Self-healing stimuli-responsive cellulose nanocrystal hydrogels

Journal Pre-proof Self-healing stimuli-responsive cellulose nanocrystal hydrogels Juntao Tang, Muhammad Umar Javaid, Chunyue Pan, Guipeng Yu, Richard M. Berry, Kam Chiu Tam

PII:

S0144-8617(19)31154-3

DOI:

https://doi.org/10.1016/j.carbpol.2019.115486

Reference:

CARP 115486

To appear in:

Carbohydrate Polymers

Received Date:

30 July 2019

Revised Date:

12 October 2019

Accepted Date:

15 October 2019

Please cite this article as: Tang J, Javaid MU, Pan C, Yu G, Berry RM, Tam KC, Self-healing stimuli-responsive cellulose nanocrystal hydrogels, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115486

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Self-healing stimuli-responsive cellulose nanocrystal hydrogels

Juntao Tang1*, Muhammad Umar Javaid1, Chunyue Pan1, Guipeng Yu1, Richard M. Berry2, Kam Chiu Tam3*

College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China

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CelluForce, Inc., 625 President-Kennedy Ave., Montreal, Quebec, Canada H3A 1K2

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Department of Chemical Engineering, Waterloo Institute for Nanotechnology, University of Waterloo,

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200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

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Emails: [email protected]; [email protected]

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Graphical Abstract

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Research Highlights:

Eco-friendly approach to prepare self-healing hydrogels

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A 10-fold increase in the storage modulus was achieved with 1.5 wt% CNC

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The system has potential applications in responsive actuators

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Abstract:

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A facile and universal approach to prepare cellulose nanocrystal reinforced functional hydrogels is proposed. An organic solvent-free and eco-friendly method was adopted,

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where both the modification and polymerization were conducted in aqueous solutions.

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Cellulose nanocrystal (CNC) and sodium alginate (SA) were first oxidized under mild conditions to introduce aldehyde groups. Subsequently, amine-containing vinyl

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functionalized monomers were introduced to the surface of CNC or backbone of oxidized SA via a dynamic Schiff-base linkage. The bio-based hydrogels were then prepared via a one-pot in-situ polymerization where the functional CNC and SA serve as novel macro-

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cross-linkers that contribute to the structural integrity and mechanical stability of the hydrogels. The hydrogels displayed uniform chemical and macroscopic structures and could self-heal within several hours at room temperature. The design of specific monomers will allow the introduction of stimuli-responsive properties to the functional hydrogels and

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a chemically robust thermally-triggered actuator was demonstrated. Due to its flexible design and practical approach, the hydrogels could find potential uses in agricultural and pharmaceutical products.

Keywords: Cellulose nanocrystal, Hydrogel, Self-healing, Stimuli responsive

Introduction 2

Polymer hydrogels, bearing some similarities to biological tissues, are soft materials that can absorb a weight of water several orders greater than their dry weight (Culver, Clegg, & Peppas, 2017; Li, Rodrigues, & Tomás, 2012; J.-Y. Sun et al., 2012; Vermonden, Censi, & Hennink, 2012). With their hydrated and porous nature, hydrogels are excellent platforms for various applications, ranging from wastewater treatment, tissue scaffold, drug delivery, biosensor and actuator (Fan, Shi, Lian, Li, & Yin, 2013; Maeda, Hara, Sakai,

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Yoshida, & Hashimoto, 2007; Merino, Martín, Kostarelos, Prato, & Vázquez, 2015; H. Wang & Heilshorn, 2015). Due to their hydrated state, they have displayed poor mechanical

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strength and toughness that have shortened their life cycle in some applications. Recently, significant attention has been devoted to the development of mechanically robust hydrogels

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and several achievements have been reported, such as particle reinforced hydrogels (Z. Hu & Chen, 2014; Q. Wang, Hou, Cheng, & Fu, 2012), double network hydrogels (Gong,

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2010), supramolecular hydrogels (Guo et al., 2014), and topological hydrogels (Kamata, Akagi, Kayasuga-Kariya, Chung, & Sakai, 2014). Among all the methodologies,

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nanocomposite hydrogels have attracted great interest due to their simple and time-saving synthetic procedures as well as easy and versatile surface functionalization. In addition to obtaining mechanically robust properties, the demand for precise control of structures or

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functions in hydrogels has increased during the past few years and great progress has been made, such as in self-healing, volumetric sensing, and responsive actuation (Cong, Wang, & Yu, 2013; Hur et al., 2014; Xia et al., 2013). The above functionalities can be realized by the careful pre-design and precise control of the chemical components as well as

