponding to the behavior of viscous solution. Then, both moduli increase suddenly and G' exceeds rapidly G" as gelation proceeds, to become largely higher than G". At the end, both moduli reach a plateau or a pseudo plateau indicating the end of gelation. These types of behavior are fully characteristic of a chemical crosslinking leading to the formation of a 3D elastic hydrogel. This clearly evidences the efficiency of the FA grafted moieties biomimetic dimerization induced by laccase via radical reaction [START_REF] Adelakun | Laccase-catalyzed dimerization of ferulic acid amplifies antioxidant activity[END_REF]Carvajal-Millan et al., 2005).

This gelation profile was also similar to other hydrogels formed through other physical or chemical crosslinks [START_REF] Chambon | Rheology of model polyurethanes at the gel point[END_REF]. The crossing point at G'=G" (tan δ = 1) has been subject of interesting scientific debate. Some researchers consider this crossover as the gel point while others define it as a characteristic point of gelation indicating the beginning of gel's formation by a transition from the purely liquid phase to the solid one dominated by a viscoelastic behavior. Our systems were not in accordance with the Winter-Chambon criterion [START_REF] Winter | Rheology of polymers near liquid-solid transitions[END_REF], following the loss tangent at different oscillation frequencies (Fig SI 5.). Consequently, we will use this point as a qualitative, noticeable indication correlated to the notion of percolation. At least these characteristic points (called gelation point) will permit to obtained a characteristic time (tgel) allowing us to compared the kinetics of various systems according to laccase activity, DSFA or PUL-FA concentration. Another characteristic parameter is the value of G' at the plateau which indicates the mechanical properties of hydrogels also correlated to the number of crosslinks.

Several crosslinking reactions with changing enzymatic activity (EA), concentration and DSFA, allowed us to obtain varieties of hydrogels with different kinetics and mechanical characteristics (Table 3). The values are quite close to those obtained with CMP-FA hydrogels [START_REF] Dulong | Enzymatic cross-linking of carboxymethylpullulan grafted with ferulic acid[END_REF].

The kinetics is largely accelerated when EA increases, with quite similar G' at the plateau (Fig. 4a). Laccase has impact only on the crosslinking kinetics and has no impact on the mechanical properties of final gels (same number of crosslinking points created), agreeing with the catalytic role of this enzyme in the crosslinking reaction [START_REF] Dulong | Enzymatic cross-linking of carboxymethylpullulan grafted with ferulic acid[END_REF].

The amount of FA grafted moieties onto pullulan affects both kinetics and final mechanical characteristics. The 3D network is formed more rapidly with stronger mechanical properties as DS FA increases (Fig. 4b). Laccase find easily and more quickly the grafted FA when their amount increases leading to a tighter network.

The increase of PUL-FA concentration during the laccase action induces a denser entanglement of polymer chains which improves the probability of FA grafted moieties meeting and reactivity, and consequently increases the crosslinking density (Fig. 4c).

Additionally, this phenomenon could be supported by the presence of intermolecular hydrophobic associations previously proved with SEC which could lead to a better reactivity between FA grafts. This slowdown of kinetics could be interpreted by this larger entanglement achieved with important concentrations which may affect the enzyme mobility.

The tan δ values (tan δ =G"/G') calculated for all final gels were from 0.01 to 0.04 (<1) confirm the presence of an elastic covalent system [START_REF] Lack | Hydrogels based on pullulan crosslinked with sodium trimetaphosphate (STMP): rheological study[END_REF]. The weakest hydrogel reached an elastic modulus of 170 Pa while the strongest one had value of 800 Pa. All of the hydrogels demonstrate stronger mechanical properties than those obtained by [START_REF] Carvajal-Millan | Impact of the structure of arabinoxylan gels on their rheological and protein transport properties[END_REF] from the water extractable arabinoxylans found in endosperm of wheat kernel (G' ranged from 5 to 41 Pa). This result proves that we have not only use the biomimetic crosslinking phenomenon found in nature but also, we have successfully improved the mechanical properties of hydrogels.

Swelling properties

Usually, the equilibrium swelling capacity of hydrogel is highly dependent on the molecular structure of polymer network, the crosslinking density as well as content interactions of hydrogel. Thus, we have investigated the swelling behavior of elaborated hydrogels as a function of substitution degree and PUL-FA concentration. Unlike the hydrogels obtained from CMP-FA [START_REF] Dulong | Enzymatic cross-linking of carboxymethylpullulan grafted with ferulic acid[END_REF]) that achieved the maximum swelling degree very fast (in only 20 s), swelling kinetics of PUL-FA hydrogels appears clearly slower reaching a pseudo plateau after 8 days (data not shown). This could be explained by the presence of charges on CMP-FA (anionic polysaccharide) which facilitates the penetration of the swelling solvent as a result of electrostatic repulsion increasing the distance between polymer chains. Moreover, the neutral character of PUL-FA hydrogel and presence of hydrophobic interactions explain the lower swelling degree than CMP-FA hydrogels (factor 7). The same effect was observed on swelling properties of hydrogels based on carboxymethylcellulose and hydroxyethylcellulose [START_REF] Sannino | Concurrent effect of microporosity and chemical structure on the equilibrium sorption properties of cellulose-based hydrogels[END_REF]. Finally, it can be concluded that swelling properties showed very low and slow equilibrium water content due to the non-ionic nature of PUL-FA hydrogels. All results are completely in agreement with the rheological studies with antagonistic tendencies between swelling and mechanical properties.

Antioxidant activity and cytotoxicity

Synthesis of ferulic acid dimers

The synthesis of FA dimers (DiFA) was performed using laccase from Trametes versicolor with appropriate amount via radical oxidation phenomenon. The details are given in the SI file.

Antioxidant properties: DPPH free radical scavenging assay

The antioxidant properties of FA, DiFA, PUL and PUL-FA derivatives were measured by DPPH free radical method based on calculating the decrease in the absorbance of DPPH•.

Obtained results are shown in Fig. 5. Results had shown that the radical scavenging effect increases slightly by increasing the analysis time from 5 to 20 min to reach the steady state within 20 min (Fig SI 2). Therefore, the time to evaluate antioxidant properties was fixed at 20 min.

For a better comparison, we have also determined for all tested samples, the half-inhibition concentration (EC50), a usual way to express the antioxidant activity of a compound which is defined as the minimum concentration of antioxidant agent able to scavenge 50% of DPPH•.

