Autonomous Chitosan-Based Self-Healing Hydrogel Formed through

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Autonomous Chitosan-Based Self-Healing Hydrogel Formed through Non-Covalent Interactions Zhongxing Zhang, Sing Shy Liow, Kun Xue, Xikui Zhang, Zibiao Li, and Xian Jun Loh ACS Appl. Polym. Mater., Just Accepted Manuscript • DOI: 10.1021/acsapm.9b00317 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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ACS Applied Polymer Materials

Autonomous Chitosan-Based Self-Healing Hydrogel Formed through Non-Covalent Interactions Zhong-Xing Zhang *,a, Sing Shy Liowa, Kun Xuea, Xikui Zhanga, Zibiao Lia, and Xian Jun Loh*, a,b a Institute

of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 2

Fusionopolis Way, Innovis, #08-03, Singapore 138634, Singapore. Email: [email protected]; [email protected] ; Tel: +65 6501 1800 b National

University of Singapore, Department of Materials Science and Engineering, Singapore 117576, Singapore

ABSTRACT: In this work, a facile strategy was developed for the formation of an autonomous chitosanbased self-healing hydrogel. This hydrogel was fabricated using in situ free radical polymerization of acrylic acid (AA) and acrylamide (AM) in the presence of chitosan in dilute acetic acid aqueous solution under mild conditions. The in situ formed hydrogel is mainly composed of chitosan graft copolymers (CS-g-P(AM-r-AA)) and a small amount of non-grafted copolymers (P(AM-r-AA)), which interact with each other through a combination of multiple non-covalent interactions, including the inter-chain electrostatic complexation between –[AA]– segments and positively charged amino groups of chitosan, the H-bonding between –[AM]– segments and the H-bonding between –[AM]– segments and chitosan backbone. Owing to the cooperation of these non-covalent interactions and the reversible nature of the non-covalent network structure, the obtained hydrogel exhibits rapid network recovery, high stretchability and efficient autonomous self-healing properties. The hydrogel can also dissolve completely in dilute acidic aqueous solution under mild conditions visibly reflecting the unique network feature of this self-healing hydrogel system.

KEYWORDS: Self-healing, hydrogel, chitosan, non-covalent interactions, in situ polymerization

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1. INTRODUCTION In nature, self-healing is a key protective feature of biological materials and systems, taking place from the single molecule level (e.g., DNA repair) to the macroscopic level (e.g., closure and healing of injured skin and blood vessels). The intrinsic self-healing processes are autonomously triggered upon the external damage and eventually the initial functions are regenerated. As a result, living organisms are able to survive in wild and prolong their lifetime. By contrast, most man-made materials are irreparably damaged by cracks at the macro- or microscale, and they eventually lose the structural integrity, mechanical and other properties with propagation of these cracks.1 Hydrogels are three-dimensional networks of hydrophilic polymer chains that are chemically and/or physically crosslinked.2-6 Hydrogels possess a lot of attractive inherent properties, such as structural similarities to biological tissue, rubbery consistency, low interfacial tension to human tissue, responsiveness to external stimuli, adaptivity, tunability, biocompatibility and biodegradability etc. that make them successful for a wide range of biomedical applications in drug delivery, tissue engineering, wound healing, and for personal health care applications etc.7-11 In recently years, an exciting trend has emerged involving the use of hydrogels in engineering materials for robotics, electronics, actuators and sensors etc. 12-17 An impetus behind this innovation is the repurposing of sustainable and innovative soft materials originally developed for biomedical applications.18-22 To fulfill the purpose, it becomes essential to enhance hydrogel’s durability by introducing autonomous self-healing ability.13 The self-healing of hydrogel is mostly achieved by constitutional dynamic chemistry, where dynamic bonds contribute to the preparation of polymer networks with the ability to undergo reversible bond breaking–reformation processes. To date, both reversible chemical bonds (including phenylboronate complexation, disulfide bonds, imine bonds, acylhydrazone bonds, reversible radical reaction and Diels–Alder reactions) and non-covalent interactions (including hydrophobic interactions, host–guest interactions, hydrogen bonds, crystallization, polymer–nanocomposite interactions and multiple weak intermolecular interactions) have been utilized to fabricate self-healing hydrogels.23 However, most of the self-healing hydrogel systems have been made based on synthetic polymers, and these systems either require sophisticated synthesis in order to incorporate the desired reversible interactions, or the components and reagents inside the systems are not biocompatible, which limited the application of self-healing hydrogels in biomedical and personal/health care areas. In recent years, self-healing hydrogels based on natural polysaccharides (such as alginate, carrageenan, 2 ACS Paragon Plus Environment