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macroscopic structures. A homogeneous distribution of functional groups within the hydrogel could increase the efficiency during applications. In the design of self-healable hydrogels, physical interactions, such as host-guest interaction, hydrogen bonding or hydrophobic interactions were utilized, however the incorporation of dynamic covalent bonds in the hydrogels may afford better dimension stability and improve the mechanical properties (Jin, Yu, Denman, & Zhang, 2013; Wei et al., 2014). 3

Cellulose nanocrystal (CNC), derived from cellulosic materials and agriculture biomass have recently attracted tremendous attentions. Due to the abundance of the material, high strength and stiffness, low weight and biodegradability, CNC is a promising candidate for a variety of applications, ranging from Pickering emulsifiers and catalyst carriers to reinforced composites (Grishkewich, Mohammed, Tang, & Tam, 2017; Tang, Berry, & Tam, 2016; Tang et al., 2014; Tang, Sisler, Grishkewich, & Tam, 2017; Tang, Shi,

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Berry, & Tam, 2015; Tang, Song, et al., 2015; J. Yang & Han, 2016). Recently, pristine and modified cellulose nanocrystals have been introduced into various polymer matrices, such

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as poly(oligo ethylene glycol methacrylate (POEGMA) (De France, Chan, Cranston, & Hoare, 2016), poly(acrylic acid) (PAA) (Yang et al., 2012), poly(acrylamide) (PAM) (Zhou,

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Wu, Yue, & Zhang, 2011), gelatin (Ooi, Ahmad, & Amin, 2016) as reinforcing agent for the preparation of robust hydrogels. However, there are limited studies describing the

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design and development of CNC-based functional and healable hydrogels. Huang et al. (Huang et al., 2018), Liu et al. (Liu, Mai, & Zhang, 2017) and Lu et al. (Lu et al., 2017)

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have reported simple pathways to fabricate self-healing hydrogels based on dynamic reversible chemistry or coordination bonds. However, hydrogels prepared previously are generally weak, hence their practical applications are limited. Yang et al. constructed a self-

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healing, tough, and resilient hydrogel based on the reversible Diels-Alder click reactions between furyl-modified CNCs and maleimide-end-functionalized polyethylene glycol (Shao, Wang, Chang, Xu, & Yang, 2017). The hydrogel displayed outstanding mechanical properties with a large elongation before fracturing. However, the modification procedures

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are complicated and organic solvents are being utilized. In this study, a universal and green approach to fabricate chemically robust and self-

healing hydrogels is proposed. To verify the protocol, cellulose nanocrystal (CNC) and sodium alginate (SA) were first oxidized to impart aldehyde functional groups that serve as reaction sites for the amine-containing vinyl functionalized monomers. The modified CNCs served as macro-cross-linkers that contribute to the structural integrity and 4

mechanical stability of the hydrogels, while the dynamic Schiff-base linkage contribute to the self-healing properties. Compared to the widely reported ionic crosslinked SA hydrogels, our designed hydrogels are much stronger and resistant to loading without breaking down into pieces, demonstrating the advantages of introducing cellulose nanocrystals. By selecting the appropriate monomers, stimuli-responsive properties can also be introduced to the hydrogel systems, and a chemical robust thermally triggered

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actuator is demonstrated. The proposed protocol of fabricating robust self-healing hydrogels will offer insights into the design of renewable and reusable functional hydrogels

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for their future application in the agricultural, and pharmaceutical sectors.

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Scheme 1 Schematic illustrating the preparation of CNC-reinforced hydrogels

Experimental

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Chemicals and Materials

Sodium alginate (SA) was purchased from FMC Biopolymer (The ratio between

guluronic and mannuronic acids is 4/6). Spray dried cellulose nanocrystals (CNC) were provided by CelluForce Inc (average dimensions of 100 nm length and 5 nm diameter). Ammonium persulfate (APS), sodium periodate (NaIO4, >99.8%), acrylamide (97.0%), N,N,N’,N’-tetramethylethylenediamine (TEMED), and silver (I) oxide (Ag2O, 99%) were purchased from Sigma Aldrich and used without further purification. Di(ethylene glycol) 5

methyl ether methacrylate (MEO2MA, 95%) and poly(ethylene glycol) methyl ether methacrylate (average Mn 300, OEGMA300) were received from Sigma Aldrich and passed through the neutral aluminum oxide columns to remove the inhibitor prior to use. Purified water from a Milli-Q Millipore system was used to prepare all the solutions. Preparation of oxidized cellulose nanocrystal (CNC-CHO) and oxidized sodium alginate (Alg-CHO)