The higher antioxidant activity, the lower EC50 and all acquired data are set out in Table 4. Both FA and DiFA present antioxidant activity against DPPH• which increases with concentration with a slightly higher activity for FA compared to its dimers (Fig. 5A). This was proved by the EC50 values which was 5.55 µg/mL (28.6 µmol/L) for FA versus 6.53 µg/mL (33.63 µmol/L) for DiFA (Table 4). Thus, FA has a slightly higher scavenging effect than its dimers [START_REF] Dulong | Antioxidant properties and bioactivity of carboxymethylpullulan grafted with ferulic acid and of their hydrogels obtained by enzymatic reaction[END_REF]. Indeed, the creation of DiFA requires the condensation of two quinones (radicals) established by the enzymatic oxidation of FA. As a result, some of FA radicals will be involved in the formation of those dimers and then no more available for scavenging DPPH free radical which can explains the decrease in antioxidant activity of DiFA.

For FA, obtained EC50 was in agreement with literature where values of 22 μmol/L, 25.1 μmol/L, 27.8 μmol/L and 28.1 μmol/L were reported [START_REF] Brand-Williams | Use of a free radical method to evaluate antioxidant activity[END_REF][START_REF] Cassano | A novel dextran hydrogel linking trans-ferulic acid for the stabilization and transdermal delivery of vitamin E[END_REF][START_REF] Dulong | Antioxidant properties and bioactivity of carboxymethylpullulan grafted with ferulic acid and of their hydrogels obtained by enzymatic reaction[END_REF][START_REF] Gortzi | Coencapsulation of ferulic and gallic acid in hp-b-cyclodextrin[END_REF]. In comparison to its counterparts of non-flavonoid phenolic compounds, FA shown a similar antioxidant activity as for its hydroxycinnamic acid counterpart, caffeic acid (27.75 μmol/L) and lower than two hydroxybenzoic acids, syringic acid (12.2 μmol/L) and gallic acid (6.82 μmol/L) [START_REF] Gülçin | Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid)[END_REF][START_REF] Salgueiro | Phenolic composition and antioxidant properties of Brazilian Honeys[END_REF]. FA possesses also lower antioxidant properties than aminoguaiacol (13.7 μmol/L) but higher than guaiacol (33.5 μmol/L) and eugenol (71.3 μmol/L) [START_REF] Kouassi | Carboxymethylpullulan grafted with aminoguaiacol: synthesis, characterization, and assessment of antibacterial and antioxidant properties[END_REF]Pérez-Rosés, Risco, Vila, Penalver & Canigueral, 2016;[START_REF] Yu | Identification of antibacterial and antioxidant constituents of the essential oils of Cynanchum chinense and Ligustrum compactum[END_REF].

If we compare the antioxidant character of PUL-FA to CMP-FA, we find that the associative behavior of PUL-FA has a negative effect [START_REF] Dulong | Antioxidant properties and bioactivity of carboxymethylpullulan grafted with ferulic acid and of their hydrogels obtained by enzymatic reaction[END_REF].

Cytotoxicity assays

To check the cytotoxicity of the FA, DiFA, and PUL-FAs, L-929 mouse fibroblast cells were put in contact with all the compounds for 24h and 48h to examine their viability and metabolic activity, respectively. From Fig. 6A, we observe that most of cells appeared in green after fluorescence microscopy inspection, reflecting that cell membranes are not damaged.

Next, to validate these qualitative observations, the metabolic activity of cells was assessed using Alamar Blue® assay after 48h of contact. As a reminder, a percentage of AB reduction below 70% of negative control indicates a cytotoxic effect. Figure 6B shows that FA and DiFA, as well as PUL-FAs were above the threshold of 70% AB reduction. Consequently, these results clearly evidence that both FA and DiFA, and PUL-FAs at a concentration of 2.5 g/L have no cytotoxic effects towards fibroblasts, whatever the DSFA (2% and 6%).

Conclusion

New advanced biomimetic pullulan hydrogels via an eco-friendly procedure (i.e. enzymatic crosslinking) with improved mechanical and viscoelastic properties were successfully prepared introducing FA moieties onto PUL backbone. Various amount of FA was grafted onto pullulan through a carbodiimidazole mediated coupling reaction via two steps. The reaction efficiencies were very low leading to substitution degrees ranged between 2 and 10% (corresponding to 23.5 and 108.1 mg/g of FA contents in PUL-FAs). All the PUL-FAs are water-soluble, even for the highest grafting amount. In each case, intramolecular hydrophobic associations between FA groups persist and lead to aggregates or isolated dense coils with a compact conformation compared to that of precursor (PUL). Thereafter, hydrogels were obtained by enzymatic crosslinking reaction using laccase from Trametes versicolor.

Enzymatic activity only impacts the gelation kinetics as a catalyst, while substitution degree and concentration both have effects on the crosslinking kinetics, the rheological and swelling properties of PUL-FA hydrogels. An increase of mechanical properties is logically observed by increasing the DSFA or concentration of PUL-FAs due to the raise of the crosslink density and of hydrophobic interactions. Comparing to CMP-FA hydrogels, PUL-FA hydrogels exhibit slower and lower swellings in water due to the neutral character and high flexibility of pullulan backbone when compared to anionic polysaccharide. Finally, based on the standard ISO 10993-5, the cytotoxicity evaluation of FA, DiFA and PUL-FAs at 2.5 g/L demonstrate that these components are not cytotoxic towards fibroblasts. All realized studies allowed us to better understand this biomimetic phenomenon and then to well control the biomimetic hydrogels for future applications. Further studies are in progress transferring our know-how to a more rigid polysaccharide with more interest in industrial sector.

Fig. 1 .

 1 Fig. 1. Synthesis of PUL-FA conjugates. Chemical generation of imidazolide-activated FA by activation with CDI (Step 1). Esterification reaction of pullulan with FA (Step 2).

Fig. 3A illustrates

  Fig. 3A illustrates the molar masses distributions of PUL and PUL-FA-10% as a function of elution volume with both light scattering (LS at 90°) and DRI profiles. Chromatograms of PUL-FA-2% and PUL-FA-6% are shown in the SI (Fig. SI-1). Average characteristic variables obtained in LiNO3 0.1mol/L at 25°C with polymer concentration at 1 g/L are collected in Table2. The mass recovery of analyzed samples (ratio between recovered sample from integrated DRI peak and injected quantity) was about 80-90% which indicates that every sample is almost totally eluted through the columns.

Fig. 2 .