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chitosan, dextran, hyaluronic acid and xanthan etc.) have aroused people’s interests due to the sustainability, biocompatibility and biodegradability of these natural resources.24-32 Among them, the most noteworthy is the chitosan-based self-healing hydrogels. Many studies have been carried out for the development of this novel soft material and application of them in a wide variety of areas like wound healing,24, 33-35 cell encapsulation,36 drug delivery37-39 and oil/water separation40 etc. Chitosan (CS) is the second most ubiquitous natural polysaccharide after cellulose on earth with a broad range of applications. It is derived from chitin by Ndeacetylation and is consequently a copolymer of N-acetylglucosamine and glucosamine. Structurally, chitosan contains a lot of amino groups on the backbone ready for derivation and modification. So far, most chitosan based self-healing hydrogels are based on dynamic covalent Schiff-Base formation.41 Other notable self-healing mechanisms based on dynamic disulfide bonds42 and Diels-Alder click reactions43 etc. have also been reported in literature. By comparison, non-covalently bonded chitosan-based self-healing systems are seldom reported. Non-covalent interactions (such as electrostatic interactions and H-bonding etc.) have been employed to fabricate chitosan-based hydrogels for many years.44-45 However, it is in very recent years that the design of chitosan based self-healing hydrogel through noncovalent interactions began to arouse people’s interest. Several examples were found in literature

including

chitosan

/polyvinyl

alcohol,

modified

chitosan/alginate

and

chitosan/polyacrylic acid systems etc.46-48 These self-healing hydrogel systems are usually composed of two components. Upon mixing with chitosan in aqueous solution, another component interacted with chitosan macromolecules through hydrophobic interactions, electrostatic interactions and H-bonding etc. so as to induce the formation of 3D network. By introducing graphite oxide or ions like Fe3+, dual-network physical chitosan-based selfhealing hydrogels were obtained.49-50 Herein we present a different chitosan-based physical self-healing hydrogel system. It was fabricated through in situ free radical polymerization of acrylic acid (AA) and acrylamide (AM) in the presence of chitosan in dilute acetic acid aqueous solution under mild condition with ammonium persulfate (APS) as initiator and N,N,N',N'-tetramethylethylenediamine (TEMED) as accelerator (Figure 1). By rational design of the gel-forming formulation and careful control of reaction conditions, the chemical crosslinking which usually occurred in normal free radical polymerization system due to radical coupling can be effectively suppressed. As a result, an autonomous chitosan-based self-healing hydrogel by only noncovalent interactions was obtained in a facile one-pot process. The hydrogel in this work 3 ACS Paragon Plus Environment


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showed rapid network recovery, high stretchability and efficient autonomous self-healing properties at high water content (up to 90 wt.%) owing to the non-covalent nature of the network structure. This hydrogel can also be totally dissolved in dilute acidic aqueous solution under mild conditions. This property will contribute to degradation and/or recycling of this soft material after use.

NH3 O O

In situ polymerization

H

N H O

O H N

Dilute HOAc

= Chitosan unit

O

CH2OH O H H OH H H

H

or

NH2

= Monomers (AM, AA)

O H H N N H H O

CH3 CO H NH H O OH H H H O CH2OH

= Synthetic chains (p(AM-r-AA))

Figure 1. Schematic cartoon showing the formation of chitosan-based physical hydrogel by in situ polymerization. 2. EXPERIMENTAL SECTION 2.1. Materials. Chitosan (deacetylation value 75.0 to 85.0 %) and N,N,N',N'tetramethylethylenediamine (TEMED, >98%) were purchased from Tokyo Chemical Industry Co., Ltd. (TCI). Ammonium persulfate (APS, >98%), acrylic acid (AA, 99%) and acrylamide (AM, >98%) were purchased from Sigma-Aldrich. All other reagents were used as received without further purification. 2.2. Fabrication of Chitosan-Based Self-Healing Hydrogel. In a typical procedure, a chitosan solution (2.5 wt%) was made by dissolving a known amount of chitosan powder in dilute acetic acid aqueous solution (1.0 wt%) with vigorous stirring at room temperature for 2 h. To the chitosan solution (8.0 g), a determined amount of AA and AM were added. The mixture was stirred and purged with nitrogen for 20 min at room temperature. Subsequently, a desired amount of TEMED and APS were added to the above mixture. The mixture was stirred and purged with nitrogen for another 10 min at room temperature. Then the mixture was sealed and placed in water bath at 30 oC for 24 h without shaking. The obtained hydrogel 4 ACS Paragon Plus Environment