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The synthesis of oxidized sodium alginate was based on a reported method with a slight modification (Yang, Bakaic, Hoare, & Cranston, 2013). For preparing CNC-CHO,

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1.5 g NaIO4 was dissolved in 100 mL of 2 wt % aqueous CNC suspension. The reaction mixture was stirred at room temperature overnight in the absence of light. After that, 0.75

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mL ethylene glycol was added to the suspension and stirred for another 1 hour to consume the excess oxidant. The mixture was then placed in a dialysis membrane (cut off MW: 12-

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14 kDa) and dialyzed against MiniQ water for over 1 week with constant change of water, until no change in the ion conductivity was detected. The obtained suspension was stored

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in the fridge (at 4 oC) for further use.

Partially oxidized sodium alginate (Alg-CHO) was prepared following a similar procedure reported previously (Pescosolido et al., 2011). Typically, 0.01 mol NaIO4 (in 5

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mL water) was added to 200 mL sodium alginate solution (2.0 wt %, by dissolving 4.0 g Alg in 200 mL water). The mixture was stirred for 4 hours at room temperature before 0.75 mL of ethylene glycol was added to the solution to quench the reaction. The mixture was also dialyzed against MiniQ water (cut off MW: 12-14 kDa) and lyophilized. The degree of

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oxidation was determined using the Purpald reagent as described by the manufacturer with a standard curve from formaldehyde. Preparation of Alg-CHO/polyacrylamide or CHC-CHO reinforced hydrogels Alg-CHO (0.025 g), acrylamide (0.5 g), and various amounts of CNC-CHO (refer to Table 1) were mixed with water (5 mL) in a 7 mL vial. The mixture was vortexed and sonicated until a translucent solution was obtained. N2 was bubbled into the mixture for at 6

least 30 min to purge the O2 inside the vial. Then APS (25 mg) was added, followed by vortexing and 10 mins bubbling with N2. After that, the vial was sealed and the reaction was kept at 70 oC for 5 hours to complete the free radical polymerization process (Scheme 1). The resulting cylindrical hydrogel was immersed in fresh water (200 mL x 5 times) for 2 days to remove unreacted reagents and residual impurities. Preparation of stimuli-responsive hydrogels

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Responsive hydrogels were synthesized following the above procedure. Briefly, 25 mg of CNC-CHO, acrylamide (0.2 g) and 0.025 g Alg-CHO were dispersed in 5 mL of

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water in a 7 mL vial, and 0.6 g MEO2MA or OEGMA300 was added to the reaction mixture (refer to Table S1). In order to dissolve the thermo-responsive monomers, 1 mL of ethanol

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was added, and N2 was introduced into the mixture for at least 30 min to purge any O2. After that, 10 μL TEMED and APS (25 mg) solution was injected into the vial and the

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polymerization was initiated. The cylindrical hydrogel was recovered after 24 hours and was immersed in fresh water (200 mL x 5 times) for 2 days to remove the unreacted

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reagents and residual impurities. The double layer hydrogel film was fabricated via a twostep procedure. The formulations of the precursor solutions were adopted from G-0.5 and TG-A (see Table 1 and S1), respectively. The precursor solution was injected into a lab-

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made polytetrafluoroethylene (PTFE) mold and polymerization was carried out at room temperature for 6 hours.

Characterization and instrumentation

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Potentiometric titration was carried out using a temperature controlled Metrohm 809

autotitrator. Before titrating with sodium hydroxide solution, the aldehyde functional groups on CNC-CHO were selectively oxidized to carboxylic acid groups using silver (I) oxide as reported previously(X. Yang et al., 2013). The sample was ground with KBr powder and compacted, and scanned using a Bruker Tensor 27 spectrometer over 400-4000 cm-1 at a resolution of 4 cm-1 with an average of 32 scans. A scanning electron microscope 7

(SEM) equipped with a LEO FESEM 1530 microscope set at 10 kV was used to observe the microstructure of the hydrogels. Rheological measurements were performed using a Bohlin rheometer equipped with circulating water system. For all gels, parallel plate geometry (20 mm diameter) was employed and all measurements were performed at a temperature of 25 °C. The strain sweep data were acquired between 0.1 and 400 % at a frequency of 0.5 Hz, while the frequency sweep data were obtained from 0.1 to 10 Hz

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under a control stress of 10 Pa (where the oscillatory deformation is within the linear regime, see Figure S1). The single and cyclic compression test was conducted on a

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cylindrical hydrogel (height of 10 mm) at a crosshead speed of 0.3 mm s-1. The stress and strain between 0.1 and 0.3 were used to calculate the initial elastic modulus (E). The

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swelling ratio (SR) of the hydrogel was also determined, where two or three samples from the hydrogel (about the same dimensions) were placed in a glass beaker and freeze dried.