 2 Fig. 2. Elution profiles in LiNO3 0.1 mol/L at 25°C: A) from refractive index (full lines) and light scattering at 90° (dashed lines) of PUL (black) and PUL-FA-10% (red) together with molar mass distribution (dotted lines). B) from refractive index (full lines) and UV at 280nm (dashed lines) of PUL (black), PUL-FA-2% (blue), PUL-FA-6% (green) and PUL-FA-10% (red).

  is largely expected considering intermolecular associations of grafted FA and regarding the first eluted fraction of PUL-FA. Aggregated structures are also confirmed when regarding the apparent DP of PUL-FAs which are much larger than real DP according to the increase of repeating units taking into accounts the DS (i.e. M0). Moreover, the decrease of the [ɳ] when DSFA increases confirms the densification of the polymer coils because of intramolecular associations[START_REF] Dulong | Enzymatic cross-linking of carboxymethylpullulan grafted with ferulic acid[END_REF]. In our case, this result seems to indicate that intramolecular associations (main fraction) are more pronounced in dilute medium. The Mark-Houwink-Sakurada (MHS) exponent (a) based on the scaling relationship between [ɳ] and M, confirms the compact conformation of PUL-FA samples. Thus, we notice a clear decrease of (a) values when DSFA increases, with values lower than 0.5 evidencing the very compact and dense structures of single coil or aggregates due to hydrophobic associations between FA. This result fully reinforced our previous conclusions.

Fig. 3 .

 3 Fig. 3. Storage (G': full symbol) and loss (G": empty symbol) moduli versus time during the crosslinking reaction: A) Influence of enzymatic activity for PUL-FA-6% at 70 g/L B)

Fig. 4 .

 4 Fig.4 . Effect of DSFA and PUL-FA concentration on the swelling properties of hydrogels in milli Q-water

Fig. 5 .

 5 Fig. 5. A) Free-radical scavenging activity of FA and DiFA after 20 min. B) Absorbance of DPPH (100 μM) and PUL (5 g/L) without DPPH and in the presence of DPPH. C) Freeradical scavenging activity of PUL-FAs after 20 min. Equivalent concentration of FA are on the right axis.

Fig. 6 .

 6 Fig. 6. Cytotoxicity evaluation of FA, DiFA and PUL-FAs. A) Live/Dead® fluorescence microscopy of L-929 cells after 24h contacting; live and dead cells appeared green and red, respectively. B) Alamar Blue® (AB) reduction of L-929 cells after 48h in contact. Results are expressed in mean ± SEM.

Table 1 :

 1 Synthesis conditions and structural characteristics of PUL-FA according to reaction shown in Fig.1

Product nFA/nAGU a DSFA th DSFA exp b FA content c Yield PUL-FA-2%

		0.15	0.15	0.02	23.5	85%
	PUL-FA-6%	0.30	0.30	0.06	67.5	87%
	PUL-FA-10%	0.60	0.60	0.10	108.1	84%

a Molar ratio of FA over PUL AGU.

Table 2 . Results obtained with SEC/MALS/DRI/VD analyses of PUL and PUL-FA derivatives (at 25°C, in LiNO3 at 0.1 M and at 1 g/L)

 2

	Sample	Mn	Mw	M0 a	DPn apparent b	[η]w	a c	Mass recovery
		(g/mol)	(g/mol)	(g/mol)		(mL/g)		(%)
	PUL	144,000 265,000	162	889	76	0.60	90
	PUL-FA-2% 231,000 670,000	165.5	1,400	70	0.43	87
	PUL-FA-6% 257,000 804,000	172.6	1,489	67	0.42	80
	PUL-FA-10% 334,000 815,000	179.6	1,859	57	0.39	70

a M0 is the molar mass of the repetition unit M0 = 162 + (176.2 x DSFA)

Table 3 . Rheological data of crosslinking reactions of PUL-FA derivatives in citrate-

 3 Values measured at the plateau at the end of the crosslinking reaction

	phosphate buffer (0.1M, pH=5.5) at 25°C
	Sample	[C]	EA (nkat) tgel (min) G' a (Pa)	G'' a (Pa)	tan δ a
		(g/L)
	PUL-FA-2%	70	0.5	55	170	2	0.01
		40	1	13	260	4	0.02
		70	0.5	33	340	9	0.03
	PUL-FA-6%	70	1	17	380	13	0.03
		70	2	8	410	17	0.04

a

Table 4 . Scavenging effects and half-inhibition concentrations of the DPPH radical (EC50) of FA, DiFA and PUL-FA derivatives

 4

	Product	[C]	[FA]eq	Scavenging effect	EC50
		(g/L)	(g/L)	(%)	(g/L)
	FA	0.01	0.01	76.4 ± 0.2	5.55 × 10 -3

Acknowledgments

We thank the French "Ministe re de l'Enseignement Supérieur, de la Recherche et de l'Innovation" for financial support of Ahdi Hadrich. We also thank the "Re ǵion Normandie" and the European Union for financial help in the purchase of equipment.

Unlike FA and its dimers, PUL did not present any antioxidant activity (Fig. 5B). We can note that the maximum of PUL precursor with DPPH• curve has an absorbance value like the DPPH• alone. This was also confirmed by the visual aspect of the analyzed samples where no change in color of DPPH• in contact of PUL was observed.

The antioxidant properties of PUL-FAs were also evaluated (Fig. 5C). Results demonstrated that all grafted PUL present antioxidant properties which increased with concentration and DSFA. As PUL shows no antioxidant activity, antioxidant properties of PUL-FAs are then provided by FA grafted moieties. Calculated values of EC50 (Table 4) of PUL-FA-2%, PUL-FA-6% and PUL-FA-10% decrease logically with DSFA increasing and were respectively 9.7 g/L (58.6 mmol/L) , 6 g/L (34.7 mmol/L) and 0.9 g/L (5 mmol/L). These values are more important than EC50 of FA which confirms a significant decrease in the antioxidant activity of FA when it's grafted onto PUL. This was also confirmed by calculating the grafted FA equivalent concentration [FA]eq in PUL-FA which was added to Fig. 5C on the right vertical axis. In comparison to the tested concentration of FA, the [FA]eq was always higher.

Nevertheless, scavenging effect of PUL-FAs was always much lower than those of FA which proves our previous conclusion. This could be attributed to the difficult accessibility of DPPH radicals to FA moieties when they are grafted onto PUL due to the greater difficulty of DPPH• diffusion into polymer solution than a molecular one.

Obtained EC50 value for PUL-FA-10% (6 g/L) can be compared to the EC50 of FA grafted starch (0.34 g/L) [START_REF] Wen | Corn starch ferulates with antioxidant properties prepared by N,N′-carbonyldiimidazole-mediated grafting procedure[END_REF]. In this case, EC50 is lower, reflecting higher antioxidant properties due to the higher FA content for starch-FA derivative (303.9 mg of FA / g of starch-FA derivative). It should be noted that for FA grafted starch, another method was used to determine antioxidant properties due to the poor water solubility of starch-FA derivative. Ferulic acid was also grafted onto chitosan with FA content (66.7 mg of FA / g of chitosan-FA derivative) approximatively similar to our PUL-FA-6% (67.5 mg of FA / g of PUL-FA derivative). The EC50 of chitosan-FA derivative (0.75 g/L) is again lower [START_REF] Liu | Free radical mediated grafting of chitosan with caffeic and ferulic acids: Structures and antioxidant activity[END_REF]. But this time, this is could be explained by the perfect solubility of this derivative in the medium used for antioxidant activity assays as well as the presence of amine function on the chitosan backbone which brought intrinsic antioxidant activity to this polysaccharide.