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was rinsed with DI water and then the water droplets were removed by filter paper. After that, the hydrogel was kept at 4 oC in a sealed petri dish in which droplets of DI water were added in order to ensure a humid environment. In this work, a series of hydrogels with different properties were synthesized by varying the reaction temperature, the initial amount of AA and AM, and the concentrations of initiator. The details of these hydrogel formulations as well as some important experimental results were listed in Table S1 (see Supporting Information). According to different test requirement, the hydrogel samples were fabricated into cylindrical shape (diameter of 10 mm and length of 50 mm) or rectangular strip (length of 35 mm, width of 25 mm and thickness of 2 mm). 2.3. General Characterization Methods. Proton nuclear magnetic resonance (1H NMR) spectra were recorded at room temperature on JEOL ECA 500 MHz NMR spectrometer operating at 500 MHz. Chemical shifts were reported in parts per million (ppm) on the δ scale, and were referenced to residual protonated solvent peaks: DMSO-d6 spectra were referenced to (CHD2)(CD3)SO at δH 2.50; CDCl3 spectra were referenced to CHCl3 at δH 7.26; D2O spectra were referenced to HDO at δH 4.70. Fourier transform infrared (FTIR) spectra of samples in KBr pellet were recorded on a Perkin-Elmer FTIR 2000 spectrometer in the region of 4000-400 cm-1; 64 scans were signal-averaged with a resolution of 2 cm-1 at room temperature. Before measurement, the hydrogel samples were washed in deionized water and ethanol for three times in order to remove any reactant residues from the sample, and then dried in vacuum oven at 50 oC for 2 days. Size exclusion chromatography (SEC) of the selfhealing hydrogel was carried out with a Shimadzu SCL-10A and LC-10AT system equipped with a Sephadex G-100 column (size: 2.5 × 32 cm), a Shimadzu RID-10A refractive index (RI) detector and a Shimadzu SPD-10Avp UV-Vis detector. Acetic acid (HOAc)-sodium acetate (NaOAc) buffer (0.3 M HOAc/0.2 M NaOAc) was used as the eluent with a flow rate of 0.5 mL/min. Freeze-dried hydrogel was dissolved in the same buffer (2.0 mg/mL) for test. Molecular weight analysis was calculated against dextran standards. Scanning electron microscopy (SEM) was carried out on a JEOL FESEM JSM6700F operating at an acceleration voltage of 5 kV, and all of the freeze-dried hydrogel samples were fractured and coated with gold in a vacuum before observation. 2.4. Monomer Conversions. Monomer conversions for in situ polymerization were calculated by 1H NMR spectroscopy based on the determination of residual acrylamide in final hydrogel using decanoic acid as an internal standard. After the in situ polymerization, a measured amount of hydrogel (usually 1.0 g) was soaked in ethanol (1.5 mL) for 12 h 5 ACS Paragon Plus Environment


(shrinking), and then allowed to hydrate in distilled water (1.5 mL) for 12 h (swelling). The final solution outside the hydrogel in each step was collected. This shrinking and swelling process was repeated 2 times. After this purification, the hydrogel sample was recycled and dried in vacuum oven at 50 oC for 2 days for solid content study. While the collected solutions were combined and the mixture was saturated with sodium chloride until the added sodium chloride did not readily dissolve. Subsequently, a measured amount of decanoic acid (usually 10 mg) in ethyl acetate (3 mL) was added as internal standard. The organic phase was separated, and the mixture was further extracted with fresh ethyl acetate (2×3 mL). The organic extracts were combined, dried over anhydrous Na2SO4 and a 3 mL aliquot was filtered, evaporated at room temperature and re-dissolved in deuterated solvent (DMSO- d6). The integral ratio of residual acrylamide/standard was calculated from 1H NMR spectrum by comparing the integrations of the three vinyl protons of acrylamide (5.50 – 6.25 ppm) to the integration of the signal at the region 0.78-0.92 ppm where three methyl protons of standard molecule resonate. The residual amount of acrylamide monomer in hydrogel and monomer conversion were then calculated from a calibration curve (see Supporting Information Figure S1, R2 = 0.999). 2.5. Self-Healing Efficiency. Self-healing efficiency of hydrogel samples was determined based on uniaxial elongation measurement according to reported methods in literature with some necessary modifications.51-52 For this test, all hydrogel samples were fabricated into a cylindrical shape with a diameter of 10 mm and length of 50 mm. The hydrogel sample was cut into two halves from the middle, and then the two halves were brought into contact and left undisturbed at room temperature for 1 day. The uniaxial elongation measurements were performed on an Instron 5567 universal testing machine (Norwood, MA, USA) with a crosshead speed of 20 mm/min at room temperature. The percentage elongation at break of the original hydrogel sample () and the healed hydrogel sample (’) were measured. The healing efficiency (f) is defined as f = ’/. Percentage elongation at break,  = (L-Lo)/Lo 100%, where L is the length of the hydrogel sample at break, and Lo is the original length of the hydrogel sample. For reproducibility, at least 3 samples were measured for each hydrogel formulation, and the results were averaged. The healing efficiency, f takes values between 0 and 1. It is obvious that the closer the value of f is to 1, the better the self-healing capability of the hydrogel is. By comparison, if the value of f is closer to zero, it indicates that the hydrogel sample has lower self-healing ability. In this work, all hydrogel samples were screened by using this method. 6 ACS Paragon Plus Environment