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Subsequently, aqueous buffer solutions at pH 7.3 (30.0 mL) were added to beakers, and the samples were left to swell. The hydrogels were weighed at prescribed time intervals after

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removing the excess water on the hydrogel surface with tissue paper. The swelling ratios were determined gravimetrically as described by Equation (1): 𝑤𝑡 −𝑤𝑑 𝑤𝑑

∗ 100 %

(1)

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SR =

where Wt is the weight of hydrogels at each time interval, Wd is the initial dried mass of hydrogel sample.

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Results and Discussion

Characterization of oxidized alginate and cellulose nanocrystals Alginates are anionic polysaccharides extracted from seaweeds, and their polymer

chains are composed of mannuronic acid (M unit) and guluronic acid (G unit) units. In aqueous solutions, the G blocks between different chains formed ionic crosslinking through divalent cations to yield a network and subsequently an alginate hydrogel (J.-Y. Sun et al., 2012). In the current study, a partially oxidized sodium alginate was synthesized and further 8

utilized to prepare the composite hydrogel. The oxidation reaction was performed with sodium periodate as reported previously (Reakasame & Boccaccini, 2018; Yang, Xie, & He, 2011). Gelation experiments were performed with calcium ions, where the gelation of sodium alginate disappeared after oxidation (Figure S2). Instead, the polymers precipitated in the presence of calcium ions, as shown in Figure S2. This result is consistent with a previous study, indicating a reduction in the chain stiffness as well as persistence length of

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the polymer (Pescosolido et al., 2011). The aldehyde content was quantified by the Purpald assay, revealing that oxidized alginate contained 21.26 mmol aldehyde/gram

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polysaccharide, which is comparable to previous studies (Grover, Braden, & Christman, 2013). It should also be noted that by introducing aldehyde functional groups to the sodium

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alginate (Alg) backbone, the Alg chains were rendered more reactive for future crosslinking

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reactions.

Figure 1 (A) FTIR spectra of cellulose nanocrystals (CNC), oxidized cellulose

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nanocrystals (CNC-CHO) and CNC-reinforced hydrogels; (B, C, D) Photographs showing gelation behavior after polymerizations (1/Black-Polyacrylamide, 2/Green-CNC and Polyacrylamide; 3/Red-CNC-CHO and Polyacrylamide).

Figure S2 shows a comparison of FTIR spectra of pristine and modified sodium alginate (Alg and Alg-CHO). The FTIR spectrum of sodium alginate possessed several 9

characteristic absorption bands at 3259 cm-1, 1596 cm-1 and 1406 cm-1, that are associated with the hydroxyl groups, and antisymmetric and symmetric stretch of carboxylate groups respectively (Jejurikar et al., 2012). After oxidation, Alg-CHO displayed a weaker OH stretching band compared to pristine Alg, as revealed by the intensity ratio of band 3259 and 1596 (I3259/I1596). New bands at 1232 and 1733 cm-1 were also observed in the spectra of Alg-CHO, in accordance with previous results (Lü et al., 2015; Sarker et al., 2014).

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However, the symmetric vibrational band at 1733 cm-1 was weak and in most cases, could not be detected due to hemiacetal formation of the free aldehyde groups (Jia et al., 2011)

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(Figure S2).

CNC-CHO were obtained through periodate oxidations, and the FTIR was used to

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analyze the modified CNC samples. The significant difference was evident at 1726 cm-1, which is attributed to the characteristic absorption band of carbonyl groups for oxidized

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CNC. Furthermore, the increase in the intensity at 896 cm-1 validated the successful introduction of aldehyde or hemiacetal groups to the CNC via sodium periodate oxidation

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(Figure 1A). The degree of functionalization on CNC-CHO nanoparticles were quantified by selective oxidation of the aldehyde to carboxyl groups using silver (I) oxide, and the conversion was confirmed by titration with sodium hydroxide solutions. Based on the

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conductivity and pH curves obtained, there was 5.1 mmol of CHO groups per gram CNCCHO (Figure S3).