Jamerson Carneiro De Oliveira

Severinne Rigolet

Claire Marichal

Vincent Roucoules

Marie-Pierre Laborie

Grafting Diels-Alder moieties on cellulose nanocrystals through carbamation

Keywords: Cellulose Nanocrystals, Carbamation, Diels-Alder, Spectroscopy Hypothesis

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cellulose nanocrystals (CNCs) are stiff cellulose nanomaterials typically isolated by the cleavage of the disordered regions of cellulose through acid hydrolysis [START_REF] Foster | Current characterization methods for cellulose nanomaterials[END_REF]. The application of cellulose nanocrystals was first explored in the context of reinforced composites [START_REF] Favier | Nanocomposite materials from latex and cellulose whiskers[END_REF] and has been ever since the subject of numerous studies for composites [START_REF] Oksman | Review of the recent developments in cellulose nanocomposite processing[END_REF]. In the early 2000s, efforts at compatibilizing CNCs with hydrophobic matrixes led to advances in their surface modifications [START_REF] Azizi Samir | Preparation of Cellulose Whiskers Reinforced Nanocomposites from an Organic Medium Suspension[END_REF][START_REF] Yuan | Surface acylation of cellulose whiskers by drying aqueous emulsion[END_REF]. This further broadened the field of applications of CNCs reaching out to papermaking, biosensors, water purification, food and pharmaceutical industries in more recent years [START_REF] Mohan | Applications of nanomaterials: Advances and key technologies[END_REF][START_REF] Xie | Applications of Cellulose Nanocrystals:A Review[END_REF].

For more advanced applications, a precise grafting of reactive species onto cellulose nanocrystals is often required. To that end, click chemistry has been implemented on CNCs based on thiol-ene reactions [START_REF] Aalbers | Post-modification of Cellulose Nanocrystal Aerogels with Thiol-Ene Click Chemistry[END_REF][START_REF] Chen | Highly alkynyl-functionalization of cellulose nanocrystals and advanced nanocomposites thereof via click chemistry[END_REF][START_REF] Huang | Functionalization of cellulose nanocrystal films via "thiol-ene" click reaction[END_REF], copper-catalyzed Huisgen reactions [START_REF] Filpponen | Regular linking of cellulose nanocrystals via click chemistry: Synthesis and formation of cellulose nanoplatelet gels[END_REF][START_REF] Sadeghifar | Production of cellulose nanocrystals using hydrobromic acid and click reactions on their surface[END_REF] and Diels-Alder reactions (García-Astrain et al., 2016;[START_REF] Shao | A Self-Healing Cellulose Nanocrystal-Poly(ethylene glycol) Nanocomposite Hydrogel via Diels-Alder Click Reaction[END_REF]. The Diels-Alder (DA) reaction is a pericyclic [4+2] cycloaddition between a conjugated diene and a dienophile [START_REF] Diels | Synthesen in der hydroaromatischen Reihe[END_REF], that has been repeatedly implemented on polysaccharides after homogeneous grafting [START_REF] Elschner | Furfuryl-and Maleimido Polysaccharides: Synthetic Strategies Toward Functional Biomaterials[END_REF]. In particular, furyl and maleimido modified polysaccharides have been successfully synthesized to deliver materials with selfhealing properties, for tissue engineering and drug release [START_REF] Elschner | Furfuryl-and Maleimido Polysaccharides: Synthetic Strategies Toward Functional Biomaterials[END_REF]. Fewer publications have addressed the grafting of Diels-Alder moieties on cellulose nanomaterials. [START_REF] Navarro | Multicolor fluorescent labeling of cellulose nanofibrils by click chemistry[END_REF] grafted furyl moieties onto cellulose nanofibrils (CNFs) that were further reacted with maleimides to produce different fluorescent probes. The presence of the moieties was proven by solid state NMR, but with a low degree of substitution (~1%). García-Astrain et al., 2016 prepared bionanocomposite hydrogels with maleimido grafted CNCs and furan modified gelatin. The hydrogel was crosslinked by Diels-Alder and higher storage moduli was observed when compared to controls not containing Diels-Alder reactive functions. [START_REF] Trovatti | Furan-modified natural rubber: A substrate for its reversible crosslinking and for clicking it onto nanocellulose[END_REF] also crosslinked a maleimide-modified nanocellulose with a furanmodified natural rubber. The crosslink between the modified nanocellulose and natural rubber was proven via an increase in storage modulus when compared to a reference sample. Furyl modified CNCs were also synthetized by [START_REF] Shao | A Self-Healing Cellulose Nanocrystal-Poly(ethylene glycol) Nanocomposite Hydrogel via Diels-Alder Click Reaction[END_REF] by nucleophilic addition of furfural on CNCs. The furyl modified CNCs were reacted with maleimido terminated poly(ethylene glycol) to create a nanocomposite hydrogel. The nanocomposites had their mechanical, morphological, swelling and self-healing properties investigated. The self-healing observed was attributed to the thermoreversibility of DA reaction. More recently, [START_REF] Kramer | Thermally reversible nanocellulose hydrogels synthesized via the furan/maleimide Diels-Alder click reaction in water[END_REF] investigated the reversibility of the DA reaction when furyl modified cellulose nanofibers were allowed to react with a bismaleimide. The thermalreversibility of the system was demonstrated through the changes in viscosity of the formed hydrogels. Due to the limited number of studies on the field, the grafting pathways for the implementation of the DA chemistry remains largely unexplored and it is of interest to revisit grafting chemistries that are commonly used for CNCs modification.