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2.6. Dynamic Oscillatory Rheological Experiments. The dynamic oscillatory rheological experiments were carried out using an AR2000 stress controlled rheometer (TA Instruments). All measurements were performed with a plate geometry (20 mm diameter) equipped with Peltier-based temperature control. Prior to test, a little water was applied to the outer edges of the hydrogel sample to prevent dehydration, and a self-made solvent trap was also employed to minimize the evaporation of water from hydrogel samples during test. All the measurements were conducted at 25±0.1 oC. Strain amplitude sweeps were usually performed first in order to determine the linear viscoelasticity region (LVR). All rheological measurements were performed in triplicate to make sure the data are reliable. The quantitative recovery test of hydrogel samples was carried out on this machine using a two-step procedure. Briefly, a hydrogel sample was subjected to increasing strain amplitude (e.g. 0.01 to 300%) at constant low frequency. After that, the recovery of storage modulus (G´) and loss modulus (G´´) were monitored with time at constant low strain amplitude and frequency. To evaluate the repeated recovery ability, the hydrogel sample was alternatively subjected to small strain amplitude (e.g. 1%) and large strain amplitude (e.g. 300%) at constant low frequency, and the recovery of storage modulus (G´) and loss modulus (G´´) were monitored with time. 2.7. Tensile Tests. Tensile tests were performed on an Instron 5567 universal testing machine (Norwood, MA, USA) at room temperature. For this test, all hydrogel specimens had a length of 35 mm, a width of 25 mm and a thickness of 2 mm. The gauge length between the two clamps was about 10 mm. The loading rate was 20 mm/min. For evaluation of self-healing ability, the hydrogel specimen was cut into two halves from the middle, and then the two halves were brought into contact and left undisturbed at room temperature for 1 day prior to test. The tensile stress–strain curves were then recorded on this computer controlled universal testing machine. 3. RESULTS AND DISCUSSION 3.1. Formation of Self-Healing Hydrogels. The in situ free radical polymerization produced random synthetic copolymer chains (P(AM-r-AA)), and most of them were grafted onto chitosan backbone forming chitosan graft copolymer (CS-g-P(AM-r-AA)) (see Supporting Information Scheme S1). The latter was the major component of the resultant hydrogel. The graft copolymer chains interacted with each other by means of multiple H-bonding interactions (synthetic polymer chain-synthetic polymer chain, synthetic polymer chainchitosan backbone, and chitosan backbone-chitosan backbone) and electrostatic interactions 7 ACS Paragon Plus Environment


between the negatively charged –[AA]- units on synthetic chains and positively charged amino groups on chitosan backbone. As a result, a hydrogel based on multiple non-covalently crosslinked networks was formed. The reversible nature of these non-covalent interactions together with the unique structural feature rendered he hydrogel designed and synthesized here possessing rapid network recovery, high stretchability and efficient autonomous selfhealing properties. Recently, Jiang et al. reported a hydrogel system by using chitosan, AA and AM, and the obtained hydrogel showed toughness, self-healing and metal ion adsorption properties.53 However, our hydrogel system exhibits great difference to that in not only appearance (our hydrogel is transparent) but also network structure and properties due to very different hydrogel composition and fabrication conditions we adopted. To investigate the role of each component on the hydrogel formation, oscillatory time sweep experiments were carried out to monitor the variation of G' and G" of a representative hydrogel-forming formulation containing 2.25 wt% of chitosan, 0.056 mol·L-1 of AA, 0.893 mol·L-1 of AM, 0.00296 mol·L-1 of APS and 0.0167 mol·L-1 of TEMED at 30 oC (Table S1, entry 13). Three control samples (Control 1, no AA; Control 2, no chitosan; and Control 3, no AM) were test under the same conditions as that of representative hydrogel-forming solution. For each sample, the oscillatory time sweep experiment was performed at a low frequency of 10 rad/s and a small strain of 1.0 %, in order to ensure that the experimental conditions did not interfere with the gelation process at 30 oC (Figure 2). The inserted photos in Figure 2 showed the appearance of each sample solution after in situ polymerization. For representative hydrogel-forming solution (Figure 2a), at the beginning, G” was larger than G’, which was expected because the sample was in a liquid state and viscous properties dominated. Both moduli increased fast with time during the first 1 h due to the starting of polymerization and grafting of the synthetic polymer chains onto chitosan backbone. According to literature, the grafting happened at the amino and hydroxyl sites along the chitosan backbone (See Supporting Information, Scheme S1). The increasing rate of G’ was higher than that of G”, which led to a G’ and G” crossover eventually. This phenomenon was related to the formation of multiple non-covalent networks as the growth of chitosan graft copolymer and the self-assembly of these macromolecules. Thus, the elastic properties eventually became dominant. The gelation time, i.e. the time required to achieve the crossover was 2.4 h according to this test. After 2.4 hours both G’ and G” continued to increase. However, the former increased much faster than the latter implying the noncovalent networks became stronger with the in situ polymerization.