Gelation and Schiff-base reactions

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Schiff-base reaction refers to the reaction between compounds containing carbonyls

(aldehydes) and amino (primary amine) groups, resulting in compounds with C=N double bonds. The reactivity and thermodynamic equilibrium may vary, depending on the carbonyl and amino groups. In order ensure that the gelation based on the Schiff-base occurred, three comparable systems were examined. To the reaction vials, 5 mL of water (black-1), 1.0 wt% of pristine CNC (green-2) and 1.0 wt% of aldehyde functionalized 10

cellulose nanocrystals (CNC-CHO) (Red-3) dispersions were mixed with 0.5 g of acrylamide respectively, followed by the addition of 25 mg of APS as initiator to trigger the polymerization reaction. As shown in Figure 1B and 1C, after polymerization, sample 1 consisted of a viscous liquid. When 1 wt% of cellulose nanocrystal was added, no gelation behavior was observed due to the absence of robust chemical reactions and number of crosslinks. However, when 1 wt% CNC-CHO was introduced to the system (denote as two component hydrogel system), gelation occurred (Figure 1C). FTIR analysis of the

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hydrogel (sample 3) revealed a characteristic peak at 1630 cm−1 showing the presence of

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imine bonds, thereby confirmation the successful formation of Schiff base linkage (Kumari & Chauhan, 2014). Further evidence of gelation is shown in Figure 1D. The product of

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Sample 2 and Sample 3 were removed from the reaction vial and placed in a polystyrene petri dish. We observed that the sample with the pristine CNC was free-flowing, consisting

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of a viscous liquid, while for the reaction mixture containing CNC-CHO, a robust gel was formed (viscoelastic material will tend to sag or creep with time) (J. Yang, Han, Zhang, Xu,

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& Sun, 2014).

From the above observations, we concluded that the aldehyde groups had reacted with the amino groups on the polyacrylamide chains, serving as nanoparticle crosslinkers

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that yielded a hydrogel. However the gel strength was weak, and this is probably due to the low efficiency of reactions between amino groups on acrylamide and aldehyde groups. Based on a similar crosslinking chemistry, Alg-CHO/polyacrylamide and CNC-CHO were used as the hydrogel matrix and reinforcing agent respectively, and a three-component

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hydrogel system was made. Figure 2A illustrates the uniform and mechanically robust hydrogels prepared using this approach, and the details of the composition are shown in Table 1. The one-pot in-situ free radical polymerization (without the addition of chemical crosslinker) is exceptionally simple and it provides a robust and universal platform to fabricate functional hydrogels in aqueous solutions. The gel could be placed on a table without a container, and their shape and dimension were maintained (Figure 2A). 11

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Figure 2 (A) Photographs showing the sol-gel transition before and after polymerizations for composite hydrogels; Photographs demonstrating the robustness of the obtained

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hydrogels with an excellent ability to withstand compressing (B) and twisting (C) (Scale

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bar is 5 cm).

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Scheme 1 shows the proposed mechanism for the preparation of nanocomposite hydrogels. Reactive aldehyde functional groups were first introduced to the polysaccharide backbone or nanoparticle surface. The vinyl-containing monomers bearing active amine groups can be used for subsequent modification via Schiff-base linkage, to be used in subsequent free radical polymerization. The functional cellulose nanocrystal acts as both a reinforcing and cross-linking agent for the hydrogel. The new composite hydrogel

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developed using this strategy could withstand high degrees of compression and deformation without any observable damage, confirming that they were mechanically

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robust, and possessed an efficient energy dissipation mechanism (Figure 2B and 2C). The elastomeric nature of the nanocomposite hydrogels is due to the long and flexible

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polyacrylamide polymer chains on the surface of CNC, and the unique network structures associated to a well-dispersed CNC acting as multifunctional cross-linker in the polymer

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matrix (De France, Hoare, & Cranston, 2017).

The self-healing properties of the two and three-component hydrogels were also

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examined. Both hydrogels were sliced into two (one stained with methylene blue) and placed close together in a container with moisture (relative humidity of 70%) (Figure 3). After 3 hours, the hydrogels fused into a single piece, and they could be lifted up without

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breaking into two pieces and no cracks were evident. As physical crosslinking, hydrogen bonding and chain entanglements also contributed to the hydrogel network, we anticipated that both the hydrogen bonds and dynamic Schiff-base linkage played important roles in the self-healing process (J. Yang & Han, 2016; Zhou et al., 2011). To further evaluate the

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self-healing properties of the prepared hydrogels, rheological studies were conducted and the storage moduli G’ of the original and healed samples were recorded and compared (Lu et al., 2017). The corresponding change (ratio of healed to original samples) of the two and three components hydrogels are shown in Figure S4.