In this context, the present work aims at exploring alternative routes to graft DA moieties on CNCs towards the preparation of materials containing clickable CNCs. As a spacer is typically required to graft reactive species on CNCs, we selected the carbamation reaction with diisocyanates to graft DA moieties. The carbamation has been repeatedly used to graft functionalities onto cellulose nanocrystals [START_REF] Abushammala | A Review of the Surface Modification of Cellulose and Nanocellulose Using Aliphatic and Aromatic Mono-and Di-Isocyanates[END_REF][START_REF] Siqueira | New process of chemical grafting of cellulose nanoparticles with a long chain isocyanate[END_REF]. In particular, diisocyanates are potent spacers for grafting desirable moieties on polysaccharidic nanocrystals [START_REF] Habibi | Highly filled bionanocomposites from functionalized polysaccharide nanocrystals[END_REF][START_REF] Thielemans | Starch nanocrystals with large chain surface modifications[END_REF][START_REF] Labet | Polymer grafting onto starch nanocrystals[END_REF][START_REF] Mano | P(CL-b-LLA) diblock copolymers grafting onto cellulosic nanocrystals[END_REF][START_REF] Shang | Hydrophobic modification of cellulose nanocrystal via covalently grafting of castor oil[END_REF][START_REF] Zoppe | Reinforcing poly(epsilon-caprolactone) nanofibers with cellulose nanocrystals[END_REF]. A slightly different approach was followed by [START_REF] Morandi | Cellulose nanocrystals grafted with polystyrene chains through surface-initiated atom transfer radical polymerization (SI-ATRP)[END_REF] with the aim to graft photolinkers onto CNCs. Recently, Abushammala et al. quantified the reactivity of toluene diisocyanate (TDI) towards cellulose nanocrystals for application in composites (Abushammala, 2019a[START_REF] Abushammala | On the Para/Ortho Reactivity of Isocyanate Groups during the Carbamation of Cellulose Nanocrystals Using 2,4-Toluene Diisocyanate[END_REF][START_REF] Abushammala | Nano-Brushes of Alcohols Grafted onto Cellulose Nanocrystals for Reinforcing Poly(Butylene Succinate): Impact of Alcohol Chain Length on Interfacial Adhesion[END_REF]. Previous literature use a procedure for modification of the CNCs through a first carbamation of the CNCs followed by the grafting of the desired molecule. In the current work we aim to explore the carbamation chemistry through an in-situ approach and access qualitatively and quantitatively the grafting efficacy.

With a view to implementing the DA chemistry on cellulose nanocrystals, this study aims at investigating in detail the efficacy of grafting furan and maleimide moieties on cellulose nanocrystals via carbamation with toluene diisocyanate. The impact of reaction conditions on the carbamation and the DA moieties grafting reaction are studied for two pairs of DA moieties.

Materials and Methods

Materials

Furfuryl alcohol (FAL, analytical standard, Sigma Aldrich), toluene 2,4-diisocyanate (TDI, analytical standard, Sigma Aldrich), 3a,4,7,7a-tetrahydro-2-(2'-hydroxyethyl)-4,7-epoxy-1H-isoindole-1,3(2H)-dione (pN2HM, 98%, Sigma Aldrich), exo-N-Hydroxy-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboximide (pNHM, 98%, Santa Cruz Biotechnology), cellulose microcrystalline (Avicel® PH-101, ~50 μm particle size, Sigma Aldrich) were used as received.

Cellulose Nanocrystals Production

Cellulose nanocrystals were produced via an adaptation of the conditions reported elsewhere [START_REF] Bondeson | Optimization of the isolation of nanocrystals from microcrystalline cellulose by acid hydrolysis[END_REF]. 13.7 g of microcrystalline cellulose were added to 200 g of a freshly prepared sulfuric acid solution (63.5 wt.%). The system was mechanically stirred for 2 hours at 44 ºC. After that, the mixture was poured into cold water and successively centrifuged (12000 rpm, 10 min -Heraeus Mutifuge X3, Thermo Fisher) with addition of water to the precipitated until a translucent suspension was collected. This suspension was dialyzed against deionized water until a constant pH was reached. CNCs dimensions measured from height profile in atomic force microscopy (AFM) were equal to 213 ± 50 nm in length and 5 ± 1 nm in width (Figure S1).

Surface Modification of Cellulose Nanocrystals

Furfuryl alcohol (FAL) and two protected maleimides (pNHM and pN2HM) were grafted onto the CNCs surface. Furan protected maleimides had to be used, as unprotected maleimides directly polymerize with TDI. The scheme below shows the ideal pathway of grafting procedure. The grafting conditions followed the same experimental plan for the three different DA moieties. CNCs dispersions were concentrated in a rotary evaporator. Addition of acetone followed by centrifugation (12000 rpm, 10 min -Heraeus Mutifuge X3, Thermo Fisher) allowed precipitation. For removal of water, acetone absolute was added to the centrifuge tube and the sample was well mixed and sonicated (30 sec., 60% power -Sonoplus HD 2200, Bandelin). The sample was once more centrifuged.

The last two steps were repeated two more times. Under argon flow, the diene or dienophile was added dropwise to dimethyl sulfoxide (DMSO) where the correspondent amount of toluene diisocyanate was previously solubilized. Regardless of the experiment, the molar ratio between TDI and diene or dienophile was always 1:1. After one hour of reaction, absolute dimethyl sulfoxide was added to the precipitated CNCs. The CNCs were well mixed and added to the reacting system. After the selected time, grafted CNCs were precipitated with acetone and centrifuged (12000 rpm, 10 min -Heraeus Mutifuge X3, Thermo Fisher). The supernatant was removed, acetone was added to the sample and the system sonicated (30 sec., 60% power -Sonoplus HD 2200, Bandelin). Centrifugation, supernatant removal, addition of acetone and sonication were performed two more times.

A factorial Design of Experiments (DOE) was used with two selected factors in two levels for each of the molecules to be grafted (Table 1). Reaction time and molar ratio between TDI and hydroxyl present on CNCs surface were selected as factors.

(+1) 3 (+1) 6x3d 2 6 (+1) 1 (-1) 6x1d 3 2 (-1) 3 (+1) 2x3d 4 2 (-1) 1 (-1) 2x1d Central point 4 (0) 2 (0) 4x2d

Determination of Surface Degree of Substitution

The surface degree of substitution (DSSurf)1 of the cellulose nanocrystals was calculated following the procedure from Eyley & Thielemans, 2014 from CHNS elemental analysis. However, some publications use XPS to calculate surface degree of substitution (DSS) of modified cellulose nanomaterials [START_REF] García-Astrain | Maleimide-grafted cellulose nanocrystals as cross-linkers for bionanocomposite hydrogels[END_REF][START_REF] Missoum | Nanofibrillated cellulose surface grafting in ionic liquid[END_REF]. Although, the design of experiments in the present work will deal with results from elemental analysis, DSSs were calculated with the intention of comparing the results from the different techniques. The DSS was determined through a similar approach as the one used by [START_REF] Andresen | Properties and characterization of hydrophobized microfibrillated cellulose[END_REF] and Le Gars, Roger, Belgacem, & Bras, 2020. The mass fraction of nitrogen on the surface can be written as:

= = × + ×
That can be rewritten as:

6 = × - ×
Where: is the molar mass of one repeating glucose unit (162 g/mol), is the mass fraction of nitrogen in the sample, is the molar mass of nitrogen atoms that are grafted according the pathway shown in Scheme 1 (28 g/mol for the diene and 42 g/mol for dienophiles) and is the molecular weight of the grafted molecule as also shown in Scheme 1 (272 g/mol for FAL, 355 g/mol for pNHM and 383 g/mol for pN2HM).