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(a) Hydrogel formulation

(b) Control 1 (no AA)

250

250 G' G"

200

150 G', G" (Pa)

G', G" (Pa)

G' G"

200

Crossover 2.4 h

150 100

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0 0

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2

3

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Time (h)

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(c) Control 2 (no CS)

(d) Control 3 (no AM)

100

100 G' G"

G' G"

50

G', G" (Pa)

G', G" (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

50

0

0

1

2

3

4

0

Time (h)

1

2

3

4

Time (h)

Figure 2. Oscillatory time sweep measurements of (a) Representative hydrogel formulation, (b) Control 1 (no AA), (c) Control 2 (no CS) and (d) Control 3 (no AM) showing the variations of G' (filled symbols) and G" (open symbols) as functions of reaction time at constant angular frequency ω of 10 rad/s and strain of 1.0 % at 30 oC. The inserted photos showed the appearance of each sample solution after in situ polymerization. Control 1 (no AA, Figure 2b) showed obvious increase in G’ and G” during the first 0.6 h which was very similar to that of hydrogel-forming formulation (Figure 2a). However, after 9 ACS Paragon Plus Environment


that, though both moduli showed further increase, G” was always larger than G’ throughout the entire reaction period (4h), showing the reaction mixture was in solution state and there was no stable polymer network formed. Another observation was that the values of both G’ and G” were much lower than those of the hydrogel-forming formulation (Figure 2a). According to these results, it was clear that AA should play a key role in the formation of stable polymer network in this system. AA introduced the electrostatic interactions between synthetic polymer chains and chitosan backbones, which not only ensured the stability but also enhanced the mechanical strength of the networks in this system. Control 2 (no chitosan, Figure 2c) showed only a little increase in G’ and G” with reaction time. In addition, the G” values were always higher than G' throughout the entire reaction period (4h) showing there was also no hydrogel formed in the circumstance of no chitosan. It was proposed, the role of chitosan is to provide cores for synthetic polymer chains to graft so as to form big graft copolymer macromolecules with many synthetic side chains able to interact with neighboring macromolecules via multiple H-bonding etc. For control 3 (no AM, Figure 2d), both G’ and G” did not show noticeable increase with reaction time. In addition, the G’ and G” became very closer to each other, but no crossover throughout the entire reaction period (4h) showing the starting reaction solution became a paste-like mixture. From these findings, it was concluded that the role of AM was to form synthetic side chains of chitosan graft copolymer. The side chains should be sufficiently long and flexible, so as to make the chitosan graft copolymer macromolecules efficiently interact with each other for stable network formation. In another hand, the existence of PAM would determine the mobility of the networks in this system. The long and flexible PAM side chains would make the functional groups accessible to each other across the cut and rejoined interfaces of the hydrogel for an efficient re-formation of non-covalent interactions.54 Through this test, it can be seen that each component is indispensable to the hydrogel formation. The fundamental differences in rheological properties between these controls and the representative hydrogel formulation clearly indicate that the formation of graft copolymer during in situ polymerization and the formation of multiple non-covalent networks are essential for the formation of physical hydrogel in this work. 3.2. Effect of Polymerization Conditions and Formulation Compositions. The effect of various parameters, including polymerization conditions and formulation compositions on hydrogel’s stretchability, self-healing ability as well as transparency was investigated. The results were listed in Table S1 (see Supporting Information). The hydrogel’s stretchability was represented by the percentage elongation at break of the original hydrogel sample. The 10 ACS Paragon Plus Environment

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self-healing efficiency was evaluated by comparing the elongation ratios at break of the original hydrogel samples and healed hydrogel samples. The mechanical properties of some hydrogels were further examined by using dynamic oscillatory rheology (see Figure S3b and Figure 3). It was found that mild polymerization temperature (optimal 30