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Figure 3 Illustration of the self-healing process of the two-component (A) and threecomponent (B) composite hydrogel systems, methylene blue was used to dye one of the

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Characterization of the composite hydrogels

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pieces in order to show the boundary and difference. (Scale bar is 5 cm)

The morphologies of hydrogel G0 and G0.5 were examined by scanning electron

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microscopy (SEM). The hydrogel specimens were freeze-dried to preserve the structure and volume of the swollen hydrogels after the solvent was removed. As shown Figure 4A and 4D, the incorporation of CNC-CHO in the network yielded more regular and uniform

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pore size. The dense porous structures indicate a dense crosslinked structure, while a fibrous structure attributed to reinforced cellulose nanocrystals is clearly evident (Figure 4F denoted by circles). Hydrogels containing 0.5 wt% CNC-CHO possessed a porous and interconnected network with pore sizes of 1.5-3.0 μm, which is consistence with previously

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published results.(Mo, Zhang, Yan, & Chang, 2018; J. Yang et al., 2014)

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Figure 4 SEM micrographs of the hydrogels indicate the formation of highly porous and

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hydrogel-G0.5 with adding 0.5 wt% CNC-CHO)

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interconnected networks (A, B, C: hydrogel-G0 without adding CNC-CHO; D, E, F:

The rheological, compression and swelling properties of oxidized alginate-

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polyacrylamide hydrogels reinforced with different amounts of CNC-CHO were quantified. The dynamic rheology was used to evaluate the viscoelastic properties, where oscillation measurements were performed within the linear viscoelastic region. Figure 5A and 5B

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present the dynamic storage modulus (G’) and loss modulus (G’’) as a function of frequency. All the hydrogels exhibited a predominantly elastic response, with the elastic modulus G’ exceeding the viscous modulus G’’ by a factor of 10 over the entire frequency range. Also the G’ values were nearly independent of frequency as would be expected for

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a covalent crosslink network, where the network relaxation time was longer than the experimental processing time. From the moduli of the different hydrogel systems, G’ increased with increasing CNC-CHO concentration yielding higher cross-link density, from the incorporation of cellulose nanocrystals thereby enhancing the hydrogel strength. The results show a 2, 5, and 10-fold increase of the storage modulus for hydrogel reinforced with 0.5, 1.0, 1.5 wt% CNC-CHO, respectively. The storage modulus (G’) of the hydrogels increased sharply from 1.9 to 20.1 kPa by increasing the amount of nanoparticles to a 15

concentration of 1.5 wt%. The concentration dependence of dynamic storage (G’) for the composite hydrogels determined at 0.1 Hz was plotted in Figure 5C. The value of G’ increased dramatically at concentrations greater than 0.4 wt% suggesting the formation of interconnected solid-like network structures, which have also been reported previously

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(Peng et al., 2018; J. Yang et al., 2012a).

Figure 5 Frequency-dependent rheological behavior of hydrogels; (A) storage modulus: (B) loss modulus; (C) complex viscosity; (D) Elastic modulus as a function of CNC-CHO

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concentration (inserted photographs show the appearance of the obtained hydrogels G0 , G0.5 and G1.0).

Figure 5D shows the complex viscosity η* as a function of frequency for the various hydrogels. For all the reaction mixtures, log η* displayed a linear function of log ω with a slope of -1, indicating that the relaxation time is much greater than the experimental time. 16

When the amount of reinforcing fillers was increased, the relaxation time of the network decreased. The loss factor was used to assess the ratio of dissipated energy to stored energy during the stress deformation process. For covalently cross-linked gels, the value of tan δ ranged from 0.05 to 0.15, which was much lower than that of physical gels (0.4-0.78). Since for a perfect chemical crosslinked network, the loss factor should approach zero; any deviation points to the presence of defects in the elastic network. The mechanical strength

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of hydrogels increased with the introduction of nanoparticles to the matrix due to the interactions between nanofillers and polymer chains, causing confinement of the polymer

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chains that retard the dynamics (Chen et al., 2015; Kumar, Rao, & Han, 2018).

The compression test was performed on the composite hydrogels with 90% water

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and the typical stress-strain curves are shown in Figure 6A. The hydrogels reinforced with CNC-CHO nanoparticles could sustain higher stress than pristine alginate-PAM hydrogel.