Solid State NMR Spectroscopy

1 H-13 C Cross Polarization Magic Angle Spinning (CPMAS) NMR spectra have been recorded at room temperature on a Bruker Avance NEO 400WB spectrometer (B0=9,4T) operating at 100.62 MHz for 13 C and 400.14 MHz for 1 H. Samples were packed in a 4mm diameter cylindrical zirconia rotor and spun at a spinning frequency of 12 kHz. 1 H-13 C CPMAS NMR experiments were performed with a proton π/2-pulse duration of 5.1 μs, a contact time of 2 ms, and a recycle delay of 3 s (according to T1 measurements) and high-power decoupling. 13 C chemical shifts are relative to Tetramethylsilane (TMS). Freeze-dried samples were used in the analysis.

Fourier-transform Infrared Spectroscopy (FTIR)

Samples were mixed in KBr (1-2 wt%) and pulverized in a mortar prior to forming pellets. The pellets were analyzed on a FT-IR 65 spectrometer (PerkinElmer) by acquiring 32 scans with a resolution of 4 cm -1 in the range from 650-4000 cm -1 . Data were recorded using Spectrum software in the slide holder mode and with an automatic background correction.

X-ray Photoelectron Spectroscopy (XPS)

X-ray photoelectron spectroscopy spectra were recorded with a VG SCIENTA SES-2002 spectrometer equipped with a concentric hemispherical analyzer. The incident radiation used was generated by a monochromatic Al Kα x-ray source (1486.6eV) operating at 420 W (14kV; 30mA). Photo-emitted electrons were collected at a takeoff angle of 90º from the surface substrate, with electron detection in the constant analyser energy mode (FAT). Wide scan spectrum signal was recorded with a pass energy of 500 eV and for high resolution spectra pass energy was set to 100 eV. The analysed surface area was approximately 24 mm 2 and the base pressure in the analyse chamber during experimentation was about 10-9 mbar. Charging effects on these isolating samples were compensated by using of a Flood Gun. The spectrometer energy scale was calibrated using the Ag 3d 5/2 , Au 4f 7/2 and Cu 2p 3/2 core level peaks, set respectively at binding energies of 368.2, 84.0 and 932.6 eV. All the data analysis was accomplished using the specific commercial software CasaXPS, version 2.3.18 (CasaXPS Software Ltd, Teignmouth, UK). The surface composition expressed in atom% was determined using integrated peak areas of each components and take into account transmission factor of the spectrometer, mean free path and Scofield sensitivity factors of each atom. All the binding energies (BE) are referenced to the C1s (C-C, CHx) at 285.0 eV and given with a precision of 0.1eV. Double-sided adhesive tape was affixed on sample holder and freeze-dried powders were pressed onto it. Samples were introduced first into the entry lock and after degassing in the analysis chamber.

X-ray Diffraction (XRD)

Wide angle X-ray diffraction measurements were carried out in a X'Pert Pro diffractometer using Cu Kα as a radiation source at 45 kV and 40 mA. Patterns were recorded in the range 2θ = 5-40º with a step size of 0.013º. Hand-milled powders were analysed in a 16mm diameter steel sample holder.

Crystallinity of cellulose was analyzed according to the procedure from [START_REF] Segal | An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer[END_REF]:

! = ! (##$) -! ( &) ! (##$) × 100
Where: ! (##$) is the signal intensity corresponding to the crystalline domain (22.5º) and ! ( &) is the signal intensity for the amorphous domain (18.3º).

CHNS Elemental Analysis

1-3 mg of freeze-dried sample were mixed with tungsten oxide and measured in a vario EL III (Elementar Analysensysteme GmbH, Germany) with a thermal conductivity detector (TDC). Oxygen was used for oxidation and helium as transport gas.

Results and Discussion

X-ray Crystallography

Figure 1 shows the diffraction patterns of cellulose nanocrystals before and after the respective grafting reactions (harshest conditions). X-ray diffraction patterns of pristine CNCs showed that cellulose did not change morphology after acid hydrolysis as the characteristic peaks from native cellulose I were observed at 14.7, 16.4, 22.4 and 34.4 °2θ corresponding to cellulose I -(11 ) 0), ( 110), ( 200) and (004), respectively. The crystalline pattern is not affected by the grafting reaction. Additionally, the apparent crystallinity Index of the pristine CNCs (76%) is maintained after the grafting reaction. Despite limitations in the crystallinity measurement approach [START_REF] Park | Cellulose crystallinity index: Measurement techniques and their impact on interpreting cellulase performance[END_REF] , the comparative value of the method is warranted. The permanence of the cellulose I morphology and the apparent crystallinity index after reaction confirm that any grafting must have occurred on the nanocrystals surface, leaving the bulk crystalline region unaffected. That conclusion is supported by the AFM image of the sample pNHM (Figure S2), which shows no considerable difference on the functionalized CNCs morphology.

Figure 1 -Diffraction pattern of pristine and modified CNCs.

Fourier-Transform Infrared Spectroscopy

FTIR spectra of pristine and grafted cellulose nanocrystals reveal the existence of urethane bonds as expected according to the TDI grafting reaction (Figure S3). The free C=O stretching of the urethane is seen at 1720 cm -1 (amide I) the ones at 1541 cm -1 and 1236 cm -1 (amide II and III, respectively) could be attributed to the amine bending and C-N stretching. The appearance of a band at 1619 cm -1 (Figure 2) possibly indicates a side reaction generating another amide I peak (urea). However, TDI presents a peak in the same region and the observed peak could come from its backbone.

Qualitative comparison of the peak intensities after a normalization shows the influence of time and concentration on the grafting of furfuryl alcohol on cellulose nanocrystals (Figure 2). The influence of the parameters for the modification with pNHM and pN2HM can be seen in Figure S4 in the supplementary information. For the TDI/NOH ratio of 2, no significant difference is seen between the samples; whereas an increase in TDI/NOH ratio to 4 or 6 increases the urethane formation. Despite the possible presence of water in the samples, a decrease in the hydroxyl signal at 3350 cm -1 , is an indication that the carbamation takes place with the cellulose nanocrystals. Similar peaks for surface grafting with TDI have been reported [START_REF] Habibi | Highly filled bionanocomposites from functionalized polysaccharide nanocrystals[END_REF][START_REF] Labet | Polymer grafting onto starch nanocrystals[END_REF][START_REF] Thielemans | Starch nanocrystals with large chain surface modifications[END_REF][START_REF] Zoppe | Reinforcing poly(epsilon-caprolactone) nanofibers with cellulose nanocrystals[END_REF]. Urethane bond formation is also clearly affected by time and concentration for pNHM modified CNCS (Figure S4(a)), with the central point experiment (4x2d) being the second strongest response. Although no quantitative analysis can be done with certainty from FTIR, these results suggest that urethane formation increases with reaction time and TDI concentration. This is also the case when grafting pN2HM on CNCs (Figure S4(b)).