oC),

lower feeding

concentration of AA (optimal 0.056 mol·L-1), and lower initiator concentration (optimal APS 0.00296 mol·L-1 with fixed TEMED 0.0167 mol·L-1) contributed to the formation of a hydrogel with good balance of self-healing ability and mechanical properties. By properly controlling the polymerization temperature (Figure S2, Supporting Information) and carefully adjusting the initiator concentration (Figure 3 and Figure S4), the possible radical coupling termination between growing chitosan graft copolymer macromolecules could be avoided. Radical coupling termination could cause covalent crosslinking of chitosan graft copolymers compromising the reversibility of polymer network, and consequently reduce hydrogel’s selfhealing ability. By following same formulation, the hydrogel formed at 40 oC (Table S1, entry 2) has the maximum elongation ratio () of ca. 182%, which was much lower than that of hydrogel formed at 30 oC ( ca. 274%) (Table S1, entry 3). At the same time, the healing efficiency of the hydrogel formed at 40 oC (f ca. 13%) is only one-third of the hydrogel formed at 30 oC (f ca. 36%). Monomer AA played a key role for the construction of a stable network during the in situ polymerization. Increasing the feeding amount of AA could introduce more bonding sites between the synthetic polymer chains and chitosan backbones via the electrostatic interactions, contributing to both self-healing ability and mechanical properties. To investigate more deeply the effect of AA on this self-healing hydrogel system, a series of hydrogels were prepared at 30 oC with varied initial AA/AM ratios in the range from 0 to 0.188 (see entry 3 and entries 5-8 in Table S1). For each formulation, the total monomer concentration was kept around 0.95 mol·L-1 to ensure the prepared hydrogels by different formulations had similar solid content for comparison. It was found, the hydrogel’s selfhealing ability and rheological properties varied significantly with AA/AM ratio (Figure S3). At AA/AM of 0 (i.e. without AA), the resultant sample showed a healing efficiency f near 50%. The sample was a very weak hydrogel with G’ around 60 Pa and Tan  over 0.5. There could be chemical crosslinking occurred partially in this case. With the increase of AA/AM to ca. 0.06, the healing efficiency showed a drop of 10% (from ca. 50% to 40%). However, the G’ of the sample significantly increased from ca. 60 Pa to ca. 2100 Pa. This observation 11 ACS Paragon Plus Environment


clearly revealed the effect of AA on the improvement of hydrogel’s mechanical property. When AA/AM further increased to ca. 0.09, the sample’s healing efficiency decreased another 3%, while the G’ further increased ca. 300 Pa. Noteworthily, upon further increase of AA/AM from ca. 0.09 to ca. 0.13, the sample’s self-healing efficiency quickly dropped down to almost 0 (Figure S3, a), while the sample’s G’ did not change obviously (Figure S3, b). The possible reason is that strong electrostatic interactions under this case compromised the flexibility and reversibility of polymer network. When the AA/Am reached 0.19, the sample’s healing efficiency was still 0. However, the G’ showed a noticeable drop. On the contrary, both the G” and Tan  increased a little. The possible reason is that in this case, too much negatively charged –[AA]- unit was incorporated into synthetic polymer chains, which caused electrostatic repulsion between synthetic polymer chains. As a result, the strength of the hydrogel sample decreased. Another interesting observation is that the transparency of the final hydrogel varied with the feeding amount of AA. At lower AA concentration (< 0.076 mol·L-1), the hydrogel was transparent (entries 7 and 8, Table S1). When the AA concentration reached 0.076 mol·L-1, the hydrogel became a little turbid (entry 3, Table S1). When the AA concentration further increased to 0.112 mol·L-1 and above, the hydrogel turned very turbid and lost its transparency (entries 5 and 6, Table S1). This phenomenon was associated with the complexation of synthetic polymer chains with chitosan backbone through electrostatic interactions. To further investigate the effect of initial APS concentrations on the properties of the formed hydrogel, the rheological experiments were carried out for the samples of entries 9-14 in Table S1. For comparison, a series of control hydrogel samples were also prepared using identical formulations and conditions to entries 9-14 in Table S1 but without AA. Herein the APS (Max) represented the maximum APS concentration of 0.0148 mol·L-1 in this study. The magnitudes of G’ and G”, and the elasticity loss factor Tan δ (Tan δ = G”/G', i.e. the viscous portion to the elastic portion) at constant strain, frequency and temperature were measured. In ideal case, if there was no occurrence of chemical crosslinking during in situ polymerization, the control samples (without AA) should always have G” higher than G’ (i.e. Tan δ > 1.0). While the normal samples (with AA) should always have G” less than G’ (i.e. Tan δ < 1.0). By observing and comparing the variations of G’, G” and Tan of both normal hydrogel samples and control hydrogel samples, the possible network structure in the resultant hydrogel and the occurrence of the chemical crosslinking during the in situ polymerization can be inferred (Figure 3). It can be seen that the control sample’s Tan δ was 12 ACS Paragon Plus Environment

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always higher than that of normal sample in this study. However, under higher APS concentrations, the control samples also formed hydrogels with Tan δ < 1.0 indicating there was chemical crosslinking happened in the polymerization system in the case of higher APS concentrations. With reducing APS, both normal sample’s and control sample’s Tan δ increased. However, normal sample’s Tan δ was always < 1.0. By comparison, at APS/APS (Max) of 20, the Tan δ of control sample was up to 0.9 showing the possible chemical crosslinking was greatly suppressed under this case. Further, at APS/APS (Max) of 15, the Tan δ of control sample was up to 1.1 showing there was no chemical crosslinking in the system. Sample G' Sample G" Control G' Control G"

G', G" (Pa)

a) 3

10

1

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20

40

b) 1.0 Tan 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80

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100

APS /APS (Max) (%)

Figure 3. Comparison of the rheological properties of resultant hydrogels prepared by formulations with AA (normal samples, entries 9-14 in Table S1) and without AA (as controls) with the variation of initial APS concentrations. (a) The changes of G’ and G” with APS. (b) The chnges of Tan with APS. Measurement conditions: 1% of strain, 10 rad/s, 25 oC.