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The measured stresses at the 80% strain level were 0.188, 0.454 and 1.129 MPa, respectively. , similar to those of other systems, such as those containing nanoclay (X. Hu

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et al., 2016), magnetic nanoparticles (Jaiswal et al., 2016), microgel or micelles (Thoniyot, Tan, Karim, Young, & Loh, 2015). Also the increment in the modulus may cause a reduction in the pore size of the hydrogels, resulting in a better distribution of applied stress

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(J. Yang & Han, 2016). As shown in the insert in Figure 6A, the hydrogels did not fracture at 98% compressive strain and recovered to original shape after the stress was removed. The consecutive cyclic compression experiments (shown in Figure S5) on the nanocomposite hydrogels resulted in reproducible and overlapping hysteresis loops,

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indicating good resilience and recoverability as well as high fatigue-resistance of the composite hydrogels. We proposed that the energy dissipation is mainly due to the intermolecular physical interactions (hydrogen bonding) between the polymers and cellulose nanocrystals (Y. Sun, Gao, Du, Cheng, & Fu, 2014; J. Yang et al., 2014; J. Yang & Xu, 2017).

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Figure 6 (A) Compressive stress-strain curves of hydrogels G0, G0.5, G1.0 and G1.5

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(inserted photograph shows the stability of hydrogel G1.0 when compressed to 95% strain); (B) The swelling properties of obtained hydrogels G0, G0.5, G1.0 and G1.5 (inserted

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photographs show the volume and transparency change before and after swelling studies).

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The swelling behavior of the hydrogel systems is shown in Figure 6B. Compared to oxidized alginate-polyacrylamide hydrogels, the reinforced hydrogels displayed low

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swelling ratios. With increasing CNC-CHO content, the equilibrium swelling ratio decreased from 18 to 10. As the swelling properties of hydrogels mainly depend on the

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hydrophilic characteristics of the functional groups and the effective cross-link density of the hydrogels, a possible reason for the change of equilibrium swelling ratio is the higher crosslinking density (X. Hu et al., 2016; Zhou et al., 2011). By incorporating nanoparticles into the polymer network, more chemical cross-links via the Schiff-base reaction as well as physical cross-linking (hydrogen bonding) through entanglement and interpenetration

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occurred (J. Yang et al., 2014).

Thermo-responsive actuators The incorporation of stimuli-responsive properties to the hydrogels is an efficient approach to transform external stimuli into user-defined functions, such as the conversion of light/heat to mechanical work. The use of thermo-responsive polymers like poly(N18

isopropylacrylamide) (PNIPAM) in actuator systems has been well-documented, however, another new family of thermo-responsive polymers comprising of oligo(ethylene glycol) methacrylate (OEGMA) has been less studied, despite its more biocompatible and sharper thermo-transition characteristics (Tang et al., 2016). In addition, the lower critical solution temperature (LCST) of the later are tunable by changing the feed ratio of the monomers, salt concentration or ionic strength. With the successful design of the composite hydrogels

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described above, we incorporated the OEGMA analogues into the hydrogel system to illustrate a possible design of a thermo-responsive actuator.

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Di(ethylene glycol) methyl ether methacrylate (MEO2MA) and poly(ethylene glycol) methyl ether methacrylate (OEGMA300) were chosen as the responsive polymer blocks. A

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summary of the composition of these different thermo-responsive hydrogels is summarized in Table S1. In order to form the Schiff-base linkage, 0.2 g acrylamide was added to the

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precursor solution. The responsive behavior of the hydrogel was characterized by measuring the transmittance at the wavelength of 500 nm. Figures 7A and 7B show the

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phase transition characteristics of the hydrogels in aqueous medium for the temperature changes ranging from 20 to 80 oC. The transmittance decreased quickly for hydrogels containing the MEO2MA moiety, while it decreased gradually for the one with OEGMA300.

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Increasing the temperature weakened the hydrogen bonds between the ether oxygen atoms and water molecules, and the hydrophobic interactions of hydrocarbon backbone were enhanced. As the shorter MEO2MA side chains are more dynamic, the hydrophobic interactions shrunk the network resulting in the hydrogel turning opaque. In addition,

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hydrogel TG-A also displayed a large volume shrinkage (shown in Figure 7C) due to the dehydration of ethylene-oxide side chains distributed along the backbone, estimated to be around 48%, which is sufficient for the design of thermo-responsive hydrogel actuators.