X-ray Photoelectron Spectroscopy

To quantitatively characterize the grafting efficiency, the surface degree of substitution (DSS) was computed from wide-scan XPS spectra (Figure 3) based on the elemental composition of the sample surface and nitrogen in particular (Table 2). [START_REF] Sapieha | X-Ray photoelectron study of the plasma fluorination of lignocellulose[END_REF][START_REF] Shang | Hydrophobic modification of cellulose nanocrystal via covalently grafting of castor oil[END_REF]. The value of 0.73 here obtained is comparable to the one reported by [START_REF] Shang | Hydrophobic modification of cellulose nanocrystal via covalently grafting of castor oil[END_REF] for cellulose nanocrystals.

In the present work, the presence of nitrogen after reaction facilitates the detection and interpretation of the XPS results. The initial evidence of grafting is the appearance of nitrogen atoms on the surface of the CNCs, absent in the pristine CNCs. Expectedly, grafting maleimides brings more nitrogen to the CNC surface than grafting furfuryl alcohol (Table 2). Further indication of the surface grafting comes from the decrease in the O/C ratio after reaction. O/C ratio for the ideal grafting moieties (TDI + diene/dienophile) present lower values than the one of cellulose: 0.38, 0.47 and 0.42 for FAL, pNHM and pN2HM respectively. Although, in theory, only two peaks should be noticed for pure cellulose, the one at 285.0 eV arises from impurities on cellulose such as lignin and fatty acids [START_REF] Sapieha | X-Ray photoelectron study of the plasma fluorination of lignocellulose[END_REF]. Six peaks were decomposed in the present work (Table 3). Evidence of grafting is given by a decrease in C2 and C3 and an increase in C1, showing that the surface is now richer in aliphatic carbons. Urethane bonds (C6) confirm the presence of reacted TDI. Deconvolutions also reveal that side reactions occur, generating the peaks at 288.3 eV in the experiment with FAL and at 289.2 eV for the reaction with pN2HM. The theoretical difference between amide and imide bonds in C1s spectrum should be of about 0.5 eV [START_REF] Beamson | High resolution XPS of organic polymers: The Scienta ESCA300 database / G. Beamson and D[END_REF], but the difference is not high enough to differentiate those bonds, with each one varying around 0.3 eV. That makes difficult to conclude about imide or amide amount on the surface. One could nevertheless infer that the increase in C4, when comparing the dienophiles to the diene, comes from the imide presence.

CHNS Elemental Analysis

CHNS Elemental Analysis of pristine CNCs was performed for three different batches (Table S1). The amount of sulfate groups did not vary significantly for all the samples analysed, showing that the isolation of the cellulose nanocrystals from the microcrystalline cellulose provided replicable results. The values of sulfur DS obtained are consistent to what was previously reported in literature for the same production method [START_REF] Brioude | Role of Cellulose Nanocrystals on the Microstructure of Maleic Anhydride Plasma Polymer Thin Films[END_REF].

The surface (DSSurf) degree of substitution for CNCs grafting with the diene/dienophiles was also determined through elemental analysis (Table 4). [START_REF] Girouard | Site-Selective Modification of Cellulose Nanocrystals with Isophorone Diisocyanate and Formation of Polyurethane-CNC Composites[END_REF]. Low values of DSsurf might influence the possibility of performing the Diels-Alder click chemistry, especially as the molecules have their reactivity possibly limited once grafted to the surface. The percentage of carbon also increased after the surface modification, as expected. It is clear that the grafting of FAL for lower TDI/NOH ratio was not efficient, confirming the results from FTIR. A possible effect of the time and the concentration on the efficiency of the grafting can also be observed by their influence in the calculated DSSurf. Several samples presented a slight increase on the mass percentage of sulfur element.

Although that might seem contradictory to the effect of grafting sulfur free molecules, the increase in that element can be easily explained by the plausible presence of adsorbed DMSO on the hydroxyl rich CNCs. When the central points of the different samples are analysed (Table S2), it confirms the trend observed in XPS where the highest substitution occurred with pNHM followed by pN2HM and FAL respectively. It is gratifying that both techniques provided consistent results.

TDI/NOH ratio was clearly a more influential factor than time for the grafting of FAL (Figure S5). Similar results were obtained for the grafting of pNHM (Figure S6(a)) and pN2HM (Figure S6(b)). The main effect of TDI/NOH ratio on FAL samples did not vary in slope between the two levels and the central point, possibly indicating that further increase in the ratio would lead to a proportional increase in the grafting. However, the change in slope of TDI/NOH ratio's effect for the samples pNHM and pN2HM suggests that further increase in surface grafting might require a disproportionally higher molar ratio to increase the grafting. Time showed to have limited effect in all the samples after 2 days.

Nuclear Magnetic Resonance Spectroscopy

13 C CP/MAS NMR spectra of samples grafted with FAL and pNHM were performed with the intention of singularizing the presence of the desired grafted moiety. Both spectra (see Figure 5 and S7) exhibit the resonances characteristic of cellulose detected between 50 and 110 ppm in agreement with the literature [START_REF] Castro | Surface grafting of cellulose nanocrystals with natural antimicrobial rosin mixture using a green process[END_REF]. In the case of FAL sample (Figure 5), additional resonances are detected around 110 ppm corresponding to CVI, CII, Cb and Cc and around 130 ppm assigned to CI, CIII, CIV and CV. Resonances detected at 144, 150 and 155 ppm can correspond to Ca, Cx and C0, respectively. The resonance at 18 ppm is assigned to CVII. Unfortunately, the signal corresponding to Cd, expected around 60 ppm, cannot be resolved from the resonances corresponding to cellulose. Nevertheless, 13 C CPMAS NMR proves that the TDI-FAL moieties are intact after reaction. Furthermore, the absence of signal at 125 ppm corresponding to isocyanate groups proves that TDI fully reacted. Note that the intensities of the resonances corresponding to the grafted moieties are significantly lower than those resulting from CNC in agreement with the low grafting yield. For the sample reacted with pNHM (Figure S7), the proximity of most of its resonances and either the cellulose or TDI complicated chemical shift assignment.