Each point represents the mean value ± SD. The X-axis represents the ratio of APS

concentration to the maximum APS concentration of 0.0148 mol·L-1. 3.3. Study on the Properties of Representative Self-Healing Hydrogel. The hydrogel prepared using a formulation containing 2.25 wt% of chitosan, 0.056 mol·L-1 of AA, 0.893 mol·L-1 of AM, 0.00296 mol·L-1 of APS and 0.0167 mol·L-1 of TEMED at 30 oC (Table S1, entry 13) was selected as representative for further study. The hydrogel could withstand stretching up to 625% of its original length before the mechanical failure, and the self-healing 13 ACS Paragon Plus Environment


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efficiency was around 88% based on uniaxial elongation measurement. Different cutting surfaces of this hydrogel also can effectively heal together. Figure 4 demonstrated the selfhealing ability qualitatively by using four different pieces of cut hydrogels. These hydrogels were prepared individually by same formulation at same conditions. When cut and brought the hydrogel segments into contact, they healed well into one in 1 hour. In another way, a freshly formed hydrogel was cut into multiple pieces first. When these pieces were brought into contact, they still healed into one in 1 hour. The ability of healing multiple hydrogel segments into one may enable this hydrogel system finding applications in soft actuators and robotic devices.

a)

b)

c)

Figure 4. Self-healing of individual hydrogel segments coming from parallel synthesis. (a) Four individual hydrogel segments (diameter 1 cm and length 2 cm; three of them were stained with dyes of methyl blue, methyl orange and rhodamine B, respectively). (b) Two segments were brought into contact for self-healing. (c) Four healed segments were arranged into a U shape. The property of the representative hydrogel was quantitatively examined by dynamic rheology measurements. A strain sweep measurement was performed to analyze the storage modulus (G’) and loss modulus (G”) of the hydrogel as a function of the oscillatory strain amplitude (Figure 5a). It can be seen, in the test range (strain 0.1 % to 300 %), the G’ values were always larger then G” values indicating elastic feature. This hydrogel also gave a steady G’, G” values in a range of strain (0.1% to 12%) at a constant oscillatory frequency of 10 rad/s. (Figure 5a). This was the linear viscoelastic region (LVR) for this hydrogel system under the test conditions. When strain > 12%, the G’ value decreased rapidly, and became closer to G” value with the increasing strain. This indicated the partial collapse of the hydrogel network under large deformation. After stopping stain, the hydrogel was found to recover its G’ and G” almost instantaneously (note: the time resolution of this measurement is ca. 6 s). Also, the recovered G’ and G” values were almost same to those within LVR. This test showed that the network can be recovered rapidly and completely in this hydrogel system


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after collapsed. This self-healing ability is attributed to reversibility of the non-covalently crosslinked network. The repeated recovery property of this hydrogel was investigated by largely straining the hydrogel (strain 300%) for 200 s and afterword monitoring the recovery at low strain (1%) for 150 s at frequency 10 rad/s (25 oC). It was found that the hydrogel system in this work can withstand several disruption-recovery cycles (at least 3) without changing its mechanical properties (Figure 5b). The robustness of this hydrogel system is attributed not only to the non-covalent crosslinking nature of the network but also the synergistic cooperation of multiple non-covalent interactions in one system. This ability could allow this hydrogel system to find application in the places where continuous motion and deformation will occur. Tensile tests were also performed to investigate the hydrogel’s self-healing ability. For comparison, the original hydrogel and self-healed hydrogel were tested under same conditions. It was found that the hydrogel’s strain at break and stress at break can be recovered up to 82% and 78%, respectively after self-healing (Figures S6 and S7), which is consistent with the elongation experimental result (f ca. 88%).

a)

700

700 G' G"

600

G', G" (Pa)

G' G"

500

500

400

400

300

300

200

200

100

100

0.1

1

10

100

0

50

100

200

250

Filled symbols: G' Open symbols: G"

Strain: 1% Tan  

1000

150

Strain: 300% Tan  

100

0

200

300

Step time (s)

Oscillation strain (%)

b)

600

LVR

G', G" (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


400

600 800 Step time (s)