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Figure 7 Transmittance versus temperature for hydrogels (A) TG-A and (B) TG-B at a wavelength of 500 nm and a heating or cooling rate of 0.5 °C/min; the inserted pictures

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demonstrated the volume and transmittance change of TG-A by increasing the temperature (Left: Room temperature., Right: 65 oC). (C) Photographs showing the transmittance and

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volume change for hydrogel TG-A at room temperature and when the temperature is raised to 65 oC; (D) photographs showing the reversible thermo-responsive properties for bilayer

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hydrogel film as indicated by transparency.

Thus, a hydrogel bilayer was prepared by sequential polymerization of the respective precursor solutions. The formulation was prepared from G-0.5 and TG-A making up of the bottom and top layers respectively. After two-step crosslinking and processing, the bilayer

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hydrogel film was removed from the hydrophobic polystyrene petri dish and rinsed with water to remove unreacted reactants. The hydrogel film was transparent (Figure S6). The optical transparency could be tuned by manipulating the temperature. As shown in Figure 7D, the film first turned opaque and finally became turbid when the temperature exceeded the phase transition temperature. We then examined the folding behavior of the hydrogel film. Exposing the thin bilayer hydrogel film to water at room temperature led to the 20

swelling of the film. When the temperature was increased beyond the phase transition temperature, the swelling properties of the hydrogel displayed a sharp change due to the dehydration of ethylene-oxide side chains distributed along the backbone. Driven by the phase and volume transition, the crosslinked bilayer film rolled up towards the TG-A side within 20 s. At the same time, the optical transmittance decreased sharply when the film was placed in water kept at 55 oC (Figure 8). After removing and placing the bilayer

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hydrogel in the water at room temperature, the hydrogel became transparent and unrolled. Thus, we successfully demonstrated a strategy for fabricating mechanically robust and self-

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healing hydrogels with fast response rate within a narrow LCST range. The results demonstrate the potential of this system which could be used in future applications, such

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as sensors and biomimetic soft robotics.

In order to demonstrate the potential application in biomedical and pharmaceutical

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applications, in-vitro release profiles (0.01 M PBS buffer) of ibuprofen (IBU) in trapped hydrogel (TG-A) at different temperatures were recorded using the procedures reported

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previously (Supramaniam, Adnan, Mohd Kaus, & Bushra, 2018). The results are shown in Figure S7. The maximum cumulative release at room temperature was found to be around 93%, which is lower than 100% as some molecules were trapped in the highly entangled

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polymer network. Besides, the release rate at room temperature was higher than at 55 oC because the polymer network collapsed when the temperature exceeded the phase transition temperature. The released behavior of IBU was retarded and a lower maximum cumulative release was obtained. By taking the advantages of this, a smart control over the released

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profile could be designed for suitable applications in controlled drug delivery.

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Figure 8 Scheme (a) and experimental (b, c) observation of rolling/unrolling of the

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crosslinked bilayer hydrogels in water.

Conclusions

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In summary, we have successfully demonstrated a universal and facile approach to prepare cellulose nanocrystal reinforced self-healing hydrogels in aqueous solutions,

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confirming the green and eco-friendly features of this system. By incorporating 1.5 wt% CNC-CHO into an oxidized sodium alginate-polyacrylamide hydrogel matrix, a 10-fold increase in the storage modulus was achieved. By further incorporating oligoethylene glycol methacrylate to the hydrogel, a thermo-responsive hydrogel was produced. The bilayer hydrogel exhibited a response within 20 s when subjected to external triggers. There

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are many applications in biomedical and pharmaceutical science, which could utilize such sustainable and biocompatible smart hydrogels.

Acknowledgement This research was supported by Hunan Provincial Natural Science Foundation of China (No. 2019JJ60073). We wish to acknowledge Celluforce Inc. for providing the cellulose 22

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nanocrystals. K.C.T. wishes to acknowledge funding from CFI and NSERC.

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Table 1 Compositions of precursor solutions for making composite hydrogels Acrylamide

APS

Alg-CHO CNC-CHO

H2O

(g)

(mg)

(g)

(mg or w/v % against water)

(mL)

G-0

0.50

25

0.025

0 (0 wt%)

5.0

G-0.5

0.50

25

0.025

25 (0.5 wt%)

5.0

G-1.0

0.50

25

0.025

50 (1.0 wt%)

5.0

G-1.5

0.50

25

0.025

75 (1.5 wt%)

5.0

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Sample

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