The presence of a resonance at 40 ppm in the spectra correspond to DMSO, which was observed as residual via elemental analysis. Nevertheless, the peak at 44.9 ppm is assigned to Cd and Ce of pNHM moieties. The broad resonance between 110-120 ppm can correspond to CII and CVI. The one at 130 ppm is assigned to CIII, CIV and CV whereas the one at 136 ppm can correspond to CI, Ca and Cb. The imide carbon could not be observed in the expected range (170-180 ppm), due to the poor magnetisation transfer from neighboring protons and the low DSS already reported. The resonance at 80 ppm corresponding to both Cf and Ce cannot be resolved from cellulose ones whereas the one between 15 and 17 ppm is assigned to the methyl groups that seems to have two distinct environments. Finally the resonance at 155 ppm corresponds to both C0 and urethane moieties created upon grafting to cellulose. In conclusion, the 13 C CPMAS NMR spectrum suggests that the grafted moieties are intact after reaction.

The superposition of the spectra (not shown) indicates that the ratio between the characteristic resonances of the cellulose and those corresponding to the FAL and pNHM moieties is greater in the case of the latter, which corroborates the grafting rates obtained from the elementary composition of the samples.

Conclusion

Grafted and pristine cellulose nanocrystals were well characterized through spectroscopic analysis. XRD revealed that the cellulose I morphology and apparent crystallinity was unaffected by the grafting reaction. FTIR, XPS, CHNS elemental analysis and 13 C CP/MAS NMR provided qualitative and quantitative evidence of the grafting efficiency. Semi quantitative analysis by FTIR and quantitative CHNS elemental analysis revealed that reaction time and TDI molar ratio had a positive impact on the degree of substitution for all three DA moieties. XPS results corroborated urethane bond formation and the favorable impact of reaction time and TDI molar ratio on the surface degree of substitution for all three DA moieties. 13 C CP/MAS NMR evidenced grafting efficiency and nature, at last for furfuryl alcohol. XPS further suggested that side reactions likely interfere with the grafting efficiency and the relatively low values of DSS might influence the performance of click chemistries on these surfaces. These findings and the precise control demanded by the application of click chemistries on materials daunt the use of the tested carbamation in that field. However, these results shed some light on the advantages and drawbacks of grafting DA moieties on CNCs through carbamation for applications beyond the more commonly explored hydrophobization.

  Scheme 1 -Ideal pathway for grafting of hydroxyl terminated molecules on cellulose nanocrystals via carbamation with toluene diisocyanate.

Figure 2 -

 2 Figure 2 -Expanded FTIR spectrum of FAL grafted CNCs.

Figure 3 -

 3 Figure 3 -Wide-scan XPS spectra of pristine and grafted CNCs.

Cellulose

  C1s carbons are commonly separated in three peaks: one at 285.0 eV due to aliphatic carbons, one at 286.6 eV due to C-O-and C-OH and one at 288.2 eV due to acetal -O-C-O-(Belgacem, Czeremuszkin, Sapieha, & Gandini, 1995) (Figure 4).

Figure 4 -

 4 Figure 4 -Deconvoluted carbon C1s XPS spectra: (a) pristine CNCs, (b) FAL grafted CNCs, (c) pNHM grafted CNCs and (d) pN2HM grafted CNCs.

Figure 5 -

 5 Figure 5 -13 C CP/MAS spectra of cellulose nanocrystals modified with furfuryl alcohol (sample 4x2d)

Table 1 -

 1

DOE for surface modification of CNCs Experiment TDI/NOH ratio Time (days) Sample Code 1 6

Table 2 -

 2 Elemental composition of pristine and grafted CNCs

	Sample	C %	O %	N %	S %	O/C	N/C	DSS
	CNC	56.8	41.7	0.0	0.7	0.73	0.00	0.00
	FAL	59.5	38.3	1.5	0.7	0.64	0.03	0.11
	pNHM	59.6	36.5	3.2	0.7	0.61	0.05	0.18
	pN2HM	60.3	36.5	2.6	0.6	0.60	0.04	0.13
	Oxygen to carbon ratio for pure cellulose should theoretically equal 0.83, but lower
	calculated values of O/C ratio are commonly reported in the literature and are
	attributed to impurities on cellulose surface

Table 3 -

 3 Functional groups deconvolution for C1s signals of grafted CNCs

		C1	C2	C3	C4	C5	C6
		C-C/C-H	C-O	O-C-O	N-C=O/C=O	O-C=O	NCOOR
	BE (ev)	285.0	286.6	288.2	288.3	289.2	289.6
	CNCs	6.4%	76.0% 17.6%	0.0%	0.0%	0.0%
	FAL	13.0%	70.3% 13.7%	1.2%	0.0%	1.8%
	pNHM	18.5%	64.2% 11.8%	2.6%	0.0%	2.9%
	pN2HM	15.8%	66.9% 10.3%	4.3%	0.9%	2.0%

Table 4 -

 4 Mass percentages values obtained through CHNS EA of grafted CNCs and calculated DSSurf DSsurf) increases with reaction time and molar ratio (Table4). It remains overall rather low in absolute value, not exceeding 0.3. In comparison, successful Huisgen click chemistry was performed by Chen et al., 2015 on cellulose nanocrystals with 81.7% surface degree of substitution (~1.2) and carbamation with isophorone diisocyanate was reported to achieve a DSsurf of 1.4

	Sample	%C	%H	%N	%S	DSsurf
	CNCs	39.45	6.25	0.00	0.91	0.00
		2x1d 42.21	6.43	0.01	0.88	0.00
		2x3d 43.03	6.36	0.03	0.51	0.00
	FAL	4x2d 43.31	6.15	0.86	1.11	0.12
		6x1d 44.97	6.16	1.13	1.03	0.16
		6x3d 46.09	6.01	1.66	0.43	0.25
		2x1d 44.96	5.98	1.07	1.17	0.10
		2x3d 44.33	6.16	1.77	1.68	0.17
	pNHM	4x2d 44.16	5.99	2.17	1.58	0.22
		6x1d 45.05	6.09	2.13	0.95	0.22
		6x3d 45.66	5.93	2.75	1.62	0.30
		2x1d 43.41	6.26	0.58	1.07	0.05
		2x3d 42.93	6.30	0.57	1.09	0.05
	pN2HM	4x2d 44.25	6.04	1.66	0.95	0.16
		6x1d 44.65	6.04	1.67	0.84	0.16
		6x3d 46.63	5.89	2.54	1.64	0.27
	Grafting efficiency (

Notation DSSurf will be used when values from surface degree of substitution are calculated from elemental analysis and DSS from XPS.

Acknowledgments

J.C.O. acknowledges the doctoral scholarship from Elisabeth and Barbara Grammel Foundation at the University of Freiburg. The authors would like to thank: Philippe Fioux for performing the XPS experiment and the deconvolution, Elke Stibal and Lothar Fischer for the technical assistance, Ludovic Josien and Laure Michelin for their assistance with the XRD measurements.