1000

100

1000


1200


Figure 5. (a) Hydrogel network recovery test by shearing a hydrogel at increasing strain from 0.1 % until 300 % at frequency 10 rad/s, and after which the recovery was monitored with time at low strain 1.0 % and frequency 10 rad/s (25 oC). (b) Repetitive disruption and recovery test by subjecting the hydrogel to alterative small oscillatory deformation (1.0 % strain) and large oscillatory deformation (300 % strain) at frequency 10 rad/s (25 oC). 3.4. Purification, Regeneration and Dissolution of Representative Self-Healing Hydrogel. The representative hydrogel prepared under the conditions in this work still contains a little amount of residual monomer (typically, monomer conversion ca. 94.6%, residual AM content ca. 0.35 wt.%). If required, the hydrogel can be purified by successively soaking with ethanol-H2O (1/1, v/v), pure H2O and sodium acetate buffer (0.1 M, pH 4.5) at room temperature (Figure S8). The method was safe and effective. After treated, the residual AM content can be greatly reduced allowing this hydrogel to pass the FDA regulation in high chance. The shape and apperance of the hydrogel can be recovered after the purification and regeneration process. Importantly, the hydrogel can still keep its self-healing and mechanical properties. This hydrogel system can also be totally dissolved in dilute acetic acid or hydrocloric acid aqueous solution under mild conditions (Table S3). This is another evidence that this hydrogel system is only physically crosslinked in nature. Such ability will contribute to the fast degradation of the hydrogel system and make the recycle after usage to be easier. 3.5. The Bulk Structure and Macromolecular Structure of Representative Self-Healing Hydrogel. The bulk structure of the chitosan-based self-healing hydrogel was investigated using a JEOL FESEM. Before examination, the hydrogel samples were freeze-dried. After that, the dried samples were cut to expose their inner structure, and then the cross-section was observed. SEM images revealed highly porous microstructure of dried hydrogel samples (Figure 6, a-1 to a-3). These pores were interconnected and the inner wall surface of the pores was smooth. The pore size was around 20 - 40 m in diameter. The freeze-dried hydrogel sample was further examined by NMR spectroscopy. For this test, the freeze-dried hydrogel sample was cut into small pieces, and then stirred in D2O containing DCl (0.10 M) at 40 oC until a clear solution was formed. The 1H NMR experiment was then carried out at 25 oC. For the representative hydrogel prepared in this work, the signals at  3.0 – 4.0 ppm and  1.25 – 2.5 ppm were attributed to the protons of chitosan and synthesized polymer chain, respectively (Figure 6b, upper). There were resonance signals at  5.5 – 6.25 ppm which were due to residual monomers. The resonance signals at  2.90 and 3.56 ppm were due to 16 ACS Paragon Plus Environment

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TEMED (the accelerator for initiator APS). The purified and freeze-dried hydrogel sample was examined by NMR in the same way. As expected, the impurities (residual monomers and TEMED) had been removed completely after purification, while the resonance peaks due to the protons of chitosan and synthesized polymer chain did not show noticeable change.

(1)

(3)

(2)

a) 40 m

20 m

Signals due to TEMED Signals due to synthetic polymer chains

Signals due to residual monomers

b)

10 m

Signals due to CS

ppm

Figure 6. (a) Typical SEM images with different magnification of the crosssection morphological structure of freeze-dried self-healing hydrogel (a-1: 250; a-2: 500; a-3: 1000). (b) 1H NMR spectra of freeze-dried self-healing hydrogel in D2O containing DCl (0.10 M) at 25 oC before (upper) and after (lower) purification, respectively.

4. CONCLUSION In summary, we have successfully developed a facile method to design and fabricate a chitosan-based self-healing hydrogel by using in situ polymerization technique. The on-site formation of chitosan graft copolymer, synergistic multiple non-covalent interactions, and only non-covalently crosslinked network are main features for this hydrogel system. The fresh hydrogel possessed high water content (up to 90%), and showed rapid network recovery 17 ACS Paragon Plus Environment


(within 6 s), high self-healing efficiency (up to 88%), and high stretchability (elongation ratio at break up to 625%). The hydrogel can withstand disruption-recovery cycle for several times. To date, chitosan-based hydrogels with high water content and outstanding self-healing ability is seldom reported. Such self-healing hydrogel system, based on synthetic-natural polymers, represents a new kind of adaptive, biomimetic soft material, which may have the potential to link living and artificial systems, and allow the development of biologically inspired soft devices in ways that are not possible with other synthetic or natural soft materials. In the next step, it is worthy to explore the integration of this self-healing hydrogel systems with other components to gain multiple functionalities such as stimuliresponsiveness, sensing and conductivity for hitherto unimagined applications.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: Calibration curve for monomer conversion measurement; Polymerization mechanism (Scheme S1); Characterization results (Table S1); Effect of the polymerization temperature, acrylic acid and APS on the properties of hydrogel; Characterization of representative hydrogel samples (PDF) AUTHOR INFORMATION Corresponding Authors * (Z.-X. Zhang) E-mail: [email protected]. * (X. J. Loh) E-mail: [email protected]. ORCID X. J. Loh: 0000-0001-8118-6502 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This study was supported by IAF-PP (HMBS Domain) grant H17/01/a0/013 (OrBID): OculaR BIomaterials and Device.


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TOC G’

Self-healing hydrogel formation

G”

Chitosan NH3 O O

H

N H O

O H N

O H H N N H H O

Monomers

In situ polymerization

Non-covalent networking


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Autonomous Chitosan-Based Self-Healing Hydrogel Formed through

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